Interleukin-2 precursor protein and its use

Abstract

The present disclosure provides an IL2 proprotein that, for example, at a tumor site, comprises an IL2 portion masked with an IL2Rα portion and a protease-cleavable linker configured such that the IL2Rα portion is released from the IL2 portion upon the action of a protease. The IL2 proprotein optionally further comprises a targeting moiety, for example, a targeting moiety that recognizes a tumor-associated antigen and directs the proprotein to the tumor site. The present disclosure further provides a pharmaceutical composition comprising the IL2 proprotein, a method of using the IL2 proprotein in therapy, as well as a nucleic acid encoding the IL2 proprotein, a recombinant cell expressing the IL2 proprotein, and a method of producing the IL2 proprotein.

Description

Technical Field

[0001] 1. Cross - reference to related applications This application claims the benefit of priority of U.S. Provisional Application No. 63 / 346,593, filed May 27, 2022; U.S. Provisional Application No. 63 / 349,078, filed Jun. 4, 2022; U.S. Provisional Application No. 63 / 355,375, filed Jun. 24, 2022; U.S. Provisional Application No. 63 / 386,999, filed Dec. 12, 2022; and U.S. Provisional Application No. 63 / 501,434, filed May 11, 2023, the contents of each of which are hereby incorporated by reference in their entirety.

[0002] 2. Sequence Listing This application includes an electronically - submitted sequence listing, which is hereby incorporated by reference in its entirety. The copy created on May 9, 2023, is named RGN - 021WO_SL.xml and is 316,721 bytes in size.

Background Art

[0003] 3. Background Interleukin - 2 (IL - 2 or IL2) is a pleiotropic cytokine mainly produced 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, facilitates the proliferation and differentiation of B cells and the synthesis of immunoglobulins by B cells, and stimulates the generation, proliferation, and activation of natural killer (NK) cells (see Non - Patent Document 1 and Non - Patent Document 2).

[0004] Due to its multifaceted effects, IL2 is not optimal for inhibiting tumor growth. The use of IL2 as an anti-tumor agent has been limited by the severe toxicity associated with the doses required for tumor response. Proleukin® (sold by Prometheus Laboratories, San Diego, Calif.) is a recombinant form of IL2 that is approved for the treatment of metastatic melanoma and metastatic renal cancer, but its side effects are very severe, so its use is recommended only in hospital settings where intensive treatment can be received. Patients receiving high-dose IL2 therapy frequently experience severe cardiovascular, pulmonary, renal, hepatic, gastrointestinal, neurological, cutaneous, hematological, and systemic adverse events that require intensive monitoring and inpatient management. The main side effect of IL2 therapy is vascular leak syndrome (VLS), which causes the accumulation of interstitial fluid 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 the treatment results are not optimal. IL2-induced pulmonary edema has been shown to be due to the direct binding of IL2 to lung endothelial cells that express low to intermediate levels of functional high-affinity IL2 receptors (Non-Patent Document 3).

[0005] For the purpose of reducing the toxicity of IL2 cancer therapy, various IL2 variants and prodrugs have been generated. However, surprisingly, such molecules have been found to have low therapeutic indices for cancer therapy. For example, the PEGylated IL2 prodrug bempegaldesleukin failed to improve the therapeutic efficacy of PD1 checkpoint inhibitors in melanoma patients in a Phase 3 clinical study (Non-Patent Document 4).

[0006] Therefore, there is a need in the art for a new IL2 therapy with improved therapeutic efficacy and safety profile.

Prior Art Documents

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

[0008] 4. Summary The present disclosure relates to an IL2 proprotein that is activated by a protease, for example, a protease expressed in a tumor microenvironment.

[0009] The IL2 proprotein comprises an IL2 portion masked by an IL2Rα moiety that is configured to have its mask removed 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.

[0010] In certain embodiments, the IL2 proprotein of the present disclosure comprises two polypeptide chains, each of which, from the N-terminus to the C-terminus, comprises an Fc domain, an optional linker (if present, preferably a non-cleavable linker), an IL2Rα portion, a protease-cleavable linker, and an IL2 portion. The IL2 proprotein may further comprise, for example, a targeting moiety (or a component thereof, such as one chain of a Fab) at the N-terminus of one or both of the Fc domains. The targeting moiety comprises, for example, an antigen-binding site ("ABS") that can bind to a target molecule (e.g., a tumor-associated antigen) present on the tumor surface or other components in the tumor microenvironment (e.g., extracellular matrix ("ECM") or tumor lymphocytes).

[0011] Exemplary IL2 portions that can be used in the IL2 proproteins of the present disclosure are described in Section 6.3. Exemplary IL2Rα portions that can be used in the IL2 proproteins of the present disclosure are described in Section 6.4.

[0012] Protease-cleavable linkers that can be used in the IL2 proproteins of the present disclosure are described in Section 6.5. Non-cleavable linkers that can be used in the IL2 proproteins of the present disclosure are described in Section 6.6.

[0013] Targeting moieties that can be used in the IL2 proproteins of the present disclosure are described in Section 6.7, and the formats of the targeting moieties are disclosed in Section 6.8. Fc domains that can be incorporated into the IL2 proproteins of the present disclosure are described in Section 6.9.

[0014] Exemplary IL2 proproteins of the present disclosure are described in Section 6.2 and numbered embodiments 1-157. The present disclosure further provides nucleic acids encoding the IL2 precursor protein of the present disclosure. The nucleic acid encoding the IL2 precursor protein can be a single nucleic acid (e.g., a vector encoding all polypeptide chains of the IL2 precursor protein) or multiple nucleic acids (e.g., two or more vectors encoding different polypeptide chains of the IL2 precursor protein). The present disclosure further provides host cells and cell lines engineered to express the nucleic acids and IL2 precursor protein of the present disclosure. The present disclosure further provides methods for producing the IL2 precursor protein of the present disclosure. Exemplary nucleic acids, host cells, and cell lines, as well as methods for producing the IL2 precursor protein, are described in Section 6.10 below, as well as in Specific Embodiments 158 - 160.

[0015] The present disclosure further provides pharmaceutical compositions comprising the IL2 precursor protein of the present disclosure. Exemplary pharmaceutical compositions are described in Section 6.11 and Numbered Embodiment 161. For example, methods of using the IL2 precursor protein and pharmaceutical compositions of the present disclosure for treating cancer are further provided herein. Exemplary methods are described in Section 6.12 and Numbered Embodiments 162 - 200.

[0016] 5. Brief Description of the Drawings

Brief Description of the Drawings

[0017]

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Mode for Carrying Out the Invention

[0018] 6. Detailed Description 6.1. Definitions As used herein, the following terms are intended to have the following meanings.

[0019] ABD chain, targeting moiety chain: The targeting moiety and antigen-binding site (ABD) within them can exist as one polypeptide chain (e.g., in the case of scFv or scFab) or form via the association of two or more polypeptide chains (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 the 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 illustrative purposes only and does not imply a particular configuration or production method. Further, references to the ABD or targeting moiety in the context of describing an IL2 proprotein include, unless the context otherwise indicates, the ABD chain or targeting moiety chain. Thus, when describing an IL2 proprotein in which an Fc domain is operably linked to a targeting moiety, the Fc domain can be directly or indirectly (e.g., via a linker) covalently linked to, for example, (1) the first ABD or targeting moiety chain of a Fab or Fv (with the other components of the Fab or Fv on the second associated ABD or targeting moiety chain), or (2) an ABD or targeting moiety chain containing an scFv or scFab.

[0020] About, approximately: Terms such as "about" and "approximately" are used throughout the specification before numerical values to indicate that the numerical value is not necessarily exact (e.g., to account for fractions, measurement precision and / or variations in precision, timing, etc.). It should be understood that the disclosure of "about X" or "approximately X" where X is a numerical value is also a disclosure of "X". Thus, for example, the disclosure of an embodiment where an array has "about X% sequence identity" to another array is also a disclosure of an embodiment where that array has "X% sequence identity" to the other array.

[0021] Activate, activation: Terms such as "activate" and "activation" refer to protease-mediated enzymatic cleavage of a protease-cleavable linker that, in conjunction with the IL2 protein of the present disclosure, results in unmasking or release of the IL2 moiety from the IL2Rα moiety.

[0022] And, or: Unless otherwise indicated, the conjunction "or" is intended to be used in its correct meaning as a Boolean operator that encompasses both the selection of features in an alternative (the selection of A is mutually exclusive from B, A or B) and the selection of combined features (both A and B are selected, A or B). In some places in the text, the term "and / or" is used for the same purpose and should not be construed to mean that "or" is used with reference to mutually exclusive alternatives.

[0023] Antibody: As used herein, the term "antibody" refers to a polypeptide (or set of polypeptides) of the immunoglobulin family that can bind non-covalently, reversibly, and specifically 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 is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, and are arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). 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. The antibody can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be understood that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, transplacental transfer, Fc receptor binding, and complement binding.By convention, the numbering of the constant region domains increases with increasing distance from the antigen-binding domain or the amino terminus of the antibody. The N-terminus is the variable region and the C-terminus is the constant region, and the CH3 and CL domains represent the carboxy termini of the heavy and light chains, respectively, of the native antibody. For convenience, and unless the context otherwise indicates, references to an antibody also refer to antibody fragments, as well as engineered antibodies that include non-natural antigen-binding domains and / or antigen-binding domains with non-natural configurations.

[0024] Antigen-binding domain: As used herein, the term “antigen-binding domain” or “ABD” refers to the portion (e.g., the targeting portion) of an antibody or antibody fragment that has the ability to bind non-covalently, reversibly, and specifically to an antigen. Examples of antibody fragments that may contain an ABD include single-chain Fv (scFv), Fab fragments, monovalent fragments consisting of VL domains, VH domains, CL domains, and CH1 domains, F(ab’)2 fragments, divalent fragments containing two Fab fragments linked by a disulfide bridge in the hinge region, Fd fragments consisting of VH and CH1 domains, Fv fragments consisting of VL and VH domains of a single arm of an antibody, dAb fragments consisting of VH domains (Ward et al., 1989, Nature 341:544-546), and isolated complementarity-determining regions (CDRs), but are not limited thereto. Thus, the term “antibody fragment” encompasses both proteolytic fragments of an antibody (e.g., Fab and F(ab)2 fragments) and engineered proteins that include one or more portions of an antibody (e.g., scFv). Antigen-binding fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetra-bodies, v-NAR, and bis-scFv (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology 23:1126-1136).

[0025] Associated: The term "associated" in the context of the IL2 protein refers to the functional relationship between two or more polypeptide chains. In particular, the term "associated" means that two or more polypeptides are associated with each other, for example, non-covalently through molecular interactions or covalently through one or more disulfide bridges or chemical bridges, to produce a functional IL2 protein. Examples of associations that may be present in the IL2 proteins of the present disclosure include associations between Fc domains that form the Fc region (homo- or heterodimers described in Section 6.9), associations between the VH and VL regions in Fab or Fv, and associations between CH1 and CL in Fab (but are not limited thereto).

[0026] Cancer: The term "cancer" refers to a disease characterized by the uncontrolled (and often rapid) growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers 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 gland cancer, autonomic ganglion cancer, biliary tract cancer, bone cancer, endometrial cancer, eye cancer, fallopian tube cancer, genital tract cancer, large intestine cancer, meninges cancer, esophageal cancer, peritoneal cancer, pituitary cancer, penile cancer, placental cancer, pleural cancer, salivary gland cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, upper aerodigestive tract cancer, urinary tract cancer, vaginal cancer, vulvar cancer, lymphoma, leukemia, lung cancer, etc., for example, any of the aforementioned types of TAA-positive cancers.

[0027] Complementary Determining Region: The term “complementary determining region” or “CDR” as used herein refers to the amino acid sequences within the antibody variable regions that confer antigen specificity and binding affinity. For example, generally, each heavy chain variable region has three CDRs (CDR-H1, CDR-H2, and CDR-H3), and each light chain variable region has three CDRs (CDR-L1, CDR-L2, and CDR-L3). The exact amino acid sequence boundaries of a given CDR can be determined using any of several 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 the 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, in 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 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 both the Kabat and Chothia CDR definitions, the CDRs consist of amino acid residues 26 - 35 (CDR-H1), 50 - 65 (CDR-H2), and 95 - 102 (CDR-H3) in human VH, 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) (numbering according to "Kabat"). Under IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0028] Effector function: The term "effector function" generally refers to the activity of an antibody molecule mediated by binding of an effector molecule or by binding through domains of the antibody other than the antigen-binding domain. Effector functions include, for example, complement-mediated effector functions mediated by binding of the C1 component of complement to the antibody. Activation of complement is important for opsonization and lysis of cellular pathogens. Activation of complement also stimulates the inflammatory response and can be involved in autoimmune hypersensitivity. Effector functions also include Fc receptor (FcR)-mediated effector functions, which can be induced upon binding of the constant domain of an antibody to an Fc receptor (FcR). Binding of an antibody to an Fc receptor on the cell surface induces a number of important and diverse biological responses, including ingestion and destruction of antibody-coated particles, removal of immune complexes, lysis of antibody-coated target cells by killer cells (referred to as antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer, and regulation of immunoglobulin production. The effector function of an antibody can be altered, for example, enhanced or reduced, by changing the affinity of the antibody for an effector molecule such as an Fc receptor or a complement component. Binding affinity generally changes by modifying the effector molecule binding site, in which case it is appropriate to position the site of interest and modify at least a portion of the site in a suitable manner. Also, a change in the binding site on the antibody for an effector molecule is not necessarily required to significantly change the overall binding affinity, but it is also envisioned that it can change the shape of the interaction that abrogates the effector mechanism, as in non-productive binding. It is further envisioned that effector functions can also be modified by modifying sites that do not directly participate in effector molecule binding but that otherwise participate in the performance of the effector function.

[0029] Epitope: An epitope, or antigenic determinant, is a portion of an antigen that is recognized by an antibody or other antigen-binding moiety described herein. Epitopes can be linear or conformational.

[0030] Fab: The term "Fab" refers to a pair of polypeptide chains, where the first polypeptide chain contains the variable heavy (VH) domain of an antibody operably linked (typically at its N-terminus) to a first constant domain (referred to herein as C1), and the second polypeptide chain contains the variable light (VL) domain at the N-terminus of an antibody operably linked (typically at its N-terminus) to a second constant domain (referred to herein as C2) that can pair with the first constant domain. In a native antibody, 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 constant domain (CL) of the light chain. The Fab of the present disclosure can be arranged according to the native orientation or can include domain substitutions or swaps that promote correct VH and VL pairing. For example, to promote correct modified Fab-chain pairing in a heterodimeric molecule, it is possible to replace the CH1 and CL domain pair in the Fab with a CH3 domain pair. It is also possible to reverse CH1 and CL such that CH1 is attached to VL and CL is attached to VH, which is generally a configuration known as Crossmab. The term "Fab" encompasses single-chain Fabs.

[0031] Fc domain and Fc region: The term "Fc domain" refers to the part of a heavy chain that pairs with the corresponding part of another heavy chain. The term "Fc region" refers to the region of what is formed by the association of two heavy-chain Fc domains. The two Fc domains within the Fc region may be the same or different from each other. In a native antibody, the Fc domains are typically the same, but one or both Fc domains can be modified, for example, via a knob-in-hole interaction, to allow for heterodimerization.

[0032] Fv: The term "Fv" refers to the smallest antibody fragment that can be derived from an immunoglobulin and contains a complete target recognition and binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain (VH-VL dimer) in a close non-covalent association. In this configuration, the three CDRs of each variable domain interact to define the target binding site on the surface of the VH-VL dimer. In many cases, the six CDRs confer target binding specificity to the antibody. However, in some instances, a single variable domain (or half of the Fv containing only the three CDRs specific for the target) may have the ability to recognize and bind to the target. References to VH-VL dimers herein are not intended to convey any particular configuration. When present on a single polypeptide chain (e.g., scFv), VH and VL are at the N-terminus or C-terminus.

[0033] Half-antibody: The term "half-antibody" refers to a molecule that contains at least one Fc domain and can associate with another molecule containing an Fc domain, for example, via a disulfide bridge or molecular interaction. A half-antibody can be composed of one polypeptide chain or two or more polypeptide chains (e.g., the two polypeptide chains of a Fab). Examples of half-antibodies are molecules that contain the heavy and light chains of an antibody (e.g., an IgG antibody). Another example of a half-antibody is a molecule that contains a first polypeptide containing a VL domain and a CL domain, and a second polypeptide containing a VH domain, a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain, wherein the VL domain and the VH domain form an ABD. Yet another example of a half-antibody is a polypeptide that contains an scFv domain, a CH2 domain, and a CH3 domain. The IL2 proprotein of the present disclosure typically contains two half-antibodies, each containing 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 of the half-antibodies in the IL2 proprotein may further contain a targeting moiety, for example, at the N-terminus of the Fc domain.

[0034] The term "half antibody" is intended for illustrative purposes only and does not imply a particular structure or production method. References to half antibodies as "first" half antibodies, "second" half antibodies, "left" half antibodies, "right" half antibodies, etc. are for convenience and illustrative purposes only.

[0035] Host cell or recombinant host cell: The terms "host cell" or "recombinant host cell" refer to cells that have been genetically engineered, for example, via the introduction of a heterologous nucleic acid. It should be understood that such terms are intended to refer not only to a particular target cell but also to the progeny of such a cell. Due to either mutations or environmental influences, certain modifications may occur in later generations, and although such progeny may not actually be identical to the parental cell, they are still included within the scope of the term "host cell" as used herein. A host cell may carry a heterologous nucleic acid, for example, transiently on an episomal heterologous expression vector or, for example, stably by integrating the heterologous nucleic acid into the host cell genome. For the purpose of expressing the IL2 proprotein of the present disclosure, host cells are preferably cell lines 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 derivatives with site-specific integration sites.

