Interleukin-2 proprotein and application thereof

Abstract

The present disclosure provides a proIL2 protein comprising an IL2 moiety masked with an IL2R [alpha] moiety and a protease cleavable linker, the proIL2 protein configured such that the IL2R [alpha] moiety is released from the IL2 moiety under the action of a protease, e.g., at a tumor site. The IL2 proprotein optionally further comprises a targeting moiety, e.g., a targeting moiety that recognizes a tumor-associated antigen and directs the proprotein to a tumor site. The disclosure further provides pharmaceutical compositions comprising the IL2 Protein and methods of using the IL2 Protein in therapy, as well as nucleic acids encoding the IL2 Protein, recombinant cells expressing the IL2 Protein, and methods of producing the IL2 Protein.

Description

[0001] 1. Cross-reference to related applications

[0002] This application claims priority to the following U.S. provisional application numbers: U.S. Provisional Application No. 63 / 349,079 filed on June 4, 2022; U.S. Provisional Application No. 63 / 355,382 filed on June 24, 2022; U.S. Provisional Application No. 63 / 387,006 filed on December 12, 2022; U.S. Provisional Application No. 63 / 481,096 filed on January 23, 2023; U.S. Provisional Application No. 63 / 493,551 filed on March 31, 2023; U.S. Patent No. 63 / 500,997 filed on May 9, 2023, the contents of each of which are incorporated herein by reference in their entirety.

[0003] 2. Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically and is hereby incorporated by reference in its entirety. The copy created on May 31, 2023 is named RGN-023WO_SL.xml and is 332,645 bytes in size. Background Art

[0005] Interleukin 2 (IL-2 or IL2) is a multipotent cytokine produced mainly by CD4+ 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, promotes 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 Waldmann, 2009, Nat Rev Immunol 6:595-601 and Malek, 2008, Annu Rev Immunol 26:453-79).

[0006] Because of its pleiotropic effects, IL2 is not optimal for inhibiting tumor growth. The use of IL2 as an antineoplastic agent is limited by the severe toxicity associated with the doses required for tumor responses. Proleukin® (marketed by Prometheus Laboratories, San Diego, Calif.), a recombinant form of IL2, has been approved for the treatment of metastatic melanoma and metastatic renal cancer, but its side effects are so severe that its use is recommended only in hospital settings that provide intensive care. Patients treated with high-dose IL2 often experience severe cardiovascular, pulmonary, renal, hepatic, gastrointestinal, neurological, skin, hematological, and systemic adverse events that require close monitoring and hospitalization. The major side effect of IL2 therapy is vascular leak syndrome (VLS), which causes interstitial fluid to accumulate in the lungs and liver, leading to pulmonary edema and liver damage. There is no treatment for VLS other than discontinuation of IL2. Low-dose IL2 regimens have been tested in patients to avoid VLS, but at the expense of poor treatment outcomes. It has been shown that IL2-induced pulmonary edema results from direct binding of IL2 to lung endothelial cells, which express low to moderate levels of functional high-affinity IL2 receptors (Krieg et al., 2010, Proc Nat Acad Sci USA 107: 11906-11).

[0007] In order to reduce the toxicity of IL2 cancer therapy, a variety of IL2 variants and prodrugs have been generated. However, it is surprising to find that such molecules have a poor therapeutic index for cancer therapy. For example, the PEGylated IL2 prodrug bempegaldesleukin failed to improve the therapeutic effect of PD1 checkpoint inhibitors on melanoma patients in a phase 3 clinical study (Mullard, 2022, Nature Reviews Drug Discovery 21:327 (doi: https: / / doi.org / 10.1038 / d41573-022-00069-3).

[0008] Therefore, there is a need in the art for new IL2 therapeutics with improved therapeutic efficacy and safety profiles. Summary of the invention

[0009] The present disclosure relates to IL2 proproteins that are activated by proteases, such as proteases expressed in a tumor environment.

[0010] The IL2 proprotein comprises an IL2 portion masked by an IL2Rα portion, the IL2 proprotein being configured such that the mask is released upon cleavage by a protease. The IL2 proprotein preferably further comprises a targeting moiety that directs the IL2 proprotein to a specific tissue or cell type.

[0011] In certain embodiments, the IL2 proprotein of the present disclosure comprises two polypeptide chains, each polypeptide chain comprising an Fc domain, a first linker that may be cleavable or non-cleavable, an IL2 portion, a second linker that may be cleavable by a protease, and an IL2Rα portion from the N-terminus to the C-terminus. The IL2 proprotein may further comprise a targeting moiety (or a component thereof, e.g., one chain of Fab) at the N-terminus of, for example, one or both Fc domains. The targeting moiety comprises an antigen binding domain ("ABD"), which antigen binding site may, for example, bind to a target molecule (e.g., a tumor-associated antigen) present on the surface of a tumor or other components (e.g., extracellular matrix) ("ECM") or tumor lymphocytes) in the tumor microenvironment.

[0012] Exemplary IL2 portions that can be used in the IL2 proproteins of the present disclosure are described in Section 6.3.

[0013] Exemplary IL2 portions that can be used in the IL2Rα proproteins of the present disclosure are described in Section 6.4.

[0014] Protease-cleavable linkers useful in the IL2 proproteins of the present disclosure are described in Section 6.5.

[0015] Non-cleavable linkers useful in the IL2 proproteins of the present disclosure are described in Section 6.6.

[0016] Targeting moieties useful in the pro-IL2 proteins of the present disclosure are described in Section 6.7 and targeting moiety formats are disclosed in Section 6.8.

[0017] Fc domains that may be incorporated into the pro-IL2 proteins of the present disclosure are described in Section 6.9.

[0018] Exemplary IL2 proproteins of the present disclosure are described in Section 6.2 and numbered Examples 1-165.

[0019] The present disclosure further provides nucleic acids encoding the IL2 proprotein of the present disclosure. The nucleic acids encoding the IL2 proprotein can be single nucleic acids (e.g., vectors encoding all polypeptide chains of the IL2 proprotein) or multiple nucleic acids (e.g., two or more vectors encoding different polypeptide chains of the IL2 proprotein). The present disclosure further provides host cells and cell lines engineered to express the nucleic acids and IL2 proprotein of the present disclosure. The present disclosure further provides methods for producing the IL2 proprotein of the present disclosure. Exemplary nucleic acids, host cells and cell lines, and methods for producing the IL2 proprotein are described in Section 6.10 and Numbered Examples 166 to 168.

[0020] The present disclosure further provides pharmaceutical compositions comprising the IL2 proprotein of the present disclosure.Exemplary pharmaceutical compositions are described in Section 6.11 and Numbered Example 169.

[0021] Further provided herein are methods of using the IL2 proproteins and pharmaceutical compositions disclosed herein, for example, to treat cancer. Exemplary methods are described in Section 6.12 and in numbered Examples 170-208. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1A to Figure 1B . Figure 1A is a schematic representation of an exemplary targeted IL2 proprotein comprising four protease cleavable linkers. Figure 1A The targeting moiety in is shown as a Fab, but the targeting moiety can be in other formats, for example, scFv or other formats described in Section 6.8. Figure 1A-1 to Figure 1A-4 Shown are close-up views of embodiments of linkers A, B, C, and D, which contain spacers on either side of a cleavable substrate (A1, A2, B1, B2, C1, C2, and D1, D2, respectively). The number of substrate and spacer sequences is for illustrative purposes only, and it is expected that protease cleavable linkers will typically have multiple substrate and spacer sequences as described in Section 6.5. Figure 1B Shown according to Figure 1A An exemplary mechanism of targeting activation of pro-IL2 protein for which the targeting moiety binds to a TAA. Targeting moieties that bind to other targets as disclosed herein may be used.

[0023] FIG. 2A to FIG. 2D . Figure 2A is a schematic representation of an exemplary targeted IL2 proprotein comprising two protease cleavable linkers. Figure 2A The targeting moiety in is shown as a Fab, but the targeting moiety can be in other formats, for example, scFv or other formats described in Section 6.8. Figure 2A-1 and Figure 2A-2 Shown are close-up views of embodiments of linkers B and D, which include spacers (B1, B2 and D1, D2, respectively) on either side of the cleavable substrate. The number of substrate and spacer sequences is for illustrative purposes only, and it is expected that protease cleavable linkers will typically have multiple substrate and spacer sequences as described in Section 6.5. FIG. 2B to FIG. 2D Shown according to Figure 2A An exemplary mechanism of activation of a targeted IL2 proprotein is described, for which the targeting moiety is bound to TCA. The resulting activated IL2 protein can bind to an IL2 receptor on a T cell (e.g., Figure 2B ), which binds to TCA on T cells via one or two targeting moieties (e.g. Figure 2C ), or can simultaneously bind to the IL2 receptor on a T cell via one or two IL2 moieties and to TCA on the same T cell via one or two targeting moieties (e.g. Figure 2D Targeting moieties that bind to other targets as disclosed herein may be used.

[0024] FIG. 3A to FIG. 3E The in vivo antitumor efficacy of EGFR-targeted or non-targeted IL2 proproteins containing cleavable and non-cleavable linkers is shown. Figure 3A is a graph comparing the mean anti-tumor efficacy of EGFR-targeted pro-IL2 protein, non-targeted pro-IL2 protein, and isotype control. FIG. 3B to FIG. 3E Each graph in Figure 2 shows the anti-tumor efficacy of a single IL2 protein or control in individual mice.

[0025] 4A to 4D The in vivo antitumor efficacy of PD1 -targeted or non-targeted IL2 proproteins containing cleavable and non-cleavable linkers is shown. Figure 4A is a graph comparing the mean anti-tumor efficacy of PD1-targeted pro-IL2 protein, non-targeted pro-IL2 protein, and isotype control. FIG. 4B to FIG. 4D Each graph in Figure 2 shows the anti-tumor efficacy of a single IL2 protein or control in individual mice.

[0026] FIG. 5A to FIG. 5B An exemplary Western blot showing uPA-digested and undigested IL2 protein original samples is shown. Figure 5A Is such as Figure 5B Western blot images of samples loaded as indicated.

[0027] 6A to 6F Shown are the in vitro activities of tumor-targeted IL2 proproteins containing protease cleavable and non-cleavable linkers in engineered CD25 KO / PD1 KO YT / STAT5-Luc reporter cells. FIG. 6A to FIG. 6D Graphs showing luciferase activity associated with CA9 targeting of IL2 proprotein, wherein each graph shows the activity of IL2 proprotein comprising a different CD9 targeting moiety ( Fig. 6A - aCD9(Ab1), Figure 6B - aCD9(Ab2), Figure 6C - aCD9 (Ab3) and Fig.6D - aCD9(Ab4)). Fig. 6E and Fig. 6F Luciferase activities associated with EGFR-targeted and PD1-targeted IL2 proproteins are shown, respectively.

[0028] 7A to 7FShown are the in vitro activities of tumor-targeted IL2 proproteins containing protease cleavable and non-cleavable linkers in engineered CD25 OE / PD1 KO YT / STAT5-Luc reporter cells. FIG. 7A to FIG. 7D are graphs showing luciferase activity associated with CD9-targeted IL2 proproteins, wherein each graph shows the activity of IL2 proproteins comprising a different CD9-targeting moiety ( Fig. 7A - aCD9(Ab1), Figure 7B - aCD9(Ab2), Figure 7C - aCD9(Ab3), Fig.7D - aCD9(Ab4)). Fig. 7E and Figure 7F Luciferase activities associated with EGFR-targeted and PD1-targeted IL2 proproteins are shown, respectively.

[0029] FIG. 8A to FIG. 8D Shown is the in vitro activity of PD1 -targeted IL2 proproteins comprising non-cleavable linkers of varying lengths. Fig. 8A It is a graph showing the activity of IL2 proprotein in PD1 OE / CD25 KO YT / STAT5-Luc reporter cells. Figure 8B It is a graph showing the activity of IL2 proprotein in PD1 KO / CD25 KO YT / STAT5-Luc reporter cells. Figure 8C It is a graph showing the activity of IL2 proprotein in PD1 OE / CD25 OE YT / STAT5-Luc reporter cells. Fig.8D It is a graph showing the activity of IL2 proprotein in PD1 KO / CD25 OE YT / STAT5-Luc reporter cells. DETAILED DESCRIPTION

[0030] 6.1. Definitions

[0031] As used herein, the following terms have the following meanings:

[0032] ABD chain, targeting moiety chain: The targeting moiety and the antigen binding site (ABD) therein can exist as one (e.g., in the case of scFv or scFab) polypeptide chain or be formed by the association of more than one polypeptide chain (e.g., in the case of Fab or Fv). As used herein, the terms "ABD chain" and "targeting moiety chain" refer to all or part of an ABD or targeting moiety present on a single polypeptide chain. The use of the terms "ABD chain" or "targeting moiety chain" is intended for convenience and descriptive purposes only and does not imply a particular configuration or method of production. In addition, reference to an ABD or targeting moiety when describing a pro-IL2 protein encompasses an ABD chain or targeting moiety chain unless the context indicates otherwise. Thus, when describing a pro-IL2 protein in which an Fc domain is operably linked to a targeting moiety, the Fc domain can be covalently linked, for example, via a peptide bond, directly or indirectly (e.g., via a linker) to, for example, (1) a first ABD or targeting moiety chain of a Fab or Fv (wherein the other components of the Fab or Fv are on a second associated ABD or targeting moiety chain) or (2) an ABD or targeting moiety chain containing a scFv or scFab.

[0033] About, approximately: Throughout the specification, the terms "about," "approximately," and the like are used in front of a number to indicate that the number is not necessarily exact (e.g., to account for fractions, variations in measurement accuracy and / or precision, timing, etc.). It should be understood that disclosing "about X" or "approximately X" where X is a number also discloses "X." Thus, for example, disclosing an embodiment in which a sequence has "about X% sequence identity" to another sequence also discloses an embodiment in which the sequence has "X% sequence identity" to the other sequence.

[0034] Activate (activation): The terms "activation" and the like as used in connection with the IL2 proprotein of the present disclosure refer to the protease-mediated enzymatic cleavage of a protease-cleavable linker which results in the unmasking or release of the IL2 portion from the IL2Rα portion.

[0035] And, or: Unless otherwise stated, the conjunction "or" shall be used in its proper sense as a Boolean logical operator, covering both the selection of features among alternatives (A or B, where the selection of A is mutually exclusive with B) and the selection of features in conjunction (A or B, where both A and B are selected). In some places in this text, the term "and / or" is used for the same purpose, which should not be interpreted as implying the use of "or" involving mutually exclusive alternatives.

[0036] Antibody: As used herein, the term "antibody" refers to a polypeptide (or polypeptide group) of the immunoglobulin family that can non-covalently, reversibly and specifically bind to an antigen. For example, a naturally occurring "antibody" of the IgG type is a tetramer comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises a domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, bispecific or multispecific antibodies, and anti-idiotype (anti-id) antibodies. Antibodies can be any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Both light and heavy chains are divided into structural and functional homologous regions. The terms "constant" and "variable" are used functionally. In this regard, it should be understood that the variable domains of both the light chain (VL) and heavy chain (VH) parts determine antigen recognition and specificity. In contrast, the constant domains of the light chain (CL) and heavy chain (CH1, CH2 or CH3) confer important biological properties, such as secretion, transplacental mobility, Fc receptor binding, complement fixation, etc. By convention, the numbering of the constant region domains increases as they become more distant from the antigen binding domain or amino terminus of the antibody. The N-terminus is the variable region, and at the C-terminus is the constant region; the CH3 and CL domains represent the carboxyl termini of the heavy and light chains of natural antibodies, respectively. For convenience, and unless the context dictates otherwise, reference to antibodies also refers to antibody fragments and engineered antibodies that include non-naturally occurring antigen binding domains and / or antigen binding domains with non-natural structures.

[0037] Antigen binding domain: As used herein, the term "antigen binding domain" or "ABD" refers to a portion of an antibody or antibody fragment (e.g., a targeting moiety) that has the ability to non-covalently, reversibly, and specifically bind to an antigen. Examples of antibody fragments that may contain an ABD include, but are not limited to, single-chain Fv (scFv), Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab)2 fragments, bivalent fragments comprising two Fab fragments connected by a disulfide bridge at the hinge region; Fd fragments, which consist of VH and CH1 domains; Fv fragments, which consist of the VL and VH domains of a single arm of an antibody; dAb fragments (Ward et al., 1989, Nature 341:544-546), which consist of a VH domain; and isolated complementary determining regions (CDRs). Therefore, the term "antibody fragment" encompasses proteolytic fragments of antibodies (e.g., Fab and F(ab)2 fragments) and engineered proteins (e.g., scFv) comprising one or more portions of an antibody. Antibody fragments may also be incorporated into single domain antibodies, macrobodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs and bis-scFvs (see, eg, Hollinger and Hudson, 2005, Nature Biotechnology 23: 1126-1136).

[0038] Association: In the context of a pro-IL2 protein, the term "association" refers to a functional relationship between two or more polypeptide chains. In particular, the term "association" means that two or more polypeptides associate with each other, for example, non-covalently through molecular interactions or covalently through one or more disulfide bridges or chemical cross-links, so as to produce a functional pro-IL2 protein. Examples of associations that may be present in a pro-IL2 protein of the present disclosure include, but are not limited to, associations between Fc domains to form an Fc region (such as homodimers or heterodimers described in Section 6.9), associations between VH and VL regions in Fab or Fv, and associations between CH1 and CL in Fab.

[0039] Cancer: The term "cancer" refers to a disease characterized by the uncontrolled (and usually rapid) growth of abnormal cells. Cancer cells can spread locally or to other parts of the body through the bloodstream and lymphatic system. Examples of various cancers are described herein, including but not limited to breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, adrenal cancer, , ganglion cancer, bile duct cancer, bone cancer, endometrial cancer, eye cancer, fallopian tube cancer, reproductive tract cancer, colorectal cancer, meningeal cancer, esophageal cancer, peritoneal cancer, pituitary cancer, penis cancer, placental cancer, pleural cancer, salivary gland cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, upper respiratory tract digestive system cancer, urinary tract cancer, vaginal cancer, vulvar cancer, lymphoma, leukemia, lung cancer, etc., for example, any TAA-positive cancer of any of the aforementioned types.

[0040] Complementarity determining region: As used herein, the term "complementarity determining region" or "CDR" refers to the sequence of amino acids within an antibody variable region that confers antigen specificity and binding affinity. For example, typically, there are three CDRs in each heavy chain variable region (e.g., CDR-H1, CDR-H2, and CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, and CDR-L3). The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al., 1991, “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (the “Kabat” numbering scheme), Al-Lazikani et al., 1997, JMB 273:927-948 (the “Chothia” numbering scheme), and ImMunoGenTics (IMGT) numbering (Lefranc, 1999, The Immunologist 7:132-136; Lefranc et al., 2003, Dev. Comp. Immunol. 27:55-77 (the “IMGT” numbering scheme). For example, for the classical form, under Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (CDR-H1), 50-65 (CDR-H2), and 51-52 (CDR-H3). (CDR-H2) and 95-102 (CDR-H3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3). Under Chothia, the CDR amino acids in VH are numbered 26-32 (CDR-H1), 52-56 (CDR-H2), and 95-102 (CDR-H3); and the amino acid residues in VL are numbered 26-32 (CDR-L1), 50-52 (CDR-L2), and 91-96 (CDR-L3). By combining the CDR definitions of both Kabat and Chothia, the CDRs are defined by amino acid residues 26-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 in human VH. (CDR-H3) and amino acid residues 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3) in human VL.Under IMGT, the CDR amino acid residues in VH are numbered approximately 26-35 (CDR-H1), 51-57 (CDR-H2), and 93-102 (CDR-H3), and the CDR amino acid residues in VL are numbered approximately 27-32 (CDR-L1), 50-52 (CDR-L2), and 89-97 (CDR-L3) (according to "Kabat" numbering). Under IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0041] Effector function: The term "effector function" refers to the activity of an antibody molecule mediated by binding of domains other than the antigen binding domain in the antibody, typically mediated by the binding of an effector molecule. Effector functions include complement-mediated effector functions, which are mediated by, for example, the binding of the C1 component of complement to the antibody. Activation of complement is important in the opsonization and lysis of cellular pathogens. Activation of complement also stimulates inflammatory responses and may also be involved in autoimmune hypersensitivity reactions. Effector functions also include Fc receptor (FcR)-mediated effector functions, which can be triggered when the constant domain of an antibody is bound to an Fc receptor (FcR). Binding of antibodies to Fc receptors on the surface of cells triggers a number of important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (known as antibody-dependent cell-mediated cytotoxicity or ADCC), release of inflammatory mediators, control of placental transfer, and immunoglobulin production. The effector function of an antibody can be altered by changing (e.g., enhancing or reducing) the affinity of the antibody for effector molecules such as Fc receptors or complement components. Binding affinity is often altered by modifying the effector molecule binding site, and in this case, the site of interest is appropriately positioned and at least a portion of the site is modified in a suitable manner. It is also envisioned that changes in the binding site on an antibody for an effector molecule need not significantly change the overall binding affinity, but may change the geometry of the interaction, rendering the effector mechanism ineffective as in non-productive binding. It is further envisioned that the effector function can also be altered by modifying sites that are not directly involved in effector molecule binding but otherwise involved in the execution of the effector function.

[0042] Epitope: An epitope or antigenic determinant is a portion of an antigen that is recognized by an antibody or other antigen binding moiety as described herein. An epitope can be linear or conformational.

[0043] Fab: The term "Fab" refers to a pair of polypeptide chains, the first polypeptide chain comprising the variable heavy chain (VH) domain of the antibody, which is operably connected (usually at the N-terminus) to the first constant domain (referred to herein as C1), and the second polypeptide chain comprising the variable light chain (VL) domain at the N-terminus of the antibody, which is operably connected (usually at the N-terminus) to the second constant domain (referred to herein as C2) capable of pairing with the first constant domain. In natural antibodies, VH is at the N-terminus of the first constant domain (CH1) of the heavy chain, and VL is at the N-terminus of the light chain constant domain (CL). The Fab of the present disclosure may be arranged according to the natural orientation or include domain substitutions or exchanges that facilitate correct VH and VL pairing. For example, it is possible to replace the CH1 and CL domain pairs in Fab with a CH3 domain pair to promote correct modified Fab chain pairing in heterodimeric molecules. It is also possible to reverse CH1 and CL so that CH1 is connected to VL and CL is connected to VH, a configuration generally referred to as Crossmab. The term "Fab" encompasses single-chain Fab.

[0044] Fc domain and Fc region: The term "Fc domain" refers to the portion of a heavy chain that pairs with the corresponding portion of another heavy chain. The term "Fc region" refers to the region formed by the association of two heavy chain Fc domains. The two Fc domains within an Fc region can be identical to or different from each other. In native antibodies, the Fc domains are usually identical, but one or both Fc domains may be modified to allow heterodimerization, for example, via knob-and-hole interactions.