[0036] Linker: The term "linker" as used herein refers to a protease-cleavable linker or a non-cleavable linker. Non-cleavable linker: A non-cleavable linker refers to a peptide whose amino acid sequence lacks the substrate sequence of a protease, such as the protease described in Section 6.5.1, that recognizes and cleaves a specific sequence motif, such as the substrate described in Section 6.5.2.

[0037] Operably linked: The term "operably linked" refers to the functional relationship between two or more peptides or polypeptide domains or nucleic acid (e.g., DNA) segments. In the context of a fusion protein or other polypeptide, the term "operably linked" means that two or more amino acid segments are linked such that a functional polypeptide is produced. For example, in the context of the IL2 protein of the present disclosure, separate components (e.g., the Fc domain and the IL2Rα portion) can be operably linked directly or via a peptide linker sequence. In the context of a nucleic acid encoding a fusion protein such as a half antibody of the IL2 protein of the present disclosure, "operably linked" means that two nucleic acids are joined such 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 the transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in an appropriate host cell or other expression system.

[0038] Polypeptide, peptide, and protein: The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues.

[0039] Proprotein: A "proprotein" is an inactive protein precursor that can be activated by proteolysis by a protease. Thus, a proprotein is "protease activatable".

[0040] Protease: As used herein, the term "protease" refers to any enzyme that catalyzes the hydrolysis of peptide bonds. Generally, proteases useful in the present disclosure, such as those described in Section 6.5.1, recognize and cleave specific sequence motifs, such as the substrates described in Section 6.5.2. Preferably, the protease is expressed at a higher level in cancer tissue compared to normal tissue.

[0041] 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 for 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.

[0042] Recognize: As used herein, the term "recognize" refers to an antibody or antibody fragment (e.g., targeting moiety) that finds its epitope and interacts with (e.g., binds to) that epitope.

[0043] Single-chain Fab or scFab: As used herein, the terms "single-chain Fab" or "scFab" refer to an antigen-binding domain (ABD) that includes a VH domain, a CH1 domain, a VL domain, a CL domain, and a linker. In some embodiments, the aforementioned domains and linker are arranged in one of the following orders in an 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 preferably a non-cleavable linker of at least 30 amino acids, preferably 32 to 50 amino acids. The single-chain Fab fragment is typically stabilized via a native disulfide bond between the CL domain and the CH1 domain. In addition, these single-chain Fab molecules can be further stabilized by the formation of interchain disulfide bonds via the insertion of cysteine residues (e.g., position 44 of the VH domain and position 100 of the VL domain according to Kabat numbering).

[0044] Single-chain Fv or scFv: As used herein, the terms "single-chain Fv" or "scFv" refer to an ABD that includes the VH and VL domains of an antibody, and these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further includes a polypeptide linker between the VH domain and the VL domain that enables the scFv to form a structure desired for antigen binding. For an overview of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds. (1994), Springer-Verlag, New York, pp. 269-315. The VH and VL are arranged in an N-terminal to C-terminal order of VH-VL or VL-VH, typically separated by a linker, such as the linkers described in Table E.

[0045] 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. A spacer can be used to separate a substrate from other domains within a molecule, e.g., an ABD. In some embodiments, the residues within the spacer minimize the action of aminopeptidases and / or exopeptidases to prevent cleavage of the N-terminal amino acid.

[0046] Specifically (or selectively) bind: The term "specifically (or selectively) binds" to an antigen or epitope refers to a binding reaction that determines the presence of a cognate antigen or epitope in a heterogeneous population of proteins and other molecules. The binding reaction can be mediated by an antibody or antibody fragment, but does not necessarily have to be. The term "specifically binds" does not exclude cross-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. Thus, such cross-reactivity per se does not change the classification of the antigen-binding domain as a "specific" binder. In certain embodiments, the antigen-binding domains of the present disclosure that specifically bind to human antigens have cross-species reactivity with one or more non-human mammalian species, e.g., primate species (including but not limited to one or more of Macaca fascicularis, Macaca mulatta, and Macaca nemestrina), or rodent species, e.g., Mus musculus.

[0047] 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, etc.). In preferred embodiments, the subject is a human.

[0048] Substrate: The term "substrate" refers to a peptide sequence on which a protease acts and within which the protease cleaves peptide bonds. Target molecule: As used herein, the term "target molecule" refers to any biomolecule (e.g., protein, carbohydrate, lipid, or combination thereof) expressed on the cell surface or in the extracellular matrix that can specifically bind to the targeting site of the IL2 protein of the present disclosure.

[0049] Targeting moiety: As used herein, the term "targeting moiety" refers to any molecule or binding portion thereof (e.g., immunoglobulin or antigen-binding fragment) that can bind to cell surface or extracellular matrix molecules at the site where the IL2 protein of the present disclosure localizes, such as on lymphocytes in a tumor cell or tumor microenvironment. In some embodiments, the targeting moiety binds to a TAA. In other embodiments, the targeting moiety binds to a TCA. The targeting moiety may also have functional activity in addition to localizing the IL2 protein to a specific site. For example, a targeting moiety that binds to a checkpoint inhibitor such as PD1 may also exhibit anti-tumor activity or enhance anti-tumor activity by IL2, e.g., by inhibiting PD1 signaling.

[0050] T cell antigen, TCA: The term "T cell antigen" or "TCA" refers to a molecule (typically a protein, carbohydrate, lipid, or combination thereof) expressed on the surface of T lymphocytes that is useful for the preferential targeting of agents 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 T lymphocytes.

[0051] Tumor: As used herein, the term "tumor" is used interchangeably with the term "cancer" herein, e.g., both terms encompass solid and liquid, e.g., diffuse or circulating tumors. As used herein, the terms "cancer" or "tumor" include pre-cancerous as well as malignant cancers and tumors.

[0052] Tumor-associated antigen, TAA: The term "tumor-associated antigen" or "TAA" refers to a molecule (typically a protein, carbohydrate, lipid, or combination thereof) that is expressed on the surface of cancer cells, either completely or as a fragment (e.g., MHC / peptide), and is useful for the preferential targeting of drugs to cancer cells. In some embodiments, the TAA is a marker expressed by both normal and cancer cells, e.g., a lineage marker. In some embodiments, the TAA is a cell surface molecule that is overexpressed in cancer cells compared to normal cells, e.g., 1-fold overexpressed, 2-fold overexpressed, 3-fold overexpressed, or more overexpressed compared to normal cells. In some embodiments, the TAA is a cell surface molecule that is inappropriately synthesized in cancer cells, e.g., a molecule that contains deletions, additions, or mutations compared to the molecule expressed on normal cells. In some embodiments, the TAA is expressed only on the cell surface of cancer cells, either completely or as a fragment (e.g., MHC / peptide), and is not synthesized or expressed on the surface of normal cells. Thus, the term "TAA" encompasses antigens specific to cancer cells and may also be known in the art as tumor-specific antigens ("TSA").

[0053] Treat, treatment, treating: As used herein, the terms "treat", "treatment", and "treating" refer to the shortening or improvement of the progression, severity, and / or duration of a proliferative disorder, or the improvement of one or more symptoms (preferably one or more distinguishable symptoms) of a proliferative disorder, resulting from administration of one or more of the IL2 proproteins of the present disclosure. In certain embodiments, the terms "treat", "treatment", and "treating" refer to the improvement of at least one measurable physical parameter of a proliferative disorder, such as tumor growth, which may not necessarily be distinguishable by the patient. In other embodiments, the terms "treat", "treatment", and "treating" refer to the inhibition of the progression of a proliferative disorder, either by stabilization of physical, e.g., physiological, parameters, such as stabilization of distinguishable symptoms, or both. In other embodiments, the terms "treat", "treatment", and "treating" refer to a decrease or stabilization in tumor size or the number of cancerous cells.

[0054] Universal light chain, ULC: As used herein, the term "universal light chain" or "ULC" refers to a variable light chain region (VL) that can pair with more than the variable heavy chain region (VL). In the context of a targeting moiety, the term "universal light chain" or "ULC" refers to a light chain polypeptide that can pair with the heavy chain region of the targeting moiety and can also pair with other heavy chain regions. The ULC may also include a constant domain, such as the CL domain of an antibody. The universal light chain is also known as the "common light chain".

[0055] 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. 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.

[0056] 6.2. IL2 Protein The present disclosure relates to an IL2 protein comprising an IL2 moiety, an IL2Rα moiety, and a protease-cleavable linker arranged such that the IL2Rα moiety reduces or blocks the activity of the IL2 moiety. The IL2 protein is configured such that when it encounters a protease, such as a protease overexpressed in the tumor environment, the protease-cleavable linker is cleaved, releasing the IL2 and stimulating cytotoxic T cell activity against tumor cells. Typically, the IL2 protein of the present disclosure is a dimer and includes two Fc domains that associate to form an Fc region, with the IL2Rα moiety, the protease-cleavable linker, and the IL2 moiety arranged in order from the N-terminus to the C-terminus at its C-terminus. The IL2 protein may further include a linker, preferably a non-cleavable linker, between the Fc domain and the IL2Rα moiety.

[0057] Accordingly, the IL2 proprotein of the present disclosure generally comprises (a) a first Fc domain and a second Fc domain that can associate to form an Fc region, (b) two IL2Rα moieties at the C-terminus of the Fc domain, optionally preceded by a linker, preferably a non-cleavable linker, (c) two protease-cleavable linkers at the C-terminus of the IL2Rα moiety, and (d) two IL2 moieties at the C-terminus of the protease-cleavable linker. The IL2 moiety in the IL2 proprotein is in an inactive form due to masking by the IL2Rα moiety, but is released, for example, in the tumor environment, after protease cleavage of the protease-cleavable linker. Examples of suitable IL2 moieties are described in Section 6.3, examples of suitable IL2Rα moieties are described in Section 6.4, and examples of suitable protease-cleavable linkers are described in Section 6.5.

[0058] Generally, the IL2 proprotein of the present disclosure comprises a plurality of linkers. Preferably, the remaining linkers other than the protease-cleavable linker whose cleavage releases the IL2 moiety are non-cleavable. Examples of non-cleavable linkers are described in Section 6.6.

[0059] The IL2 proprotein may further comprise one or more targeting moieties, and in some embodiments, may comprise two targeting moieties at the N-terminus of the Fc domain. Examples of targeting moieties are described in 6.7, and suitable formats of targeting moieties are described in Section 6.8. In some embodiments, the Fc domain comprises a hinge domain at its N-terminus. Suitable Fc domains are described in Section 6.9, regardless of the presence or absence of a hinge sequence.

[0060] An exemplary IL2 proprotein is depicted in Figure 1A. The IL2 proprotein a) The heavy chain of the Fab, which associates with the light chain of the Fab on a separate polypeptide chain to together form a first targeting moiety, followed by an Fc domain containing a hinge domain, followed by a linker ( "Linker A"), which is preferably a non-cleavable linker, followed by an IL2Rα moiety, followed by a protease-cleavable linker ( "Linker B"), followed by an IL2 moiety, and a first polypeptide chain, b) The heavy chain of the Fab, which associates with the light chain of the Fab on a separate polypeptide chain to together form a second targeting moiety, followed by an Fc domain containing a hinge domain, followed by a linker ( "Linker C"), which is preferably a non-cleavable linker, followed by an IL2Rα moiety, followed by a protease-cleavable linker ( "Linker D"), followed by an IL2 moiety, and a second polypeptide chain, and includes.

[0061] 6.3. IL2 moiety The IL2 moiety of the IL2 protein of the present disclosure includes a wild-type or variant IL2 moiety.

[0062] In eukaryotic cells, human IL2 is synthesized as a 153-amino acid precursor polypeptide, from which 20 amino acids are removed to produce mature secreted IL2 (Taniguchi et al., 1983, Nature 302(5906):305-10). Mature human IL2 has the following amino acid sequence: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 2) In some embodiments, the IL2 moiety of the present disclosure is not CD122-directed. For example, the IL2 moiety does not have an amino acid substitution in the IL2 moiety that preferentially binds the IL2 moiety to IL2Rβ as compared to IL2Rα.

[0063] In some embodiments, the IL2 portion of the present disclosure is CD25-directed. For example, the IL2 portion has one or more amino acid substitutions that preferentially bind the IL2 portion to IL2Rα as compared to IL2Rβ.

[0064] In certain embodiments, the IL2 protein of the present disclosure has one or more amino acid substitutions in the IL2 portion that reduce binding to IL2Rβ. For example, in some embodiments, the IL2 portion can be reduced in binding to human IL2Rβ by up to 50-fold (and in some embodiments, up to 100-fold) to 1,000-fold as compared to wild-type human IL2.

[0065] The IL2 portion with reduced binding to IL2Rβ can retain its affinity for IL2Rα or binding to IL2Rα can be reduced. For example, in some embodiments, the IL2 portion can be reduced in binding to human IL2Rα by up to 50-fold as compared to wild-type human IL2.

[0066] Other features of useful IL2 variants can include the ability to induce the 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 improvement in the therapeutic index.

[0067] In one embodiment, the IL2 portion includes one or more amino acid substitutions that reduce affinity for IL2Rβ and maintain affinity for IL2Rα. An exemplary amino acid substitution is N88D. Other amino acid substitutions that reduce or abolish the affinity of IL2 for IL2Rβ are D20T, N88R, N88D, or Q126D (see, e.g., U.S. Patent Publication No. 2007 / 0036752).

[0068] In one embodiment, the IL2 moiety comprises one or more amino acid substitutions that reduce the affinity for IL2Rα and maintain the affinity for IL2Rβ or reduce the affinity to a lesser extent, resulting in a CD122-directed IL2 moiety. An exemplary CD122-directed IL2 moiety comprises both the H16A and F42A substitutions. Thus, in some embodiments, the IL2 moiety comprises the amino acid sequence of human IL2 having the H16A and F42A substitutions, as shown below: SAPTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 124) In certain embodiments, the IL2 moiety comprises an amino acid substitution that eliminates the O-glycosylation site of IL2 at the 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 particular embodiment, the substitution is T3A.

[0069] The IL2 moiety is preferably an essentially full-length IL2 molecule, such as a human IL2 molecule. In certain embodiments, the IL2 moiety is a human IL-2 molecule. As described in U.S. Patent No. 4,518,584, C125 can be replaced with S, V, or A to reduce protein aggregation.

[0070] As described therein, the N-terminal alanine residue of IL2 can also be deleted to result in des-A1 IL2. Furthermore, the IL2 moiety can comprise a substitution of methionine 104 with a neutral amino acid such as alanine, as described in U.S. Patent No. 5,206,344.

[0071] Thus, the IL2 portion of the present disclosure can have amino acid deletions and / or substitutions selected from des-A1 M104A IL2, des-A1 M104A C125S IL2, M104A IL2, M104A C125A IL2, des-A1 M104A C125A IL2, or M104A C125S IL2, in addition to other variations that modify the binding of IL2 to its receptor. These and other variants can be found in U.S. Patent No. 5,116,943 and Weiger et al., 1989, Eur J Biochem 180:295-300.

[0072] In various embodiments, any of the aforementioned IL2 portions can include 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.

[0073] 6.4. IL2Rα Portion The IL2 protein of the present disclosure includes an IL2Rα portion that includes, or consists of, the IL2 binding domain of IL2Rα, such as the extracellular domain of IL2Rα. The sequence of the mature human IL2Rα extracellular domain (corresponding to amino acids 22-272 of human IL2Rα) is as follows.

[0074]

Chemical formula

[0075] The sequence of the IL2 binding portion of the human IL2Rα extracellular domain (including two "sushi" domains corresponding to amino acids 22-186 of human IL2Rα) is as follows.

[0076]

Chemical formula

[0077] The sequence of an alternative IL2-binding portion of the extracellular domain of human IL2Rα corresponding to amino acids 22-240 of human IL2Rα is as follows.

[0078]

Chemical formula

[0079] The IL2Rα portion preferably contains 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 one of the above sequences, i.e., amino acids 22-186 of IL2Rα, amino acids 22-240 of IL2Rα, or amino acids 22-272 of IL2Rα, or any one of its optional IL2-binding portions.

[0080] In certain embodiments, the IL2Rα portion can contain 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, 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) a maximum of 251, 240, 230, 220, 210, 200, 190, 180, or 170 amino acids of the extracellular domain of human IL2Rα. In certain embodiments, the portion of human IL2Rα is linked by any one of (a) and (b) above, for example, at least 160 and a maximum of 180 amino acids from human IL2Rα, at least 162 and a maximum of 200 amino acids from human IL2Rα, at least 160 and a maximum of 220 amino acids from human IL2Rα, at least 164 and a maximum of 190 amino acids from human IL2Rα, etc.

[0081] In some embodiments, the IL2Rα portion has, or does not have, up to 5 additional amino acids, up to 10 additional amino acids, up to 15 additional amino acids, up to 20 additional amino acids, up to 30 additional amino acids, or up to 40 additional amino acids at the C-terminus of amino acid residue 186 of IL2Rα, and 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.

[0082] In certain embodiments, the IL2Rα moiety 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 substitutions 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 from asparagine. In some embodiments, one or more substitutions are substitutions 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 from threonine. In some embodiments, one or more substitutions are 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, T70Q, T70E, T70G, T70H, T70I, T70L, T70K, T70M, T70F, T70P, T70W, T70Y, or T70V, amino acid C192 (e.g., C192R, C192N, C192D, C192Q, C192E, C192G, C192H, C192I, C192L, C192K, C192M, C192F, C192P, C192W, C192Y, or C192V), or any combination thereof.

[0083] 6.5. Protease-Cleavable Linker The IL2 protein of the present disclosure typically has one or more substrates for proteases located between the IL2Rα portion and the IL2 portion, and includes a linker whose cleavage results in activated IL2.

[0084] The protease-cleavable linker can range from 20 amino acids to 80 amino acids or more. In certain embodiments, the protease-cleavable linker ranges 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.

[0085] The protease-cleavable linker includes one or more substrate sequences for one or more proteases, such as one or more of the proteases described in Section 6.5.1. One or more substrate sequences, such as one or more of the substrate sequences described in Section 6.5.2, are typically adjacent to one or more spacer sequences, such as the spacer sequences described in Section 6.5.3. Each protease-cleavable linker can include 1, 2, 3 or more substrate sequences. The spacer sequences may be adjacent, overlapping, or separated by spacer sequences. Preferably, the C-terminus and N-terminus of the protease-cleavable linker include spacer sequences.

[0086] Exemplary protease-cleavable linker sequences are described in Section 6.5.4. 6.5.1. Proteases Exemplary proteases into which substrate sequences can be incorporated into protease-cleavable linkers are listed in Table A below.

[0087]

Table 1-1

[0088]

Table 1-2

[0089] In certain embodiments, the protease is matrix metalloprotease (MMP)-2, MMP-9, legumain, thrombin, fibroblast activation protease (FAP), MMP-1, MMP-3, MMP-7, MMP-8, MMP-12, MMP-13, MMP-14, membrane type 1 matrix metalloprotease (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), disintegrin and metalloprotease (ADAM) 10, ADAM12, ADAM17, ADAM with thrombospondin motif (ADAMTS), ADAMTS5, beta-secretase (BACE), granzyme A, granzyme B, guanidino benzoatase, hepsin, matriptase, matriptase 2, meprin, neprilysin, prostate-specific membrane antigen (PSMA), tumor necrosis factor converting enzyme (TACE), kallikrein-related peptidase (KLK) 3, KLK5, KLK7, KLK11, hepatitis C virus NS3 / 4 protease (HCV-NS3 / 4), tissue plasminogen activator (tPA), calpain, calpain 2, glutamate carboxypeptidase II, plasma kallikrein, AMSH-like protease, AMSH, gamma-secretase component, anti-plasmin cleavage enzyme (APCE), decidin 1, apoptosis-related cysteine peptidase, or N-acetylated alpha-linked acidic dipeptidase-like 1.

[0090] 6.5.2. Substrate Exemplary substrate sequences that are cleavable by tumor proteases and can be incorporated into protease-cleavable linkers are set forth in Table B below.

[0091]

Table 2-1

[0092]

Table 2-2

[0093]

Table 2-3

[0094]

Table 2-4

[0095]

Table 2-5

[0096] 6.5.3. Spacer Exemplary spacer arrays that can be incorporated into protease-cleavable linkers are described in Table C below. In addition to the spacer arrays described in Table C, any of the non-cleavable linker arrays described in Section 6.6, for example, the non-cleavable linker arrays described in Table E, or portions thereof, can be used as spacer arrays.

[0097]

Table 3-1

[0098]

Table 3-2

[0099] In some embodiments, as used in Table C above, n is an integer from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. 6.5.4. Exemplary Protease-Cleavable Linkers Exemplary protease-cleavable linkers containing one or more substrate sequences and spacer sequences are described in Table D below.

[0100] [Table 4-1]

[0101] [Table 4-2]

[0102] [Table 4-3]

[0103] [Table 4-4]

[0104] [Table 4-5]

[0105] In certain embodiments, 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 substitution compared to the sequences described in Table D. Thus, in some embodiments, the protease-cleavable linker comprises or consists of any amino acid sequence of Table D having 1 to 5 amino acid substitutions compared to the sequences described in Table D.

[0106] 6.6. Non-Cleavable Linkers In certain embodiments, the present disclosure provides an IL2 protein in which two or more components of the IL2 protein are connected to each other by a peptide linker. By way of example and not limitation, linkers can be used to connect the Fc domain and the IL2Rα portion, or different domains within a targeting portion (e.g., the VH and VL domains in an scFv).

[0107] Preferably, all linkers in the IL2 protein other than the protease-cleavable linker whose cleavage results in activation of IL2 are non-cleavable linkers (NCLs).

[0108] Non-cleavable linkers can range from 2 amino acids to 60 or more amino acids, and in certain embodiments, non-cleavable linkers 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.

[0109] In certain embodiments, non-cleavable linkers are at least 5 amino acids, at least 6 amino acids, or at least 7 amino acids in length, and optionally, up to 30 amino acids, up to 40 amino acids, up to 50 amino acids, or up to 60 amino acids in length.

[0110] In some of the foregoing embodiments, non-cleavable linkers range from 5 amino acids to 50 amino acids in length, e.g., in the range of 5 - 50, 5 - 45, 5 - 40, 5 - 35, 5 - 30, 5 - 25, or 5 - 20 amino acids in length. In other foregoing embodiments, non-cleavable linkers range from 6 amino acids to 50 amino acids in length, e.g., in the range of 6 - 50, 6 - 45, 6 - 40, 6 - 35, 6 - 30, 6 - 25, or 6 - 20 amino acids in length. In still other foregoing embodiments, non-cleavable linkers range from 7 amino acids to 50 amino acids in length, e.g., in the range of 7 - 50, 7 - 45, 7 - 40, 7 - 35, 7 - 30, 7 - 25, or 7 - 20 amino acids in length.

[0111] Charges (e.g., charged hydrophilic linkers) and / or flexible linkers are particularly preferred. Examples of flexible linkers that can be used in the IL2 proprotein of the present disclosure include those disclosed in Chen et al., 2013, Adv Drug Deliv Rev. 65(10): 1357-1369 and Klein et al., 2014, Protein Engineering, Design & Selection 27(10):325-330. Particularly useful flexible non-cleavable linkers are repeats of glycine and serine, e.g., G n S (SEQ ID NO: 266) or SG n (SEQ ID NO: 267) monomers or multimers, or those containing them, where n is an integer from 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the non-cleavable linker is a repeat of G4S (SEQ ID NO: 1), e.g., (GGGGS) n (SEQ ID NO: 153) monomers or multimers, or those containing it.

[0112] Polyglycine non-cleavable linkers can be suitably used in the IL2 proprotein of the present disclosure. In some embodiments, the peptide non-cleavable linker contains two consecutive glycines (2Gly), three consecutive glycines (3Gly), four consecutive glycines (4Gly) (SEQ ID NO: 268), five consecutive glycines (5Gly) (SEQ ID NO: 269), six consecutive glycines (6Gly) (SEQ ID NO: 270), seven consecutive glycines (7Gly) (SEQ ID NO: 271), eight consecutive glycines (8Gly) (SEQ ID NO: 272), or nine consecutive glycines (9Gly) (SEQ ID NO: 273).

[0113] Exemplary non-cleavable linker sequences are listed in Table E below.

[0114]

Table 5-1

[0115]

Table 5-2

[0116]

Table 5-3

[0117] In certain embodiments, the IL2 proprotein of the present disclosure may comprise a polypeptide chain containing, in an N-terminal to C-terminal orientation, a targeting moiety (or targeting moiety chain), a hinge domain and a CH2 domain, and a CH3 domain. Thus, the hinge domain can be said to connect the targeting moiety to the CH2 domain and constitute a kind of linker. Exemplary hinge domains are described in Section 6.9.3.

[0118] 6.7. Targeting moiety By incorporating a targeting moiety into the IL2 proprotein of the present disclosure, delivery of high concentrations of IL2 to the tumor microenvironment is enabled, while at the same time systemic exposure is reduced and there are fewer side effects than those obtained using unmasked IL2.

[0119] It is contemplated that any target molecule that is present at a particular site or tissue or that can drive the IL2 proprotein can be targeted by the IL2 proprotein of the present disclosure. In some embodiments, the IL2 proprotein is intended to treat cancer, for example, by inducing a local immune response against tumor tissue. Thus, the targeting molecule can be any local tumor and associated target molecule. Target molecules recognized by the targeting moiety of the IL2 proprotein of the present disclosure are generally found, for example, on the surface of activated T cells, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, in free form in serum, in the extracellular matrix (ECM), or on immune cells present at the target site, such as tumor-reactive lymphocytes.

[0120] In various aspects, the targeting moiety can bind 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"). Those skilled in the art will recognize that the foregoing categories of target molecules are not mutually exclusive, and thus, a given target molecule may be classified into multiple of the foregoing categories of target molecules. For example, some molecules may be considered both a TAA and an ECM protein, and other molecules may be considered both a TCA and a checkpoint inhibitor.

[0121] Exemplary types of cancer that can be targeted include acute lymphoblastic leukemia, acute myeloid leukemia, biliary cancer, B cell leukemia, B cell lymphoma, biliary cancer, bone cancer, brain cancer, breast cancer, triple-negative breast cancer, cervical cancer, Burkitt 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 lymphoma, liver cancer, lung cancer, medullary thyroid cancer, melanoma, multiple myeloma, ovarian cancer, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer, lung adenocarcinoma, kidney cancer, sarcoma, skin cancer, testicular cancer, urothelial cancer, and other bladder cancers. However, those skilled in the art will recognize that TAAs and other target molecules related to the tumor microenvironment are known for substantially any type of cancer.

[0122] Non-limiting examples of ECM antigens include syndecan, heparanase, integrin, osteopontin, link, cadherin, laminin, laminin EGF-type, lectin, fibronectin, notch, nectin (e.g., nectin-4), tenascin, collagen (e.g., collagen type X), and matrilysin.

[0123] Other target molecules are cell surface molecules of tumors or viral lymphocytes, such as T cell costimulatory 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.

[0124] In certain embodiments, the target molecule is a checkpoint inhibitor, such as CTLA-4, PD1, PDL1, PDL2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2. In certain embodiments, the target molecule is PD1. In other embodiments, the target molecule is LAG3.

[0125] Antibodies and antigen-binding portions can generally bind to specific antigenic determinants and direct the IL2 proprotein to a target site, such as a specific tumor cell type or tumor stroma bearing the antigenic determinant. In certain embodiments, the targeting moiety 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 (TNCA2), fibronectin extra domain B (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 (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T cell receptor / CD3-zeta chain, MAGE-tumor antigen family (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, 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-tumor antigen family (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, alpha-fetoprotein, E-cadherin, alpha-catenin, beta-catenin, and gamma-catenin, p120ctn, gp100Pmel117, 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 family of tumor antigens, Imp-1, P1A, Epstein-Barr virus-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, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, LAGA-1a, p53, prostein, PSMA, survival and telomerase, prostate cancer tumor antigen-1 (PCTA-1), ELF2M, neutrophil elastase, Ephrin B2, insulin-like 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 A1 domain of tenascin-C (TnC A1).

[0126] Suitable formats for the targeting moiety 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, such as an scFv described in Section 6.8.2, or a Fab described in Section 6.8.1.

[0127] In some embodiments, the targeting moiety targets an exemplary target molecule described in Table F below, along with a reference to an exemplary antibody or antibody sequence on which the targeting moiety can be based.

[0128] [Table 6-1]

[0129] [Table 6-2]

[0130] [Table 6-3]

[0131] [Table 6-4]

[0132] [Table 6-5]

[0133] [Table 6-6]

[0134] [Table 6-7]

[0135] [Table 6-8]

[0136]

Table 6-9

[0137]

Table 6-10

[0138]

Table 6-11

[0139]

Table 6-12

[0140]

Table 6-13

[0141]

Table 6-14

[0142] In some embodiments, the targeting moiety competes with the antibodies described in Table F for binding to the target molecule. In further embodiments, the targeting moiety comprises a CDR that has the CDR sequences of the antibodies described in Table F. In some embodiments, the targeting moiety comprises all six CDR sequences of the antibodies described in Table F. In other embodiments, the targeting moiety comprises at least the heavy chain CDR sequences (CDR-H1, CDR-H2, CDR-H3, and the light chain CDR sequences of the universal light chain). In further embodiments, the targeting moiety comprises a VH that comprises the amino acid sequence of the VH of the antibodies described in Table F. In some embodiments, the targeting moiety further comprises a VL that comprises the amino acid sequence of the VL of the antibodies described in Table F. In other embodiments, the targeting moiety further comprises a universal light chain VL sequence.

[0143] In some embodiments, the targeting moiety is non-blocking or low-blocking of ligand-receptor binding. Examples of non-blocking or low-blocking anti-PD1 antibodies include the VH / VL amino acid sequences of SEQ ID NO: 2 / 10 of PCT Publication No. WO2015 / 112800A1, Blocking 16 / 17 of U.S. Patent 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. Patent No. 10,294,299 B2. Examples of non-blocking or low-blocking anti-LAG3 antibodies include antibodies having the VH / VL amino acid sequences of SEQ ID NO: 23 / 24, 3 / 4, and 11 / 12 of U.S. Publication No. 2022 / 0056126 A1.

[0144] Additional target molecules that can be targeted by the IL2 proprotein are disclosed in Table I below, for example, in Hafeez et al., 2020, Molecules 25:4764, doi:10.3390 / molecules25204764, particularly in Table 1. Table 1 of Hafeez et al. is hereby incorporated by reference in its entirety.

[0145] 6.8. Format of the targeting moiety In certain embodiments, 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 an epitope. In one embodiment, the antigen-binding portion 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 ) fragments, VL (or V L ) fragments, Fab fragments, F(ab’)2 fragments, scFv fragments, Fv fragments, minibodies, diabodies, triabodies, and tetra-bodies.

[0146] 6.8.1. Fab Fab domains have traditionally been produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain. Fab domains can include constant domain sequences and variable region sequences from any suitable species and can thus be murine, chimeric, human, or humanized.

[0147] Fab domains typically include a CH1 domain attached to the VH domain, which pairs with a CL domain attached to the VL domain. In wild-type immunoglobulins, the VH domain pairs with the VL domain to form the Fv region, and the CH1 domain pairs with the CL domain to further stabilize the binding site. A disulfide bond between the two constant domains can further stabilize the Fab domain.

[0148] 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 enable correct association of Fab domains belonging to the same targeting moiety and minimize aberrant pairing of Fab domains belonging to different targeting moieties. For example, the Fab heterodimerization strategies shown in Table G below can be used.

[0149]

Table 7

[0150] Thus, in certain embodiments, correct association between the two polypeptides of the Fab is facilitated, for example, by exchanging the VL and VH domains of the Fab or by exchanging the CH1 and CL domains of the Fab, as described in WO2009 / 080251.

[0151] Correct Fab pairing can also be facilitated 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 by introducing one or more amino acid modifications in the VH domain and one or more amino acid modifications in the VL domain. The amino acids to be modified are typically part of the VH:VL and CH1:CL interfaces such that the Fab components preferentially pair with each other rather than with the components of other Fabs.

[0152] In one embodiment, the one or more amino acid modifications are limited to conserved framework residues of the variable (VH, VL) and constant (CH1, CL) domains, 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.

[0153] In one embodiment, the modifications introduced in the VH domain and CH1 domain and / or in the VL domain and CL domain 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 combinations of various interactions. Complementarity between protein surfaces has been widely described in the literature in terms of lock and key fit, knob into hole, protrusion and cavity, donor and acceptor, etc., all of which imply the nature of the structural and chemical correspondence between two interacting surfaces.

[0154] In one embodiment, the one or more introduced modifications introduce new hydrogen bonds across the interface of the Fab components. In one embodiment, the one or more introduced modifications introduce new salt bridges across the interface of the Fab components. Exemplary substitutions are described in WO2014 / 150973 and WO2014 / 082179, the contents of which are incorporated herein by reference.

[0155] In some embodiments, the Fab domain comprises a 192E substitution in the CH1 domain and 114A and 137K substitutions in the CL domain, thereby introducing a salt bridge between the CH1 domain and the CL domain (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0156] In some embodiments, the Fab domain comprises 143Q and 188V substitutions within the CH1 domain and 113T and 176V substitutions within the CL domain, which serves to exchange the hydrophobic and polar contact regions between the CH1 domain and the CL domain (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0157] In some embodiments, the Fab domain can include modifications in some or all of the VH, CH1, VL, and CL domains to introduce an orthogonal Fab interface that promotes correct assembly of the Fab domain (Lewis et al., 2014 Nature Biotechnology 32:191-198). In embodiments, 39K and 62E modifications are introduced into the VH domain, H172A and F174G modifications are introduced into the CH1 domain, 1R, 38D, (36F) modifications are introduced into the VL domain, and L135Y and S176W modifications are introduced into the CL domain. In another embodiment, a 39Y modification is introduced into the VH domain and a 38R modification is introduced into the VL domain.

[0158] The Fab domain can also be modified to replace the native CH1:CL disulfide bond with an engineered disulfide bond, thereby increasing the efficiency of Fab component pairing. For example, the engineered disulfide bond can be introduced by introducing 126C into the CH1 domain and 121C into the CL domain (see, e.g., Mazor et al., 2015, MAbs 7:377-89).

[0159] The Fab domain can also be modified by replacing the CH1 and CL domains with alternative domains that promote correct assembly. For example, Wu et al., 2015, MAbs 7:364-76 replaced the CH1 domain with the constant domain of the T cell receptor and the CL domain with the b domain of the T cell receptor, and described that these domain replacements were paired with additional charge-charge interactions between the VL and VH domains by introducing a 38D modification into the VL domain and a 39K modification into the VH domain.

[0160] Instead of, or in addition to, using a Fab heterodimerization strategy 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 the IL2 proprotein of the present disclosure. In various embodiments, using a common light chain as described herein reduces the number of inappropriate species in the IL2 proprotein compared to using the original cognate 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 comprises a human heavy chain variable gene segment that is rearranged in vivo within a mouse B cell engineered to express a limited human light chain repertoire or a single human light chain cognate to the human heavy chain, generating an antibody repertoire comprising a plurality of human VHs that are cognate to one of one, or two possible human VLs in response to exposure to the antigen of interest, and the antibody repertoire is specific for the antigen of interest. The common light chain is derived from a rearranged human Vκ1-39Jκ5 sequence or a rearranged human Vκ3-20Jκ1 sequence and includes somatic variants (e.g., affinity matured forms). See, for example, U.S. Patent No. 10,412,940.