[0045] Fv: The term "Fv" refers to the smallest antibody fragment derived from an immunoglobulin that contains a complete target recognition and binding site. This region consists of a dimer consisting of one heavy chain variable domain and one light chain variable domain, which are in a tight non-covalent association (VH-VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define the target binding site on the surface of the VH-VL dimer. Typically, six CDRs confer target binding specificity to antibodies. However, in some cases, even a single variable domain (or half of an Fv containing only three target-specific CDRs) can have the ability to recognize and bind to a target. Reference to VH-VL dimers herein is not intended to express any particular configuration. When present on a single polypeptide chain (e.g., scFv), VH is located at the N-terminus or C-terminus of VL.

[0046] Half antibody: The term "half antibody" refers to a molecule that comprises at least one Fc domain and can associate with another molecule comprising Fc through, for example, a disulfide bridge or a molecular interaction. A half antibody can be composed of one polypeptide chain or more than one polypeptide chain (e.g., two polypeptide chains of Fab). An example of a half antibody is a molecule comprising a heavy chain and a light chain of an antibody (e.g., an IgG antibody). Another example of a half antibody is a molecule comprising a first polypeptide comprising a VL domain and a CL domain and a second polypeptide comprising a VH domain, a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain, wherein the VL and VH domains form an ABD. Another example of a half antibody is a polypeptide comprising a scFv domain, a CH2 domain, and a CH3 domain. The IL2 protein of the present disclosure generally comprises two half antibodies, each half antibody comprising an Fc domain, an IL2Rα portion at the C-terminus of the Fc domain, a protease cleavable linker at the C-terminus of the IL2Rα portion, and an IL2 portion at the C-terminus of the protease cleavable linker. One or both half antibodies of the IL2 proprotein may further comprise a targeting moiety, for example, at the N-terminus of the Fc domain.

[0047] The term "half antibody" is used for descriptive purposes only and does not imply a particular configuration or method of production. Describing half antibodies as the "first" half antibody, the "second" half antibody, the "left" half antibody, the "right" half antibody, etc. is for convenience and descriptive purposes only.

[0048] Host cell or recombinant host cell: The term "host cell" or "recombinant host cell" refers to a cell that has been genetically engineered, for example, by the introduction of a heterologous nucleic acid. It should be understood that such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be the same as the parent cell, but are still included within the scope of the term "host cell" as used herein. The host cell may carry the heterologous nucleic acid transiently, for example, on an extrachromosomal heterologous expression vector, or stably, for example, by integrating the heterologous nucleic acid into the host cell genome. For the purpose of expressing the IL2 protein progenitor of the present disclosure, the host cell is preferably a cell line of mammalian origin or mammalian-like characteristics, such as monkey kidney cells (COS, e.g., COS-1, COS-7), HEK293), baby hamster kidney (BHK, e.g., BHK21), Chinese hamster ovary (CHO), NSO, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2), SP2 / 0, HeLa, Madin-Darby bovine kidney (MDBK), myeloma and lymphoma cells or derivatives and / or engineered variants thereof. Engineered variants include, for example, derivatives that grow at a higher density than the original cell line and / or derivatives with modified glycan profiles and / or site-specific integration site derivatives.

[0049] Linker: As used herein, the term "linker" refers to a protease cleavable linker or a non-cleavable linker.

[0050] Non-cleavable linker: A non-cleavable linker refers to a peptide whose amino acid sequence lacks a substrate sequence for a protease (eg, a protease described in Section 6.5.1) that recognizes and cleaves a specific sequence motif (eg, a substrate described in Section 6.5.2).

[0051] Operably linked: The term "operably linked" refers to a functional relationship between two or more peptide or polypeptide domains or nucleic acid (e.g., DNA) segments. In the context of fusion proteins or other polypeptides, the term "operably linked" means that two or more amino acid fragments are linked to produce a functional polypeptide. For example, in the context of the IL2 proprotein of the present disclosure, the separate components (e.g., the Fc domain and the IL2Rα portion) can be operably linked directly or through a peptide linker sequence. In the context of nucleic acids encoding fusion proteins (such as half antibodies of the IL2 proprotein of the present disclosure), "operably linked" means that the two nucleic acids are linked so that the amino acid sequences encoded by the two nucleic acids remain in frame. In the context of transcriptional regulation, the term refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, if a promoter or enhancer sequence stimulates or regulates the transcription of a coding sequence in an appropriate host cell or other expression system, then the promoter or enhancer sequence is operably linked to a coding sequence.

[0052] Polypeptide, peptide and protein: The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues.

[0053] Proprotein: A "proprotein" is an inactive protein precursor and which can be activated by the proteolytic action of a protease. Thus, a proprotein is "protease-activatable."

[0054] Protease: As used herein, the term "protease" refers to any enzyme that catalyzes the hydrolysis of peptide bonds. In general, proteases useful in the present disclosure, e.g., the proteases described in Section 6.5.1, recognize and cleave specific sequence motifs, e.g., substrates described in Section 6.5.2. Preferably, the protease is expressed at higher levels in cancer tissues than in normal tissues.

[0055] Protease cleavable linker: As used herein, the term "protease cleavable linker" or "PCL" refers to a peptide whose amino acid sequence contains one or more (e.g., two, three or more) substrate sequences of one or more proteases. Exemplary protease cleavable linkers are described in Section 6.5 and exemplary protease cleavable linker sequences are disclosed in Section 6.5.4.

[0056] Recognition: As used herein, the term "recognition" refers to an antibody or antibody fragment (eg, targeting moiety) that finds and interacts with (eg, binds to) its epitope.

[0057] Single-chain Fab or scFab: As used herein, the term "single-chain Fab" or "scFab" refers to an ABD comprising a VH domain, a CH1 domain, a VL domain, a CL domain, and a linker. In some embodiments, the aforementioned domains and linkers are arranged in one of the following orders in the N-terminal to C-terminal orientation: (a) VH-CH1-linker-VL-CL, (b) VL-CL-linker-VH-CH1, (c) VH-CL-linker-VL-CH1 or (d) VL-CH1-linker-VH-CL. The linker is suitably a non-cleavable linker of at least 30 amino acids, preferably 32 to 50 amino acids. Single-chain Fab fragments are generally stabilized via a natural disulfide bond between the CL domain and the CH1 domain. In addition, these single-chain Fab molecules can be further stabilized by generating interchain disulfide bonds via the insertion of cysteine ​​residues (e.g., at position 44 in the VH domain and position 100 in the VL domain according to Kabat numbering).

[0058] Single-chain Fv or scFv: As used herein, the term "single-chain Fv" or "scFv" refers to an ABD comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, ed. (1994), Springer-Verlag, New York, pp. 269-315. VH and VL can be arranged in the order of N-terminus to C-terminus (i.e., VH-VL or VL-VH), usually separated by a linker, such as the linkers listed in Table E.

[0059] Spacer: As used herein, the term "spacer" refers to a peptide incorporated into a linker containing a substrate, the amino acid sequence of which is not a substrate for a protease. The spacer can be used to separate the substrate from other domains in the molecule (e.g., ABD). In some aspects, the residues in the spacer minimize the action of aminopeptidases and / or exopeptidases to prevent cleavage of the N-terminal amino acid.

[0060] Specific (or selective) binding: The term "specific (or selective) binding" to an antigen or epitope refers to a binding reaction that determines the presence of a homologous antigen or epitope in a heterogeneous population of proteins and other molecules. The binding reaction can, but need not, be mediated by an antibody or antibody fragment. The term "specific binding" does not exclude cross-species reactivity. For example, an antigen-binding domain (e.g., an antigen-binding fragment of an antibody) that "specifically binds" to an antigen from one species may also "specifically bind" to that antigen in one or more other species. Therefore, this cross-species reactivity itself does not change the classification of the antigen-binding domain as a "specific" binder. In certain embodiments, the antigen-binding domain of the present disclosure that specifically binds to a human antigen has cross-species reactivity with one or more non-human mammalian species (e.g., primate species (including but not limited to one or more of cynomolgus monkeys, macaques, and pig-tailed monkeys)) or rodents (e.g., house mice).

[0061] Subject: The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. In a preferred embodiment, the subject is a human.

[0062] Substrate: The term "substrate" refers to the peptide sequence upon which a protease will act and in which the protease will cleave peptide bonds.

[0063] Target molecule: As used herein, the term "target molecule" refers to any biological molecule (eg, protein, carbohydrate, lipid, or combination thereof) expressed on the cell surface or in the extracellular matrix to which a targeting moiety in a pro-IL2 protein of the present disclosure can specifically bind.

[0064] Targeting moiety: As used herein, the term "targeting moiety" refers to any molecule or binding moiety (e.g., an immunoglobulin or antigen binding fragment) that can bind to a cell surface or extracellular matrix molecule (e.g., on a tumor cell or on a lymphocyte in a tumor microenvironment) at the site to which the IL2 proprotein of the present disclosure is to be localized. In some embodiments, the targeting moiety binds to a TAA. In other embodiments, the targeting moiety binds to a TCA. In addition to localizing the IL2 proprotein to a specific site, the targeting moiety can also have functional activity. For example, a targeting moiety that binds to a checkpoint inhibitor such as PD1 can also exhibit anti-tumor activity or enhance the anti-tumor activity of IL2, for example by inhibiting PD1 signaling.

[0065] T cell antigen, TCA: The term "T cell antigen" or "TCA" refers to a molecule (usually a protein, carbohydrate, lipid, or some combination thereof) expressed on the surface of T lymphocytes and can be used to preferentially target an agent to a specific site. In some embodiments, the site is cancer tissue and / or the T cell antigen is a tumor reactive lymphocyte antigen, a cell surface molecule of a tumor or viral lymphocyte, or a checkpoint inhibitor expressed on a T lymphocyte.

[0066] Tumor: The term "tumor" is used interchangeably herein with the term "cancer", e.g., both terms encompass solid and liquid, e.g., diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" includes precancerous as well as malignant cancers and tumors.

[0067] Tumor-associated antigen, TAA: The term "tumor-associated antigen" or "TAA" refers to a molecule (typically a protein, carbohydrate, lipid, or some combination thereof) that is expressed on the surface of a cancer cell, either in its entirety or as a fragment (e.g., MHC / peptide), and which can be used to preferentially target an agent to a cancer cell. In some embodiments, a TAA is a marker expressed by both normal cells and cancer cells (e.g., a lineage marker). In some embodiments, a TAA is a cell surface molecule that is overexpressed in cancer cells compared to normal cells, e.g., 1-fold overexpression, 2-fold overexpression, 3-fold overexpression, or more compared to normal cells. In some embodiments, a TAA is a cell surface molecule that is inappropriately synthesized in a cancer cell, e.g., a molecule that contains a deletion, addition, or mutation compared to a molecule expressed on a normal cell. In some embodiments, a TAA will be expressed exclusively on the cell surface of a cancer cell, either in its entirety or as a fragment (e.g., MHC / peptide), and is not synthesized or expressed on the surface of a normal cell. Thus, the term "TAA" encompasses antigens that are specific to cancer cells, sometimes referred to in the art as tumor-specific antigens ("TSAs").

[0068] Treat (treat / treatment / treating): As used herein, the term "treat / treatment / treating" refers to reducing or ameliorating the progression, severity and / or duration of a proliferative disorder, or the improvement of one or more symptoms (preferably, one or more discernible symptoms) of a proliferative disorder caused by the administration of one or more IL2 pro-proteins of the present disclosure. In specific embodiments, the terms "treat, treatment and treating" refer to improving at least one measurable physical parameter of a proliferative disorder (such as the growth of a tumor) that is not necessarily discernible by the patient. In other embodiments, the term "treat" refers to inhibiting the progression of a proliferative disorder physically, for example, by stabilizing discernible symptoms, physiologically, for example, by stabilizing physical parameters, or both. In other embodiments, the terms "treat, treatment and treating" refer to a reduction or stabilization of tumor size or cancer cell count.

[0069] Universal light chain, ULC: As used herein, the term "universal light chain" or "ULC" refers to a light chain variable region (VL) that can be paired with more than one heavy chain variable region (VL). In the context of a targeting moiety, the term "universal light chain" or "ULC" refers to a light chain polypeptide that is capable of pairing with a heavy chain region of a targeting moiety and is also capable of pairing with other heavy chain regions. The ULC may also include a constant domain, such as the CL domain of an antibody. Universal light chains are also referred to as "common light chains."

[0070] VH: The term "VH" refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, dsFv or Fab.

[0071] VL: The term "VL" refers to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv, or Fab.

[0072] 6.2.IL2 Proprotein

[0073] The present disclosure relates to an IL2 proprotein comprising an IL2 portion, an IL2Rα portion and a protease cleavable linker, the IL2 proprotein being arranged so that IL2Rα reduces or blocks the activity of the IL2 portion. The IL2 proprotein is configured so that when encountering a protease (e.g., a protease overexpressed in a tumor environment), the protease cleavable linker is cleaved and IL2 is released and stimulates cytotoxic T cell activity against tumor cells. Typically, the IL2 proprotein of the present disclosure is a dimer and comprises two Fc domains that associate to form an Fc region, the C-terminus of a protease cleavable or non-cleavable linker, the IL2 portion, an additional linker that is protease cleavable, and an IL2Rα portion arranged in N-terminal to C-terminal order.

[0074] In some embodiments, the IL2 protein of the present disclosure generally comprises: (a) a first Fc domain and a second Fc domain that are capable of associating to form an Fc region; (b) two linkers located at the C-terminus of the Fc domain that may be cleavable or non-cleavable by a protease, as described in Figure 1A and Figure 2A (c) at the C-terminus of the first and third linkers are two (first and second) IL2 moieties; (c) at the C-terminus of the IL2 moieties are two additional linkers cleavable by a protease, and with reference to the numbered embodiments below, these linkers correspond to linkers A and C and with reference to the numbered embodiments below, these linkers correspond to the first linker and the third linker) Figure 1A and Figure 2A , these linkers correspond to linker B and linker D, and with reference to the numbered examples below, these linkers correspond to the second linker and the fourth linker; and (d) two (first and second) IL2Rα portions located at the C-terminus of the second and fourth linkers. The IL2 portion in the pro-IL2 protein is in an inactive form due to being masked by the IL2Rα portion, but is released after protease cleavage of one or more protease cleavable linkers at a site where a protease capable of cleaving one or more of the protease cleavable linkers is expressed (e.g., in a tumor environment).

[0075] The IL2 proprotein may further comprise one or more targeting moieties, and in some embodiments comprises two targeting moieties located at the N-terminus of the Fc domain. Examples of targeting moieties are described in Section 6.7 and suitable targeting moiety formats are described in Section 6.8. Figure 1A and Figure 2A In the embodiment depicted in , the IL2 protein may contain two Fab domains at its N-terminus. In some embodiments, such as Figure 1A and Figure 2A As depicted in , the Fc domain comprises a hinge domain at its N-terminus.

[0076] Examples of suitable IL2 portions for incorporation into IL2 proproteins are described in Section 6.3, examples of suitable IL2Rα portions are described in Section 6.4, and examples of suitable protease-cleavable linkers are described in Section 6.5.

[0077] In general, the IL2 proproteins of the present disclosure contain multiple linkers. Preferably, when present, linkers other than the specified protease cleavable linkers are non-cleavable. Examples of non-cleavable linkers are described in Section 6.6.

[0078] Suitable Fc domains, with or without hinge sequences, are described in Section 6.9.

[0079] Figure 1A An exemplary IL2 proprotein is depicted in . The IL2 proprotein comprises:

[0080] a) a first polypeptide chain comprising the heavy chain of a Fab associated with the light chain of the Fab on a separate polypeptide chain, together forming a first targeting moiety, followed by an Fc domain comprising a hinge domain, followed by a first protease cleavable linker ("Linker A"), followed by an IL2 moiety, followed by a second protease cleavable linker ("Linker B"), followed by an IL2Rα moiety; and

[0081] b) a second polypeptide chain comprising the heavy chain of the Fab associated with the light chain of the Fab on a separate polypeptide chain, together forming a second targeting moiety, followed by an Fc domain comprising a hinge domain, followed by a first protease cleavable linker ("Linker C"), followed by the IL2 portion, followed by a second protease cleavable linker ("Linker D"), followed by the IL2Rα portion.

[0082] Figure 2A Another exemplary IL2 proprotein is depicted in . The IL2 proprotein comprises:

[0083] a) a first polypeptide chain comprising the heavy chain of a Fab associated with the light chain of the Fab on a separate polypeptide chain, together forming a first targeting moiety, followed by an Fc domain comprising a hinge domain, followed by a non-cleavable linker ("Linker A"), followed by the IL2 portion, followed by a protease cleavable linker ("Linker B"), followed by the IL2Rα portion; and

[0084] b) a second polypeptide chain comprising the heavy chain of the Fab associated with the light chain of the Fab on a separate polypeptide chain, together forming a second targeting moiety, followed by an Fc domain comprising a hinge domain, followed by a non-cleavable linker ("Linker C"), followed by the IL2 portion, followed by a protease cleavable linker ("Linker D"), followed by the IL2Rα portion.

[0085] Thus, the IL2 proprotein may include four protease-cleavable linkers (e.g. Figure 1A ), or two protease-cleavable linkers (as Figure 2A as shown).

[0086] Cleavage of all protease cleavable linkers in an IL2 proprotein having four protease cleavable linkers results in the release of an activated IL2 protein comprising an IL2 portion and lacking an Fc portion, an IL2Rα portion, and a targeting portion (if present). In some embodiments, this configuration is advantageously used for an IL2 proprotein comprising a targeting portion that binds to a TAA or ECM target molecule expressed in a tumor environment. Figure 1B As shown, and without intending to be bound by theory, the inventors believe that in this configuration, the targeting moiety targets the IL2 protein precursor to the tumor environment, wherein the protease cleavage protease can cleave the linker, resulting in the release of the IL2 protein comprising the IL2 moiety and the linker sequence. This locally activated IL2 protein then induces an immune response against cancer cells by stimulating T lymphocytes in the tumor environment.

[0087] Cleavage of the two protease cleavable linkers in the IL2 proprotein with two protease cleavable linkers results in the release of an activated IL2 protein comprising the IL2 portion, the Fc portion, and the targeting portion (if present), but lacking the IL2Rα portion. In some embodiments, this configuration is advantageously used for IL2 proproteins comprising a targeting portion bound to a TCA, particularly a TCA expressed on antigen-activated T cells (e.g., PD1, Lag3, 41BB, etc.). FIG. 2B to FIG. 2D As shown, and without intending to be bound by theory, the inventors believe that in this configuration, cleavage of the protease-cleavable linker in the tumor environment results in the release of the IL2 protein comprising the IL2 portion and the T cell targeting portion. This locally activated T cell-targeted IL2 protein then induces an enhanced immune response against cancer cells by stimulating T lymphocytes in the tumor environment.

[0088] Importantly, without being bound by theory, the inventors believe that the inclusion of a protease cleavable linker between the IL2 portion and the IL2Rα portion of the IL2 proprotein of the present disclosure is important for optimal stimulation of cytotoxic T cell activity against tumor cells and induction of an immune response enhanced by IL2. In contrast, molecules having components arranged as in the IL2 proprotein of the present disclosure but having a non-cleavable linker separating the IL2 portion and the IL2Rα portion showed low tumor growth control (see US 2022 / 0402989 paragraph

[0253] and Figures 6G and 6H).

[0089] 6.3.IL2 section

[0090] The IL2 portion of the IL2 proprotein of the present disclosure comprises a wild-type or variant IL2 portion.

[0091] In eukaryotic cells, human IL2 is synthesized as a 153 amino acid precursor polypeptide, of which 20 amino acids are removed to generate mature secretory IL2 (Taniguchi et al., 1983, Nature 302(5906):305-10). Mature human IL2 has the following amino acid sequence:

[0092] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:15)

[0093] In some embodiments, the IL2 portions of the disclosure are not CD122-directed, e.g., they do not have amino acid substitutions in the IL2 portion that cause them to preferentially bind IL2Rβ over IL2Rα.

[0094] In some embodiments, the IL2 portions of the disclosure are CD25-directed, e.g., they have one or more amino acid substitutions in the IL2 portion that cause them to preferentially bind to IL2Rα over IL2Rβ.

[0095] In certain embodiments, the IL2 proproteins of the present disclosure have one or more amino acid substitutions in the IL2 portion that reduce binding to IL2Rβ. For example, in some embodiments, the IL2 portion can have up to 50-fold (and in some embodiments up to 100-fold) to 1,000-fold reduced binding to human IL2Rβ compared to wild-type human IL2.

[0096] The IL2 portion with reduced binding to IL2Rβ can retain its affinity for IL2Rα, or have reduced binding to IL2Rα. For example, in some embodiments, the IL2 portion can have up to 50-fold reduced binding to human ffα compared to wild-type human IL2.

[0097] Other characteristics of useful IL2 variants may include the ability to induce proliferation of IL2Rα-bearing CD8+ T cells in tumors, the ability to induce IL2 signaling in IL2Rα-bearing CD8+ T cells in tumors, and an improved therapeutic index.

[0098] In one embodiment, the IL2 portion comprises one or more amino acid substitutions that reduce affinity for IL2Rβ and retain affinity for IL2Rα. An exemplary amino acid substitution is N88D. Other amino acid substitutions that reduce or eliminate IL2 affinity for IL2Rβ are D20T, N88R, N88D, or Q126D (see, e.g., U.S. Patent Publication No. US2007 / 0036752).

[0099] In one embodiment, the IL2 portion comprises one or more amino acid substitutions that reduce affinity for IL2Rα and retain or reduce affinity for IL2Rβ to a lesser extent, resulting in a CD122-directed IL2 portion. Exemplary CD122-directed IL2 portions are those comprising H16A and F42A substitutions. Thus, in some embodiments, the IL2 portion comprises the amino acid sequence of human IL2 having H16A and F42A substitutions, as shown below:

[0100] SAPTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ IDNO:124)

[0101] In certain embodiments, the IL2 portion comprises 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 at T3 are T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P. In a specific embodiment, the substitution is T3A.

[0102] The IL2 portion is preferably substantially a full-length IL2 molecule, e.g., a human IL2 molecule. In certain embodiments, the IL2 portion is a human IL-2 molecule.

[0103] C125 can be substituted with S, V or A to reduce protein aggregation as described in US Pat. No. 4,518,584.

[0104] As described herein, the N-terminal alanine residue of IL2 can also be deleted to generate des-A1 IL2.

[0105] Additionally, the IL2 portion can include a replacement of methionine 104 with a neutral amino acid such as alanine, as described in US Pat. No. 5,206,344.

[0106] Thus, the IL2 portion of the present 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 alter the binding of IL2 to its receptor. These and other mutants can be found in U.S. Pat. No. 5,116,943 and in Weiger et al., 1989, Eur J Biochem 180:295-300.

[0107] In various aspects, 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 to mature human IL2.