[0161] 6.8.2.scFv A single-chain Fv or "scFv" antibody fragment contains the VH and VL domains of an antibody within a single polypeptide chain, can be expressed as a single-chain polypeptide, and retains the specificity of the intact antibody from which they are derived. Generally, an scFv polypeptide further includes 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 linking the VH and VL chains of an scFv are the non-cleavable linkers identified in Section v.

[0162] As used herein, unless otherwise specified, an scFv may have the VL and VH variable regions in either order with respect to the N-terminus and C-terminus of the polypeptide, and the scFv may include VL-linker-VH, or may include VH-linker-VL.

[0163] An scFv can include VH and VL sequences from any suitable species, such as mouse, human, or humanized VH and VL sequences. To generate an scFv-encoding nucleic acid, the VH and VL-encoding DNA fragments are operably linked to another fragment encoding a linker (e.g., the linker described in Section 6.6, typically amino acids glycine and serine, such as a repeat of a sequence containing the amino acid sequence (Gly4-Ser)3 (SEQ ID NO: 157)) 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, for example, 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).

[0164] 6.9. Fc Region The IL2 protein of the present disclosure typically includes a pair of Fc domains that associate to form an Fc region. In natural antibodies, the Fc region includes a hinge region at their N-terminus to form the constant domain. Throughout the present disclosure, unless otherwise specified, references to the Fc domain include an Fc domain having a hinge domain at its N-terminus.

[0165] The Fc domain can be derived from any suitable species operably linked 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 site or its components can be fused to the N-terminus and / or C-terminus of the IgG Fc domain.

[0166] 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 derived from IgG1, IgG2, IgG3, and IgG4 are provided in Table Y below.

[0167]

Table 8

[0168] In some embodiments, 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: 346. If the Fc domain comprises at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 346 (e.g., 90% - 99% sequence identity to SEQ ID NO: 346), the Fc domain may also comprise one or more of the 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).

[0169] In some embodiments, 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: 347. If the Fc domain comprises at least 90% sequence identity and less than 100% sequence identity to SEQ ID NO: 347 (e.g., 90% - 99% sequence identity to SEQ ID NO: 347), the Fc domain may also comprise one or more of the 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).

[0170] In some embodiments, 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: 348. If the Fc domain comprises at least 90% and less than 100% sequence identity to SEQ ID NO: 348 (e.g., 90% - 99% sequence identity to SEQ ID NO: 348), the Fc domain may also comprise one or more amino acid substitutions described herein, e.g., 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).

[0171] In some embodiments, 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: 349. If the Fc domain comprises at least 90% and less than 100% sequence identity to SEQ ID NO: 349 (e.g., 90% - 99% sequence identity to SEQ ID NO: 349), the Fc domain may also comprise one or more amino acid substitutions described herein, e.g., 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).

[0172] The two Fc domains within the Fc region may be the same as or different from each other. In a natural antibody, the Fc domains are typically the same, but for the purpose of producing a multispecific binding molecule, e.g., an IL2 proprotein of the present disclosure and an MBM produced by its activation, the Fc domains may advantageously be different to allow for heterodimerization as described in Section 6.9.2 below.

[0173] In natural antibodies, the heavy-chain Fc domains of IgA, IgD, and IgG are composed of two heavy-chain constant domains (CH2 and CH3), and the domains of IgE and IgM are composed of three heavy-chain constant domains (CH2, CH3, and CH4). These dimerize to create the Fc region.

[0174] In the IL2 proprotein of the present disclosure, the Fc region, and / or the Fc domain thereof, may include heavy-chain constant domains from one or more different classes of antibodies, for example, from 1, 2, or 3 different classes.

[0175] In one embodiment, the Fc region includes the CH2 and CH3 domains derived from IgG1. In one embodiment, the Fc region includes the CH2 and CH3 domains derived from IgG2. In one embodiment, the Fc region includes the CH2 and CH3 domains derived from IgG3.

[0176] In one embodiment, the Fc region includes the CH2 and CH3 domains derived from IgG4. In one embodiment, the Fc region includes the CH4 domain derived from IgM. The IgM CH4 domain is typically located at the C-terminus of the CH3 domain.

[0177] In one embodiment, the Fc region includes the CH2 and CH3 domains derived from IgG, and the CH4 domain derived from IgM. It will be understood that the heavy chain constant domains for use in producing the Fc region for the IL2 protein of the present disclosure may include variants of the naturally occurring constant domains described above. Such variants may include one or more amino acid mutations compared to the wild-type constant domain. In one example, the Fc region of the present disclosure includes at least one constant domain that differs in sequence from the wild-type constant domain. It will be understood that the variant constant domain may be longer or shorter than the wild-type constant domain. Preferably, the variant constant domain is at least 60% identical or similar to the wild-type constant domain. In another example, the variant constant domain is at least 70% identical or similar. In 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.

[0178] IgM and IgA are naturally present in humans as covalently linked multimers of a common H2L2 antibody unit. IgM exists as a pentamer when the J chain is incorporated and as a hexamer when the J chain is absent. IgA exists as monomeric and dimeric forms. The heavy chains of IgM and IgA have an 18-amino acid extension to the C-terminal constant domain known as the tailpiece. The tailpiece contains cysteine residues that form disulfide bonds between the heavy chains within the polymer and is thought to play an important role in polymerization. The tailpiece also contains a glycosylation site. In certain embodiments, the IL2 protein of the present disclosure does not include a tailpiece.

[0179] The Fc domain incorporated into the IL2 protein of the present disclosure may include one or more modifications that alter the functional properties of the protein, such as binding to Fc receptors such as FcRn or leukocyte receptors, binding to complement, an altered disulfide bond structure, or an altered glycosylation pattern. Exemplary Fc modifications that alter effector function are described in Section 6.9.1.

[0180] The Fc domain can also be modified to include modifications that improve the manufacturability of asymmetric IL2 proproteins, for example, by enabling heterodimerization, which is the preferential pairing of non-identical Fc domains to the same Fc domain. Heterodimerization enables the production of IL2 proproteins in which different polypeptide components are connected to each other by Fc regions containing Fc domains with different sequences. Examples of heterodimerization strategies are illustrated in Section 6.9.2.

[0181] It will be understood that any of the above modifications can be combined in any suitable manner to achieve the desired functional properties and / or to modify the properties of the IL2 proprotein in combination with other modifications.

[0182] 6.9.1. Fc Domains with Altered Effector Function In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and / or effector functions.

[0183] In certain embodiments, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In certain embodiments, the Fc receptor is an activating 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 cell phagocytosis (ADCP), and cytokine secretion. In certain embodiments, the effector function is ADCC.

[0184] In one embodiment, the Fc domain (e.g., the Fc domain of an IL2 proprotein half-antibody) or Fc region (e.g., one or both Fc domains of an IL2 proprotein that can associate to form an Fc region) comprises an amino acid substitution at a position selected from the group 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 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 such an embodiment, the Fc domain or region is an Igd Fc domain or region, particularly 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 (numbering according to the Kabat EU index). In one embodiment, the Fc domain or Fc region comprises an amino acid substitution at position P329 and further amino acid substitutions at positions selected from E233, L234, L235, N297, and P331 (numbering according to the Kabat EU index). In a more specific embodiment, the further amino acid substitutions are E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a particular embodiment, the Fc domain or Fc region comprises amino acid substitutions at positions P329, L234, and L235 (numbering according to the Kabat EU index). In a more specific embodiment, the Fc domain comprises the amino acid mutations L234A, L235A, and P329G (“P329G LALA”, “PGLALA” or “LALAPG”).

[0185] Typically, the same one or more amino acid substitutions are present in each of the two Fc domains of the Fc region. Thus, in certain embodiments, each Fc domain of the Fc region comprises the amino acid substitutions L234A, L235A, and P329G (Kabat EU indexing), i.e., in each of the first and second Fc domains of the Fc region, the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A), and the proline residue at position 329 is replaced with a glycine residue (P329G) (numbering according to the Kabat EU index).

[0186] In one embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In some embodiments, the IgG1 Fc domain is a variant IgG1 comprising the D265A, N297A mutations (EU numbering) that reduce effector function.

[0187] In another embodiment, the Fc domain is an IgG4 Fc domain with reduced binding to Fc receptors. An exemplary IgG4 Fc domain with reduced binding to Fc receptors may comprise an amino acid sequence selected from Table H below. In some embodiments, the Fc domain comprises only the bolded portion of the sequences shown below:

[0188]

Table 9-1

[0189]

Table 9-2

[0190]

Table 9-3

[0191]

Table 9-4

[0192]

Table 9-5

[0193] In certain embodiments, IgG4 having reduced effector function includes the bolded portion of the amino acid sequence of SEQ ID NO: 31 of WO2014 / 121087 and may also be referred to herein as IgG4s or hIgG4s.

[0194] For the heterodimeric Fc region, it is possible to incorporate combinations of the variant IgG4 Fc sequences described above, for example, an Fc region comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 30 of WO2014 / 121087 (or the bolded portion thereof) and an Fc domain comprising the amino acid sequence of SEQ ID NO: 37 of WO2014 / 121087 (or the bolded portion thereof), or an Fc region comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 31 of WO2014 / 121087 (or the bolded portion thereof) and an Fc domain comprising the amino acid sequence of SEQ ID NO: 38 of WO2014 / 121087 (or the bolded portion thereof).

[0195] 6.9.2. Fc Heterodimerization Variant A particular IL2 proprotein dimerizes between two Fc domains that are operably linked to non-identical N-terminal or C-terminal regions, unlike native immunoglobulins. Insufficient heterodimerization of the two Fc domains to form the Fc region can be an obstacle to increasing the yield of the desired heterodimeric molecule and can pose a challenge for purification. For example, various approaches available in the art can be used in enhancing dimerization of the Fc domains that may be present in the IL2 proprotein of the present disclosure, as disclosed in EP1870459A1, 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. 2006 / 204493A1, and PCT Publication No. WO2009 / 089004A1.

[0196] In some embodiments, the present disclosure provides an IL2 proprotein comprising an Fc heterodimer, i.e., an Fc region comprising heterologous, non-identical Fc domains. Typically, each Fc domain in the Fc heterodimer comprises the CH3 domain of an antibody. The CH3 domain is derived from the constant region of an antibody of any isotype, class, or subclass, preferably an IgG (IgG1, IgG2, IgG3, and IgG4) class antibody, as described in the foregoing section.

[0197] Heterodimerization of two different heavy chains at the CH3 domain results in the desired IL2 proprotein, while homodimerization of the same heavy chain reduces the yield of the desired IL2 proprotein. Thus, in preferred embodiments, the polypeptides that associate to form the IL2 proprotein of the present disclosure comprise CH3 domains having modifications that are advantageous for heterodimeric association relative to unmodified Fc domains.

[0198] In certain embodiments, the modification that promotes the formation of the Fc heterodimer is a so-called "knob-into-hole" or "knob-in-hole" modification that includes a "knob" modification on one of the Fc domains and a "hole" modification on the other Fc domain. The knob-into-hole technology is described, for example, in U.S. Patent No. 5,731,168, US7,695,936, Ridgway et al., 1996, Prot Eng 9:617-621, and Carter, 2001, Immunol Meth 248:7-15. Generally, this method involves introducing a protrusion ("knob") at the interface of the first polypeptide and a corresponding cavity ("hole") at the interface of the second polypeptide so that the protrusion can be placed within the cavity to promote heterodimer formation and impede homodimer formation. The protrusion is constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). A compensatory cavity of the same or similar size as the protrusion is created at the interface of the second polypeptide by replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine).

[0199] Thus, in some embodiments, the amino acid residues within the CH3 domain of the first subunit of the Fc domain are replaced with amino acid residues having a larger side chain volume, thereby creating a protrusion within the CH3 domain of the first subunit that can be disposed within the cavity within the CH3 domain of the second subunit, and the amino acid residues within the CH3 domain of the second subunit of the Fc domain are replaced with amino acid residues having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit in which the protrusion within the CH3 domain of the first subunit can be disposed. Preferably, the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protrusion and the cavity can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis. An exemplary substitution is Y470T.

[0200] In certain such embodiments, in the first Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), in the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V), optionally, the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the Kabat EU index). In a further embodiment, in the first Fc domain, additionally, the serine residue at position 354 is replaced with a cysteine residue (S354C), or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (in particular, the serine residue at position 354 is replaced with a cysteine residue). In the second Fc domain, additionally, the tyrosine residue at position 349 is replaced with a cysteine residue (Y349C) (numbering according to the Kabat EU index). In certain embodiments, the first Fc domain comprises the amino acid substitutions S354C and T366W, and the second Fc domain comprises the amino acid substitutions Y349C, T366S, L368A, and Y407V (numbering according to the Kabat EU index).

[0201] In some embodiments, electrostatic steering (e.g., as described in Gunasekaran et al., 2010, J Biol Chem 285(25):19637-46) can be used to promote the association of the first and second Fc domains of the Fc region.

[0202] As an alternative to, or in addition to, the use of an Fc domain engineered to promote heterodimerization, the Fc domain can be engineered to enable a purification strategy that allows for the selection of Fc heterodimers. In one such embodiment, one polypeptide comprises a modified Fc domain that inhibits its binding to Protein A, thus enabling a purification method that yields a heterodimeric protein. See, e.g., U.S. Patent No. 8,586,713. Thus, an IL2 proprotein comprises a first CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from each other by at least one amino acid, and the at least one amino acid difference reduces the binding of the IL2 proprotein to Protein A as compared to the corresponding IL2 proprotein lacking the amino acid difference. In one embodiment, the first CH3 domain binds to Protein A and the second CH3 domain comprises a mutation / modification that reduces or abolishes Protein A binding, such as an H95R modification (H435R according to EU numbering by IMGT exon numbering). The second CH3 may further comprise a Y96F modification (Y436F according to EU by IMGT). Modifications of this class are referred to herein as "star" mutations.

[0203] In some embodiments, the Fc can comprise one or more mutations (e.g., knob and hole mutations) to promote heterodimerization, as well as star mutations to facilitate purification. 6.9.3. Hinge Domain The IL2 protein of the present disclosure may include an Fc domain containing a hinge domain at its N-terminus. The hinge region can be a natural hinge region or a modified hinge region. The hinge region is typically found at the N-terminus of the Fc region. The term "hinge domain" refers to a naturally occurring or non-natural hinge sequence that is a monomer hinge domain in the context of a single or monomer polypeptide chain, and in the context of a dimeric polypeptide (e.g., a homodimer or heterodimer IL2 protein formed by the association of two Fc domains), may include two associated hinge sequences on separate polypeptide chains. Sometimes, the two associated hinge sequences are referred to as the "hinge region."

[0204] The natural hinge region is typically the hinge region found between the Fab domain and the Fc domain in a naturally occurring antibody. A modified hinge region is any hinge that differs in length and / or composition from the natural hinge region. Such a hinge can include hinge regions from other species such as human, mouse, rat, rabbit, shark, pig, hamster, camel, llama, or goat. Other modified hinge regions can include the complete hinge region derived from an antibody of a different class or subclass than that of the heavy chain Fc domain or Fc region. Alternatively, a modified hinge region can include a portion or repeating unit of the natural hinge, with each repeating unit being derived from the natural hinge region. In a further alternative, the natural hinge region can be altered by converting one or more cysteines or other residues to neutral residues such as serine or alanine, or by converting appropriately positioned residues to cysteine residues. By such means, the number of cysteine residues in the hinge region can be increased or decreased. Other modified hinge regions may be completely synthetic and may be designed to have desired properties such as length, cysteine composition, and flexibility.

[0205] Some modified hinge regions are already described, for example, in U.S. Patent No. 5,677,425, WO99 / 15549, WO2005 / 003170, WO2005 / 003169, WO2005 / 003170, WO98 / 25971, and WO2005 / 003171, which are incorporated herein by reference.

[0206] In one embodiment, the IL2 protein of the present disclosure includes an Fc region in which one or both Fc domains have an intact hinge domain at their N-terminus. In various embodiments, positions 233-236 within the hinge region can be G, G, G, and a gap; G, G, a gap, and a gap; G, a gap, a gap, and a gap; or all gaps, and the positions are numbered by EU numbering.

[0207] In some embodiments, the IL2 protein of the present disclosure includes a modified hinge region that reduces the binding affinity for Fcγ receptors relative to the wild-type hinge region of the same isotype (e.g., human IgG1 or human IgG4).

[0208] In one embodiment, the IL2 protein of the present disclosure includes an Fc region in which each Fc domain has an intact hinge domain at its N-terminus, each Fc domain and hinge domain are derived from IgG4, and each hinge domain includes the modified sequence CPPC. The core hinge region of human IgG4 includes the sequence CPSC as compared to IgG1 that includes the sequence CPPC. The serine residue present in the IgG4 sequence results in an increase in flexibility in this region, and thus, a proportion of the molecules form disulfide bonds (intra-chain disulfides) within the same protein chain rather than cross-linking to other heavy chains within the IgG molecule to form inter-chain disulfides. (Angel et al., 1993, Mol Immunol 30(1):105-108). By changing the serine residue to proline to obtain the same core sequence as IgG1, complete formation of inter-chain disulfides is enabled within the IgG4 hinge region, and thus, the heterogeneity in the purified product is reduced. This altered isotype is called IgG4P.

[0209] 6.9.3.1. Chimeric hinge sequence The hinge domain can be a chimeric hinge domain. For example, a chimeric hinge can include an "upper hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region combined with a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region.

[0210] In certain embodiments, the chimeric hinge region comprises the amino acid sequence EPKSCDKTHTCPPCPAPPVA (SEQ ID NO: 326, previously disclosed as SEQ ID NO: 8 in WO2014 / 121087, which is hereby incorporated by reference in its entirety) or ESKYGPPCPPCPAPPVA (SEQ ID NO: 327, previously disclosed as SEQ ID NO: 9 in WO2014 / 121087). Such chimeric hinge sequences can be suitably linked to the IgG4 CH2 region (e.g., incorporated into an IgG4 Fc domain, e.g., a human or mouse Fc domain, which can be further modified in the CH2 and / or CH3 domains to reduce effector function, as described, for example, in Section 6.9.1).