[0108] IL2Rα

[0109] The IL2 proprotein of the present disclosure comprises an IL2Rα portion, which comprises an IL2 binding domain of IL2Rα or the IL2 binding domain composition, for example, an extracellular domain of IL2Rα. The sequence of the mature human IL2Rα extracellular domain (corresponding to amino acids 22-272 of human IL2Rα) is:

[0110] Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys

[0111] Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg

[0112] Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly

[0113] Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser

[0114] Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln

[0115] Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp

[0116] Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn

[0117] Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr

[0118] Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu

[0119] Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln

[0120] Leu Ile Cys Thr Gly Glu Met Glu Thr Ser Gln Phe Pro Gly Glu Glu

[0121] Lys Pro Gln Ala Ser Pro Glu Gly Arg Pro Glu Ser Glu Thr Ser Cys

[0122] Leu Val Thr Thr Thr Asp Phe Gln Ile Gln Thr Glu Met Ala Ala Thr

[0123] Met Glu Thr Ser Ile Phe Thr Thr Glu Tyr Gln Val Ala Val Ala Gly

[0124] Cys Val Phe Leu Leu Ile Ser Val Leu Leu Leu Ser Gly Leu Thr Trp

[0125] Gln Arg Arg Gln Arg Lys Ser Arg Arg Thr Ile (SEQ ID NO: 8)

[0126] The sequence of the IL2-binding portion of the human IL2Rα extracellular domain (comprising two "sushi" domains, which correspond to amino acids 22-186 of human IL2Rα) is:

[0127] Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys

[0128] Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg

[0129] Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly

[0130] Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser

[0131] Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln

[0132] Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp

[0133] Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn

[0134] Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr

[0135] Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu

[0136] Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln

[0137] Leu Ile Cys Thr Gly(SEQ ID NO: 9)

[0138] The sequence of the alternative IL2 binding portion of the human IL2Rα extracellular domain (corresponding to amino acids 22-240 of human IL2Rα) is:

[0139] Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys

[0140] Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg

[0141] Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly

[0142] Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser

[0143] Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln

[0144] Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp

[0145] Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn

[0146] Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr

[0147] Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu

[0148] Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln

[0149] Leu Ile Cys Thr Gly Glu Met Glu Thr Ser Gln Phe Pro Gly Glu Glu

[0150] Lys Pro Gln Ala Ser Pro Glu Gly Arg Pro Glu Ser Glu Thr Ser Cys

[0151] Leu Val Thr Thr Thr Asp Phe Gln Ile Gln Thr Glu Met Ala Ala Thr

[0152] Met Glu Thr Ser Ile Phe Thr Thr Glu Tyr Gln (SEQ ID NO: 10)

[0153] The IL2Rα portion preferably 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 to any of the above sequences, i.e., any of amino acids 22-186 of IL2Rα, amino acids 22-240 of IL2Rα, or amino acids 22-272 of IL2Rα, or any IL2 binding portion thereof.

[0154] In certain aspects, the IL2Rα portion can comprise or consist 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 to the IL2 binding portion of human IL2Rα, optionally wherein 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) at most 251, at most 240, at most 230, at most 220, at most 210, at most 200, at most 190, at most 180 or at most 170 amino acids of the extracellular domain of human IL2Rα. In specific embodiments, the portion of human IL2Rα is defined by any of (a) and (b) in the previous sentence, e.g., at least 160 and at most 180 amino acids from human IL2Rα, at least 162 and at most 200 amino acids from human IL2Rα, at least 160 and at most 220 amino acids from human IL2Rα, at least 164 and at most 190 amino acids from human IL2Rα, etc.

[0155] In some embodiments, the IL2Rα 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 to amino acids 22-186, with or without an additional up to 5 amino acids, up to 10 amino acids, up to 15 amino acids, up to 20 amino acids, up to 30 amino acids, or up to 40 amino acids at the C-terminus of amino acid residue 186 of IL2Rα.

[0156] In certain embodiments, the IL2Rα portion has at least one less O-glycosylation and / or N-glycosylation compared to the extracellular domain of native IL2Rα, for example by substitution at one or more of amino acid N49, amino acid N68, amino acid T74, amino acid T85, amino acid T197, amino acid T203, amino acid T208, and amino acid 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, the one or more substitutions are from threonine to 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, the one or more substitutions are at amino acid S50 (e.g., S50P), amino acid S51 (e.g., S51R, S51N, S51D, S51C, S51Q, S51E, S51G, S51H, S51I, S51L, S51K, S51M, S51F, S51P, S51W, S51Y, or S51V), amino acid T69 (e.g., T69P), amino acid T70 (e.g., T70R, T70N, T70D, T70C, T70 Q, 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.

[0157] 6.5. Protease-cleavable linkers

[0158] The IL2 proproteins of the present disclosure typically comprise four linkers, referred to in the numbered embodiments below as the first, second, third, and fourth linkers, wherein the first and second linkers are located on one polypeptide chain and the third and fourth linkers are located on another polypeptide chain. Figure 1A and Figure 2A In the embodiment shown in , the first and second joints are referred to as joint A and joint B and the third and fourth joints are referred to as joint C and joint D.

[0159] In other embodiments, all four linkers (the first, second, third, and fourth linkers (corresponding to Linker A, Linker B, Linker C, and Linker D) are protease cleavable. Figure 1A An exemplary IL2 proprotein configured according to such embodiments is shown in FIG.

[0160] In some embodiments, the second and fourth linkers (corresponding to Linker B and Linker D) are protease cleavable and the first and third linkers (corresponding to Linker A and Linker C) are non-cleavable. Figure 2A An exemplary IL2 proprotein configured according to such embodiments is shown in FIG.

[0161] Protease cleavable linkers can range from 20 amino acids to 80 or more amino acids, and in certain aspects, non-cleavable peptide linkers can range from 20 amino acids to 60 amino acids, 20 amino acids to 40 amino acids, 30 amino acids to 50 amino acids, 20 amino acids to 80 amino acids, or 30 amino acids to 70 amino acids in length.

[0162] The protease cleavable linker comprises one or more substrate sequences for one or more proteases, such as one or more proteases listed in Section 6.5.1. One or more substrate sequences, such as one or more substrate sequences listed in Section 6.5.2, are usually flanked by one or more spacer sequences, such as the spacer sequences described in Section 6.5.3. Each protease cleavable linker can include one, two, three or more substrate sequences. The spacer sequences can be adjacent, overlapping or separated by spacer sequences. Preferably, the C-terminus and N-terminus of the protease cleavable linker contain spacer sequences.

[0163] In various aspects of the IL2 proprotein comprising four protease cleavable linkers, the first and third protease cleavable linkers (corresponding to Figure 1A The linker A and linker C in the embodiment of the present invention are cleavable by the same protease and / or the second and fourth protease cleavable linkers (corresponding to Figure 1A In some embodiments, the linker B and linker D) are cleavable by the same protease. In some embodiments, the protease is a protease listed in Table A.

[0164] In a further aspect of the IL2 proprotein comprising four protease cleavable linkers, the first and third protease cleavable linkers (corresponding to Figure 1A The linker A and linker C in the embodiment of the present invention comprise the same substrate sequence and / or the second and fourth protease cleavable linkers (corresponding to Figure 1A In some embodiments, the first and third protease cleavable linkers (corresponding to Figure 1A The linker A and linker C in the embodiment of the present invention also comprise the same spacer sequence and / or the second and fourth protease cleavable linkers (corresponding to Figure 1A In some embodiments, the spacer sequences are listed in Table C.

[0165] In a further aspect of the IL2 proprotein comprising four protease cleavable linkers, the first and third protease cleavable linkers (corresponding to Figure 1A The linker A and linker C in the embodiment of the present invention comprise the same linker sequence and / or the second and fourth protease cleavable linkers (corresponding to Figure 1A In some embodiments, the linker sequences are listed in Table D.

[0166] In some embodiments of the IL2 proprotein comprising four protease cleavable linkers, the first and third protease cleavable linkers (corresponding to Figure 1A The embodiment of the linker A and linker C) and the second and fourth protease cleavable linkers (corresponding to Figure 1A The connector B and connector D in the embodiment are the same.

[0167] In other embodiments of the IL2 proprotein comprising four protease cleavable linkers, the first and third protease cleavable linkers (corresponding to Figure 1A The linkers A and C in the embodiment of the present invention are different from the second and fourth protease cleavable linkers (corresponding to Figure 1A Connector B and connector D in the embodiment of the present invention).

[0168] In a cell containing two protease-cleavable linkers (corresponding to Figure 2A In some embodiments of the IL2 protein precursor (linker B and linker D in the embodiments of the present invention), the two protease cleavable linkers are the same. In other embodiments, the two protease cleavable linkers are different.

[0169] In the foregoing aspects and embodiments of the pro-IL2 protein comprising four protease cleavable linkers and the pro-IL2 protein comprising two protease cleavable linkers, the different linkers may be cleavable by the same protease, different proteases, or when the linker comprises multiple substrate sequences, the different linkers may be cleavable by multiple proteases, one or more of which are common and one or more of which are different.

[0170] Exemplary protease-cleavable linker sequences are listed in Section 6.5.4.

[0171] 6.5.1. Proteases

[0172] Exemplary proteases whose substrate sequences can be incorporated into a protease-cleavable linker are listed in Table A below.

[0173]

[0174] In a specific embodiment, the protease is matrix metalloproteinase (MMP)-2, MMP-9, asparaginase, thrombin, fibroblast activation protease (FAP), MMP-1, MMP-3, MMP-7, MMP-8, MMP-12, MMP-13, MMP-14, membrane type 1 matrix metalloproteinase (MT1-MMP), plasmin, transmembrane protease, serine (TMPRSS-3 / 4), cathepsin A, cathepsin B, cathepsin D, cathepsin E, cathepsin F, cathepsin H, cathepsin K, cathepsin L , cathepsin L2, cathepsin O, cathepsin S, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, caspase 14, human neutrophil elastase, urokinase / urokinase-type plasminogen activator (uPA), a disintegrin and metalloproteinase (ADAM) 10, ADAM12, ADAM17, ADAM with thrombospondin motif (ADAMTS), ADAMTS5, beta-secretase (BACE), granzyme A, granzyme B, guanidinobenzoic acidase, hepsin, matriptase, matriptase 2, transmembrane peptidase, neprilysin, prostate-specific membrane antigen (PSMA), tumor necrosis factor-converting enzyme (TACE), kallikrein-related peptidase (KLK) 3, KLK5, KLK7, KLK11, NS3 / 4 protease of hepatitis C virus (HCV-NS3 / 4), tissue plasminogen activator (tPA), calpain, calpain 2, glutamate carboxypeptidase II, plasma kallikrein, AMSH-like protease, AMSH, gamma-secretase component, antiplasmin cleaving enzyme (APCE), decysteine ​​1, apoptosis-associated cysteine ​​peptidase or N-acetylated alpha-linked acid dipeptidase-like 1.

[0175] 6.5.2. Substrate

[0176] Exemplary substrate sequences that are cleavable by tumor proteases and that can incorporate a protease-cleavable linker are listed in Table B below.

[0177]

[0178]

[0179]

[0180]

[0181] 6.5.3. Spacers

[0182] Exemplary spacer sequences that can be incorporated into a protease cleavable linker are listed below in Table C. In addition to the spacer sequences listed in Table C, any non-cleavable linker sequence described in Section 6.6, e.g., a non-cleavable linker sequence listed in Table E, or a portion thereof, can be used as a spacer sequence.

[0183]

[0184] In some embodiments, as used in Table C above, n is an integer from 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0185] 6.5.4. Exemplary protease-cleavable linkers

[0186] Exemplary protease-cleavable linkers comprising one or more substrate sequences and a spacer sequence are listed in Table D below.

[0187]

[0188]

[0189]

[0190]

[0191] In certain aspects, the protease cleavable linker comprises an amino acid sequence having up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid substitutions compared to a sequence listed in Table D. Thus, in some embodiments, the protease cleavable linker comprises or consists of any amino acid sequence in Table D having 1-5 amino acid substitutions compared to a sequence listed in Table D.

[0192] 6.6. Non-cleavable linkers

[0193] In certain aspects, the present disclosure provides an IL2 proprotein in which two or more components of the IL2 proprotein are connected to each other via a peptide linker. By way of example and not limitation, a linker can be used to connect an Fc domain and a targeting moiety or different domains within a targeting moiety (e.g., VH and VL domains in an scFv).

[0194] Preferably, all linkers in the IL2 proprotein, except for the protease cleavable linker whose cleavage results in IL2 activation, are non-cleavable linkers (NCL).

[0195] The non-cleavable linker can range from 2 amino acids to 60 or more amino acids, and in certain aspects the non-cleavable peptide linker can range from 3 amino acids to 50 amino acids, 4 to 30 amino acids, 5 to 25 amino acids, 10 to 25 amino acids, 10 amino acids to 60 amino acids, 12 amino acids to 20 amino acids, 20 amino acids to 50 amino acids, or 25 amino acids to 35 amino acids in length.

[0196] In particular aspects, the non-cleavable linker is at least 5 amino acids, at least 6 amino acids, or at least 7 amino acids in length, and optionally is up to 30 amino acids, up to 40 amino acids, up to 50 amino acids, or up to 60 amino acids in length.

[0197] In some of the foregoing embodiments, the non-cleavable linker ranges from 5 amino acids to 50 amino acids in length, for example, ranging from 5 to 50 amino acids, 5 to 45 amino acids, 5 to 40 amino acids, 5 to 35 amino acids, 5 to 30 amino acids, 5 to 25 amino acids, or 5 to 20 amino acids in length. In other embodiments of the foregoing, the non-cleavable linker ranges from 6 amino acids to 50 amino acids in length, for example, ranging from 6 to 50 amino acids, 6 to 45 amino acids, 6 to 40 amino acids, 6 to 35 amino acids, 6 to 30 amino acids, 6 to 25 amino acids, or 6 to 20 amino acids in length. In yet other embodiments, the non-cleavable linker ranges from 7 amino acids to 50 amino acids in length, for example, ranging from 7 to 50 amino acids, 7 to 45 amino acids, 7 to 40 amino acids, 7 to 35 amino acids, 7 to 30 amino acids, 7 to 25 amino acids, or 7 to 20 amino acids in length.

[0198] Charged (eg, charged hydrophilic linkers) and / or flexible non-cleavable linkers are particularly preferred.

[0199] Examples of flexible non-cleavable linkers that can be used in the IL2 proproteins of the present disclosure include those disclosed by 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 non-cleavable linkers are or contain repeats of glycine and serine, e.g., G n S (SEQ ID NO: 292) or SG n(SEQ ID NO: 293), wherein 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 non-cleavable linker is or comprises a monomer or multimer of G4S (SEQ ID NO: 294) repeats, for example, (GGGGS) n (SEQ ID NO: 295).

[0200] Polyglycine non-cleavable linkers can be suitably used in the IL2 proproteins of the present disclosure. In some embodiments, the peptide non-cleavable linker comprises two consecutive glycines (2Gly), three consecutive glycines (3Gly), four consecutive glycines (4Gly) (SEQ ID NO: 296), five consecutive glycines (5Gly) (SEQ ID NO: 297), six consecutive glycines (6Gly) (SEQ ID NO: 298), seven consecutive glycines (7Gly) (SEQ ID NO: 299), eight consecutive glycines (8Gly) (SEQ ID NO: 300) or nine consecutive glycines (9Gly) (SEQ ID NO: 301).

[0201] Exemplary non-cleavable linker sequences are listed in Table E below.

[0202]

[0203]

[0204] In certain aspects, the IL2 proprotein of the present disclosure may comprise a polypeptide chain comprising a targeting moiety (or a targeting moiety chain), a hinge domain and a CH2 domain and a CH3 domain in the N-terminal to C-terminal direction. Thus, the hinge domain connects the targeting moiety to the CH2 domain and can be said to constitute a type of linker. Exemplary hinge domains are described in Section 6.9.3.

[0205] 6.7. Targeting moiety

[0206] Incorporation of targeting moieties in the disclosed pro-IL2 proteins allows for the delivery of high concentrations of IL2 to the tumor microenvironment while reducing systemic exposure, resulting in fewer side effects than obtained with unmasked IL2 molecules.

[0207] Any type of target molecule that is expected to exist or can drive IL2 proprotein at a specific site or tissue can be targeted by IL2 proprotein of the present invention. In certain embodiments, IL2 proprotein is intended to treat cancer, for example, by inducing a local immune response against tumor tissue. Therefore, the targeting molecule can be any local tumor and a related target molecule. The target molecule identified by the targeting portion of IL2 proprotein of the present invention is usually present in immune cells such as activated T cells, on tumor cells, on virus-infected cells, on other diseased cells, in free state in serum, in extracellular matrix (ECM) or in target sites, such as tumor reactive lymphocytes.

[0208] Among the various extracellular matrix ("ECM") antigens, tumor reactive lymphocyte antigens, cell surface molecules of tumor or viral lymphocytes, T cell antigens ("TCAs"), checkpoint inhibitors, or tumor associated antigens ("TAAs"). The skilled artisan will recognize that the aforementioned categories of target molecules are not mutually exclusive, and thus a given target molecule may fall into more than one of the aforementioned categories of target molecules. For example, some molecules may be considered as TAAs and ECM proteins, while other molecules may be considered as TCAs and checkpoint inhibitors.

[0209] Exemplary cancer types that can be targeted include acute lymphocytic leukemia, acute myeloid leukemia, cholangiocarcinoma, B cell leukemia, B cell lymphoma, bile duct cancer, bone cancer, brain cancer, breast cancer, triple negative breast cancer, cervical cancer, Burkitt's lymphoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancer, endometrial cancer, esophageal cancer, gallbladder cancer, gastric cancer, gastrointestinal cancer, glioma, hairy cell leukemia, head and neck cancer, Hodgkin's lymphoma, liver cancer, lung cancer, medullary thyroid cancer, melanoma, multiple myeloma, ovarian cancer, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, lung cancer, kidney cancer, sarcoma, skin cancer, testicular cancer, urothelial carcinoma and other bladder cancers. However, those skilled in the art will recognize that TAAs and other target molecules associated with the tumor microenvironment are known for almost any type of cancer.

[0210] Non-limiting examples of ECM antigens include syndecan, heparanase, integrin, osteopontin, connexin, cadherin, laminin, laminin-type EGF, lectin, fibronectin, notch proteins, adhesion proteins (e.g., adhesion protein-4), tenascin, procollagen (e.g., type X collagen), and matrix proteins.

[0211] Other target molecules are cell surface molecules of tumor or viral lymphocytes, for example T cell co-stimulatory proteins such as CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C and B7-H3.

[0212] In specific 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, CHK2. In specific embodiments, the target molecule is PD1. In other embodiments, the target molecule is LAG3.

[0213] Antibodies and antigen binding portions generally bind to specific antigenic determinants and are capable of directing the IL2 proprotein to a target site, such as to a specific type of tumor cell or tumor stroma carrying the antigenic determinant. In specific embodiments, the targeting portion recognizes a tumor-associated antigen (TAA). Preferably, the TAA is a human TAA. The antigen may or may not be present on normal cells. In certain embodiments, the TAA is preferentially expressed or upregulated on tumor cells compared to normal cells. In other embodiments, the TAA is a lineage marker. Exemplary TAAs include fibroblast activation protein (FAP), the A1 domain of tenascin-C (TNC A1), the A2 domain of tenascin-C (TNC A2), extra domain B of fibronectin (EDB), melanoma-associated chondroitin sulfate proteoglycan (MCSP), MART-1 / Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase binding protein (ADAbp), cyclophilin 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 (PS A) and their immunogenic epitopes PSA-1, PSA-2 and PSA-3, prostate specific membrane antigen (PSMA), T cell receptor / CD3-ζ chain, MAGE family of tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-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 ​​family of tumor antigens (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, alpha-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn, gp100 Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papillomavirus proteins, Smad tumor family antigens, Imp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brain 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 (heteromultimer associated with TCR), CD22 (B cell receptor), CD23 (low affinity IgE receptor), CD30 (cytokine receptor), CD33 (myeloid cell surface antigen), CD40 (tumor necrosis factor receptor), IL-6R- (IL6 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 IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, muthsp70-2, M-CSF, prostate enzyme, prostate enzyme-specific antigen (PSA), PAP, LAGA-1a, p53, prostate-specific protein (prostein), PSMA, survivin 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 of tenascin-C (TnC A1).

[0214] Suitable targeting moiety formats are described in Section 6.8. The targeting moiety is preferably an antigen binding moiety, such as an antibody or an antigen binding portion of an antibody, e.g., a scFv as described in Section 6.8.2 or a Fab as described in Section 6.8.1.

[0215] In some embodiments, the targeting moiety targets the exemplary target molecules listed in Table F below, with reference to exemplary antibodies or antibody sequences on which the targeting moiety can be based.

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227] In some aspects, the targeting moiety competes with the antibodies listed in Table F for binding to the target molecule. In a further aspect, the targeting moiety comprises a CDR having a CDR sequence of an antibody listed in Table F. In some embodiments, the targeting moiety comprises all 6 CDR sequences of the antibodies listed in Table F. In other embodiments, the targeting moiety comprises at least a heavy chain CDR sequence (CDR-H1, CDR-H2, CDR-H3 and a light chain CDR sequence of a universal light chain. In a further aspect, the targeting moiety comprises a VH comprising the amino acid sequence of the VH of the antibody shown in Table F. In some embodiments, the targeting moiety further comprises a VL comprising the amino acid sequence of the VL of the antibody listed in Table F. In other embodiments, the targeting moiety further comprises a universal light chain VL sequence.

[0228] In some embodiments, the targeting moiety is a non-blocking or weakly blocking ligand-receptor binding. Examples of non-blocking or weakly blocking anti-PD1 antibodies include antibodies having the following VH / VL amino acid sequences: SEQ ID No: 2 / 10 of PCT Publication No. WO2015 / 112800A1; SEQ ID No: 16 / 17 of U.S. Pat. No. 11,034,765 B2; SEQ ID No. 164 / 178, 165 / 179, 166 / 180, 167 / 181, 168 / 182, 169 / 183, 170 / 184, 171 / 185, 172 / 186, 173 / 187, 174 / 188, 175 / 189, 176 / 190 and 177 / 190 of U.S. Pat. No. 10,294,299 B2. Examples of non-blocking or weakly blocking anti-LAG3 antibodies include antibodies having the VH / VL amino acid sequences of SEQ ID Nos 23 / 24, 3 / 4, and 11 / 12 of US Publication No. US2022 / 0056126A1.

[0229] Other target molecules that can be targeted by the IL2 proprotein are disclosed in Table 1 below and in, for example, Hafeez et al., 2020, Molecules 25:4764, doi: 10.3390 / molecules25204764, particularly in Table 1. The entire contents of Table 1 of Hafeez et al. are incorporated herein by reference.

[0230] 6.8. Targeting Section Format

[0231] In certain aspects, the targeting moiety of the IL2 proprotein of the present disclosure can be any type of antibody or fragment thereof that retains specific binding to the antigenic determinant. In one embodiment, the targeting moiety is an immunoglobulin molecule or fragment thereof, particularly an IgG class immunoglobulin molecule, more particularly an IgG1 or IgG4 immunoglobulin molecule. Antibody fragments include, but are not limited to, VH (or V H ) fragment, VL (or V L ) fragment, Fab fragment, F(ab')2 fragment, scFv fragment, Fv fragment, miniantibody, diabody, triabody and tetrabody.