[0211] 6.9.3.2. Hinge sequence with reduced effector function In further embodiments, the hinge region can be modified to reduce effector function, as described in WO2016 / 161010A2, which is hereby incorporated by reference in its entirety. In various embodiments, positions 233 - 236 of the modified hinge region can be G, G, G, and blank; G, G, blank, and blank; G, blank, blank, and blank; or all blank, where the positions are numbered according to EU numbering (shown in Figure 1 of WO2016161010A2). These segments can be represented as GGG-, GG--, G---, or ----, where "-" represents a blank position.

[0212] Position 236 is vacant in canonical human IgG2 but occupied in other canonical human IgG isotypes. Positions 233 - 235 are occupied by residues other than G in all four human isotypes (as shown in Figure 1 of WO2016161010A2).

[0213] The hinge modifications within positions 233 - 236 can be combined with position 228, which is occupied by P in native human IgG1 and IgG2, but by S in human IgG4 and by R in human IgG3. The S228P mutation in IgG4 antibodies is advantageous for stabilizing IgG4 antibodies and reducing the exchange of heavy chain-light chain pairs between exogenous and endogenous antibodies. Preferably, positions 226 - 229 are occupied by C, P, P, and C, respectively.

[0214] Exemplary hinge regions have residues 226 - 236, sometimes referred to as the middle (or core) and lower hinge, and are occupied by modified hinge sequences designated as GGG-(233 - 236), GG--(233 - 236), G---(233 - 236), and no G (233 - 236). Optionally, the hinge domain amino acid sequence comprises CPPCPAPGGG-GPSVF (SEQ ID NO: 328) (previously disclosed as SEQ ID NO: 1 in WO2016161010A2), CPPCPAPGG--GPSVF (SEQ ID NO: 329) (previously disclosed as SEQ ID NO: 2 in WO2016161010A2), CPPCPAPG---GPSVF (SEQ ID NO: 330) (previously disclosed as SEQ ID NO: 3 in WO2016161010A2), or CPPCPAP----GPSVF (SEQ ID NO: 331) (previously disclosed as SEQ ID NO: 4 in WO2016161010A2).

[0215] The modified hinge region described above can be incorporated into the heavy chain constant region, which typically includes CH2 and CH3 domains and may have an additional hinge segment (e.g., an upper hinge) adjacent to the designated region. Such additional constant region segments that are present are typically of the same isotype, preferably a human isotype, but may also be hybrids of different isotypes. The isotype of such additional human constant region segments is preferably human IgG4, but may also be human IgG1, IgG2, or IgG3 or hybrids thereof with different domains. Exemplary sequences of human IgG1, IgG2, and IgG4 are shown in Figures 2-4 of WO2016161010A2.

[0216] In certain embodiments, the modified hinge sequence can be linked to the IgG4 CH2 region (e.g., incorporated into an IgG4 Fc domain, e.g., a human or mouse Fc domain, which can be further modified in the CH2 and / or CH3 domains to reduce effector function, as described, for example, in Section 6.9.1).

[0217] 6.10. Nucleic Acids and Host Cells In another aspect, the disclosure provides a nucleic acid encoding the IL2 proprotein of the 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., 2, 3, 4 or more) nucleic acids.

[0218] A single nucleic acid can encode a portion of an IL2 proprotein that contains a single polypeptide chain, an IL2 proprotein that contains two or more polypeptide chains, or an IL2 proprotein that contains three or more polypeptide chains (e.g., a single nucleic acid can encode two polypeptide chains of an IL2 proprotein that contains 3, 4 or more polypeptide chains, or three polypeptide chains of an IL2 proprotein that contains 4 or more polypeptide chains). To control expression separately, open reading frames encoding two or more polypeptide chains can be placed under the control of separate transcriptional regulatory elements (e.g., a promoter and / or enhancer). Open reading frames encoding two or more polypeptides can also be controlled by the same transcriptional regulatory element and separated by an internal ribosome entry site (IRES) sequence to allow translation into separate polypeptides.

[0219] In some embodiments, an IL2 proprotein that contains two or more polypeptide chains is encoded by two or more nucleic acids. The number of nucleic acids encoding the IL2 proprotein can be less than or equal to the number of polypeptide chains in the IL2 proprotein (e.g., when two or more polypeptide chains are encoded by a single nucleic acid).

[0220] The nucleic acids of the present disclosure can be DNA or RNA (e.g., mRNA). In another aspect, the present disclosure provides host cells and vectors comprising the nucleic acids of the present disclosure. The nucleic acids can be present in a single vector, as described in more detail below herein, or in separate vectors present in the same host cell or separate host cells.

[0221] 6.10.1. Vector The present disclosure provides a vector comprising a nucleotide sequence encoding an IL2 protein or a component thereof described herein, for example, one or two of the polypeptide chains of a half-antibody of the IL2 protein. The vector includes, but is not limited to, a virus, plasmid, cosmid, lambda phage, or yeast artificial chromosome (YAC).

[0222] A number of vector systems can be used. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retroviruses (Rous sarcoma virus, MMTV or MOMLV), or SV40 virus. Another class of vectors utilizes RNA elements derived from RNA viruses such as Semliki Forest virus, Eastern equine encephalitis virus, and flavivirus.

[0223] Additionally, cells with stably integrated DNA into the chromosome can be selected by introducing one or more markers that enable the selection of transfected host cells. The markers can provide, for example, prototropy for auxotrophic hosts, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be either directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. These elements can include splice signals, as well as transcriptional promoters, enhancers, and termination signals.

[0224] When a DNA sequence containing an expression vector or construct is prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. To achieve this, various techniques can be used, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection, or other conventional techniques. Methods and conditions for culturing the resulting transfected cells and recovering the expressed polypeptide are known to those skilled in the art and can vary or be optimized based on the specific expression vector and mammalian host cell used, based on this specification.

[0225] 6.10.2. Cells The present disclosure also provides a host cell comprising a nucleic acid of the present disclosure. In one embodiment, the host cell is genetically engineered to contain one or more nucleic acids described herein.

[0226] In one embodiment, the host cell is genetically engineered by using an expression cassette. The phrase "expression cassette" refers to a nucleotide sequence capable of affecting the expression of a gene in a host compatible with such a sequence. Such cassettes can include a promoter, an open reading frame with or without introns, and a termination signal. Additional factors necessary or useful for bringing about expression, such as an inducible promoter, may also be used.

[0227] The present disclosure also provides a host cell comprising a vector described herein. The cell can be, but is not limited to, a eukaryotic cell, a bacterial cell, an insect cell, or a human cell. 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.

[0228] 6.11. Pharmaceutical Compositions The IL2 protein of the present disclosure can be in the form of a composition comprising the IL2 protein and one or more carriers, excipients, and / or diluents. The composition can be formulated for a specific use such as veterinary use or pharmaceutical use in humans. The form of the composition (e.g., dry powder, liquid formulation, etc.), as well as the excipients, diluents, and / or carriers used, depend on the intended use of the IL2 protein and, in the case of therapeutic use, the mode of administration.

[0229] In the case of therapeutic use, the composition can be provided as part of a sterile pharmaceutical composition comprising a pharmaceutically acceptable carrier. This composition can be in any suitable form (depending on the desired method of administration to the patient). The pharmaceutical composition can be administered to the patient by various routes such as oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, intrathecal, topical, or local. The most suitable route for administration in any given case will depend on the particular IL2 protein, the subject, and the nature and severity of the disease, as well as the physical condition of the subject. Typically, the pharmaceutical composition will be administered intravenously or subcutaneously.

[0230] The pharmaceutical composition can conveniently be presented in unit dosage form containing a predetermined amount of the IL2 protein of the present disclosure per dose. The amount of IL2 protein contained in a unit dose will depend on the disease being treated and other factors well known in the art. Such unit dosages can be in the form of a lyophilized dry powder or in liquid form containing an amount of IL2 protein suitable for a single administration. The dry powder unit dosage form can be packaged in a kit together with a syringe, a suitable amount of diluent, and / or other components useful for administration. The unit dosage in liquid form can be conveniently supplied in the form of a syringe pre-filled with an amount of IL2 protein suitable for a single administration.

[0231] The pharmaceutical composition can also be supplied in bulk since it contains an amount of IL2 protein suitable for multiple administrations. The pharmaceutical composition can be prepared for storage as a lyophilized formulation or an aqueous solution by mixing the IL2 proprotein having the desired purity with any optional pharmaceutically acceptable carrier, excipient, or stabilizer (all of which are referred to herein as “carriers”), i.e., buffers, stabilizers, preservatives, isotonic agents, nonionic detergents, antioxidants, and various other additives. See Remington’s Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives should be non-toxic to the recipient at the dosages and concentrations employed.

[0232] Buffers serve to maintain the pH within a range close to physiological conditions. They can be present at a wide variety of concentrations, but will typically be present at concentrations in the range of about 2 mM to about 50 mM. Suitable buffers for use in the present disclosure include both organic and inorganic acids and their salts, such as citrate buffers (e.g., sodium citrate - disodium citrate mixtures, citric acid - trisodium citrate mixtures, citric acid - sodium citrate mixtures, etc.), succinate buffers (e.g., succinic acid - sodium 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 - sodium fumarate mixtures, disodium fumarate mixtures, sodium fumarate - disodium fumarate mixtures, etc.), gluconate buffers (e.g., gluconic acid - sodium glyconate mixtures, gluconic acid - sodium hydroxide mixtures, gluconic acid - potassium glyconate mixtures, etc.), oxalate buffers (e.g., oxalic acid - sodium oxalate mixtures, oxalic acid - sodium hydroxide mixtures, oxalic acid - potassium oxalate mixtures, etc.), lactate buffers (e.g., lactic acid - sodium lactate mixtures, lactic acid - sodium hydroxide mixtures, lactic acid - potassium lactate mixtures, etc.), and acetate buffers (e.g., acetic acid - sodium acetate mixtures, acetic acid - sodium hydroxide mixtures, etc.). Additionally, phosphate buffers, histidine buffers, and trimethylamine salts (e.g., Tris) can be used.

[0233] A preservative may be added to delay the growth of microorganisms and may be added in an amount in the range of about 0.2% to 1% (w / v). Suitable preservatives for use in the present disclosure include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzylammonium chloride, benzalconium halide (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkyl parabens (e.g., methyl or propyl paraben), catechol, resorcinol, cyclohexanol, and 3-pentanol. Isotonic agents, sometimes known as “stabilizers,” may be added to ensure the isotonicity of the liquid compositions of the present disclosure and include polyhydric sugar alcohols, such as sugar alcohols having three or more hydroxyl groups (e.g., glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol). A stabilizer, in its function, refers to a broad category of excipients that can range from bulking agents to additives and that serve to solubilize a therapeutic agent or prevent its denaturation or adhesion to the container walls.Typical stabilizers can be polyhydric sugar alcohols (listed above), amino acids (e.g., arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.), organic sugars or sugar alcohols (e.g., lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, etc., including cyclitols such as inositol), polyethylene glycol, amino acid polymers, sulfur-containing reducing agents (e.g., urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglycerol, and sodium thiosulfate, etc.), low molecular weight polypeptides (e.g., peptides of 10 residues or less), proteins (e.g., human serum albumin, bovine serum albumin, gelatin, or immunoglobulins, etc.), hydrophilic polymers (e.g., polyvinylpyrrolidone, etc.), monosaccharides (e.g., xylose, mannose, fructose, glucose, etc.), disaccharides (e.g., lactose, maltose, sucrose, and trehalose, etc.), trisaccharides (e.g., raffinose, etc.), and polysaccharides (e.g., dextran, etc.). The stabilizer can be present in an amount in the range of 0.5 to 10% by weight per weight of the IL2 proprotein.

[0234] Adding a non-ionic surfactant or detergent (also known as a "wetting agent") can help solubilize the glycoprotein and protect the glycoprotein from agitation-induced aggregation, thereby allowing the formulation to be exposed to a shear plane under load without causing protein denaturation. Suitable non-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188, etc.), and pluronic polyols. The non-ionic surfactant can be present in the range of about 0.05 mg / mL to about 1.0 mg / mL (e.g., about 0.07 mg / mL to about 0.2 mg / mL).

[0235] Examples of various additional excipients include bulking agents (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), and co-solvents.

[0236] The IL2 proprotein of the present disclosure can be formulated, for example, as a pharmaceutical composition comprising the IL2 proprotein, which contains one or more pharmaceutically acceptable excipients or carriers. To prepare a pharmaceutical composition or a 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.

[0237] For example, a formulation of an IL2 protein can be prepared by mixing the IL2 protein, for example, in the form of a lyophilized powder, slurry, aqueous solution, lotion, or suspension, with a physiologically acceptable carrier, excipient, or stabilizer (see, e.g., Hardman et al., 2001, Goodman and Gilman’s The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro, 2000, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wlkins, New York, N.Y., 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, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y.).

[0238] The effective amount for a particular subject can vary depending on factors such as the condition being treated, the overall health of the subject, the route and dosage of administration of the method, 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).

[0239] The compositions of the present disclosure can be administered via one or more routes of administration using one or more of the various methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired result. Routes of administration selected for the IL2 protein include, for example, intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, intraspinal, or other common routes of administration by injection or infusion. General administration may sometimes refer to a mode of administration other than enteral and topical administration by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, intrathecal, intraspinal, epidural, and intrathoracic injections and infusions. Alternatively, the compositions of the present disclosure can be administered via local, epithelial, or mucosal routes of administration, such as non-general routes such as intranasal, oral, vaginal, rectal, sublingual, or topical. In one embodiment, the IL2 protein is administered by infusion. In another embodiment, the IL2 protein of the present disclosure is administered subcutaneously.

[0240] 6.12. Therapeutic Indications and Methods of Use The present disclosure provides methods for using and applying the IL2 proteins of the present disclosure. In certain aspects, the present disclosure provides a method of treating cancer, comprising administering to a subject in need thereof an IL2 protein or pharmaceutical composition described herein. In some embodiments, the activated IL2 protein comprising an IL2 moiety is produced by cleavage of one or more protease-cleavable linkers in the IL2 protein by one or more proteases expressed by cancer tissue. Thus, the IL2 protein is selectively activated in cancer tissue.

[0241] In some embodiments, the present disclosure provides a method of 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 or pharmaceutical composition described herein, wherein the IL2 proprotein has one or more protease-cleavable linkers, each of which comprises one or more substrates for one or more proteases expressed by the cancer tissue in which the IL2 protein is intended. Thus, the activated IL2 protein comprising the IL2 moiety is produced by cleavage of one or more protease-cleavable linkers in the IL2 proprotein by one or more proteases in the cancer tissue.

[0242] The present disclosure further provides a method of local delivery of an IL2 protein, the method comprising administering to a subject the IL2 proprotein or pharmaceutical composition described herein, wherein the IL2 proprotein has one or more protease-cleavable linkers, each of which comprises one or more substrates for one or more proteases expressed by the tissue to which the IL2 protein is locally delivered. As used herein, the term "locally delivered" does not require local administration, but rather refers to the activation of the protein at the intended site by proteases active at the intended site, in conjunction with optional targeting of the active component of the IL2 proprotein to the intended local site using a targeting moiety that recognizes a target molecule expressed by the tissue.

[0243] The present disclosure provides 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 an IL2 protein or pharmaceutical composition described herein, wherein the IL2 protein has one or more protease-cleavable linkers, each of which comprises one or more substrates for one or more proteases expressed by the tissue where the IL2 therapy is desired and / or intended. The method further provides a method having one or more protease-cleavable linkers, each of which comprises one or more substrates for one or more proteases expressed by the tissue where the IL2 therapy is desired and / or intended.

[0244] Accordingly, the method described above enables an IL2 therapy with reduced off-target side effects by preferentially activating the IL2 protein at the site intended for IL2 treatment.

[0245] In some embodiments of the method described above, the IL2 protein is also targeted and comprises one or more targeting moieties that recognize a target molecule expressed at the site (e.g., by a tissue) intended for treatment.

[0246] Accordingly, the present disclosure provides a method for targeted delivery of an activated IL2 protein to a site intended for treatment, such as cancer tissue, the method comprising administering to the subject an IL2 protein or pharmaceutical composition described herein, wherein the IL2 comprises one or more targeting moieties that recognize a target molecule expressed locally or by the tissue (e.g., cancer tissue) intended for treatment, and wherein the IL2 has one or more protease-cleavable linkers, each of which comprises one or more substrates for one or more proteases expressed by the tissue where the IL2 therapy is desired and / or intended.

[0247] The present disclosure further provides a method for locally inducing an immune response in a target tissue, comprising administering to a subject an IL2 protein or a pharmaceutical composition described herein, wherein the IL2 protein or the pharmaceutical composition described herein comprises 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 comprising one or more substrates for one or more proteases expressed in the target tissue. And the activated IL2 protein comprising the IL2 moiety can be produced by cleavage of one or more protease-cleavable linkers in the IL2 protein by one or more proteases in the target tissue. And the resulting activated IL2 protein can induce an immune response against at least one cell type in the target tissue.

[0248] In some embodiments, the administration is not local to the tissue. For example, if the target tissue is cancer tissue, the administration can be systemic or subcutaneous. The IL2 protein of the present disclosure can be used for the treatment of any proliferative disorder (e.g., cancer) that expresses a target molecule (either on tumor cells or in the tumor microenvironment, e.g., extracellular matrix or tumor lymphocytes). In certain embodiments, the cancer is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical cancer, anal cancer, appendiceal cancer, astrocytoma, basal cell cancer, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, Burkitt lymphoma, cancer of unknown primary origin, cardiac tumor, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative tumor, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal cancer, germinoma, endometrial cancer, epithelioma, esophageal cancer, ganglioneuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hairy cell leukemia, hepatocellular cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell cancer, mesothelioma, occult primary metastatic squamous neck cancer, midline cancer involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative tumor, nasal and paranasal cancer, nasopharyngeal cancer, 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, rectal cancer, renal cell cancer, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sézary 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.

[0249] Table I below shows exemplary indications for which an IL2 proprotein targeting a specific target molecule can be used.

[0250] [Table 10-1]

[0251] [Table 10-2]

[0252] [Table 10-3]

[0253] Additional target molecules and corresponding indications are disclosed, for example, in Hafeez et al., 2020, Molecules 25:4764, doi:10.3390 / molecules25204764, particularly in Table 1. Table 1 is hereby incorporated by reference in its entirety.

[0254] 7. Sequences The sequences of certain IL2 proproteins of the present disclosure are provided in Table S below.

[0255] [Table 11-1]

[0256] [Table 11-2]

[0257] 8. Numbered Embodiments Although various specific embodiments have been illustrated and described, it will be understood that various changes can be made without departing from the spirit and scope of the present disclosure(s). The present disclosure is illustrated by the numbered embodiments described below.

[0258] In the following numbered embodiments, the targeting moiety preferably binds to a mammalian target molecule, the IL2 and IL2Rα moieties preferably are derived from mammalian IL2 and IL2Rα, the Fc domain preferably is derived from a mammalian antibody, and the subject preferably is a mammal. More preferably, the mammal is a human.

[0259] 1. An IL2 proprotein, comprising: (a) a first polypeptide chain comprising: (i) a first Fc domain; (ii) an optional first non-cleavable linker (NCL); (iii) a first IL2Rα moiety; (iv) a first protease-cleavable linker (PCL); and (v) a first IL2 moiety; and (b) a second polypeptide chain comprising: (i) a second Fc domain capable of associating with the first Fc domain to form an Fc region; (ii) an optional second non-cleavable linker (NCL); (iii) a second IL2Rα moiety; (iv) a second protease-cleavable linker (PCL); and (v) a second IL2 moiety.

[0260] 2. The IL2 proprotein according to embodiment 1, wherein the IL2 moiety comprises an amino acid sequence having at least about 90% sequence identity to mature human IL2. 3. The IL2 proprotein according to embodiment 1, wherein the IL2 moiety comprises an amino acid sequence having about 95% sequence identity to mature human IL2.

[0261] 4. The IL2 proprotein according to any one of embodiments 1 to 3, wherein the IL2 moiety comprises an amino acid sequence having an N-terminal alanine deletion compared to mature human IL2. 5. The IL2 protein according to 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, and optionally, the amino acid substitution is N88D.

[0262] 6. The IL2 protein according to 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.

[0263] 7. The IL2 protein according to any one of embodiments 1 to 6, wherein the IL2Rα portion comprises, or consists of, an amino acid sequence having at least about 90% sequence identity to the IL2-binding portion of human IL2Rα.

[0264] 8. The IL2 protein according to any one of embodiments 1 to 6, wherein the IL2Rα portion comprises, or consists of, an amino acid sequence having at least about 95% sequence identity to the IL2-binding portion of human IL2Rα.

[0265] 9. The IL2 protein according to 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.

[0266] 10. The IL2 protein according to 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.

[0267] 11. The IL2 protein according to any one of embodiments 1 to 6, wherein the IL2Rα portion comprises, or consists of, an amino acid sequence having at least about 99% sequence identity to the IL2-binding portion of human IL2Rα.

[0268] 12. The IL2 protein according to any one of embodiments 1 to 6, wherein the IL2Rα moiety comprises or consists of an amino acid sequence having 100% sequence identity to the IL2-binding portion of human IL2Rα.

[0269] 13. The IL2 protein according to any one of embodiments 7 to 12, wherein the IL2Rα moiety comprises an amino acid sequence having at least 90% sequence identity to (a) amino acids 22 to 186 of IL2Rα, (b) amino acids 22 to 240 of IL2Rα, and / or (c) amino acids 22 to 272 of human IL2Rα.

[0270] 14. The IL2 protein according to any one of embodiments 7 to 12, wherein the IL2Rα moiety comprises an amino acid sequence having at least 95% sequence identity to (a) amino acids 22 to 186 of IL2Rα, (b) amino acids 22 to 240 of IL2Rα, and / or (c) amino acids 22 to 272 of human IL2Rα.

[0271] 15. The IL2 protein according to any one of embodiments 7 to 12, wherein the IL2Rα moiety comprises an amino acid sequence having at least 96% sequence identity to (a) amino acids 22 to 186 of IL2Rα, (b) amino acids 22 to 240 of IL2Rα, and / or (c) amino acids 22 to 272 of human IL2Rα.

[0272] 16. The IL2 protein according to any one of embodiments 7 to 12, wherein the IL2Rα moiety comprises an amino acid sequence having at least 97% sequence identity to (a) amino acids 22 to 186 of IL2Rα, (b) amino acids 22 to 240 of IL2Rα, and / or (c) amino acids 22 to 272 of human IL2Rα.

[0273] 17. The IL2 protein according to any one of embodiments 7 to 12, wherein the IL2Rα moiety comprises an amino acid sequence having at least 98% sequence identity to (a) amino acids 22 to 186 of IL2Rα, (b) amino acids 22 to 240 of IL2Rα, and / or (c) amino acids 22 to 272 of human IL2Rα.

[0274] 18. The IL2 protein according to any one of embodiments 7 to 12, wherein the IL2Rα moiety comprises an amino acid sequence having at least 99% sequence identity to (a) amino acids 22 to 186 of IL2Rα, (b) amino acids 22 to 240 of IL2Rα, and / or (c) amino acids 22 to 272 of human IL2Rα.

[0275] 19. The IL2 protein according to any one of embodiments 1 to 18, wherein the first PCL and / or the second PCL comprises a substrate sequence cleavable by any protease described in Table A.

[0276] 20. The IL2 protein according to any one of embodiments 1 to 19, wherein the first PCL and / or the second PCL comprises one or more substrate sequences selected from the metastable sequences described in Table B.

[0277] 21. The IL2 protein according to any one of embodiments 1 to 20, wherein the first PCL and / or the second PCL comprises one or more spacer sequences selected from the metastable sequences described in Table C.

[0278] 22. The IL2 protein according to any one of embodiments 1 to 21, wherein the first PCL and / or the second PCL comprises the amino acid sequence of any of the PCL sequences described in Table D, or a variant thereof having a maximum of 5 amino acid substitutions, for example, a variant having 1 amino acid substitution, 2 amino acid substitutions, 3 amino acid substitutions, 4 amino acid substitutions, or 5 amino acid substitutions.

[0279] 23. The IL2 proprotein according to any one of Embodiments 1 to 22, wherein the first PCL and / or the second PCL comprises or consists of the amino acid sequence GGGISSGLLSGRSDNHGGGISSGLLSGRSDNHGGS (SEQ ID NO: 185).

[0280] 24. The IL2 proprotein according to any one of Embodiments 1 to 22, wherein the first PCL and / or the second PCL comprises or consists of the amino acid sequence GGGISSGLLSGRSDNHGGGISSGLLSGRSDNHGGS GGGISSGLLSGRSDNHGGGISSGLLSGRSDNHGGS (SEQ ID NO: 186).

[0281] 25. The IL2 proprotein according to any one of Embodiments 1 to 22, wherein the first PCL and / or the second PCL comprises or consists of the amino acid sequence GGSGGSIPVSLRSGGGISSGLLSGRSDNHGGSGGS (SEQ ID NO: 187).

[0282] 26. The IL2 proprotein according to any one of Embodiments 1 to 22, wherein the first PCL and / or the second PCL comprises or consists of the amino acid sequence GGSGGSVPLSLYSGGGISSGLLSGRSDNHGGSGGS (SEQ ID NO: 188).

[0283] 27. The IL2 proprotein according to any one of Embodiments 1 to 22, wherein the first PCL and / or the second PCL comprises or consists of the amino acid sequence GGSHPVGLLARGGGHPVGLLARGGGHPVGLLARGS (SEQ ID NO: 189).

[0284] 28. The IL2 proprotein according to any one of Embodiments 1 to 22, wherein the first PCL and / or the second PCL comprises or consists of the amino acid sequence GGSHPVGLLARGGGHPVGLLARGGSGRSAGGSGRSA (SEQ ID NO: 190).

[0285] 29. The IL2 proprotein according to any one of Embodiments 1 to 28, wherein the first NCL and / or the second NCL comprises an amino acid sequence of any one of the NCL sequences set forth in Table E, or a variant thereof having a maximum of 5 amino acid substitutions, for example, 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.

[0286] 30. The IL2 proprotein according to any one of Embodiments 1 to 28, wherein the first and second non-cleavable linkers range from 5 to 25 amino acids in length. 31. The IL2 proprotein according to any one of Embodiments 1 to 28, wherein the first and second non-cleavable linkers range from 7 to 20 amino acids in length.

[0287] 32. The IL2 proprotein according to any one of Embodiments 1 to 28, wherein the first and second non-cleavable linkers are at least 5 amino acids in length. 33. The IL2 proprotein according to any one of Embodiments 1 to 28, wherein the first and second non-cleavable linkers are at least 7 amino acids in length.

[0288] 34. The IL2 proprotein according to any one of Embodiments 1 to 34, wherein the first and second protease-cleavable linkers range from 20 amino acids to 80 amino acids in length.

[0289] 35. The IL2 proprotein according to any one of Embodiments 1 to 34, wherein the first and second protease-cleavable linkers range from 20 amino acids to 60 amino acids in length.

[0290] 36. The IL2 proprotein according to any one of Embodiments 1 to 35, wherein the first Fc domain and / or the second Fc domain comprises a hinge domain. 37. The IL2 proprotein according to any one of Embodiments 1 to 30, further comprising one or more targeting moieties that bind to one or more target molecules.

[0291] 38. The IL2 proprotein according to embodiment 37, comprising a first targeting moiety and / or a second targeting moiety. 39. The IL2 proprotein according to embodiment 38, 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.

[0292] 40. The IL2 proprotein according to embodiment 37 or embodiment 39, wherein the first targeting moiety and / or the second targeting moiety is a Fab. 41. The IL2 proprotein according to embodiment 37 or embodiment 39, wherein the first targeting moiety and / or the second targeting moiety is a scFv.

[0293] 42. The IL2 proprotein according to any one of embodiments 37 to 41, wherein the first targeting moiety and / or the second targeting moiety can bind to an extracellular matrix (ECM) antigen, a tumor-reactive lymphocyte antigen, a cell surface molecule of a tumor or viral lymphocyte, or a checkpoint inhibitor, or a tumor-associated antigen (TAA).

[0294] 43. The IL2 proprotein according to any one of embodiments 37 to 42, wherein the first targeting moiety and / or the second targeting moiety can bind to any target molecule specified in Section 6.7.

[0295] 44. The IL2 proprotein according to any one of embodiments 37 to 43, wherein the first targeting moiety and / or the second targeting moiety (a) comprises the (i) CDR or (ii) VH and VL sequences of the antibodies listed in Table F, or (b) competes with the antibodies listed in Table F for binding to the target molecule.

[0296] 45. The first targeting moiety and / or the second targeting moiety can bind to an ECM antigen, and the ECM antigen is optionally selected from syndecan, heparanase, integrin, osteopontin, link, cadherin, laminin, laminin EGF-like, lectin, fibronectin, Notch, nectin (e.g., nectin-4), tenascin, collagen (e.g., type X collagen), and matrilysin, the IL2 proprotein according to any one of embodiments 37 to 43.

[0297] 46. The first targeting moiety and / or the second targeting moiety can bind to nectin, e.g., nectin 4, the IL2 proprotein according to embodiment 45. 47. The first targeting moiety and / or the second targeting moiety can bind to collagen, e.g., type X collagen, the IL2 proprotein according to embodiment 45.

[0298] 48. The first targeting moiety and / or the second targeting moiety can bind to cell surface molecules of tumors or viral lymphocytes, the IL2 proprotein according to any one of embodiments 37 to 43.

[0299] 49. The antigen is a T cell costimulatory protein, the IL2 proprotein according to embodiment 48. 50. The T cell costimulatory protein is 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, the IL2 proprotein according to embodiment 49.

[0300] 51. The T cell costimulatory protein is B7-H3, the IL2 proprotein according to embodiment 50. 52. The first targeting moiety and / or the second targeting moiety can bind to a checkpoint inhibitor, the IL2 proprotein according to any one of embodiments 37 to 43.

[0301] 53. The IL2 proprotein according to embodiment 52, 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.

[0302] 54. The IL2 proprotein according to embodiment 53, wherein the checkpoint inhibitor is PDL1. 55. The IL2 proprotein according to any one of embodiments 37 to 43, wherein the first targeting moiety and / or the second targeting moiety is capable of binding to a tumor-associated antigen (''TAA'').

[0303] 56. The first targeting moiety and / or the second targeting moiety is selected from AFP, ALK, BAGE protein, BIRC5 (survivin), BIRC7, β-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 protein (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 protein (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-Nouvel antigen (Tn), TRP-1, TRP-2, tyrosinase, or uroplakin-3, the IL2 proprotein according to embodiment 55.

[0304] 57. The IL2 proprotein according to embodiment 56, wherein the TAA is EGFR. 58. The IL2 proprotein according to embodiment 56, wherein the TAA is HER2. 59. The IL2 proprotein according to embodiment 56, wherein the TAA is EPCAM.

[0305] 60. The IL2 proprotein according to embodiment 56, wherein the TAA is CEACAM5. 61. The IL2 protein according to embodiment 56, wherein TAA is CD20.

[0306] 62. The IL2 protein according to any one of embodiments 1 to 61, wherein the Fc region is a homodimer. 63. The IL2 protein according to any one of embodiments 1 to 61, wherein the Fc region is a heterodimer.

[0307] 64. (a) In the N-terminal to C-terminal orientation, (i) a first Fc domain, (ii) optionally, a first non-cleavable linker, (iii) a first IL2Rα portion, (iv) a first protease-cleavable linker, and (v) a first polypeptide chain comprising a first IL2 portion, and (b) In the N-terminal to C-terminal orientation, (i) a second Fc domain, (ii) optionally, a second non-cleavable linker, (iii) a second IL2Rα portion, (iv) a second protease-cleavable linker, and (v) a second polypeptide chain comprising a second IL2 portion, the IL2 protein according to any one of embodiments 1 to 63.

[0308] 65. An IL2 protein, wherein the IL2 protein is optionally the IL2 protein according to any one of embodiments 1 to 64, and the IL2 protein is (a) a first Fc domain and a second Fc domain that can associate to form an Fc region, and (b) optionally, a linker, optionally, two IL2Rα portions at the C-terminus of the Fc domain preceded by a non-cleavable linker, and (c) two protease-cleavable linkers at the C-terminus of the IL2Rα portion, (d) An IL2 protein comprising two IL2 moieties at the C-terminus of a protease-cleavable linker.

[0309] 66. (a) The first polypeptide comprises a first Fc domain, optionally followed by a first non-cleavable linker, followed by a first IL2Rα moiety, followed by a first protease-cleavable linker, followed by a first IL2 moiety, and (b) The second polypeptide comprises a second Fc domain, optionally followed by a second non-cleavable linker, followed by a second IL2Rα moiety, followed by a second protease-cleavable linker, followed by a second IL2 moiety, the IL2 protein according to any one of embodiments 1 to 64.

[0310] 67. The IL2 protein according to any one of embodiments 1 to 64, having the configuration depicted in Figure 1A. 68. An IL2 protein, wherein the IL2 protein is optionally an IL2 protein according to any one of embodiments 1 to 64, and the IL2 protein is (a) In the order from the N-terminus to the C-terminus, (i) A first amino acid sequence having at least about 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 346, 347, 348, 349, 350, and 351, (ii) A non-cleavable linker / non-cleavable means for connecting the first amino acid sequence to a third amino acid sequence, optionally wherein the linker / means comprises or consists of a second amino acid sequence comprising the amino acid sequence set forth in Table E, the non-cleavable linker / non-cleavable means, (iii) A third amino acid sequence having at least about 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 3, 4, and 5, (iv) A fourth amino acid sequence comprising one or more of the amino acid sequences set forth in Table B or Table D, and (v) a first polypeptide chain comprising a fifth amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 2, (b) in the order from the N-terminus to the C-terminus, (i) a sixth amino acid sequence having at least about 95% sequence identity to any one of SEQ ID NOs: 346, 347, 348, 349, 350, and 351, (ii) a non-cleavable linker / non-cleavable means for connecting the sixth amino acid sequence to an eighth amino acid sequence, optionally, the linker / means comprising or consisting of a seventh amino acid sequence comprising the amino acid sequence set forth in Table E, a non-cleavable linker / non-cleavable means, (iii) an eighth amino acid sequence having at least about 95% sequence identity to any one of SEQ ID NOs: 3, 4, and 5, (iv) a ninth amino acid sequence comprising one or more amino acid sequences set forth in Table B or Table D, and (v) a second polypeptide chain comprising a tenth amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO: 2, an IL2 proprotein.

[0311] 69. The IL2 proprotein according to embodiment 68, wherein the first polypeptide comprises, at the N-terminus of the first amino acid sequence, an eleventh amino acid sequence having at least about 95% sequence identity to any one of SEQ ID NOs: 352, 353, 354, and 355.

[0312] 70. The IL2 proprotein according to embodiment 69, wherein the eleventh amino acid sequence is the amino acid sequence of SEQ ID NO: 352. 71. The IL2 proprotein according to embodiment 69, wherein the eleventh amino acid sequence is the amino acid sequence of SEQ ID NO: 353.