[0232] 6.8.1.Fab

[0233] Fab domains are traditionally produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain.The Fab domain may comprise constant domain and variable region sequences from any suitable species, and may therefore be murine, chimeric, human or humanized.

[0234] The Fab domain typically comprises a CH1 domain connected to a VH domain, which is paired with a CL domain connected to a VL domain. In wild-type immunoglobulins, the VH domain is paired with the VL domain to form the Fv region, and the CH1 domain is paired with the CL domain to further stabilize the binding site. The disulfide bond between the two constant domains can further stabilize the Fab domain.

[0235] For the IL2 proprotein of the present disclosure, particularly when the light chain of the targeting moiety is not a common or universal light chain, it is advantageous to use a Fab heterodimerization strategy to allow the correct association of Fab domains belonging to the same targeting moiety and minimize the abnormal pairing of Fab domains belonging to different targeting moieties. For example, the Fab heterodimerization strategy shown in Table G below can be used:

[0236]

[0237] Thus, in certain embodiments, the correct association between the two polypeptides of the Fab is promoted by exchanging the VL and VH domains of the Fab with each other or by exchanging the CH1 and CL domains with each other, as described, for example, in WO 2009 / 080251.

[0238] Correct Fab pairing can also be promoted by introducing one or more amino acid modifications in the CH1 domain of the Fab and one or more amino acid modifications in the CL domain and / or one or more amino acid modifications in the VH domain and one or more amino acid modifications in the VL domain. The modified amino acids are typically part of the VH:VL and CH1:CL interfaces, such that the Fab components preferentially pair with each other rather than with components of other Fabs.

[0239] In one embodiment, one or more amino acid modifications are limited to conserved framework residues of the variable domains (VH, VL) and constant domains (CH1, CL), as 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.

[0240] In one embodiment, the modifications introduced in the VH and CH1 and / or VL and CL domains are complementary to each other. Complementarity at the heavy and light chain interfaces can be achieved based on steric and hydrophobic contacts, electrostatic / charge interactions, or a combination of various interactions. Complementarity between protein surfaces is widely described in the literature as lock and key fits, knobs into sockets, protrusions and cavities, donors and acceptors, etc., all of which imply the nature of structural and chemical matching between two interacting surfaces.

[0241] In one embodiment, the one or more modifications introduced introduce new hydrogen bonds at the interface of the Fab component. In one embodiment, the one or more modifications introduced introduce new salt bridges across the interface of the Fab component. Exemplary substitutions are described in WO 2014 / 150973 and WO 2014 / 082179, the contents of which are incorporated herein by reference.

[0242] In some embodiments, the Fab domain comprises a 192E substitution in the CH1 domain and 114A and 137K substitutions in the CL domain, which introduces a salt bridge between the CH1 and CL domains (see, eg, Golay et al., 2016, J Immunol 196:3199-211).

[0243] In some embodiments, the Fab domain comprises 143Q and 188V substitutions in the CH1 domain and 113T and 176V substitutions in the CL domain, which serve to exchange the hydrophobic and polar regions of contact between the CH1 and CL domains (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0244] In some embodiments, the Fab domain may include modifications in some or all of the VH, CH1, VL, and CL domains to introduce an orthogonal Fab interface that promotes the correct assembly of the Fab domain (Lewis et al., 2014 Nature Biotechnology 32: 191-198). In an embodiment, 39K and 62E modifications are introduced in the VH domain, H172A and F174G modifications are introduced in the CH1 domain, 1 R, 38D, (36F) modifications are introduced in the VL domain, and L135Y and S176W modifications are introduced in the CL domain. In another embodiment, 39Y modifications are introduced in the VH domain, and 38R modifications are introduced in the VL domain.

[0245] The Fab domain can also be modified to replace the native CH1:CL disulfide bond with an engineered disulfide bond to increase the efficiency of Fab component pairing. For example, an engineered disulfide bond can be introduced by introducing 126C in the CH1 domain and 121C in the CL domain (see, e.g., Mazor et al., 2015, MAbs 7:377-89).

[0246] The Fab domain can also be modified by replacing the CH1 domain and the CL domain with alternative domains that promote 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 replacing the CL domain with the b domain of the T cell receptor, and pairing these domain replacements with additional charge-charge interactions between the VL and VH domains by introducing a 38D modification in the VL domain and a 39K modification in the VH domain.

[0247] Instead of or in addition to using Fab heterodimerization strategies to promote correct VH-VL pairing, the VL of a common light chain (also referred to as a universal light chain) can be used for each unique ABD in a disclosed IL2 proprotein. In various embodiments, the use of a common light chain as described herein reduces the number of inappropriate species in the IL2 proprotein compared to using the original homologous VL. In various embodiments, the VL domain of the ABD is identified from a monospecific antibody comprising a common light chain. In various embodiments, the VH region of the ABD in the IL2 proprotein is included in a human heavy chain variable gene fragment rearranged in vivo in a mouse B cell, and these mouse B cells were previously engineered to express a limited human light chain library (or a single human light chain) homologous to a human heavy chain, and in response to exposure to an antigen of interest, an antibody library comprising multiple human VHs is produced, and these multiple human VHs are homologous to one of two possible human VLs or one human VL, wherein the antibody has a library specific to an antigen of interest. The common light chain is a light chain derived from a rearranged human Vκ1-39Jκ5 sequence or a rearranged human Vκ3-20Jκ1 sequence, and includes somatically mutated (eg, affinity matured) versions. See, eg, US Pat. No. 10,412,940.

[0248] 6.8.2.scFv

[0249] Single-chain Fv or "scFv" antibody fragments contain the VH and VL domains of an antibody in a single polypeptide chain, can be expressed as a single polypeptide chain, and retain the specificity of the intact antibody from which it is derived. In general, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for target binding. Examples of linkers suitable for connecting the VH and VL chains of the scFv are the non-cleavable linkers identified in Section v.

[0250] Unless otherwise indicated, as used herein, a scFv can have the VL and VH variable regions in either order, for example, relative to the N-terminus and C-terminus of the polypeptide, a scFv can comprise VL-linker-VH or can comprise VH-linker-VL.

[0251] The scFv may comprise VH and VL sequences from any suitable species, such as murine, human or humanized VH and VL sequences.

[0252] To generate a nucleic acid encoding an scFv, the DNA fragments encoding VH and VL are operably linked to another fragment encoding a linker, e.g., a fragment encoding any linker described in Section 6.6 (typically a repeat of a sequence containing the amino acids glycine and serine, such as the amino acid sequence (Gly4-Ser)3 (SEQ ID NO: 170) such that 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).

[0253] 6.9. Fc Region

[0254] The IL2 protein of the present disclosure generally includes a pair of Fc domains that associate to form an Fc region. In a native antibody, the Fc region includes a hinge region at its N-terminus to form a constant domain. Throughout the present disclosure, unless otherwise indicated, reference to an Fc domain encompasses an Fc domain having a hinge domain at its N-terminus.

[0255] The Fc domain can be derived from any suitable species operably connected to the ABD or its components. In one embodiment, the Fc domain is derived from a human Fc domain. In a preferred embodiment, the targeting moiety or its components are fused to an IgG Fc molecule. The targeting moiety or its components can be fused to the N-terminal or C-terminal or both of the IgG Fc domain.

[0256] The Fc domain can be derived from any suitable class of antibodies, 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. Exemplary sequences of Fc domains from IgG1, IgG2, IgG3 and IgG4 are provided in Table Y below.

[0257]

[0258] In some aspects, the Fc domain 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 to SEQ ID NO: 1. In cases where the Fc domain has at least 90% sequence identity to SEQ ID NO: 1 and has less than 100% sequence identity (e.g., between 90% and 99% sequence identity to SEQ ID NO: 1), the Fc domain may further comprise one or more amino acid substitutions described herein, such as one or more substitutions that reduce effector function (e.g., as described in Section 6.9.1) and / or one or more substitutions that promote Fc heterodimerization (e.g., as described in Section 6.9.2).

[0259] In some aspects, the Fc domain 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 to SEQ ID NO: 2. In cases where the Fc domain has at least 90% sequence identity to SEQ ID NO: 2 and has less than 100% sequence identity (e.g., between 90% and 99% sequence identity to SEQ ID NO: 2), the Fc domain may further comprise one or more amino acid substitutions described herein, such as one or more substitutions that reduce effector function (e.g., as described in Section 6.9.1) and / or one or more substitutions that promote Fc heterodimerization (e.g., as described in Section 6.9.2).

[0260] In some aspects, the Fc domain 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 to SEQ ID NO: 3. In cases where the Fc domain has at least 90% sequence identity to SEQ ID NO: 3 and has less than 100% sequence identity (e.g., between 90% and 99% sequence identity to SEQ ID NO: 3), the Fc domain may further comprise one or more amino acid substitutions described herein, such as one or more substitutions that reduce effector function (e.g., as described in Section 6.9.1) and / or one or more substitutions that promote Fc heterodimerization (e.g., as described in Section 6.9.2).

[0261] In some aspects, the Fc domain 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 to SEQ ID NO: 4. In cases where the Fc domain has at least 90% sequence identity to SEQ ID NO: 4 and has less than 100% sequence identity (e.g., between 90% and 99% sequence identity to SEQ ID NO: 4), the Fc domain may further comprise one or more amino acid substitutions described herein, such as one or more substitutions that reduce effector function (e.g., as described in Section 6.9.1) and / or one or more substitutions that promote Fc heterodimerization (e.g., as described in Section 6.9.2).

[0262] The two Fc domains within the Fc region may be identical or different from each other. In native antibodies, the Fc domains are usually identical, but to generate multispecific binding molecules, e.g., the IL2 proprotein of the present disclosure and the MBM generated by its activation, the Fc domains may advantageously be different to allow heterodimerization, as described in Section 6.9.2 below.

[0263] In natural antibodies, the heavy chain Fc domain of IgA, IgD, and IgG consists of two heavy chain constant domains (CH2 and CH3), while the heavy chain Fc domain of IgE and IgM consists of three heavy chain constant domains (CH2, CH3, and CH4). These dimers form the Fc region.

[0264] In the pro-IL2 proteins of the present disclosure, the Fc region and / or Fc domains therein may comprise heavy chain constant domains from one or more different types of antibodies, such as one, two or three different types.

[0265] In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG1.

[0266] In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG2.

[0267] In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG3.

[0268] In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG4.

[0269] In one embodiment, the Fc region comprises a CH4 domain from IgM. The IgM CH4 domain is typically located at the C-terminus of the CH3 domain.

[0270] In one embodiment, the Fc region comprises CH2 and CH3 domains derived from IgG and a CH4 domain derived from IgM.

[0271] It should be understood that the heavy chain constant domain of the Fc region used to produce the IL2 proprotein of the present disclosure may include variants of the above-mentioned naturally occurring constant domains. Such variants may include one or more amino acid variations compared to the wild-type constant domain. In one example, the Fc region of the present disclosure includes at least one constant domain whose sequence is different from the wild-type constant domain. It should 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 another example, the variant constant domain is at least 80% identical or similar. In another example, the variant constant domain is at least 90% identical or similar. In another example, the variant constant domain is at least 95% identical or similar.

[0272] IgM and IgA naturally exist in the human body as covalent polymers of the common H2L2 antibody unit. When IgM incorporates a J chain, it appears as a pentamer; or when IgM lacks a J chain, it appears as a hexamer. IgA appears as a monomer and a dimer. The heavy chains of IgM and IgA have 18 amino acids extending to the C-terminal constant domain, called the tail segment. The tail segment contains cysteine ​​residues that form disulfide bonds between the heavy chains of the polymer and are believed to play an important role in polymerization. The tail segment also contains a glycosylation site. In certain embodiments, the IL2 protein of the present disclosure does not contain a tail segment.

[0273] The Fc domain incorporated into the pro-IL2 proteins of the present disclosure may comprise one or more modifications that alter the functional properties of the protein, e.g., binding to Fc receptors such as FcRn or leukocyte receptors, binding to complement, modified disulfide bond structures, or altered glycosylation patterns. Exemplary Fc modifications that alter effector function are described in Section 6.9.1.

[0274] The Fc domain can also be altered to include modifications that improve the manufacturability of an asymmetric IL2 proprotein, such as by allowing heterodimerization, which is a preferential pairing of different Fc domains relative to the same Fc domain. Heterodimerization allows the production of an IL2 proprotein in which different polypeptide components are connected to each other via an Fc region containing Fc domains that differ in sequence. Examples of heterodimerization strategies are illustrated in Section 6.9.2.

[0275] It will be appreciated that any of the above modifications may be combined in any suitable manner to achieve desired functional properties and / or combined with other modifications to alter the properties of the pro-IL2 protein.

[0276] 6.9.1. Fc domains with altered effector functions

[0277] In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and / or effector function.

[0278] In a specific embodiment, 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 a specific embodiment, 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 the group consisting of complement dependent cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and cytokine secretion. In a specific embodiment, the effector function is ADCC.

[0279] In one embodiment, the Fc domain (e.g., the Fc domain of the IL2 proprotein half antibody) or Fc region (e.g., one or two Fc domains of the IL2 proprotein that can associate to form an Fc region) comprises an amino acid substitution at a position selected from the group consisting of E233, L234, L235, N297, P331, and P329 (numbering according to the Kabat EU index). In a more specific embodiment, the Fc domain or Fc region comprises an amino acid substitution at a position selected from the group consisting of L234, L235, and P329 (numbering according to the Kabat EU index). In some embodiments, the Fc domain or Fc region comprises the amino acid substitutions L234A and L235A (numbering according to the Kabat EU index). In one such embodiment, the Fc domain or region is an Igd Fc domain or region, in particular a human Igd Fc domain or region. In one embodiment, the Fc domain or Fc region comprises 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 domain or Fc region comprises an amino acid substitution at position P329 and comprises a further amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (numbered according to the Kabat EU index). In a more specific embodiment, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In a specific embodiment, the Fc domain or Fc region comprises an amino acid substitution at position P329, L234 and L235 (numbered according to the Kabat EU index). In a more specific embodiment, the Fc domain comprises amino acid mutations L234A, L235A and P329G ("P329G LALA", "PGLALA" or "LALAPG").

[0280] Typically, the same one or more amino acid substitutions are present in each of the two Fc domains of the Fc region. Thus, in a specific embodiment, each Fc domain of the Fc region comprises amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering), i.e., in each of the first Fc domain and the second Fc domain of the Fc region, the leucine residue at position 234 is substituted with an alanine residue (L234A), the leucine residue at position 235 is substituted with an alanine residue (L235A), and the proline residue at position 329 is substituted with a glycine residue (P329G) (numbering according to the Kabat EU index).

[0281] In one embodiment, the Fc domain is an IgG1 Fc domain, in particular a human IgG1 Fc domain. In some embodiments, the IgG1 Fc domain is a variant IgG1 comprising D265A, N297A mutations (EU numbering) to reduce effector function.

[0282] In another embodiment, the Fc domain is an IgG4 Fc domain with reduced binding to an Fc receptor. An exemplary IgG4 Fc domain with reduced binding to an Fc receptor may comprise an amino acid sequence selected from Table H below: In some embodiments, the Fc domain includes only the bold portion of the sequence shown below:

[0283]

[0284]

[0285]

[0286] In specific embodiments, the IgG4 with reduced effector function comprises the bold portion of the amino acid sequence of SEQ ID NO: 31 of WO2014 / 121087, sometimes referred to herein as IgG4 or hIgG4.

[0287] For the heterodimeric Fc region, a combination of the above-mentioned variant IgG4 Fc sequences can be incorporated, for example, an Fc region comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 30 of WO2014 / 121087 (or its bold portion) and an Fc domain comprising the amino acid sequence of SEQ ID NO: 37 of WO2014 / 121087 (or its bold portion), or an Fc region comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 31 of WO2014 / 121087 (or its bold portion) and an Fc domain comprising the amino acid sequence of SEQ ID NO: 38 of WO2014 / 121087 (or its bold portion).

[0288] 6.9.2. Fc Heterodimerization Variants

[0289] Some IL2 proproteins require dimerization between two Fc domains, which, unlike natural immunoglobulins, are operably linked to different N-terminal or C-terminal regions. Insufficient heterodimerization of the two Fc domains to form an Fc region may be an obstacle to increasing the yield of the desired heterodimeric molecule and poses a challenge to purification. Various methods available in the art can be used to enhance the dimerization of the Fc domains that may be present in the IL2 proproteins of the present disclosure, such as disclosed in EP 1870459A1; U.S. Patent No. 5,582,996; U.S. Patent No. 5,731,168; U.S. Patent No. 5,910,573; U.S. Patent No. 5,932,448; U.S. Patent No. 6,833,441; U.S. Patent No. 7,183,076; U.S. Patent Application Publication No. 2006204493A1; and PCT Publication No. WO 2009 / 089004A1.

[0290] In some embodiments, the present disclosure provides an IL2 protein progeny comprising an Fc heterodimer, i.e., an Fc region comprising heterologous, non-identical Fc domains. Typically, each Fc domain in the Fc heterodimer comprises a CH3 domain of an antibody. The CH3 domain is derived from the constant region of an antibody of any isotype, class or subclass, and preferably the constant region of an antibody of the IgG (IgG1, IgG2, IgG3 and IgG4) class, as described in the previous section.

[0291] Heterodimerization of two different heavy chains of the CH3 domain produces the desired IL2 proprotein, while homodimerization of the same heavy chain will reduce the yield of the desired IL2 proprotein. Therefore, in a preferred embodiment, the polypeptides that associate to form the IL2 proprotein of the present disclosure will contain a CH3 domain with a modification that favors heterodimer association relative to an unmodified Fc domain.

[0292] In a specific embodiment, the modification that promotes the formation of Fc heterodimers is a so-called "knob-in-hole" or "knob-in-hole" modification, comprising a "knob" modification in one Fc domain and a "hole" modification in another Fc domain. Knob-in-hole technology is described in the following literature: for example, U.S. Patent No. 5,731,168; US 7,695,936; Ridgway et al., 1996, Prot Eng 9:617-621, and Carter, 2001, Immunol Meth 248:7-15. In general, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and introducing a corresponding cavity ("hole") in the interface of a second polypeptide, so that the protrusion can be positioned in the cavity, thereby promoting heterodimer formation and hindering homodimer formation. Protrusions are constructed by replacing small amino acid side chains from the interface of a first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of the same or similar size to the protrusions are created in the interface of the second polypeptide by replacing larger amino acid side chains with smaller ones (eg, alanine or threonine).

[0293] Therefore, in some embodiments, the amino acid residue in the CH3 domain of the first subunit of the Fc domain is replaced by an amino acid residue with a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit, and the protrusion can be positioned in the cavity in the CH3 domain of the second subunit, and the amino acid residue in the CH3 domain of the second subunit of the Fc domain is replaced by an amino acid residue with a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit, and the protrusion in the CH3 domain of the first subunit can be positioned in the cavity. Preferably, the amino acid residue with 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 with a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T) and valine (V). Protrusions and cavities can be prepared by changing the nucleic acid encoding a polypeptide, such as by site-specific mutation formation or by peptide synthesis. Exemplary substitutions are Y470T.

[0294] In a specific such embodiment, in the first Fc domain, the threonine residue at position 366 is substituted with a tryptophan residue (T366W), and in the Fc domain, the tyrosine residue at position 407 is substituted with a valine residue (Y407V), and optionally, the threonine residue at position 366 is substituted with a serine residue (T366S) and the leucine residue at position 368 is substituted with an alanine residue (L368A) (numbering according to the Kabat EU index). In a further embodiment, in the first Fc domain, the serine residue at position 354 is additionally substituted with a cysteine ​​residue (S354C), or the glutamic acid residue at position 356 is substituted with a cysteine ​​residue (E356C) (particularly the serine residue at position 354 is substituted with a cysteine ​​residue), and in the second Fc domain, the tyrosine residue at position 349 is additionally substituted with a cysteine ​​residue (Y349C) (numbering according to the Kabat EU index). In a specific embodiment, the first Fc domain comprises amino acid substitutions S354C and T366W, and the second Fc domain comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (numbering according to the Kabat EU index).

[0295] In some embodiments, electrostatic steering (eg, as described in Gunasekaran et al., 2010, J Biol Chem 285(25): 19637-46) can be used to promote binding of the first and second Fc domains of the Fc region.

[0296] As an alternative or addition to using an Fc domain modified to promote heterodimerization, the Fc domain can be modified to allow a purification strategy that can select Fc heterodimers. In one such embodiment, a polypeptide comprises a modified Fc domain that eliminates its binding to protein A, thereby enabling a purification method for heterodimeric proteins. See, for example, U.S. Patent No. 8,586,713. Therefore, the IL2 proprotein comprises a first CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains are different from each other by at least one amino acid, and wherein at least one amino acid difference reduces the binding of the IL2 proprotein to protein A compared to the corresponding IL2 proprotein lacking the amino acid difference. In one embodiment, the first CH3 domain binds protein A and the second CH3 domain contains a mutation / modification that reduces or eliminates protein A binding, such as an H95R modification (by IMGT exon numbering; by EU numbering H435R). The second CH3 may further include a Y96F modification (by IMGT; by EU Y436F). Such modifications are referred to herein as "star" mutations.

[0297] In some embodiments, the Fc may contain one or more mutations (eg, knob and hole mutations) to promote heterodimerization and a star mutation to promote purification.

[0298] 6.9.3. Hinge domain

[0299] The IL2 proprotein of the present disclosure may include an Fc domain, which includes a hinge domain at its N-terminus. The hinge region may be a natural or modified hinge region. The hinge region is usually located at the N-terminus of the Fc region. Unless the context otherwise provides, the term "hinge domain" refers to a hinge sequence that occurs naturally or non-naturally, which is a monomer hinge domain in the context of a single-chain or monomeric polypeptide chain and may include two related hinge sequences on a separate polypeptide chain in the context of a dimeric polypeptide (e.g., a homodimer or heterodimer IL2 proprotein formed by the association of two Fc domains). Sometimes, two related hinge sequences are referred to as "hinge regions".

[0300] The native hinge region is the hinge region usually found between the Fab and Fc domains of naturally occurring antibodies. The modified hinge region is any hinge that is different from the native hinge region in length and / or component. Such hinges can include hinge regions from other species, such as humans, mice, rats, rabbits, sharks, pigs, hamsters, camels, llamas or goat hinge regions. Other modified hinge regions can include a complete hinge region derived from antibodies of different classes or subclasses from the heavy chain Fc domain or Fc region. Alternatively, the modified hinge region can include a part of a native hinge or a repeating unit, wherein each unit in the repeat is derived from a native hinge region. In other alternatives, the native hinge region can be changed by converting one or more cysteine ​​or other residues into neutral residues, such as serine or alanine, or by converting appropriately placed residues into cysteine ​​residues. In this way, the number of cysteine ​​residues in the hinge region can increase or decrease. Other modified hinge regions can be fully synthetic and can be designed to have desired properties, such as length, cysteine ​​composition and flexibility.

[0301] Many modified hinge regions have been described in, for example, US Pat. No. 5,677,425, WO 99 / 15549, WO 2005 / 003170, WO 2005 / 003169, WO 2005 / 003170, WO 98 / 25971, and WO 2005 / 003171 and the contents of these are incorporated herein by reference.

[0302] In one embodiment, an IL2 proprotein of the present disclosure comprises an Fc region, wherein one or both Fc domains have an intact hinge domain at their N-termini.

[0303] In various embodiments, positions 233-236 within the hinge region can be G, G, G, and unoccupied; G, G, unoccupied, and unoccupied; G, unoccupied, unoccupied, and unoccupied; or all unoccupied, where the positions are numbered by EU numbering.

[0304] In some embodiments, a pro-IL2 protein of the present disclosure comprises a modified hinge region that reduces binding affinity to an Fcγ receptor relative to a wild-type hinge region of the same isotype (eg, human IgG1 or human IgG4).

[0305] In one embodiment, the IL2 protein of the present disclosure comprises an Fc region, wherein each Fc domain has a complete hinge domain at its N-terminus, wherein each Fc domain and hinge domain are derived from IgG4 and each hinge domain comprises a modified sequence CPPC. Compared with IgG1 comprising the sequence CPPC, the core hinge region of human IgG4 comprises the sequence CPSC. The serine residues present in the IgG4 sequence lead to increased flexibility in this region, so that a portion of the molecules form disulfide bonds (intrachain disulfide bonds) within the same protein chain, rather than bridging to other heavy chains in the IgG molecule to form interchain disulfide bonds. (Angel et al., 1993, Mol Immunol 30 (1): 105-108). Changing the serine residue to proline to obtain the same core sequence as IgG1 can completely form interchain disulfide bonds in the IgG4 hinge region, thereby reducing heterogeneity in the purified product. This altered isotype is referred to as IgG4P.

[0306] 6.9.3.1. Chimeric Hinge Sequences

[0307] The hinge domain may be a chimeric hinge domain.

[0308] For example, a chimeric hinge may comprise an "upper hinge" sequence derived from a human IgGl, human IgG2, or human IgG4 hinge region combined with a "lower hinge" sequence derived from a human IgGl, human IgG2, or human IgG4 hinge region.

[0309] In certain embodiments, the chimeric hinge region comprises the amino acid sequence EPKSCDKTHTCPPCPAPPVA (SEQ ID NO: 364) (previously disclosed as SEQ ID NO: 8 of WO2014 / 121087, which is incorporated herein by reference in its entirety) or ESKYGPPCPPCPAPPVA (SEQ ID NO: 365) (previously disclosed as SEQ ID NO: 9 of WO2014 / 121087). Such chimeric hinge sequences can be suitably linked to an IgG4 CH2 region (e.g., by incorporation of an IgG4 Fc domain, e.g., a human or murine Fc domain, which can be further modified in the CH2 and / or CH3 domains to reduce effector function, e.g., as described in Section 6.9.1).

[0310] 6.9.3.2. Hinge sequences with reduced effector function

[0311] In further embodiments, the hinge region may be modified to reduce effector function, for example as described in WO2016161010A2, which is incorporated herein by reference in its entirety. 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, and the positions are numbered according to EU numbering (as shown in FIG. 1 of WO2016161010A2). These fragments may be represented as GGG-, GG--, G---, or ----, where "-" represents an unoccupied position.

[0312] Position 236 is unoccupied in canonical human IgG2, but is occupied in other canonical human IgG isotypes. In all four human isotypes, positions 233-235 are occupied by residues other than G (as shown in Figure 1 of WO2016161010A2).

[0313] Hinge modifications within positions 233-236 can be combined with position 228 occupied by P. Position 228 is naturally occupied by P in human IgG1 and IgG2, but occupied by S in human IgG4 and occupied by R in human IgG3. The S228P mutation in IgG4 antibodies is beneficial for stabilizing IgG4 antibodies and reducing the exchange of heavy chain light chain pairs between exogenous antibodies and endogenous antibodies. Preferably, positions 226-229 are occupied by C, P, P and C, respectively.

[0314] Exemplary hinge regions have residues 226-236, sometimes referred to as the middle (or core) and lower hinges, occupied by modified hinge sequences designated GGG-(233-236), GG--(233-236), G---(233-236), and no G(233-236). Optionally, the hinge domain amino acid sequence includes CPPCPAP GG-GPSVF (SEQ ID NO: 366) (previously disclosed as SEQ ID NO: 1 of WO2016161010A2), CPPCPAP G--GPSVF (SEQ ID NO: 367) (previously disclosed as SEQ ID NO: 2 of WO2016161010A2), CPPCPAP---GPSVF (SEQ ID NO: 368) (previously disclosed as SEQ ID NO: 3 of WO2016161010A2) or CPPCPAP----GPSVF (SEQ ID NO: 369) (previously disclosed as SEQ ID NO: 4 of WO2016161010A2).

[0315] The hinge region modified above can be incorporated into a heavy chain constant region, which generally includes CH2 and CH3 domains, and which may have additional hinge segments (e.g., upper hinges) flanking the specified region. Such additional constant region fragments present generally have the same isotype, preferably a human isotype, although they may be hybrids of different isotypes. The isotype of such additional human constant region fragments is preferably human IgG4, but may also be human IgG1, IgG2 or IgG3 or a hybrid with different isotypes of its domains. Exemplary sequences of human IgG1, IgG2 and IgG4 are shown in Figures 2 to 4 of WO2016161010A2.

[0316] In particular embodiments, the modified hinge sequence can be linked to an IgG4 CH2 region (e.g., by incorporating an IgG4 Fc domain, such as a human or murine Fc domain, which can be further modified in the CH2 and / or CH3 domains to reduce effector function, e.g., as described in Section 6.9.1).

[0317] 6.10. Nucleic Acids and Host Cells

[0318] In another aspect, the present disclosure provides nucleic acids encoding the IL2 proproteins of the present disclosure. In some embodiments, the IL2 proprotein is encoded by a single nucleic acid. In other embodiments, the IL2 proprotein may be encoded by multiple (e.g., two, three, four or more) nucleic acids.

[0319] A single nucleic acid can encode a portion of an IL2 proprotein comprising a single polypeptide chain, an IL2 proprotein comprising two or more polypeptide chains, or an IL2 proprotein comprising more than two polypeptide chains (e.g., a single nucleic acid can encode two polypeptide chains of an IL2 proprotein comprising three, four or more polypeptide chains, or three polypeptide chains of an IL2 proprotein comprising four or more polypeptide chains). For separate control of expression, the open reading frames encoding the two or more polypeptide chains can be under the control of separate transcriptional regulatory elements (e.g., promoters and / or enhancers). The open reading frames encoding the two or more polypeptides can also be controlled by the same transcriptional regulatory elements and separated by an internal ribosome entry site (IRES) sequence, thereby allowing translation into separate polypeptides.

[0320] In some embodiments, the IL2 proprotein comprising two or more polypeptide chains is encoded by two or more nucleic acids. The number of nucleic acids encoding the IL2 proprotein may be equal to or less than the number of polypeptide chains in the IL2 proprotein (e.g., when more than one polypeptide chain is encoded by a single nucleic acid).

[0321] The nucleic acid of the present disclosure can be DNA or RNA (eg, mRNA).

[0322] 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 in the same host cell or in separate host cells, as described in more detail below.

[0323] 6.10.1. Carrier

[0324] The present disclosure provides a vector comprising a nucleotide sequence encoding an IL2 proprotein or a component thereof as described herein, such as one or two polypeptide chains of a half antibody of the IL2 proprotein. The vector includes, but is not limited to, a virus, a plasmid, a cosmid, a lambda phage, or a yeast artificial chromosome (YAC).

[0325] A variety of vector systems can be used. For example, one type of vector utilizes DNA elements derived from animal viruses, such as, for example, bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (Rous sarcoma virus, MMTV or MOMLV) or SV40 virus. Another type of vector utilizes RNA elements derived from RNA viruses, such as Semliki Forest virus, Eastern equine encephalitis virus and flavivirus.

[0326] In addition, can select the cell that DNA is stably integrated in its chromosome by introducing one or more markers that allow to select transfected host cells.Mark can provide for example the original plasm of auxotrophic host, biocide resistance (for example, antibiotic) or resistance to heavy metal (for example copper) etc.Selectable marker gene can be directly linked to DNA sequence dna to be expressed, or is introduced in the same cell by cotransformation.The best synthesis of mRNA may also need additional elements.These elements can comprise splicing signal and transcription promoter, enhancer and termination signal.

[0327] Once the expression vector or DNA sequence containing the construct is prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. Various techniques can be used to achieve this, such as, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques. Methods and components for culturing the resulting transfected cells and for recovering the expressed polypeptide are known to those skilled in the art and can be varied or optimized based on the present specification according to the specific expression vector and mammalian host cell used.

[0328] 6.10.2. Cells

[0329] The present disclosure also provides host cells comprising the nucleic acids of the present disclosure.

[0330] In one embodiment, a host cell is genetically engineered to contain one or more nucleic acids described herein.

[0331] In one embodiment, host cells are genetically engineered by using an expression cassette. The phrase "expression cassette" refers to a nucleotide sequence that can affect the expression of a gene in a host compatible with such sequence. Such cassettes may include a promoter, an open reading frame with or without introns, and a termination signal. Additional factors that are necessary or helpful to affect expression may also be used, such as, for example, an inducible promoter.

[0332] The present disclosure also provides host cells comprising the vectors described herein.

[0333] Cells may be, but are not limited to, eukaryotic cells, bacterial cells, insect cells or human cells. 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.

[0334] 6.11. Pharmaceutical compositions

[0335] The IL2 proprotein of the present disclosure may be in the form of a composition comprising the IL2 proprotein and one or more carriers, excipients and / or diluents. The composition may be formulated for a specific use, such as for veterinary use or human pharmaceutical use. The form of the composition (e.g., dry powder, liquid formulation, etc.) and the excipients, diluents and / or carriers used will depend on the intended use of the IL2 proprotein and the mode of administration for therapeutic use.

[0336] For therapeutic use, the composition can be provided as part of a sterile pharmaceutical composition comprising a pharmaceutically acceptable carrier. The composition can be in any suitable form (depending on the desired method of applying it to the patient). The pharmaceutical composition can be applied to the patient by a variety of routes, such as oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, intrathecal, external application or topical. The most suitable route of administration in any given case will depend on the specific IL2 protein precursor, the subject, and the nature and severity of the disease and the physical condition of the subject. Typically, the pharmaceutical composition will be administered intravenously or subcutaneously.

[0337] The pharmaceutical composition can conveniently exist in a unit dosage form containing a predetermined amount of the IL2 proprotein of the present disclosure per dose. The amount of the IL2 proprotein contained in the unit dose will depend on the disease being treated and other factors well known in the art. Such unit doses can be in the form of a lyophilized dry powder containing a certain amount of IL2 proprotein suitable for a single administration, or in liquid form. The dry powder unit dosage form can be packaged in a kit together with a syringe, an appropriate amount of diluent and / or other components for administration. The unit dose in liquid form can be conveniently supplied in the form of a syringe pre-filled with a certain amount of IL2 proprotein suitable for a single administration.

[0338] The pharmaceutical composition may also be supplied in bulk containing an amount of IL2 protein suitable for multiple administrations.

[0339] Pharmaceutical compositions can be prepared by mixing the IL2 protein precursor having the desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (i.e., buffers, stabilizers, preservatives, isotonic agents, nonionic detergents, antioxidants and other various additives) commonly used in the art (all of which are referred to herein as "carriers") for storage as lyophilized preparations or aqueous solutions. See Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives should be nontoxic to the recipient at the dosages and concentrations employed.

[0340] Buffers help maintain pH values ​​within a range close to physiological conditions. They can be present in a variety of concentrations, but are generally present in a concentration range of about 2 mM to about 50 mM. Buffers suitable for use in the present disclosure include organic and inorganic acids and salts thereof, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixtures, citric acid-trisodium citrate mixtures, citric acid-monosodium citrate mixtures, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixtures, succinic acid-sodium hydroxide mixtures, succinic acid-disodium succinate mixtures, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixtures, tartaric acid-potassium tartrate mixtures, tartaric acid-sodium hydroxide mixtures, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixtures, In some embodiments, the present invention provides the present invention relates to a kind of buffered saline (for example, a sodium hydroxide mixture, a gluconic acid-sodium oxalate mixture, a sodium hydroxide mixture, a potassium oxalate mixture, etc.), a sodium hydroxide buffer (for example, a sodium hydroxide mixture, a sodium hydroxide mixture, a potassium oxalate mixture, etc.), a sodium hydroxide buffer (for example, a sodium hydroxide mixture, a sodium hydroxide mixture, a sodium hydroxide mixture, etc.), a sodium hydroxide buffer (for example ...

[0341] Preservatives can be added to delay microbial growth, and can be added in an amount within the range of about 0.2% to 1% (w / v). Preservatives suitable for the present disclosure include phenol, benzyl alcohol, metacresol, methyl parahydroxybenzoate, propyl parahydroxybenzoate, octadecyl dimethyl benzyl ammonium chloride, benzalkonium chloride halides (e.g., chloride, bromide and iodide), hexamethyl ammonium chloride and alkyl parahydroxybenzoates (such as methyl parahydroxybenzoate or propyl ester), catechol, resorcinol, cyclohexanol and 3-pentanol. Isotonic agents (sometimes referred to as "stabilizers") can be added to ensure the isotonicity of the liquid composition of the present disclosure, and isotonic agents include polyols, such as trivalent or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol and mannitol. Stabilizers refer to a large class of excipients, and their functional range ranges from fillers to dissolving therapeutic agents or helping to prevent denaturation or adhesion to the additive of the container wall. Typical stabilizers may be polyols (listed above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, inositol, galactitol, glycerol, etc., including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents such as urea. , glutathione, lipoic acid, sodium thioacetate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (e.g., peptides of 10 residues or less); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose, glucose; disaccharides such as lactose, maltose, sucrose and trehalose; and trisaccharides such as raffinose; and polysaccharides such as dextran. The stabilizer may be present in an amount ranging from 0.5 to 10 wt% per wt IL2 protein.

[0342] Nonionic surfactants or detergents (also referred to as "wetting agents") may be added to help dissolve the glycoprotein and protect the glycoprotein from agitation-induced aggregation, which also allows the formulation to be exposed to shear surface stress without causing protein denaturation. Suitable nonionic surfactants include polysorbates (20, 80, etc.), polyoxathamers (184, 188, etc.), and pluronic polyols. The nonionic surfactant may be present in a range of about 0.05 mg / mL to about 1.0 mg / mL, for example, about 0.07 mg / mL to about 0.2 mg / mL.

[0343] Other miscellaneous excipients include fillers (eg, starch), chelating agents (eg, EDTA), antioxidants (eg, ascorbic acid, methionine, vitamin E), and co-solvents.

[0344] The IL2 proprotein of the present disclosure can be formulated as a pharmaceutical composition comprising the IL2 proprotein, for example, containing one or more pharmaceutically acceptable excipients or carriers. In order to prepare a pharmaceutical or sterile composition comprising the IL2 proprotein of the present disclosure, the IL2 proprotein preparation can be combined with one or more pharmaceutically acceptable excipients or carriers.

[0345] For example, formulations of the IL2 proprotein can be prepared by mixing the IL2 proprotein with a physiologically acceptable carrier, excipient or stabilizer in the form of, for example, a lyophilized powder, a slurry, an aqueous solution, a lotion or a suspension (see, e.g., Hardman et al., 2001, Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro, 2000, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wlkins, New York, NY; Avis, et al. (eds.), 1993, Pharmaceutical Dosage Forms: General Medications, Marcel Dekker, NY; Lieberman, et al. (eds.), 1990, Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.), 1990, Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie, 2000, ExcipientToxicity and Safety, Marcel Dekker, Inc., New York, NY).

[0346] The effective amount for a particular subject may vary depending on a variety of factors, such as the condition being treated, the subject's overall health, the route and dosage of administration, and the severity of side effects (see, e.g., Maynard et al. (1996) A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001) Good Laboratory and Good Clinical Practice, Urch Publ., London, UK).

[0347] Compositions of the present disclosure can also be used by one or more routes of administration using one or more methods in a variety of methods known in the art. As will be appreciated by the technician, routes of administration and / or mode will vary according to the desired result. The selected route of administration of IL2 proprotein includes intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other general routes of administration, such as by injection or infusion. General administration can represent the mode of administration except enteral and topical administration, usually by injection, and includes but is not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, compositions of the present disclosure can be used by non-general routes, such as topical, epidermal or mucosal routes of administration, for example, intranasal, oral, vaginal, rectal, sublingual or local. In one embodiment, IL2 proprotein is administered by infusion. In another embodiment, subcutaneous administration of IL2 proprotein of the present disclosure.

[0348] 6.12. Therapeutic Indications and Directions of Use

[0349] The present disclosure provides methods of use and applications of the IL2 proprotein of the present disclosure.

[0350] In certain aspects, the present disclosure provides a method for treating cancer, comprising administering to a subject in need thereof a pro-IL2 protein or a pharmaceutical composition as described herein. In some embodiments, an activated IL2 protein comprising an IL2 portion is produced by cleaving one or more protease cleavable linkers in the pro-IL2 protein by one or more proteases expressed by cancer tissue. Thus, the pro-IL2 protein is selectively activated in cancer tissue.

[0351] In some embodiments, the present disclosure provides a method for treating cancer with an IL2 protein selectively activated in cancer tissue, comprising administering to a subject in need thereof a pro-IL2 protein or a pharmaceutical composition as described herein, wherein the pro-IL2 protein has one or more protease cleavable linkers, each comprising one or more substrates of one or more proteases expressed by the cancer tissue targeted by the IL2 protein. Thus, the activated IL2 protein comprising the IL2 portion is produced by cleaving one or more protease cleavable linkers in the pro-IL2 protein by one or more proteases in the cancer tissue.

[0352] The present disclosure also provides a method for local delivery of IL2 protein, comprising administering to a subject a pro-IL2 protein or a pharmaceutical composition as described herein, wherein the pro-IL2 protein has one or more protease cleavable linkers, each linker comprising one or more substrates for one or more proteases expressed by a tissue to which the IL2 protein is to be locally delivered. As used herein, the term "local delivery" does not require local administration, but rather means that the active component of the pro-IL2 protein refers to activation of the protein at a site of interest by a protease active at the intended site, optionally with targeting to the site of interest using a targeting moiety that recognizes a target molecule expressed by the tissue.

[0353] The present disclosure further provides methods of administering to a subject an IL2 therapy with reduced systemic exposure and / or reduced systemic toxicity, comprising administering to the subject an IL2 therapy in the form of a pro-IL2 protein or a pharmaceutical composition described herein, wherein the pro-IL2 protein has one or more protease-cleavable linkers, each linker comprising one or more substrates for one or more proteases expressed by a tissue for which the IL2 therapy is in need and / or intended.

[0354] Thus, the above methods allow for IL2 therapy with reduced off-target side effects by preferentially activating the IL2 proprotein at the site for IL2 treatment.

[0355] In some embodiments of the foregoing methods, the pro-IL2 protein is also targeted and comprises one or more targeting moieties that recognize a target molecule expressed at the site of intended treatment (eg, by a tissue).

[0356] Thus, the present disclosure provides methods for targeted delivery of activated IL2 protein to a site of intended treatment (e.g., cancer tissue), comprising administering to a subject a pro-IL2 protein or a pharmaceutical composition as described herein, wherein the IL2 comprises one or more targeting moieties that recognize a target molecule expressed locally in or by the tissue intended to be treated (e.g., cancer tissue) and has one or more protease-cleavable linkers, each linker comprising a substrate for one or more proteases expressed by the tissue for which IL2 therapy is needed and / or intended.

[0357] The present disclosure further provides a method for inducing an immune response locally in a target tissue, comprising administering to a subject an IL2 proprotein or pharmaceutical composition as described herein, wherein the IL2 proprotein or pharmaceutical composition has one or more targeting moieties capable of binding to a target molecule expressed in the target tissue and one or more protease cleavable linkers, each linker comprising one or more substrates for one or more proteases expressed in the target tissue. The one or more protease cleavable linkers in the IL2 proprotein can then be cleaved by one or more proteases in the target tissue to produce an activated IL2 protein comprising an IL2 moiety. The activated IL2 protein thus produced can induce an immune response against at least one cell type in the target tissue.

[0358] In some embodiments, administration is not local to a tissue. For example, when the target tissue is cancer tissue, administration may be systemic or subcutaneous.

[0359] The IL2 proproteins disclosed herein can be used to treat any proliferative disorder (e.g., cancer) expressing a target molecule (on tumor cells or in the tumor microenvironment, e.g., extracellular matrix or tumor lymphocytes). In specific embodiments, the cancer is acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, Burkitt's lymphoma, cancer of unknown primary, heart tumor, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative tumor, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, lead Tube cancer, embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, fibrous histiocytoma, Ewing's sarcoma, eye cancer, germ cell tumors, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, glioma, head and neck cancer, hairy cell leukemia, hepatocellular carcinoma, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cancer, liver cancer, lobular primordial tumor carcinoma of the nasal cavity, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck carcinoma with occult primary, midline tract cancer involving the NUT gene, oral cancer, multiple endocrine neoplasms syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, nasal and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis , paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, colorectal cancer, renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphoma, teratoma, testicular cancer, pharyngeal cancer, thymoma and thymic cancer, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer or Wilms tumor.

[0360] Table I below shows exemplary indications for which pro-IL2 proteins targeted to specific target molecules may be used.

[0361]

[0362]

[0363]

[0364] Other target molecules and corresponding indications are disclosed in, for example, Hafeez et al., 2020, Molecules 25:4764, doi: 10.3390 / molecules25204764, in particular in Table 1. Table 1 is incorporated herein by reference in its entirety.

[0365] 7. Sequence

[0366] The sequences of certain IL2 proproteins of the present disclosure are provided in Table S below.

[0367]

[0368]

[0369] 8. Numbered Examples

[0370] Although various specific embodiments have been shown and described, it should be understood that various changes can be made without departing from the spirit and scope of the present disclosure. The present disclosure is illustrated by the numbered embodiments set forth below.

[0371] In the subsequent numbered embodiments, the targeting moiety preferably binds to a mammalian target molecule, the IL2 and IL2Rα moieties are preferably derived from mammalian IL2 and IL2Rα, the Fc domain is preferably derived from a mammalian antibody, and the subject is preferably a mammal. More preferably, the mammal is a human.