[0313] 72. The IL2 proprotein according to embodiment 69, wherein the eleventh amino acid sequence is the amino acid sequence of SEQ ID NO: 354. 73. The IL2 proprotein according to embodiment 69, wherein the 11th amino acid sequence is the amino acid sequence of SEQ ID NO: 355.

[0314] 74. The IL2 proprotein according to any one of embodiments 68 to 73, wherein the second polypeptide comprises, at the N-terminus of the first amino acid sequence, a 12th amino acid sequence having at least about 95% sequence identity to any one of SEQ ID NOs: 352, 353, 354, and 355.

[0315] 75. The IL2 proprotein according to embodiment 74, wherein the 12th amino acid sequence is the amino acid sequence of SEQ ID NO: 352. 76. The IL2 proprotein according to embodiment 74, wherein the 12th amino acid sequence is the amino acid sequence of SEQ ID NO: 353.

[0316] 77. The IL2 proprotein according to embodiment 74, wherein the 12th amino acid sequence is the amino acid sequence of SEQ ID NO: 354. 78. The IL2 proprotein according to embodiment 74, wherein the 12th amino acid sequence is the amino acid sequence of SEQ ID NO: 355.

[0317] 79. The IL2 proprotein according to any one of embodiments 68 to 78, wherein the first amino acid sequence has at least about 98% sequence identity to any one of SEQ ID NOs: 346, 347, 348, 349, 350, and 351.

[0318] 80. The IL2 proprotein according to any one of embodiments 68 to 78, wherein the first amino acid sequence is the amino acid sequence of any one of SEQ ID NOs: 346, 347, 348, 349, 350, and 351.

[0319] 81. The IL2 proprotein according to embodiment 80, wherein the first amino acid sequence is the amino acid sequence of SEQ ID NO: 350. 82. The IL2 proprotein according to embodiment 80, wherein the first amino acid sequence is the amino acid sequence of SEQ ID NO: 351.

[0320] 83. The IL2 proprotein according to any one of Embodiments 68 to 82, wherein the first amino acid sequence is 350 amino acids or less in length. 84. The IL2 proprotein according to any one of Embodiments 68 to 82, wherein the first amino acid sequence is 330 amino acids or less in length.

[0321] 85. The IL2 proprotein according to any one of Embodiments 68 to 84, wherein the sixth amino acid sequence has at least about 98% sequence identity to any one of SEQ ID NOs: 346, 347, 348, 349, 350, and 351.

[0322] 86. The IL2 proprotein according to any one of Embodiments 68 to 84, wherein the sixth amino acid sequence is the amino acid sequence of any one of SEQ ID NOs: 346, 347, 348, 349, 350, and 351.

[0323] 87. The IL2 proprotein according to Embodiment 86, wherein the sixth amino acid sequence is the amino acid sequence of SEQ ID NO: 350. 88. The IL2 proprotein according to Embodiment 86, wherein the sixth amino acid sequence is the amino acid sequence of SEQ ID NO: 351.

[0324] 89. The IL2 proprotein according to any one of Embodiments 68 to 88, wherein the sixth amino acid sequence is 350 amino acids or less in length. 90. The IL2 proprotein according to any one of Embodiments 68 to 88, wherein the sixth amino acid sequence is 330 amino acids or less in length.

[0325] 91. The IL2 proprotein according to any one of Embodiments 68 to 90, wherein the second amino acid sequence is the amino acid sequence set forth in Table E. 92. The IL2 proprotein according to Embodiment 91, wherein the second amino acid sequence is the amino acid sequence (GGGGS) n where n is 1, 2, 3, 4, or 5.

[0326] 93. The IL2 proprotein according to any one of embodiments 68 to 92, wherein the second amino acid sequence is 25 amino acids or less in length. 94. The IL2 proprotein according to any one of embodiments 68 to 92, wherein the second amino acid sequence is 15 amino acids or less in length.

[0327] 95. The IL2 proprotein according to any one of embodiments 68 to 92, wherein the second amino acid sequence is 6 amino acids or less in length. 96. The IL2 proprotein according to any one of embodiments 68 to 95, wherein the seventh amino acid sequence is the amino acid sequence set forth in Table E.

[0328] 97. The IL2 proprotein according to embodiment 96, wherein the seventh amino acid sequence is the amino acid sequence (GGGGS) n and n is 1, 2, 3, 4, or 5. 98. The IL2 proprotein according to any one of embodiments 68 to 97, wherein the seventh amino acid sequence is 25 amino acids or less in length.

[0329] 99. The IL2 proprotein according to any one of embodiments 68 to 97, wherein the seventh amino acid sequence is 15 amino acids or less in length. 100. The IL2 proprotein according to any one of embodiments 68 to 97, wherein the seventh amino acid sequence is 6 amino acids or less in length.

[0330] 101. The IL2 proprotein according to any one of embodiments 68 to 100, wherein the third amino acid sequence has at least about 98% sequence identity to any one of SEQ ID NOs: 3, 4, and 5.

[0331] 102. The IL2 proprotein according to any one of embodiments 68 to 100, wherein the third amino acid sequence is the amino acid sequence of any one of SEQ ID NOs: 3, 4, and 5.

[0332] 103. The IL2 proprotein according to embodiment 102, wherein the third amino acid sequence is the amino acid sequence of SEQ ID NO: 3. 104. The IL2 proprotein according to embodiment 102, wherein the fourth amino acid sequence is the amino acid sequence of SEQ ID NO: 3.

[0333] 105. The IL2 proprotein according to embodiment 102, wherein the fifth amino acid sequence is the amino acid sequence of SEQ ID NO: 3. 106. The IL2 proprotein according to any one of embodiments 68 to 105, wherein the third amino acid sequence is 255 amino acids or shorter in length.

[0334] 107. The IL2 proprotein according to any one of embodiments 68 to 105, wherein the third amino acid sequence is 225 amino acids or shorter in length. 108. The IL2 proprotein according to any one of embodiments 68 to 105, wherein the third amino acid sequence is 170 amino acids or shorter in length.

[0335] 109. The IL2 proprotein according to any one of embodiments 68 to 108, wherein the eighth amino acid sequence has at least about 98% sequence identity to any one of SEQ ID NOs: 3, 4, and 5.

[0336] 110. The IL2 proprotein according to any one of embodiments 68 to 109, wherein the eighth amino acid sequence is the amino acid sequence of any one of SEQ ID NOs: 3, 4, and 5.

[0337] 111. The IL2 proprotein according to embodiment 110, wherein the eighth amino acid sequence is the amino acid sequence of SEQ ID NO: 3. 112. The IL2 proprotein according to embodiment 110, wherein the eighth amino acid sequence is the amino acid sequence of SEQ ID NO: 4.

[0338] 113. The IL2 proprotein according to embodiment 110, wherein the eighth amino acid sequence is the amino acid sequence of SEQ ID NO: 5. 114. The IL2 proprotein according to any one of Embodiments 68 to 113, wherein the eighth amino acid sequence is 255 amino acids or shorter in length.

[0339] 115. The IL2 proprotein according to any one of Embodiments 68 to 113, wherein the eighth amino acid sequence is 225 amino acids or shorter in length. 116. The IL2 proprotein according to any one of Embodiments 68 to 113, wherein the eighth amino acid sequence is 170 amino acids or shorter in length.

[0340] 117. The IL2 proprotein according to any one of Embodiments 68 to 116, wherein the fourth amino acid sequence contains one or more amino acid sequences described in Table B. 118. The IL2 proprotein according to Embodiment 117, wherein the fourth amino acid sequence contains the amino acid sequence HPVGLLAR (SEQ ID NO: 261).

[0341] 119. The IL2 proprotein according to Embodiment 117, wherein the fourth amino acid sequence contains the amino acid sequence VPLSLYSG (SEQ ID NO: 149). 120. The IL2 proprotein according to Embodiment 117, wherein the fourth amino acid sequence contains the amino acid sequence ISSGLLS (SEQ ID NO: 64).

[0342] 121. The IL2 proprotein according to Embodiment 117, wherein the fourth amino acid sequence contains the amino acid sequence PLGLWSQ (SEQ ID NO: 105). 122. The IL2 proprotein according to any one of Embodiments 68 to 116, wherein the fourth amino acid sequence is the amino acid sequence described in Table D.

[0343] 123. The IL2 proprotein according to Embodiment 122, wherein the fourth amino acid sequence is the amino acid sequence GGGISSGLLSGRSDNHGGGISSGLLSGRSDNHGGS (SEQ ID NO: 185).

[0344] 124. The IL2 proprotein according to embodiment 122, wherein the fourth amino acid sequence is the amino acid sequence GGSHPVGLLARGGGHPVGLLARGGGHPVGLLARGS (SEQ ID NO: 189).

[0345] 125. The IL2 proprotein according to embodiment 122, wherein the fourth amino acid sequence is the amino acid sequence GGGHPVGLLARGGGS (SEQ ID NO: 339). 126. The IL2 proprotein according to embodiment 122, wherein the fourth amino acid sequence is the amino acid sequence GGGISSGLLSGRSDNHGGGS (SEQ ID NO: 338).

[0346] 127. The IL2 proprotein according to embodiment 122, wherein the fourth amino acid sequence is the amino acid sequence GGGGSGGGGSGGGGSVPLSLYSGGGSGGSGGSGS (SEQ ID NO: 207).

[0347] 128. The IL2 proprotein according to any one of embodiments 68 to 127, wherein the ninth amino acid sequence comprises one or more of the amino acid sequences set forth in Table B. 129. The IL2 proprotein according to embodiment 128, wherein the ninth amino acid sequence comprises the amino acid sequence HPVGLLAR (SEQ ID NO: 261).

[0348] 130. The IL2 proprotein according to embodiment 128, wherein the ninth amino acid sequence comprises the amino acid sequence VPLSLYSG (SEQ ID NO: 149). 131. The IL2 proprotein according to embodiment 128, wherein the ninth amino acid sequence comprises the amino acid sequence ISSGLLS (SEQ ID NO: 64).

[0349] 132. The IL2 proprotein according to embodiment 128, wherein the ninth amino acid sequence comprises the amino acid sequence PLGLWSQ (SEQ ID NO: 105). 133. The IL2 proprotein according to any one of embodiments 68 to 127, wherein the ninth amino acid sequence is the amino acid sequence set forth in Table D.

[0350] 134. The IL2 proprotein according to embodiment 133, wherein the 9th amino acid sequence is the amino acid sequence GGGISSGLLSGRSDNHGGGISSGLLSGRSDNHGGS (SEQ ID NO: 185).

[0351] 135. The IL2 proprotein according to embodiment 133, wherein the 9th amino acid sequence is the amino acid sequence GGSHPVGLLARGGGHPVGLLARGGGHPVGLLARGS (SEQ ID NO: 189).

[0352] 136. The IL2 proprotein according to embodiment 133, wherein the 9th amino acid sequence is the amino acid sequence GGGHPVGLLARGGGS (SEQ ID NO: 339). 137. The IL2 proprotein according to embodiment 13, wherein the 9th amino acid sequence is the amino acid sequence GGGISSGLLSGRSDNHGGGS (SEQ ID NO: 338).

[0353] 138. The IL2 proprotein according to embodiment 133, wherein the 9th amino acid sequence is the amino acid sequence GGGGSGGGGSGGGGSVPLSLYSGGGSGGSGGSGS (SEQ ID NO: 207).

[0354] 139. The IL2 proprotein according to any one of embodiments 68 to 138, wherein the 5th amino acid sequence has at least about 98% sequence identity to SEQ ID NO: 2. 140. The IL2 proprotein according to any one of embodiments 68 to 138, wherein the 5th amino acid sequence is the amino acid sequence of SEQ ID NO: 2.

[0355] 141. The IL2 proprotein according to any one of embodiments 68 to 140, wherein the 5th amino acid sequence is 150 amino acids or less in length. 142. The IL2 proprotein according to any one of embodiments 68 to 140, wherein the 5th amino acid sequence is 135 amino acids or less in length.

[0356] 143. The IL2 proprotein according to any one of embodiments 68 to 142, wherein the 10th amino acid sequence has at least about 98% sequence identity to SEQ ID NO: 2.

[0357] 144. The IL2 proprotein according to any one of embodiments 68 to 142, wherein the 10th amino acid sequence is the amino acid sequence of SEQ ID NO: 2. 145. The IL2 proprotein according to any one of embodiments 68 to 144, wherein the 10th amino acid sequence is 150 amino acids or shorter in length.

[0358] 146. The IL2 proprotein according to any one of embodiments 68 to 144, wherein the 10th amino acid sequence is 135 amino acids or shorter in length. 147. The IL2 proprotein according to any one of embodiments 68 to 146, wherein the first polypeptide chain lacks an additional sequence at the C-terminus of the first amino acid sequence.

[0359] 148. The IL2 proprotein according to any one of embodiments 68 to 147, wherein the first polypeptide chain lacks an additional sequence between the first amino acid sequence and the second amino acid sequence.

[0360] 149. The IL2 proprotein according to any one of embodiments 68 to 148, wherein the first polypeptide chain lacks an additional sequence between the second amino acid sequence and the third amino acid sequence.

[0361] 150. The IL2 proprotein according to any one of embodiments 68 to 149, wherein the first polypeptide chain lacks an additional sequence between the third amino acid sequence and the fourth amino acid sequence.

[0362] 151. The IL2 proprotein according to any one of embodiments 68 to 150, wherein the first polypeptide chain lacks an additional sequence between the fourth amino acid sequence and the fifth amino acid sequence.

[0363] 152. The IL2 proprotein according to any one of embodiments 68 to 151, wherein the second polypeptide chain lacks an additional sequence at the C-terminus of the sixth amino acid sequence. 153. The IL2 proprotein according to any one of embodiments 68 to 152, wherein the second polypeptide chain lacks an additional sequence between the sixth amino acid sequence and the seventh amino acid sequence.

[0364] 154. The IL2 proprotein according to any one of embodiments 68 to 153, wherein the second polypeptide chain lacks an additional sequence between the seventh amino acid sequence and the eighth amino acid sequence.

[0365] 155. The IL2 proprotein according to any one of embodiments 68 to 154, wherein the second polypeptide chain lacks an additional sequence between the eighth amino acid sequence and the ninth amino acid sequence.

[0366] 156. The IL2 proprotein according to any one of embodiments 68 to 155, wherein the second polypeptide chain lacks an additional sequence between the ninth amino acid sequence and the tenth amino acid sequence.

[0367] 157. The IL2 proprotein according to any one of embodiments 68 to 156, wherein the first polypeptide and the second polypeptide are identical. 158. A nucleic acid or nucleic acids encoding the IL2 proprotein according to any one of embodiments 1 to 157.

[0368] 159. A host cell engineered to express the IL2 proprotein according to any one of embodiments 1 to 157 or the nucleic acid(s) of embodiment 64. 160. A method for producing the IL2 proprotein according to any one of embodiments 1 to 157, comprising culturing the host cell of embodiment 159 and recovering the IL2 proprotein expressed thereby.

[0369] A pharmaceutical composition comprising the IL2 proprotein according to any one of Embodiments 1 to 157 and an excipient. A method for treating cancer, comprising administering to a subject in need thereof the IL2 proprotein according to any one of Embodiments 1 to 157 or the pharmaceutical composition of Embodiment 161.

[0370] The method according to Embodiment 162, wherein the IL2 proprotein comprises at least one targeting moiety capable of binding to a target molecule, and the cancer is associated with the expression of the target molecule described in Table I, for example, TAA and related cancers.

[0371] The method according to Embodiment 163, wherein the activated IL2 protein comprising an IL2 moiety is produced by cleavage of one or more protease-cleavable linkers in the IL2 proprotein by one or more proteases expressed by cancer tissue.

[0372] The method according to Embodiment 164, wherein the IL2 protein is selectively activated in cancer tissue. A method for local delivery of an IL2 protein, comprising administering to a subject the IL2 proprotein according to any one of Embodiments 1 to 157 (or a pharmaceutical composition comprising the IL2 proprotein and an excipient), wherein the IL2 protein has one or more protease-cleavable linkers, each of which comprises one or more substrates for one or more proteases expressed by the tissue to which the IL2 protein is locally delivered.

[0373] The method according to Embodiment 166, wherein the IL2 proprotein comprises one or more targeting moieties that recognize target molecules expressed by the tissue. The method according to Embodiment 167, wherein the IL2 proprotein comprises two targeting moieties that each recognize a target molecule expressed by the tissue.

[0374] 169. The method according to embodiment 167 or embodiment 168, wherein the tissue is a cancer tissue. 170. The method according to embodiment 169, wherein 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).

[0375] 171. The method according to any one of embodiments 166 to 170, wherein the activated IL2 protein comprising an IL2 moiety is produced by cleavage of one or more protease-cleavable linkers in the IL2 proprotein by one or more proteases in the tissue.

[0376] 172. A method of treating cancer with an IL2 protein that is selectively activated in cancer tissue, comprising administering to a subject in need thereof the IL2 proprotein (or a pharmaceutical composition comprising the IL2 proprotein and an excipient) according to any one of embodiments 1 to 157, wherein the IL2 protein has one or more protease-cleavable linkers, each of which comprises one or more substrates for one or more proteases expressed by the cancer tissue to the IL2 protein.

[0377] 173. The method according to embodiment 172, wherein the IL2 proprotein comprises one or more targeting moieties that recognize a target molecule expressed by the cancer tissue. 174. The method according to embodiment 173, wherein the IL2 proprotein comprises two targeting moieties that each recognize a target molecule expressed by the cancer tissue.

[0378] 175. The method according to embodiment 173 or embodiment 174, wherein 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).

[0379] 176. The method according to any one of embodiments 172 to 175, wherein an activated IL2 protein comprising an IL2 moiety is produced by cleavage of one or more protease-cleavable linkers in the IL2 proprotein by one or more proteases in a cancer tissue.