[0372] 1. An IL2 protein, comprising:

[0373] (a) a first polypeptide chain comprising:

[0374] (i) a first Fc domain;

[0375] (ii) a first linker, which is a protease cleavable linker (PCL) or a non-cleavable linker ("NCL");

[0376] (iii) the first IL2Rα portion;

[0377] (iv) a second linker which is a protease cleavable linker ("PCL"); and

[0378] (v) a first IL2 portion; and

[0379] (b) a second polypeptide chain comprising:

[0380] (i) a second Fc domain, which is capable of associating with said first Fc domain to form an Fc region;

[0381] (ii) a third linker, which is a protease cleavable linker (PCL) or a non-cleavable linker ("NCL");

[0382] (iii) a second IL2Rα portion;

[0383] (iv) a fourth linker which is a protease cleavable linker ("PCL"); and

[0384] (v) Second IL2 portion.

[0385] 2. The IL2 proprotein of embodiment 1, wherein the IL2 portion comprises an amino acid sequence having at least about 90% sequence identity to mature human IL2.

[0386] 3. The IL2 proprotein of embodiment 1, wherein the IL2 portion comprises an amino acid sequence having about 95% sequence identity to mature human IL2.

[0387] 4. The IL2 proprotein of any one of embodiments 1 to 3, wherein the IL2 portion comprises an amino acid sequence having an N-terminal alanine deletion compared to mature human IL2.

[0388] 5. The IL2 proprotein of any one of embodiments 1 to 4, wherein the IL2 portion comprises an amino acid sequence having an amino acid substitution at position N88 compared to wild-type IL2, optionally wherein the amino acid substitution is N88D.

[0389] 6. The IL2 proprotein of any one of embodiments 1 to 5, wherein the IL2 portion comprises an amino acid sequence having an amino acid substitution C125S, C125A or C125V compared to wild-type IL2.

[0390] 7. The IL2 proprotein of any one of embodiments 1 to 6, wherein the IL2Rα portion comprises or consists of an amino acid sequence that has at least about 90% sequence identity to the IL2 binding portion of human IL2Rα.

[0391] 8. The IL2 proprotein of any one of embodiments 1 to 6, wherein the IL2Rα portion comprises or consists of an amino acid sequence that has at least about 95% sequence identity to the IL2 binding portion of human IL2Rα.

[0392] 9. The IL2 proprotein of any one of embodiments 1 to 6, wherein the IL2Rα portion comprises or consists of an amino acid sequence having at least about 97% sequence identity to the IL2 binding portion of human IL2Rα.

[0393] 10. The IL2 proprotein of any one of embodiments 1 to 6, wherein the IL2Rα portion comprises or consists of an amino acid sequence having at least about 98% sequence identity to the IL2 binding portion of human IL2Rα.

[0394] 11. The IL2 proprotein of any one of embodiments 1 to 6, wherein the IL2Rα portion comprises or consists of an amino acid sequence that has at least about 99% sequence identity to the IL2 binding portion of human IL2Rα.

[0395] 12. The IL2 proprotein of any one of embodiments 1 to 6, wherein the IL2Rα portion comprises or consists of an amino acid sequence that has 100% sequence identity to the IL2 binding portion of human IL2Rα.

[0396] 13. The IL2 proprotein of any one of embodiments 7 to 12, wherein the IL2Rα portion comprises an amino acid sequence having at least 90% sequence identity to (a) amino acids 22-186 of IL2Rα, (b) amino acids 22-240 of IL2Rα, and / or (c) amino acids 22-272 of human IL2Rα.

[0397] 14. The IL2 proprotein of any one of embodiments 7 to 12, wherein the IL2Rα portion comprises an amino acid sequence having at least 95% sequence identity to (a) amino acids 22-186 of IL2Rα, (b) amino acids 22-240 of IL2Rα, and / or (c) amino acids 22-272 of human IL2Rα.

[0398] 15. The IL2 proprotein of any one of embodiments 7 to 12, wherein the IL2Rα portion comprises an amino acid sequence having at least 96% sequence identity to (a) amino acids 22-186 of IL2Rα, (b) amino acids 22-240 of IL2Rα, and / or (c) amino acids 22-272 of human IL2Rα.

[0399] 16. The IL2 proprotein of any one of embodiments 7 to 12, wherein the IL2Rα portion comprises an amino acid sequence having at least 97% sequence identity to (a) amino acids 22-186 of IL2Rα, (b) amino acids 22-240 of IL2Rα, and / or (c) amino acids 22-272 of human IL2Rα.

[0400] 17. The IL2 proprotein of any one of embodiments 7 to 12, wherein the IL2Rα portion comprises an amino acid sequence having at least 98% sequence identity to (a) amino acids 22-186 of IL2Rα, (b) amino acids 22-240 of IL2Rα, and / or (c) amino acids 22-272 of human IL2Rα.

[0401] 18. The IL2 proprotein of any one of embodiments 7 to 12, wherein the IL2Rα portion comprises an amino acid sequence having at least 99% sequence identity to (a) amino acids 22-186 of IL2Rα, (b) amino acids 22-240 of IL2Rα, and / or (c) amino acids 22-272 of human IL2Rα.

[0402] 19. The IL2 proprotein of any one of embodiments 1 to 18, wherein the second linker, the fourth linker, optionally the first linker, optionally the third linker, or any combination of two or more or all of the foregoing (e.g., (i) the first and third linkers, (ii) the second and fourth linkers, (iii) the first and second linkers, (iv) the third and fourth linkers, (v) the first, second, third, and fourth linkers) comprises a substrate sequence cleavable by any protease listed in Table A.

[0403] 20. The IL2 proprotein of any one of embodiments 1 to 19, wherein the second linker, the fourth linker, optionally the first linker, optionally the third linker, or any combination of two or more or all of the foregoing (e.g., (i) the first and third linkers, (ii) the second and fourth linkers, (iii) the first and second linkers, (iv) the third and fourth linkers, (v) the first, second, third, and fourth linkers) comprises one or more substrate sequences selected from the substrate sequences listed in Table B.

[0404] 21. The IL2 proprotein of any one of embodiments 1 to 20, wherein the second linker, the fourth linker, optionally the first linker, optionally the third linker, or any combination of two or more or all of the foregoing (e.g., (i) the first and third linkers, (ii) the second and fourth linkers, (iii) the first and second linkers, (iv) the third and fourth linkers, (v) the first, second, third, and fourth linkers) comprises one or more spacer sequences selected from the substrate sequences listed in Table C.

[0405] 22. The IL2 proprotein of any one of embodiments 1 to 21, wherein the second linker, the fourth linker, optionally the first linker, optionally the third linker, or any combination of two or more or all of the foregoing (e.g., (i) the first and third linkers, (ii) the second and fourth linkers, (iii) the first and second linkers, (iv) the third and fourth linkers, (v) the first, second, third and fourth linkers) comprises the amino acid sequence of any one of the PCL sequences listed in Table D or a variant thereof having up to 5 amino acid substitutions, e.g., a variant thereof having 1 amino acid substitution, 2 amino acid substitutions, 3 amino acid substitutions, 4 amino acid substitutions, or 5 amino acid substitutions.

[0406] 23. The IL2 proprotein of any one of embodiments 1 to 22, wherein said first and third linkers are the same and / or said third and fourth linkers are the same.

[0407] 24. The IL2 proprotein of embodiment 23, wherein the first, second, third and fourth linkers are the same.

[0408] 25. The IL2 proprotein of any one of embodiments 1 to 23, wherein said first and third linkers are non-cleavable linkers.

[0409] 26. The IL2 proprotein of embodiment 25, wherein said non-cleavable linker comprises or consists of any one of said NCL sequences listed in Table E.

[0410] 27. The IL2 proprotein of embodiment 25, wherein the first and third linkers range from 2 to 60 amino acids in length.

[0411] 28. The IL2 proprotein of embodiment 25, wherein the first and third linkers range from 5 to 25 amino acids in length.

[0412] 29. The IL2 proprotein of embodiment 25, wherein the first and third linkers range from 7 to 20 amino acids in length.

[0413] 30. The IL2 proprotein of embodiment 25, wherein said first and third linkers are at least 5 amino acids in length.

[0414] 31. The IL2 proprotein of any one of embodiments 1 to 23, wherein said first, second, third and fourth linkers are protease cleavable linkers.

[0415] 32. The IL2 proprotein of embodiment 31, wherein the first, second, third and fourth linkers range from 20 amino acids to 80 amino acids in length.

[0416] 33. The IL2 proprotein of embodiment 31, wherein the first, second, third and fourth linkers range from 20 amino acids to 60 amino acids in length.

[0417] 34. The IL2 proprotein of any one of embodiments 1 to 33, wherein said first Fc domain and / or said second Fc domain comprises a hinge domain.

[0418] 35. The IL2 proprotein of any one of embodiments 1 to 34, further comprising one or more targeting moieties that bind to one or more target molecules.

[0419] 36. The IL2 proprotein of embodiment 35, comprising a first targeting moiety and / or a second targeting moiety.

[0420] 37. The IL2 proprotein of embodiment 36, comprising a first targeting moiety or a component thereof (e.g., VH of Fab) at the N-terminus of the first Fc domain and / or a second targeting moiety or a component thereof (e.g., VH of Fab) at the N-terminus of the second Fc domain.

[0421] 38. The IL2 proprotein of embodiment 35 or embodiment 37, wherein the first targeting moiety and / or the second targeting moiety is a Fab.

[0422] 39. The IL2 proprotein of embodiment 35 or embodiment 37, wherein the first targeting moiety and / or the second targeting moiety is a scFv.

[0423] 40. The IL2 proprotein of any one of embodiments 35 to 39, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to an extracellular matrix ("ECM") antigen, a tumor reactive lymphocyte antigen, a cell surface molecule of a tumor or a viral lymphocyte, a T cell antigen ("TCA"), a checkpoint inhibitor, or a tumor associated antigen ("TAA").

[0424] 41. The IL2 proprotein of any one of embodiments 35 to 40, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to any target molecule identified in Section 6.7.

[0425] 42. The IL2 proprotein of any one of embodiments 35 to 41, wherein the first targeting moiety and / or the second targeting moiety (a) comprises (i) CDRs or (ii) VH and VL sequences of an antibody listed in Table F or (b) competes with an antibody listed in Table F for binding to the target molecule.

[0426] 43. The IL2 protein of any one of embodiments 35 to 41, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to an ECM antigen, the ECM antigen being optionally selected from syndecan, heparanase, integrin, osteopontin, connexin, cadherin, laminin, laminin-type EGF, lectin, fibronectin, notch proteins, adhesion proteins (e.g., adhesion protein-4), tenascin, collagen (e.g., type X collagen), and matrix proteins.

[0427] 44. The IL2 proprotein of embodiment 43, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to an adhesion protein, eg, adhesion protein 4.

[0428] 45. The IL2 proprotein of embodiment 43, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to collagen, eg, collagen X.

[0429] 46. ​​The IL2 proprotein of any one of embodiments 35 to 41, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to a cell surface molecule of a tumor or viral lymphocyte.

[0430] 47. The IL2 proprotein of embodiment 46, wherein the antigen is a T cell co-stimulatory protein.

[0431] 48. The IL2 protein of embodiment 47, wherein the T cell co-stimulatory protein is CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C or B7-H3.

[0432] 49. The IL2 proprotein of embodiment 48, wherein the T cell co-stimulatory protein is B7-H3.

[0433] 50. The IL2 proprotein of any one of embodiments 35 to 41, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to a check point inhibitor.

[0434] 51. The IL2 protein of embodiment 50, wherein the checkpoint inhibitor is CTLA-4, PD1, PDL1, PDL2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1 or CHK2.

[0435] 52. The IL2 proprotein of embodiment 51, wherein the checkpoint inhibitor is PDL1.

[0436] 53. The IL2 proprotein of embodiment 51, wherein the checkpoint inhibitor is PD1.

[0437] 54. The IL2 proprotein of embodiment 51, wherein the checkpoint inhibitor is LAG3.

[0438] 55. The IL2 proprotein of any one of embodiments 35 to 41, wherein said first targeting moiety and / or second targeting moiety is capable of binding to a tumor associated antigen ("TAA").

[0439] 56. The IL2 protein according to embodiment 55, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to AFP, ALK, BAGE protein, BIRC5 (survivin), BIRC7, beta-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CEACAM5 (also known as carcinoembryonic antigen or CEA), CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE ​​proteins (e.g., GAGE-1 or -2), GD2, GD3, GloboH, Glypican-3, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, LMP2, MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE protein, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase, or uroplasmin-3.

[0440] 57. The IL2 proprotein of embodiment 56, wherein the TAA is EGFR.

[0441] 58. The IL2 proprotein of embodiment 56, wherein the TAA is HER2.

[0442] 59. The IL2 proprotein of embodiment 56, wherein the TAA is EPCAM.

[0443] 60. The IL2 proprotein of embodiment 56, wherein the TAA is CEACAM5.

[0444] 61. The IL2 proprotein of embodiment 56, wherein the TAA is CD20.

[0445] 62. The IL2 proprotein of any one of embodiments 1 to 61, wherein the Fc region is a homodimer.

[0446] 63. The IL2 proprotein of any one of embodiments 1 to 61, wherein the Fc region is a heterodimer.

[0447] 64. The IL2 proprotein of any one of embodiments 1 to 63, wherein:

[0448] (a) the first polypeptide chain comprises, from the N-terminus to the C-terminus:

[0449] (i) the first Fc domain;

[0450] (ii) the first connector;

[0451] (iii) the first IL2 portion;

[0452] (iv) the second connector; and

[0453] (v) said first IL2Rα portion; and

[0454] (b) the second polypeptide chain comprises in the N-terminal to C-terminal direction;

[0455] (i) the second Fc domain;

[0456] (ii) the third connector;

[0457] (iii) the second IL2 portion;

[0458] (iv) the fourth connector; and

[0459] (v) the second IL2Rα portion.

[0460] 65. IL2 proprotein, which is optionally the IL2 proprotein according to any one of embodiments 1 to 64, wherein the IL2 proprotein comprises:

[0461] (a) a first Fc domain and a second Fc domain capable of associating to form an Fc region;

[0462] (b) two linkers, which are located at the C-terminus of the Fc domain and are non-cleavable or protease-cleavable;

[0463] (c) two IL2 moieties located C-terminally to the first and third linkers;

[0464] (d) two additional linkers, located at the C-terminus of the IL2 portion, which are cleavable by a protease; and

[0465] (e) Two IL2Rα moieties located C-terminally to the second and fourth linkers.

[0466] 66. The IL2 proprotein of any one of embodiments 1 to 63, wherein

[0467] (a) the first polypeptide chain comprises: the first Fc domain, followed by the first linker wherein the first linker is a protease cleavable linker, followed by the first IL2 portion, followed by the second linker, followed by the first IL2Rα portion; and

[0468] (b) said second polypeptide chain comprises: said second Fc domain, followed by said third linker, wherein said third linker is a protease cleavable linker, followed by said second IL2 portion, followed by said fourth linker, followed by said second IL2Rα portion.

[0469] 67. The IL2 proprotein of any one of embodiments 1 to 63, wherein

[0470] (a) the first polypeptide chain comprises: the first Fc domain, followed by the first linker, wherein the first linker is a non-cleavable linker, followed by the first IL2 portion, followed by the second linker, followed by the first IL2Rα portion; and

[0471] (b) said second polypeptide chain comprises: said second Fc domain, followed by said third linker, wherein said third linker is a non-cleavable linker, followed by said second IL2 portion, followed by said fourth linker, followed by said second IL2Rα portion.

[0472] 68. The IL2 proprotein according to any one of embodiments 1 to 63, having Figure 1A The configuration depicted in .

[0473] 69. The IL2 proprotein according to any one of embodiments 1 to 63, having Figure 2A The configuration depicted in .

[0474] 70. IL2 proprotein, which is optionally the IL2 proprotein according to any one of embodiments 1 to 64, wherein said IL2 proprotein comprises:

[0475] (a) a first polypeptide chain comprising, in order from N-terminus to C-terminus:

[0476] (i) a first amino acid sequence having at least about 95% sequence identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, and 6;

[0477] (ii) (1) a cleavable linker / cleavable tool for linking the first amino acid sequence to a third amino acid sequence, optionally wherein the cleavable linker / cleavable tool comprises or consists of a second amino acid sequence comprising one or more sequences listed in Table B or Table D; or (2) a non-cleavable linker / non-cleavable tool for linking the first amino acid sequence to a third amino acid sequence, optionally wherein the non-cleavable linker / non-cleavable tool comprises or consists of a sequence listed in Table E;

[0478] (iii) the third amino acid sequence, which has at least about 95% sequence identity to SEQ ID NO: 7;

[0479] (iv) a fourth amino acid sequence comprising the sequence listed in Table B; and

[0480] (v) a fifth amino acid sequence having at least about 95% sequence identity to any one of SEQ ID NOs: 8, 9, and 10; and

[0481] (b) a second polypeptide chain comprising, in order from N-terminus to C-terminus:

[0482] (i) a sixth amino acid sequence having at least about 95% sequence identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, and 6;

[0483] (ii) (1) a cleavable linker / cleavable tool for connecting the sixth amino acid sequence to an eighth amino acid sequence, optionally wherein the cleavable linker / cleavable tool comprises or consists of a seventh amino acid sequence, the seventh amino acid sequence comprising one or more sequences listed in Table B or Table D; or (2) a non-cleavable linker / non-cleavable tool for connecting the sixth amino acid sequence to an eighth amino acid sequence, optionally wherein the non-cleavable linker / non-cleavable tool comprises or consists of a sequence listed in Table E;

[0484] (iii) the eighth amino acid sequence, which has at least about 95% sequence identity to SEQ ID NO: 7;

[0485] (iv) a ninth amino acid sequence comprising the sequence listed in Table B; and

[0486] (v) a tenth amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 8, 9 or 10.

[0487] 71. The IL2 proprotein of embodiment 70, wherein the first polypeptide comprises an eleventh amino acid sequence at the N-terminus of the first amino acid sequence, the eleventh amino acid sequence having at least about 95% sequence identity to any one of SEQ ID NOs: 11, 12, 13 or 14.

[0488] 72. The IL2 proprotein of embodiment 71, wherein the eleventh amino acid sequence is the amino acid sequence of SEQ ID NO:11.

[0489] 73. The IL2 proprotein according to embodiment 71, wherein the eleventh amino acid sequence is the amino acid sequence of SEQ ID NO:12.

[0490] 74. The IL2 proprotein of embodiment 71, wherein the eleventh amino acid sequence is the amino acid sequence of SEQ ID NO:13.

[0491] 75. The IL2 proprotein of embodiment 71, wherein the eleventh amino acid sequence is the amino acid sequence of SEQ ID NO:14.

[0492] 76. The IL2 proprotein of any one of embodiments 70 to 75, wherein said second polypeptide comprises a twelfth amino acid sequence at the N-terminus of said first amino acid sequence, said twelfth amino acid sequence having at least about 95% sequence identity to any one of SEQ ID NOs: 11, 12, 13 or 14.

[0493] 77. The IL2 proprotein of embodiment 76, wherein the twelfth amino acid sequence is the amino acid sequence of SEQ ID NO:11.

[0494] 78. The IL2 proprotein of embodiment 76, wherein the twelfth amino acid sequence is the amino acid sequence of SEQ ID NO:12.

[0495] 79. The IL2 proprotein of embodiment 76, wherein the twelfth amino acid sequence is the amino acid sequence of SEQ ID NO:13.

[0496] 80. The IL2 proprotein of embodiment 76, wherein the twelfth amino acid sequence is the amino acid sequence of SEQ ID NO:14.

[0497] 81. The IL2 proprotein of any one of embodiments 70 to 80, wherein said first amino acid sequence has at least about 98% sequence identity to any one of SEQ ID NO: 1, 2, 3, 4, 5 or 6.

[0498] 82. The IL2 proprotein of any one of embodiments 70 to 80, wherein said first amino acid sequence is the amino acid sequence of any one of SEQ ID NO: 1, 2, 3, 4, 5 or 6.

[0499] 83. The IL2 proprotein of embodiment 82, wherein the first amino acid sequence is the amino acid sequence of SEQ ID NO: 5.

[0500] 84. The IL2 proprotein of embodiment 82, wherein the first amino acid sequence is the amino acid sequence of SEQ ID NO: 6.

[0501] 85. The IL2 proprotein of any one of embodiments 70 to 84, wherein said first amino acid sequence is 350 or fewer amino acids in length.

[0502] 86. The IL2 proprotein of any one of embodiments 70 to 84, wherein said first amino acid sequence is 330 or fewer amino acids in length.

[0503] 87. The IL2 proprotein of any one of embodiments 70 to 86, wherein said sixth amino acid sequence has at least about 98% sequence identity to any one of SEQ ID NO: 1, 2, 3, 4, 5 or 6.

[0504] 88. The IL2 proprotein of any one of embodiments 70 to 80, wherein said sixth amino acid sequence is the amino acid sequence of any one of SEQ ID NO: 1, 2, 3, 4, 5 or 6.

[0505] 89. The IL2 proprotein of embodiment 88, wherein the sixth amino acid sequence is the amino acid sequence of SEQ ID NO:5.

[0506] 90. The IL2 proprotein of embodiment 88, wherein the sixth amino acid sequence is the amino acid sequence of SEQ ID NO:6.

[0507] 91. The IL2 proprotein of any one of embodiments 70 to 90, wherein said sixth amino acid sequence is 350 or fewer amino acids in length.

[0508] 92. The IL2 proprotein of any one of embodiments 70 to 90, wherein said sixth amino acid sequence is 330 or fewer amino acids in length.

[0509] 93. The IL2 proprotein of any one of embodiments 70 to 92, wherein said second amino acid sequence comprises one or more amino acid sequences listed in Table B.

[0510] 94. The IL2 proprotein of embodiment 93, wherein the second amino acid sequence comprises the sequence HPVGLLAR (SEQ ID NO: 163).

[0511] 95. The IL2 proprotein of embodiment 93, wherein the second amino acid sequence comprises the sequence VPLSLYSG (SEQ ID NO: 159).

[0512] 96. The IL2 proprotein of embodiment 93, wherein the second amino acid sequence comprises the sequence ISSGLLS (SEQ ID NO: 370).

[0513] 97. The IL2 proprotein of embodiment 93, wherein the second amino acid sequence comprises the sequence PLGLWSQ (SEQ ID NO: 115).

[0514] 98. The IL2 proprotein of any one of embodiments 70 to 93, wherein said second amino acid sequence is an amino acid sequence listed in Table D.

[0515] 99. The IL2 proprotein of embodiment 98, wherein the second amino acid sequence is the amino acid sequence GGGISSGLLSGRSDNHGGGISSGLLSGRSDNHGGS (SEQ ID NO: 199).

[0516] 100. The IL2 proprotein of embodiment 98, wherein the second amino acid sequence is the amino acid sequence GGSHPVGLLARGGGHPVGLLARGGGHPVGLLARGS (SEQ ID NO: 203).

[0517] 101. The IL2 proprotein of embodiment 98, wherein the second amino acid sequence is the amino acid sequence GGGHPVGLLARGGGS (SEQ ID NO: 285).

[0518] 102. The IL2 proprotein of embodiment 98, wherein the second amino acid sequence is the amino acid sequence GISSGLLSGRSDNHG (SEQ ID NO: 282).

[0519] 103. The IL2 proprotein of embodiment 98, wherein the second amino acid sequence is the amino acid sequence GGGSISSGLLSGRSDNHGGGS (SEQ ID NO: 283).

[0520] 104. The IL2 proprotein of embodiment 98, wherein the second amino acid sequence is the amino acid sequence GGGISSGLLSGRSDNHGGGS (SEQ ID NO: 284).

[0521] 105. The IL2 proprotein of embodiment 98, wherein the second amino acid sequence is the amino acid sequence GGGGSGGGGSGGGGSVPLSLYSGGGSGGSGGSGS (SEQ ID NO: 221).

[0522] 106. The IL2 proprotein of any one of embodiments 70 to 92, wherein said second amino acid sequence is an amino acid sequence listed in Table E.

[0523] 107. The IL2 proprotein of embodiment 106, wherein the second amino acid sequence is the amino acid sequence (GGGGS) n , wherein n is 1, 2, 3, 4 or 5 (SEQ ID NO: 357).

[0524] 108. The IL2 proprotein of any one of embodiments 93 to 107, wherein said second amino acid sequence is 25 or fewer amino acids in length.

[0525] 109. The IL2 proprotein of any one of embodiments 93 to 107, wherein said second amino acid sequence is 15 or fewer amino acids in length.

[0526] 110. The IL2 proprotein of any one of embodiments 93 to 107, wherein said second amino acid sequence is 6 or fewer amino acids in length.

[0527] 111. The IL2 proprotein of any one of embodiments 70 to 110, wherein said seventh amino acid sequence comprises one or more amino acid sequences listed in Table B.

[0528] 112. The IL2 proprotein of embodiment 111, wherein the seventh amino acid sequence comprises the sequence HPVGLLAR (SEQ ID NO: 163).

[0529] 113. The IL2 proprotein of embodiment 111, wherein the seventh amino acid sequence comprises the sequence VPLSLYSG (SEQ ID NO: 159).

[0530] 114. The IL2 proprotein of embodiment 111, wherein the seventh amino acid sequence comprises the sequence ISSGLLS (SEQ ID NO: 370).

[0531] 115. The IL2 proprotein of embodiment 111, wherein the seventh amino acid sequence comprises the sequence PLGLWSQ (SEQ ID NO: 115).

[0532] 116. The IL2 proprotein of any one of embodiments 70 to 111, wherein said seventh amino acid sequence is an amino acid sequence listed in Table D.

[0533] 117. The IL2 proprotein of embodiment 116, wherein the second amino acid sequence is the amino acid sequence GGGISSGLLSGRSDNHGGGISSGLLSGRSDNHGGS (SEQ ID NO: 199).

[0534] 118. The IL2 proprotein of embodiment 116, wherein the second amino acid sequence is the amino acid sequence GGSHPVGLLARGGGHPVGLLARGGGHPVGLLARGS (SEQ ID NO: 203).

[0535] 119. The IL2 proprotein of embodiment 116, wherein the second amino acid sequence is the amino acid sequence GGGHPVGLLARGGGS (SEQ ID NO: 285).

[0536] 120. The IL2 proprotein of embodiment 116, wherein the second amino acid sequence is the amino acid sequence GISSGLLSGRSDNHG (SEQ ID NO: 282).

[0537] 121. The IL2 proprotein of embodiment 116, wherein the second amino acid sequence is the amino acid sequence GGGSISSGLLSGRSDNHGGGS (SEQ ID NO: 283).

[0538] 122. The IL2 proprotein of embodiment 116, wherein the second amino acid sequence is the amino acid sequence GGGISSGLLSGRSDNHGGGS (SEQ ID NO: 284).

[0539] 123. The IL2 proprotein of embodiment 116, wherein the second amino acid sequence is the amino acid sequence GGGGSGGGGSGGGGSVPLSLYSGGGSGGSGGSGS (SEQ ID NO: 221).

[0540] 124. The IL2 proprotein of any one of embodiments 70 to 110, wherein said seventh amino acid sequence is an amino acid sequence listed in Table E.

[0541] 125. The IL2 proprotein of embodiment 124, wherein the second amino acid sequence is the amino acid sequence (GGGGS) n , wherein n is 1, 2, 3, 4 or 5 (SEQ ID NO: 357).

[0542] 126. The IL2 proprotein of any one of embodiments 111 to 125, wherein said seventh amino acid sequence is 25 or fewer amino acids in length.

[0543] 127. The IL2 proprotein of any one of embodiments 111 to 125, wherein said seventh amino acid sequence is 15 or fewer amino acids in length.

[0544] 128. The IL2 proprotein of any one of embodiments 111 to 125, wherein said seventh amino acid sequence is 6 or fewer amino acids in length.

[0545] 129. The IL2 proprotein of any one of embodiments 70 to 128, wherein said third amino acid sequence has at least about 98% sequence identity to SEQ ID NO:7.

[0546] 130. The IL2 proprotein of any one of embodiments 70 to 128, wherein said third amino acid sequence is the amino acid sequence of SEQ ID NO:7.

[0547] 131. The IL2 proprotein of any one of embodiments 70 to 130, wherein said third amino acid sequence is 150 or fewer amino acids in length.

[0548] 132. The IL2 proprotein of any one of embodiments 70 to 130, wherein said third amino acid sequence is 135 or fewer amino acids in length.

[0549] 133. The IL2 proprotein of any one of embodiments 70 to 132, wherein said eighth amino acid sequence has at least about 98% sequence identity to SEQ ID NO:7.

[0550] 134. The IL2 proprotein of any one of embodiments 70 to 132, wherein said eighth amino acid sequence is the amino acid sequence of SEQ ID NO:7.

[0551] 135. The IL2 proprotein of any one of embodiments 70 to 134, wherein said eighth amino acid sequence is 150 or fewer amino acids in length.

[0552] 136. The IL2 proprotein of any one of embodiments 70 to 134, wherein said eighth amino acid sequence is 135 or fewer amino acids in length.

[0553] 137. The IL2 proprotein of any one of embodiments 70 to 136, wherein said fourth amino acid sequence comprises one or more amino acid sequences listed in Table B.

[0554] 138. The IL2 proprotein of any one of embodiments 70 to 136, wherein said fourth amino acid sequence is an amino acid sequence listed in Table D.

[0555] 139. The IL2 proprotein of any one of embodiments 70 to 138, wherein said ninth amino acid sequence comprises one or more amino acid sequences listed in Table B.

[0556] 140. The IL2 proprotein of any one of embodiments 70 to 139, wherein said ninth amino acid sequence is an amino acid sequence listed in Table D.

[0557] 141. The IL2 proprotein of any one of embodiments 70 to 140, wherein said fifth amino acid sequence has at least about 98% sequence identity to SEQ ID NO: 8, 9 or 10.

[0558] 142. The IL2 proprotein of embodiment 141, wherein the fifth amino acid sequence is the amino acid sequence of SEQ ID NO:8.

[0559] 143. The IL2 proprotein of embodiment 141, wherein the fifth amino acid sequence is the amino acid sequence of SEQ ID NO:9.

[0560] 144. The IL2 proprotein of embodiment 141, wherein the fifth amino acid sequence is the amino acid sequence of SEQ ID NO:10.

[0561] 145. The IL2 proprotein of any one of embodiments 70 to 144, wherein said fifth amino acid sequence is 255 or fewer amino acids in length.

[0562] 146. The IL2 proprotein of any one of embodiments 70 to 144, wherein said fifth amino acid sequence is 225 or fewer amino acids in length.

[0563] 147. The IL2 proprotein of any one of embodiments 70 to 144, wherein said fifth amino acid sequence is 170 or fewer amino acids in length.

[0564] 148. The IL2 proprotein of any one of embodiments 70 to 147, wherein said tenth amino acid sequence has at least about 98% sequence identity to SEQ ID NO: 8, 9 or 10.

[0565] 149. The IL2 proprotein of embodiment 148, wherein the tenth amino acid sequence is the amino acid sequence of SEQ ID NO:8.

[0566] 150. The IL2 proprotein of embodiment 148, wherein the tenth amino acid sequence is the amino acid sequence of SEQ ID NO:9.

[0567] 151. The IL2 proprotein of embodiment 148, wherein the tenth amino acid sequence is the amino acid sequence of SEQ ID NO:10.

[0568] 152. The IL2 proprotein of any one of embodiments 70 to 151, wherein said tenth amino acid sequence is 255 or fewer amino acids in length.

[0569] 153. The IL2 proprotein of any one of embodiments 70 to 151, wherein said tenth amino acid sequence is 225 or fewer amino acids in length.

[0570] 154. The IL2 proprotein of any one of embodiments 70 to 151, wherein said tenth amino acid sequence is 170 or fewer amino acids in length.

[0571] 155. The IL2 proprotein of any one of embodiments 70 to 154, wherein said first polypeptide chain lacks additional sequence at the C-terminus of said first amino acid sequence.

[0572] 156. The IL2 proprotein of any one of embodiments 70 to 155, wherein said first polypeptide chain lacks an additional sequence between said first and second amino acid sequences.

[0573] 157. The IL2 proprotein of any one of embodiments 70 to 156, wherein said first polypeptide chain lacks an additional sequence between said second and third amino acid sequences.

[0574] 158. The IL2 proprotein of any one of embodiments 70 to 157, wherein said first polypeptide chain lacks additional sequences between said third amino acid sequence and said fourth amino acid sequence.

[0575] 159. The IL2 proprotein of any one of embodiments 70 to 158, wherein said first polypeptide chain lacks additional sequences between said fourth amino acid sequence and said fifth amino acid sequence.

[0576] 160. The IL2 proprotein of any one of embodiments 70 to 159, wherein said second polypeptide chain lacks an additional sequence at the C-terminus of said sixth amino acid sequence.

[0577] 161. The IL2 proprotein of any one of embodiments 70 to 160, wherein said second polypeptide chain lacks additional sequence between said sixth amino acid sequence and said seventh amino acid sequence.

[0578] 162. The IL2 proprotein of any one of embodiments 70 to 161, wherein said second polypeptide chain lacks additional sequence between said seventh amino acid sequence and said eighth amino acid sequence.

[0579] 163. The IL2 proprotein of any one of embodiments 70 to 162, wherein said second polypeptide chain lacks additional sequence between said eighth amino acid sequence and said ninth amino acid sequence.

[0580] 164. The IL2 proprotein of any one of embodiments 70 to 163, wherein said second polypeptide chain lacks additional sequence between said ninth amino acid sequence and said tenth amino acid sequence.

[0581] 165. The IL2 proprotein of any one of embodiments 70 to 164, wherein said first polypeptide and said second polypeptide are identical.

[0582] 166. A nucleic acid or nucleic acids encoding the IL2 proprotein of any one of embodiments 1 to 165.

[0583] 167. A host cell engineered to express the IL2 proprotein of any one of embodiments 1 to 165 or the nucleic acid of embodiment 166.

[0584] 168. A method of producing the IL2 proprotein of any one of embodiments 1 to 165, the method comprising culturing the host cell of embodiment 167 and recovering the IL2 proprotein expressed thereby.

[0585] 169. A pharmaceutical composition comprising the IL2 proprotein according to any one of embodiments 1 to 165 and an excipient.

[0586] 170. A method of treating cancer, comprising administering the pro-IL2 protein of any one of embodiments 1 to 165 or the pharmaceutical composition of embodiment 169 to a subject in need thereof.

[0587] 171. A method according to embodiment 170, wherein the IL2 protein comprises at least one targeting portion capable of binding to a target molecule and wherein the cancer is associated with the expression of the target molecule, for example, the TAAs and related cancers listed in Table I.

[0588] 172. A method according to embodiment 171, wherein the activated IL2 protein comprising an IL2 portion is produced by cleaving one or more protease cleavable linkers in the IL2 protein precursor by one or more proteases expressed by the cancer tissue.

[0589] 173. A method according to embodiment 172, wherein the IL2 protein is selectively activated in the cancer tissue.

[0590] 174. A method for local delivery of an IL2 protein, the method comprising administering to a subject the pro-IL2 protein of any one of embodiments 1 to 165 (or a pharmaceutical composition comprising the pro-IL2 protein and an excipient), the pro-IL2 protein having one or more protease cleavable linkers, each protease cleavable linker comprising one or more substrates for one or more proteases expressed by a tissue to which the IL2 protein is to be locally delivered.

[0591] 175. A method according to embodiment 174, wherein the IL2 protein contains one or more targeting moieties that recognize a target molecule expressed by the tissue.

[0592] 176. A method according to embodiment 175, wherein the IL2 protein contains two targeting moieties, each targeting moiety recognizing a target molecule expressed by the tissue.

[0593] 177. The method of embodiment 175 or embodiment 176, wherein the tissue is cancer tissue.

[0594] 178. The method of embodiment 177, wherein the one or more targeting moieties are capable of binding to an extracellular matrix ("ECM") antigen, a tumor-reactive lymphocyte antigen, a cell surface molecule of a tumor or viral lymphocyte, a T cell antigen ("TCA"), a checkpoint inhibitor, or a tumor-associated antigen ("TAA").

[0595] 179. A method according to any one of embodiments 174 to 178, wherein the activated IL2 protein comprising an IL2 portion is produced by cleavage of one or more protease cleavable linkers in the IL2 protein by one or more proteases in the tissue.

[0596] 180. A method for treating cancer with an IL2 protein that is selectively activated in cancer tissue, the method comprising administering to a subject in need thereof the pro-IL2 protein of any one of embodiments 1 to 165 (or a pharmaceutical composition comprising the pro-IL2 protein and an excipient), wherein the pro-IL2 protein has one or more protease cleavable linkers, each protease cleavable linker comprising one or more substrates of one or more proteases expressed by the cancer tissue targeted by the IL2 protein.

[0597] 181. A method according to embodiment 180, wherein the IL2 protein contains one or more targeting moieties that recognize a target molecule expressed by the cancer tissue.

[0598] 182. A method according to embodiment 181, wherein the IL2 protein comprises two targeting moieties, each targeting moiety recognizing a target molecule expressed by the cancer tissue.

[0599] 183. The method of embodiment 181 or embodiment 182, wherein the one or more targeting moieties are capable of binding to an extracellular matrix ("ECM") antigen, a tumor-reactive lymphocyte antigen, a cell surface molecule of a tumor or viral lymphocyte, a T cell antigen ("TCA"), a checkpoint inhibitor, or a tumor-associated antigen ("TAA").

[0600] 184. A method according to any one of embodiments 180 to 183, wherein the activated IL2 protein comprising an IL2 portion is produced by cleaving one or more protease-cleavable linkers in the IL2 protein by one or more proteases in the cancer tissue.

[0601] 185. A method of administering an IL2 therapy with reduced systemic exposure and / or reduced systemic toxicity to a subject, the method comprising administering to the subject an IL2 therapy in the form of a proprotein of any one of embodiments 1 to 165 (or a pharmaceutical composition comprising a proprotein of IL2 and an excipient), wherein the proprotein of IL2 has one or more protease cleavable linkers, each protease cleavable linker comprising one or more substrates for one or more proteases expressed by a tissue for which the IL2 therapy is required and / or expected.

[0602] 186. A method according to embodiment 185, wherein the IL2 protein contains one or more targeting portions that recognize a target molecule expressed by the tissue.

[0603] 187. A method according to embodiment 186, wherein the IL2 protein comprises two targeting moieties, each targeting moiety recognizing a target molecule expressed by the tissue.

[0604] 188. The method of any one of embodiments 185 to 187, wherein the tissue is cancer tissue.

[0605] 189. A method according to embodiment 188, wherein the one or more targeting moieties are capable of binding to an extracellular matrix ("ECM") antigen, a tumor-reactive lymphocyte antigen, a cell surface molecule of a tumor or viral lymphocyte, a T cell antigen ("TCA"), a checkpoint inhibitor, or a tumor-associated antigen ("TAA").

[0606] 190. A method according to any one of embodiments ‎184 to ‎189, wherein the activated IL2 protein comprising the IL2 portion is produced by cleavage of one or more protease cleavable linkers in the IL2 protein by one or more proteases in the tissue.

[0607] 191. A method for treating cancer with an IL2 protein that is selectively activated in cancer tissue, the method comprising administering to a subject in need thereof the IL2 proprotein of any one of embodiments 1 to 165 (or a pharmaceutical composition comprising the IL2 proprotein and an excipient), the IL2 proprotein having one or more protease cleavable linkers, each protease cleavable linker comprising one or more substrates of one or more proteases expressed by the cancer tissue.

[0608] 192. A method according to embodiment 191, wherein the IL2 protein contains one or more targeting portions that recognize a target molecule expressed by the cancer tissue.

[0609] 193. A method according to embodiment 192, wherein the IL2 protein comprises two targeting moieties, each targeting moiety recognizing a target molecule expressed by the cancer tissue.

[0610] 194. The method of Example 191 or Example 192, wherein the one or more targeting moieties are capable of binding to an extracellular matrix ("ECM") antigen, a tumor-reactive lymphocyte antigen, a cell surface molecule of a tumor or viral lymphocyte, a T cell antigen ("TCA"), a checkpoint inhibitor, or a tumor-associated antigen ("TAA").

[0611] 195. A method according to any one of embodiments 191 to 194, wherein the activated IL2 protein comprising an IL2 portion is produced by cleavage of one or more protease cleavable linkers in the IL2 protein by one or more proteases in the cancer tissue.

[0612] 196. A method for targeted delivery of activated IL2 protein to cancer tissue, the method comprising administering to a subject the pro-IL2 protein (or a pharmaceutical composition comprising the pro-IL2 protein and an excipient) according to any one of embodiments 1 to 165, wherein the pro-IL2 protein:

[0613] (a) comprising one or more targeting moieties that recognize a target molecule expressed by said cancer tissue; and

[0614] (b) having one or more protease cleavable linkers, each protease cleavable linker comprising one or more substrates for one or more proteases expressed by a tissue for which IL2 therapy is required and / or desired.

[0615] 197. A method according to embodiment 196, wherein the IL2 protein comprises two targeting moieties, each targeting moiety recognizing a target molecule expressed by the cancer tissue.

[0616] 198. A method according to Example 196 or Example 197, wherein the one or more targeting moieties are capable of binding to an extracellular matrix ("ECM") antigen, a tumor-reactive lymphocyte antigen, a cell surface molecule of a tumor or viral lymphocyte, a T cell antigen ("TCA"), a checkpoint inhibitor, or a tumor-associated antigen ("TAA").

[0617] 199. A method according to any one of embodiments 196 to 198, wherein the activated IL2 protein comprising an IL2 portion is produced by cleavage of one or more protease cleavable linkers in the IL2 protein by one or more proteases in the cancer tissue.

[0618] 200. A method for inducing an immune response locally in a target tissue, the method comprising administering to a subject an IL2 proprotein (or a pharmaceutical composition comprising an IL2 proprotein and an excipient) according to any one of embodiments 1 to 165, wherein the IL2 proprotein has one or more targeting moieties capable of binding to a target molecule expressed in the target tissue and one or more protease cleavable linkers, each linker comprising one or more substrates for one or more proteases expressed in the target tissue.

[0619] 201. A method according to embodiment 200, wherein the IL2 protein comprises two targeting moieties, each targeting moiety recognizing a target molecule expressed in the target tissue.

[0620] 202. The method of embodiment 200 or embodiment 201, wherein the target tissue is cancer tissue.

[0621] 203. A method according to any one of embodiments 200 to 202, wherein the one or more targeting moieties are capable of binding to an extracellular matrix ("ECM") antigen, a tumor-reactive lymphocyte antigen, a cell surface molecule of a tumor or viral lymphocyte, a T cell antigen ("TCA"), a checkpoint inhibitor, or a tumor-associated antigen ("TAA").

[0622] 204. The method of any one of embodiments 200 to 203, wherein the activated IL2 protein comprising an IL2 portion is produced by cleavage of one or more protease cleavable linkers in the original IL2 protein by one or more proteases in the target tissue.

[0623] 205. The method of embodiment 204, wherein said IL2 protein induces said immune response against at least one cell type in said target tissue.

[0624] 206. The method of any one of embodiments 170 to 205, wherein said administration is non-topical.

[0625] 207. The method of embodiment 206, wherein said administration is systemic.

[0626] 208. The method of embodiment 206, wherein said administration is subcutaneous.

[0627] 9. Examples

[0628] 9.1. Example 1: Production of IL2 Proprotein

[0629] The construct encoding the IL2 proprotein containing the targeting portion, the Fc domain, the IL2 and IL2Rα portions and the cleavable and / or non-cleavable linker is synthesized as a DNA fragment and cloned into a suitable expression vector. A 29 amino acid signal sequence from the mouse inactivated tyrosine protein kinase transmembrane receptor ROR1 (mROR1) is added to the N-terminus of the construct. All IL2 proproteins are expressed as proproteins containing the signal sequence. The signal sequence is cleaved by intracellular processing to produce mature protein.

[0630] Following the manufacturer's protocol, the construct was cloned into Expi293F TM Transient expression in cells (ThermoFisher). Using the ProteinMaker system (Protein BioSolutions, Gaithersburg, MD) and HiTrap TM Proteins in the supernatant of Expi293F were purified using Protein GHP or MabSelect SuRe pcc columns (Cytiva). After a single elution step, the antibodies were neutralized, dialyzed into a final buffer of phosphate-buffered saline (PBS) containing 5% glycerol, aliquoted and stored at -80°C.

[0631] Table 1 below provides an overview of the IL2 proproteins encoded by the generated constructs. Table 1 describes a single half-antibody for each IL2 proprotein, wherein each IL2 proprotein comprises two identical half-antibodies.

[0632]

[0633] 9.2. Example 2: Antitumor activity of EGFR-targeted pro-IL2 protein

[0634] The antitumor activity of EGFR-targeted IL2 proprotein was evaluated in the MC38 tumor model. Briefly, 7 × 10 5 MC38 tumor cells were implanted subcutaneously into the right hind flank of 8- to 10-week-old female mice expressing humanized EGFR protein on day 0. On day 9, when the average tumor size reached 90 mm 3 At 14:00, mice inoculated with tumors were randomly divided into treatment groups. Mice in each randomized group received two intraperitoneal injections on days 9 and 12. Tumor size was measured semiweekly using a digital caliper and calculated as length x width. 2 / 2. Figure 3A The mean tumor volume (mm) after tumor implantation in each treatment group is shown in 3 - / + SEM). FIG. 3B to FIG. 3E Individual tumor growth curves for each treatment group are depicted in .

[0635] When tumor growth curves for each treatment group were averaged, the EGFR-targeted IL2 proprotein construct with a cleavable linker exhibited more potent tumor growth inhibition compared to the targeted IL2 proprotein construct with a non-cleavable linker and the non-targeted IL2 proprotein construct with a cleavable linker and isotype control ( Figure 3A : Arrows indicate treatment days). FIG. 3B to FIG. 3E Individual tumor growth curves for each treatment group are shown in . Overall, these results indicate that both TAA targeting and cleavable linkers significantly enhance the in vivo anti-tumor efficacy of EGFR-targeted IL2 proprotein constructs.

[0636] 9.3. Example 3: Anti-tumor activity of PD1 targeting pro-IL2 protein

[0637] The antitumor activity of PD1-targeted IL2 protease was evaluated in the MC38 tumor model. On day 0, 3 x 10 5 MC38 tumor cells (ACL8874) were implanted subcutaneously into the right flank of 8- to 10-week-old female mice expressing humanized PD-1. On day 9, when the average tumor size reached 90 mm 3 At 14:00, mice inoculated with tumors were randomized into treatment groups. Mice in each randomized group received two intraperitoneal injections of 1.5 mg / kg of the indicated protein on days 9 and 12. Tumor size was measured semiweekly using a digital caliper and calculated as length x width. 2 / 2. The average tumor volume (mm) in each treatment group after drug administration was plotted 3 - / + SEM)( Figure 4A ).

[0638] When tumor growth curves for each treatment group were averaged, tumor-inoculated mice treated with the PD1-targeted IL2 proprotein construct with a cleavable linker showed reduced tumor growth, whereas mice treated with the non-targeted IL2 proprotein with a cleavable linker or the isotype control showed no inhibition of tumor growth ( Figure 4A ). Individual tumor growth curves for each treatment group showed that none of the isotype-treated mice exhibited tumor suppression ( Figure 4B Similarly, mice treated with the nontargeted pro-IL2 protein showed no tumor suppression ( Figure 4C However, four out of five mice treated with the PD1-targeted pro-IL2 protein remained tumor-free during the evaluation period ( Figure 4D ).

[0639] Compared with isotype control or non-targeted IL2 proprotein, PD1-targeted IL2 proprotein with a cleavable linker exhibited robust tumor growth inhibition, and treated mice experienced complete tumor regression at a higher frequency, indicating that TCA targeting enhanced the in vivo antitumor efficacy of PD1-targeted IL2 proprotein with a cleavable linker.

[0640] 9.4. Example 4: Evaluation of the cleavage capacity of IL2 proproteins containing cleavable linkers

[0641] In order to evaluate whether the protease cleavable linker in the original IL2 protein can be digested by recombinant protease, two constructs mAb-PCL (15AA) -IL2-PCL (15AA) -IL2Rα and mAb-PCL (15AA) -IL2-PCL (20AA) -IL2Rα were selected to be digested using recombinant human protease. In brief, each construct was incubated at 37°C for 24 hours with 200 ng uPA (R&D catalog number 1310-SE) in assay buffer according to the manufacturer's protocol. Without the addition of protease, no digestion control was incubated for the same duration under the same conditions. After digestion, SDS loading buffer containing a reducing agent was added to each sample. Next, the sample was boiled and SDS PAGE was performed on an Invitrogen 4-20% Tris-glycine gel. Then, the protein was transferred to a PVDF membrane using the iBLot 2 dry transfer method. The membrane was then probed with biotinylated anti-hIL2 (R&D Catalog No. BAF202) followed by streptavidin-HRP detection (Cytiva (Catalog No. RPN1231)).

[0642] Digestion of the IL2 proprotein using uPA protease resulted in the release of free IL2 from both IL2 proproteins, although the IL2 proprotein construct containing a 20AA linker between the IL2 and IL2Rα domains showed more complete linker digestion and IL2 release relative to the construct containing a 15AA linker between IL2 and IL2Rα ( Figure 5A and Figure 5B ). These observations suggest that the extent of protease digestion depends on the length of the protease-cleavable linker.

[0643] 9.5. Example 5: In vitro activity of tumor-targeted pro-IL2 protein

[0644] The in vitro activity of protease-digested and undigested IL2 proproteins containing protease-cleavable or non-cleavable linkers was assessed by luciferase reporter assay as described in Section 9.5.1.2, using one of the engineered reporter cell lines generated as described in Section 9.5.1.1.

[0645] 9.5.1. Methods

[0646] 9.5.1.1. Engineering of YT / STAT5-Luc reporter cells

[0647] The human T / NK-like leukemia YT cell line was electroporated with a signal transducer and activator of transcription 5 (STAT5)-driven luciferase reporter construct and maintained in Iscove's modified Dulbecco's medium supplemented with 2 mM L-glutamine / penicillin / streptomycin + 20% fetal bovine serum (FBS) + 200 µg / ml hygromycin. A single cell clone with high responsiveness to IL2 was identified and renamed YT / STAT5-Luc cl.4. IL2Rα (CD25) was knocked out in this clone using CRISPR-Cas9 technology, and the resulting cell line YT / STAT5-Luc / IL2Rα KO (referred to as CD25 KO for simplicity) was verified by flow cytometry.

[0648] Human IL2Rα was then stably reintroduced into the CD25 KO cell line (amino acids M1-I272 of accession number NP_000408.1), and the resulting cell line was verified as CD25 OE by flow cytometry and maintained in Iscove’s modified Dulbecco’s medium supplemented with 2 mM L-glutamine / penicillin / streptomycin + 20% FBS + 200 µg / ml hygromycin + 15 µg / mL blasticidin.

[0649] Since YT cells endogenously express PD1, CD25 KO and CD25 OE cells were transformed using CRISPR / Cas9 technology to knock out PD1 expression, and the resulting cell lines CD25 KO / PD1 KO and CD25OE / PD1 KO were verified by flow cytometry.

[0650] 9.5.1.2. Luciferase reporter gene assay

[0651] One day before screening, engineered YT / STAT5-Luc reporter cells CD25 KO / PD1 KO and CD25 OE / PD1KO were diluted to 3 x 10 in RPMI1640 medium containing 2 mM L-glutamine / penicillin / streptomycin + 10% FBS. 5 cells / mL.

[0652] The IL2 protein was digested in a digestion buffer (50 mM Tris, 0.01% (v / v) Tween ®20, pH 8.5) with or without recombinant human uPA (R&D Cat. No. 1310-SE) for overnight digestion. 20 nM enzyme was added for every 200 nM fusion protein and digested, then diluted into assay medium (RPMI1640 medium supplemented with 2 mM L-glutamine / penicillin / streptomycin + 10% FBS) for bioassay.

[0653] On the day of the assay, cells were centrifuged and resuspended in assay medium at 2.5 x 10 4 Reporter cells / well were plated in 96-well white flat-bottom plates and incubated with recombinant IL2, uPA-digested pro-IL2 protein, or undigested pro-IL2 protein. Each construct was serially diluted (1:5) over an 11-point titration range (50 nM to 5.12 fM), with the 12th point containing no protein. After incubation of the plates at 37°C, 5% CO2 for 4 hours and 30 minutes, 100 µL of ONE-Glo™ (Promega) reagent was added to the wells to lyse the cells and detect luciferase activity. The emitted light was measured in RLU on an Envision multilabel plate reader (PerkinElmer).

[0654] 9.5.2. Results

[0655] Pro-IL2 proteins containing different tumor targeting moieties (e.g., anti-CA9, anti-EGFR, or anti-PD1 Fab moieties) were first evaluated using CD25 KO / PD1 KO cells. For all constructs tested, each protease cleavable linker (PCL) was 15 amino acids in length.

[0656] In each case, uPA-digested pro-IL2 protein produced reporter activity similar to that associated with recombinant IL2, regardless of the targeting moiety ( 6A to 6F Undigested IL2 protein resulted in little to no luciferase activity ( 6A to 6F Similarly, none of the IL2 proproteins with a non-cleavable linker showed luciferase activity ( FIG. 6A to FIG. 6D ).

[0657] Next, the same IL2 proproteins were evaluated using CD25 OE / PD1 KO cells. In this evaluation, all constructs were conjugated with detectable luciferase activity ( 7A to 7F However, recombinant IL2 and uPA-digested IL2 proproteins containing a protease-cleavable linker showed relatively high and comparable potency, which was several orders of magnitude higher than that observed with undigested IL2 proprotein or constructs containing a non-cleavable linker ( 7A to 7F ).

[0658] Altogether, these results indicate that pro-IL2 proteins exhibit minimal activity unless their IL2 moiety is released following protease digestion. Furthermore, IL2 released from pro-IL2 proteins is as potent as recombinant IL2.

[0659] 9.6. Example 6: Effect of linker length on IL2 protein activity

[0660] To reduce treatment-related side effects, it is important to minimize the activity of pro-IL2 proteins until they reach the target tissue. The effect of linker length on attenuating pro-IL2 protein activity was evaluated in a luciferase reporter assay using engineered YT / STAT5-Luc reporter cells.

[0661] RPMI1640 medium supplemented with 2mM L-glutamine / penicillin / streptomycin + 10% fetal bovine serum (FBS) was used as the assay medium to prepare cell suspensions and protein dilutions. One day before screening, engineered YT / STAT5-Luc reporter cells (CD25 KO / PD1 KO, CD25 KO / PD1 OE, CD25 OE / PD1 OE, and CD25 OE / PD1KO) were diluted at 3 x 10 5 On the day of the assay, cells were centrifuged and resuspended in assay medium at 2.5 x 10 4 Reporter cells / well were plated in 96-well white flat-bottom plates and incubated with recombinant IL2 or PD1-targeted IL2 proproteins of varying lengths with non-cleavable linkers. Constructs were serially diluted (1:5) over an 11-point titration range (50 nM to 5.12 fM), with the 12th point containing no protein. The plates were incubated at 37°C and 5% CO2 for 4 hours and 30 minutes, after which 100 mL of ONE-Glo™ (Promega) reagent was added to the wells to lyse the cells and detect luciferase activity. The emitted light was measured in RLU on an Envision multilabel plate reader (PerkinElmer).

[0662] In general, IL2 proproteins with longer linker lengths exhibited greater luciferase activity than IL2 proproteins with shorter linker lengths ( FIG. 8A to FIG. 8D This linker length-dependent reduction in activity was most pronounced in PD1 OE / CD25KO cells ( Fig. 8A ).

[0663] 10. References

[0664] All publications, patents, patent applications, or other documents cited in this application are hereby incorporated by reference in their entirety for all purposes, to the same extent as if each individual publication, patent, patent application, and / or other document was individually indicated to be incorporated by reference for all purposes. If there is an inconsistency between the teachings of one or more references incorporated herein and the present disclosure, the teachings of the present specification are intended to control.

Claims

1. An IL2 protein, comprising: (a) a first polypeptide chain comprising, in order from N-terminus to C-terminus: (i) a first targeting moiety or a component thereof; (ii) a first Fc domain; (iii) a first linker, which is a protease cleavable linker (PCL) or a non-cleavable linker ("NCL"); (iv) a first IL2 portion; (v) a second linker which is a protease cleavable linker ("PCL"); and (vi) a first IL2Rα portion; and (b) a second polypeptide chain comprising, in order from N-terminus to C-terminus: (i) a second targeting moiety or a component thereof; (ii) a second Fc domain, which is capable of associating with the first Fc domain to form an Fc region; (iii) a third linker, which is a protease cleavable linker (PCL) or a non-cleavable linker ("NCL"); (iv) a second IL2 portion; (v) a fourth linker which is a protease cleavable linker ("PCL"); and (vi) A second IL2Rα portion.

2. The IL2 proprotein of claim 1, wherein the IL2 portion comprises an amino acid sequence having at least about 90% or about 95% sequence identity to mature human IL2.

3. The IL2 proprotein according to claim 1 or 2, wherein the IL2 portion comprises an amino acid sequence having an N-terminal alanine deletion compared to mature human IL2.

4. The IL2 proprotein of any one of claims 1 to 3, wherein the IL2 portion comprises an amino acid sequence having an amino acid substitution at position N88 compared to wild-type IL2, optionally wherein the amino acid substitution is N88D, and / or (b) an amino acid sequence having an amino acid substitution C125S, C125A or C125V compared to wild-type IL2.

5. The IL2 proprotein of any one of claims 1 to 4, wherein the IL2Rα portion comprises or consists of an amino acid sequence having at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the IL2 binding portion of human IL2Rα.

6. The IL2 proprotein of claim 5, wherein the IL2Rα portion comprises an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to (a) amino acids 22-186 of IL2Rα, (b) amino acids 22-240 of IL2Rα, and / or (c) amino acids 22-272 of human IL2Rα.

7. The IL2 proprotein of any one of claims 1 to 6, wherein the second linker, the fourth linker, optionally the first linker, optionally the third linker or any combination of two or more or all of the foregoing (e.g., (i) the first and third linkers, (ii) the second and fourth linkers, (iii) the first and second linkers, (iv) the third and fourth linkers, (v) the first, second, third and fourth linkers) comprises (a) a substrate sequence cleavable by any protease listed in Table A, (b) one or more substrate sequences selected from the substrate sequences listed in Table B, (c) one or more spacer sequences selected from the substrate sequences listed in Table C, and / or (d) an amino acid sequence of any one of the PCL sequences listed in Table D, or a variant thereof having up to 5 amino acid substitutions, e.g., a variant thereof having 1 amino acid substitution, 2 amino acid substitutions, 3 amino acid substitutions, 4 amino acid substitutions or 5 amino acid substitutions.

8. The IL2 proprotein according to any one of claims 1 to 7, wherein the first and third linkers are identical and / or the third and fourth linkers are identical.

9. The IL2 proprotein of claim 8, wherein the first, second, third and fourth linkers are identical.

10. The IL2 proprotein of any one of claims 1 to 8, wherein the first and third linkers are non-cleavable linkers.

11. The IL2 proprotein according to claim 10, wherein the non-cleavable linker comprises or consists of any one of the NCL sequences listed in Table E.

12. The IL2 proprotein of claim 10, wherein the first and third linkers (a) range from 2 to 60 amino acids, 5 to 25 amino acids, or 7 to 20 amino acids in length, or (b) are at least 5 amino acids in length.

13. The IL2 proprotein of any one of claims 1 to 8, wherein the first, second, third and fourth linkers are protease cleavable linkers.

14. The IL2 proprotein of claim 13, wherein the first, second, third and fourth linkers range from 20 amino acids to 80 amino acids or from 20 amino acids to 60 amino acids in length.

15. The IL2 proprotein according to any one of claims 1 to 14, wherein the first Fc domain and / or the second Fc domain comprises a hinge domain.

16. The IL2 proprotein according to any one of claims 1 to 15, wherein the first targeting moiety and / or the second targeting moiety is a Fab.

17. The IL2 proprotein according to any one of claims 1 to 15, wherein the first targeting moiety and / or the second targeting moiety is a scFv.

18. The IL2 proprotein according to any one of claims 1 to 17, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to any target molecule identified in Section 6.

7.

19. The IL2 proprotein of any one of claims 1 to 18, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to an ECM antigen, the ECM antigen being optionally selected from syndecan, heparanase, integrin, osteopontin, connexin, cadherin, laminin, laminin-type EGF, lectin, fibronectin, notch proteins, adhesion proteins (e.g., adhesion protein-4), tenascin, collagen (e.g., type X collagen), and matrix proteins.

20. The IL2 proprotein according to any one of claims 1 to 19, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to a cell surface molecule of a tumor or viral lymphocyte.

21. The IL2 protein of claim 20, wherein the antigen is a T cell co-stimulatory protein, which is optionally selected from CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C and B7-H3.

22. The IL2 protein of any one of claims 1 to 21, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to a check point inhibitor, optionally selected from CTLA-4, PD1, PDL1, PDL2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1 and CHK2.

23. The IL2 proprotein of any one of claims 1 to 22, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to a tumor associated antigen ("TAA"), optionally selected from AFP, ALK, BAGE protein, BIRC5 (survivin), BIRC7, β-catenin, bcr-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CEACAM5 (also known as carcinoembryonic antigen or CEA), CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE ​​proteins (e.g., GAGE-1 or -2), GD2, GD3, GloboH, Glypican-3, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, LMP2, MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase, and uroplasmin-3.

24. The IL2 proprotein of any one of claims 1 to 23, wherein the Fc region is a homodimer.

25. The IL2 proprotein of any one of claims 1 to 23, wherein the Fc region is a heterodimer.

26. The IL2 proprotein according to any one of claims 1 to 25, having the configuration depicted in Figure 1A.

27. The IL2 proprotein according to any one of claims 1 to 25, having the configuration depicted in Figure 2A.

28. An IL2 proprotein, optionally the IL2 proprotein according to any one of claims 1 to 27, wherein the IL2 proprotein comprises: (a) a first polypeptide chain comprising, in order from N-terminus to C-terminus: (i) a first amino acid sequence having at least about 95% sequence identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, and 6; (ii) a second amino acid sequence comprising: (1) one or more sequences listed in Table B or (2) a sequence listed in Table E; (iii) a third amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 7; (iv) a fourth amino acid sequence comprising the sequence listed in Table B; and (v) a fifth amino acid sequence having at least about 95% sequence identity to any one of SEQ ID NOs: 8, 9, and 10; and (b) a second polypeptide chain comprising, in order from N-terminus to C-terminus: (i) a sixth amino acid sequence having at least about 95% sequence identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, and 6; (ii) a seventh amino acid sequence comprising: (1) one or more sequences listed in Table B or (2) a sequence listed in Table E; (iii) an eighth amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 7; (iv) a ninth amino acid sequence comprising the sequence listed in Table B; and (v) a tenth amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 8, 9 or 10.

29. The IL2 proprotein of claim 28, wherein the first polypeptide comprises an eleventh amino acid sequence at the N-terminus of the first amino acid sequence, the eleventh amino acid sequence having at least about 95%, at least about 98% or 100% sequence identity to any one of SEQ ID NOs: 11, 12, 13 or 14.

30. The IL2 proprotein of claim 28 or 29, wherein the second polypeptide comprises a twelfth amino acid sequence at the N-terminus of the first amino acid sequence, the twelfth amino acid sequence having at least about 95%, at least about 98% or 100% sequence identity to any one of SEQ ID NOs: 11, 12, 13 or 14.

31. The IL2 proprotein of any one of claims 28 to 30, wherein the first amino acid sequence has at least about 98% or 100% sequence identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, or 6.

32. The IL2 proprotein of any one of claims 28 to 31, wherein the sixth amino acid sequence has at least about 98% or 100% sequence identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, or 6.

33. The IL2 proprotein according to any one of claims 28 to 32, wherein the second amino acid sequence comprises (a) one or more of the amino acid sequences listed in Table B or (b) an amino acid sequence listed in Table D.

34. The IL2 proprotein according to any one of claims 28 to 32, wherein the second amino acid sequence is an amino acid sequence listed in Table E.

35. The IL2 proprotein of any one of claims 28 to 34, wherein the second amino acid sequence is 25 or fewer, 15 or fewer, or 6 or fewer amino acids in length.

36. The IL2 proprotein according to any one of claims 28 to 35, wherein the seventh amino acid sequence comprises (a) one or more of the amino acid sequences listed in Table B or (b) an amino acid sequence listed in Table D.

37. The IL2 proprotein according to any one of claims 28 to 35, wherein the seventh amino acid sequence is an amino acid sequence listed in Table E.

38. The IL2 proprotein of any one of claims 28 to 37, wherein the seventh amino acid sequence is 25 or fewer, 15 or fewer, or 6 or fewer amino acids in length.

39. The IL2 proprotein of any one of claims 28 to 38, wherein the third amino acid sequence has at least about 98% or 100% sequence identity to SEQ ID NO:

7.

40. The IL2 proprotein of any one of claims 28 to 39, wherein the eighth amino acid sequence has at least about 98% or 100% sequence identity to SEQ ID NO:

7.

41. The IL2 proprotein of any one of claims 28 to 40, wherein the fourth amino acid sequence comprises (a) one or more of the amino acid sequences listed in Table B or (b) an amino acid sequence listed in Table D.

42. The IL2 proprotein of any one of claims 28 to 41, wherein the ninth amino acid sequence comprises (a) one or more amino acid sequences listed in Table B or (b) an amino acid sequence listed in Table D.

43. The IL2 proprotein of any one of claims 28 to 42, wherein the ninth amino acid sequence is an amino acid sequence listed in Table D.

44. The IL2 proprotein of any one of claims 28 to 43, wherein the fifth amino acid sequence has at least about 98% or 100% sequence identity to SEQ ID NO: 8, 9 or 10.

45. The IL2 proprotein of any one of claims 28 to 44, wherein the tenth amino acid sequence has at least about 98% or 100% sequence identity with SEQ ID NO: 8, 9 or 10.

46. ​​The IL2 proprotein of any one of claims 28 to 45, wherein the first polypeptide chain lacks additional sequences at the C-terminus of the first amino acid sequence.

47. The IL2 proprotein of any one of claims 28 to 46, wherein the first polypeptide chain (a) lacks an additional sequence between the first and second amino acid sequences, (b) lacks an additional sequence between the second and third amino acid sequences, (c) lacks an additional sequence between the third and second amino acid sequences, and / or (d) lacks an additional sequence between the fourth and fifth amino acid sequences.

48. The IL2 proprotein of any one of claims 28 to 47, wherein the second polypeptide chain lacks an additional sequence at the C-terminus of the sixth amino acid sequence.

49. The IL2 proprotein of any one of claims 28 to 48, wherein the second polypeptide chain (a) lacks additional sequence between the sixth and seventh amino acid sequences, (b) lacks additional sequence between the seventh and eighth amino acid sequences, (c) lacks additional sequence between the eighth and ninth amino acid sequences, and / or (d) lacks additional sequence between the ninth and tenth amino acid sequences.

50. A nucleic acid or nucleic acids encoding the IL2 proprotein according to any one of claims 1 to 49.

51. A host cell engineered to express the IL2 proprotein of any one of claims 1 to 49 or the nucleic acid of claim 50.

52. A method of producing an IL2 proprotein according to any one of claims 1 to 49, said method comprising culturing the host cell according to claim 51 and recovering the IL2 proprotein expressed thereby.

53. A pharmaceutical composition comprising the IL2 proprotein according to any one of claims 1 to 49 and an excipient.

54. A method of treating cancer, the method comprising administering to a subject in need thereof the pro-IL2 protein of any one of claims 1 to 49 or the pharmaceutical composition of claim 53.

55. A method for local delivery of an IL2 protein, treating cancer with an IL2 protein selectively activated in cancer tissue, administering to the subject an IL2 therapy with reduced systemic exposure and / or reduced systemic toxicity, targeted delivery of an activated IL2 protein to cancer tissue, or inducing an immune response locally in a target tissue, the method comprising administering to the subject an IL2 proprotein according to any one of claims 1 to 49 (or a pharmaceutical composition comprising the IL2 proprotein and an excipient), the IL2 proprotein having one or more protease cleavable linkers, each protease cleavable linker comprising one or more substrates for one or more proteases expressed by a tissue (e.g., a cancer tissue) to which the IL2 protein is to be locally delivered.

56. The method of claim 54 or 55, wherein administration is non-topical.

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