[0380] 177. 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 the IL2 proprotein (or a pharmaceutical composition comprising the IL2 proprotein and an excipient) according to any one of embodiments 1 to 157, wherein the IL2 protein has one or more protease-cleavable linkers, each comprising one or more substrates for one or more proteases expressed by a tissue where the IL2 therapy is desired and / or intended.

[0381] 178. The method according to embodiment 177, wherein the IL2 proprotein comprises one or more targeting moieties that recognize a target molecule expressed by a tissue. 179. The method according to embodiment 178, wherein the IL2 proprotein comprises two targeting moieties that each recognize a target molecule expressed by a tissue.

[0382] 180. The method according to any one of embodiments 177 to 179, wherein the tissue is a cancer tissue. 181. The method according to embodiment 180, 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).

[0383] The method according to any one of embodiments 177 to 181, wherein an activated IL2 protein comprising an IL2 portion is produced by cleavage of one or more protease-cleavable linkers in the IL2 proprotein by one or more proteases in a tissue.

[0384] 183. A method of 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 (or a pharmaceutical composition comprising the IL2 proprotein and an excipient) according to any one of embodiments 1 to 157, wherein the IL2 protein has one or more protease-cleavable linkers, each of which comprises one or more substrates for one or more proteases expressed by the cancer tissue.

[0385] 184. The method according to embodiment 183, wherein the IL2 proprotein comprises one or more targeting moieties that recognize a target molecule expressed by the cancer tissue. 185. The method according to embodiment 184, wherein the IL2 proprotein comprises two targeting moieties that each recognize a target molecule expressed by the cancer tissue.

[0386] 186. The method according to embodiment 184 or embodiment 185, 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).

[0387] 187. The method according to any one of embodiments 183 to 186, wherein an activated IL2 protein comprising an IL2 portion is produced by cleavage of one or more protease-cleavable linkers in the IL2 proprotein by one or more proteases in the cancer tissue.

[0388] 188. A method for targeted delivery of an activated IL2 protein to a cancer tissue, comprising administering to a subject the IL2 proprotein (or a pharmaceutical composition comprising the IL2 proprotein and an excipient) according to any one of Embodiments 1 to 157, wherein the IL2 protein is (a) comprises one or more targeting moieties that recognize a target molecule expressed by the cancer tissue, and (b) has one or more protease-cleavable linkers, each comprising one or more substrates for one or more proteases expressed by the tissue where IL2 therapy is desired and / or intended.

[0389] 189. The method according to Embodiment 188, wherein the IL2 proprotein comprises two targeting moieties that each recognize a target molecule expressed by the cancer tissue. 190. The method according to Embodiment 188 or Embodiment 189, wherein the one or more targeting moieties can bind 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).

[0390] 191. The method according to any one of Embodiments 188 to 190, wherein the activated IL2 protein comprising the IL2 moiety is produced by cleavage of one or more protease-cleavable linkers in the IL2 proprotein by one or more proteases in the cancer tissue.

[0391] A method for locally inducing an immune response in a target tissue, comprising administering to a subject the IL2 proprotein described in any one of Embodiments 1 to 157 (or a pharmaceutical composition comprising the IL2 proprotein and an excipient), wherein the IL2 protein 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 comprising one or more substrates for one or more proteases expressed in the target tissue.

[0392] 193. The method according to Embodiment 192, wherein the IL2 proprotein comprises two targeting moieties each recognizing a target molecule expressed in the target tissue. 194. The method according to Embodiment 192 or Embodiment 98, wherein the target tissue is a cancer tissue.

[0393] 195. The method according to any one of Embodiments 192 to 194, wherein one or more targeting moieties can bind 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").

[0394] 196. The method according to any one of Embodiments 192 to 195, wherein the activated IL2 protein comprising the IL2 moiety is produced by cleavage of one or more protease-cleavable linkers in the IL2 proprotein by one or more proteases in the target tissue.

[0395] 197. The method according to Embodiment 196, wherein the IL2 protein induces an immune response against at least one cell type in the target tissue. 198. The method according to any one of Embodiments 162 to 197, wherein the administration is non-local.

[0396] 199. The method according to Embodiment 198, wherein the administration is systemic. The method according to embodiment 198, wherein the administration is subcutaneous.

Example

[0397] 8.1. Example 8.1.1. Example 1: Digestion of a protease-cleavable linker using a recombinant protease Various IL2 proprotein constructs were generated in the format of targeting moiety - IL2Rα moiety - linker 2 - IL2 moiety. An overview of the IL2 proproteins encoded by the generated constructs is provided in Table 1 below. Table 1 describes a single half-antibody of each IL2 proprotein, and each IL2 proprotein contains two identical half-antibodies.

[0398]

Table 12

[0399] The term "targeting moiety" related to the experimental construct is an abbreviation for an antibody having an Fc domain and an N-terminal Fab domain and a non-cleavable linker at the C-terminus of the Fc domain (having the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 157)). The IL2Rα moiety has the amino acid sequence ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTG (SEQ ID NO: 4). The IL2 moiety has the amino acid sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 2). Various constructs include protease-cleavable linkers PCL1, PCL2, PCL3, PCL4, PCL5, and PCL6, and non-cleavable linkers NCL1 and NCL2 between the IL2Rα moiety and IL2Rα.

[0400] To test whether some of the protease-cleavable linkers in the targeting moiety-IL2Rα moiety-linker-IL2 moiety were accessible for digestion by recombinant proteases, the above constructs were digested using various recombinant human proteases such as uPA, MT-SP1, MMP2, and MMP9 (purchased from R&D Systems).

[0401] Briefly stated, 2 μg of each construct (including PCL1, PCL2, PCL3, PCL4, PCL5, PCL6, NCL1, and NCL2 between IL2Rα and IL2) was digested at 37 °C for 24 h with each of 50 ng of recombinant protease in the assay buffer recommended by the manufacturer or without using them. After digestion, SDS sample loading buffer was added to each sample and run on an Invitrogen 4–12% Tris-Glycine gel for SDS PAGE analysis. For each protease, different assay buffers were used: (a) 100 μl of assay buffer 50 mM Tris, 0.01% (v / v) Tween® 20, pH 8.5 without enzyme (b) 100 μl of assay buffer 50 mM Tris, 0.01% (v / v) Tween® 20, pH 8.5 with 50 ng of uPA enzyme (c) 100 μl of assay buffer 50 mM Tris, 50 mM NaCl, 0.01% (v / v) Tween® 20, pH 9.0 with 50 ng of matriptase / ST14 enzyme (d) 100 μl of assay buffer 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w / v) Brij35, pH 7.5 (TCNB) with 50 ng of APMA-activated MMP2 enzyme (e) 100 μl of assay buffer 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij35 (w / v), pH 7.5 (TCNB) with 50 ng of APMA-activated MMP9 enzyme To visualize the bands, the gel was stained using SimplyBlue Stain from Invitrogen.

[0402] The undigested full-length construct migrates at 225 KDa (lane 1 of Figures 2 and 3). The correctly digested targeted portion-IL2Rα where both IL2s are cleaved at the linker sequence migrates at approximately 200 KDa (lane 2 of Figures 2 and 3), suggesting that both linker sites are accessible for protease cleavage. Most of the protease substrates tested can be cleaved by multiple proteases sharing similar substrate sequence requirements. Some proteases show cleavage outside the cleavable linker sequence.

[0403] 8.1.2. Example 2: Activity of protease-released IL2 in the YT / STAT5 luc luciferase assay To test whether protease-released IL2 is active, a recombinant protease-digested cleavable linker construct was tested in the YT / STAT5 luc luciferase assay. Briefly, 2 μg of αCD20-IL2Rα-PCL-IL2 was digested with 50 ng of matriptase (R&D, catalog number 3946-SEB-010) or uPA (R&D, catalog number 1310-SE) in 100 μl of assay buffer 50 mM Tris, 0.01% (v / v) Tween® 20, pH 8.5 at 37 °C for 24 hours. YT / pGL452-STAT5 luc / CL4 / hIL7Ra cells were incubated at 37 °C, 5% CO2 for 4 - 6 hours with titrations of IL2(WT)-mmh, αCD20-IL2Rα-G4S-IL2, and MT-SP1 or uPA digested and undigested CD20-IL2Rα-PCL-IL2. After incubation, the plates and One-Glow luciferase substrate were equilibrated at room temperature. 100 μl of One-Glo substrate was added per well and incubated for 3 - 5 minutes. Luminescence was measured with Envision.

[0404] Referring to Figure 4, protease-mediated cleavage of IL2 from CD20-IL2Rα-PCL-IL2 results in improved activity in the YT / STAT5 luc / CL4 luciferase assay. IL2 in the targeted moiety-IL2Rα-IL2 format exists in a self-inhibitory trans-dimer format with minimal luciferase activity, as shown by CD20-IL2Rα-G4S-IL2. Upon cleavage, after digestion with MT-SP1 or uPA, the cleaved IL2 has STAT5 luc activity similar to IL2(WT)-mmh, suggesting that protease-cleaved IL2 is fully bioactive.

[0405] 8.1.3. Example 3: In Vivo Efficacy of Tumor-Targeted IL2Rα-IL2 with a Protease-Cleavable Linker The schematic diagram in Figure 5A shows that 0.85×10 6 MC38 / hCD20 tumor cells were subcutaneously implanted into the right posterior flank of 8- to 10-week-old female C57BL / 6 mice (Jackson Laboratory). Mice were randomized when the tumor size reached approximately 110 mm 3 and intraperitoneally treated with various proteins. The treatment was administered intraperitoneally once every 2 weeks for a total of 4 doses. Tumors were measured twice a week using digital calipers, and the tumor size was calculated as length x width 2 / 2. Measurements were taken until the tumor size reached 2250 mm 3 or until it was necessary to euthanize any mouse in any group due to ulceration. Changes in body weight were monitored throughout the course of the study. Observations were extended until day 42.

[0406] Figures 5B to 5N show the anti-tumor efficacy of TAA-targeted IL2Rα-IL2 with or without a cleavable linker. The αCD20-targeted construct with a cleavable linker showed significantly better anti-tumor efficacy and tumor-free survival until the end of the study compared to the CD20-targeted construct with a non-cleavable linker at the doses tested in the MC38 / hCD20 mouse model. Bivalent αCD20 with effectorless Fc and non-targeted isotype groups did not show tumor growth control. As shown in Figures 5M and 5N, there was no significant change in body weight in any of the groups of treated mice, and the mice did not show any weight loss at the time of treatment. Figures 5D to 5L show the individual tumor curves for all the groups tested in the study.

[0407] 8.1.4. Example 4: Stability of Protease-Cleavable Linker In Vivo As shown in Figure 6A, 8 - 10 week-old female C57BL / 6 mice (Jackson Laboratory) without tumors were dosed with a single 5 mpk subcutaneous injection of αCD20-IL2Rα-linker-IL2 protein having three different cleavable linkers and two non-cleavable linker controls (n = 2 mice per group). Plasma was collected from the mice 16 hours, 40 hours, and 64 hours after treatment, and the stability of the dosed protein in serum was checked by Western blot analysis (Figure 6B). 1 ul of serum was run on a 4 - 12% Tris-Glycine gel together with undigested and recombinant protease-digested CD20-IL2Rα-PCL1-IL2 as a control, followed by Western blotting using goat anti-human IgG-Fc specific, HRP-conjugate (Sigma A0170-1ML) for detection. In non-tumor-bearing mice, a short 3-day in vivo stability study was performed. The three cleavable linkers tested showed different cleavage patterns in serum over the 3-day period. αCD20-IL2Rα-PCL2-IL2 was the most stable at the three time points tested, 16 hours, 40 hours, and 64 hours. CD20-IL2Rα-PCL4-IL2 showed moderate stability, and CD20-IL2Rα-PCL5-IL2 was the least stable, showing cleavage of the cleavable linker and IL2 even at the 16-hour time point. The non-cleavable controls showed minimal cleavage of IL2 as expected. The undigested full-length construct runs at 225 KDa (Figure 6B, lane 1). The fully digested targeted moiety-IL2Rα with both IL2s cleaved at the linker sequence runs at approximately 200 KDa (Figure 6B, lane 2).

[0408] 8.1.5. Example 5: Contribution of Tumor Targeting to the Efficacy of αCD20-IL2Rα-Cleavable Linker-IL2 Figure 7 shows the implantation and treatment protocol of 8 - 10 week-old female C57BL / 6 mice (Jackson Laboratory) in the MC38 / hCD20 model where 1 × 10 6 MC38 / hCD20 tumor cells were implanted subcutaneously in the right hind flank. When the tumor size reached approximately 110 mm 3When the mice reached [a certain condition], they were randomized and intraperitoneally treated with various proteins. The treatment was administered intraperitoneally once a week for a total of 2 doses. Tumors were measured twice a week using digital calipers, and the tumor size was calculated as length x width 2 / 2 (Figure 8). Measurements were taken until the tumor size reached 2250 mm 3 , or until it was necessary to euthanize any mouse in any group due to ulceration. Changes in body weight were monitored throughout the course of the study.

[0409] The inventors evaluated the contribution of tumor targeting in the anti-tumor effect of TAA-targeted IL2Rα-IL2 with a cleavable linker. As shown in Figure 9, the CD20-targeted IL2Rα-IL2 protein with a cleavable linker showed significantly better anti-tumor efficacy and tumor-free survival compared to the non-targeted IL2Rα-IL2 protein with a cleavable linker and the CD20-targeted IL2Rα-IL2 protein with a non-cleavable G4S linker in the MC38 / hCD20 mouse model. Bivalent αCD20 with an effectorless Fc and non-targeted isotype groups did not show tumor growth control. There was no significant change in body weight in any of the groups of treated mice, and the mice did not show any weight loss at the time of treatment.

[0410] 9. Citation of References All publications, patents, patent applications, and 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, or other document was individually indicated to be incorporated by reference for all purposes. In the event of any conflict between one or more of the teachings incorporated by reference herein and the present disclosure, the teachings of this specification are intended.

Claims

1. It is an IL2 proprotein, (a) In order from the N-terminus to the C-terminus, (i) the first targeting portion or its components, (ii) The first FC domain, (iii) Optional first non-disconnectable linker (NCL), (iv) The first IL2Rα portion, (v) First protease-cleavable linker (PCL), and (vi) A first polypeptide chain including the first IL2 portion, (b) In order from the N-terminus to the C-terminus, (i) Second targeting portion or its components, (ii) A second Fc domain that can associate with the first Fc domain to form an Fc region, (iii) Optional second non-disconnectable linker (NCL), (iv) The second IL2Rα portion, (v) A second protease-cleavable linker (PCL), and (vi) an IL2 proprotein comprising a second polypeptide chain containing a second IL2 moiety.

2. The aforementioned IL2 portion, (a) an amino acid sequence having at least about 90% or at least about 95% sequence identity with mature human IL2, (b) an amino acid sequence having an N-terminal alanine deletion compared to mature human IL2, and / or (c) an amino acid substitution at the N88 position compared to (a) wild-type IL2, wherein the amino acid substitution is optionally N88D, and / or (b) an amino acid sequence having the amino acid substitution C125S, C125A, or C125V compared to wild-type IL2 The IL2 proprotein according to claim 1, comprising:

3. The IL2 proprotein according to claim 1, wherein the IL2Rα portion contains or comprises 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 with respect to the IL2 binding portion of human IL2Rα.

4. The IL2 proprotein according to claim 3, 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 with (a) amino acids 22-186 of IL2Rα, (b) amino acids 22-240 of IL2Rα, and / or (c) amino acids 22-272 of human IL2Rα.

5. The IL2 proprotein according to claim 1, wherein the first PCL and / or the second PCL comprises (a) a substrate sequence that can be cleaved by any protease listed in Table A, (b) one or more substrate sequences selected from the metastable sequences listed in Table B, (c) one or more spacer sequences selected from the metastable sequences listed in Table C, and / or (d) any amino acid sequence from among the PCL sequences listed in Table D, or a variant thereof having up to five amino acid substitutions, for example, a variant having one amino acid substitution, two amino acid substitutions, three amino acid substitutions, four amino acid substitutions, or five amino acid substitutions.

6. The IL2 proprotein according to claim 1, wherein the first NCL and / or the second NCL comprises any amino acid sequence from the NCL sequences listed in Table E, or a variant thereof having up to five amino acid substitutions, for example, a variant having one amino acid substitution, two amino acid substitutions, three amino acid substitutions, four amino acid substitutions, or five amino acid substitutions.

7. The IL2 proprotein according to claim 1, wherein the first and second non-cleavable linkers are (a) in the range of 5 to 25 amino acids or 7 to 20 amino acids in length, or (b) at least 5 amino acids or at least 7 amino acids in length.

8. The IL2 proprotein according to claim 1, wherein the first and second protease-cleavable linkers are in the range of 20 to 80 amino acids or 20 to 60 amino acid lengths.

9. The IL2 proprotein according to claim 1, wherein the first Fc domain and / or the second Fc domain comprises a hinge domain.

10. The IL2 proprotein according to claim 1, wherein the first targeting portion and / or the second targeting portion is Fab.

11. The IL2 proprotein according to claim 1, wherein the first targeting portion and / or the second targeting portion is scFv.

12. The IL2 proprotein according to claim 1, wherein the first targeting portion and / or the second targeting portion can bind to any target molecule specified in Section 6.

7.

13. The first targeting portion and / or the second targeting portion (a) ECM antigens, which are optionally selected from syndecan, heparanase, integrin, osteopontin, lynx, cadherin, laminin, laminin EGF type, lectin, fibronectin, notch, nectin (e.g., nectin-4), tenascin, collagen (e.g., collagen type X), and matrixin, (b) cell surface molecules of tumors or viral lymphocytes, (c) T cell costimulatory 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, (d) A checkpoint inhibitor, which is 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, and / or (e) Tumor-associated antigens ("TAAs"), which are 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, C DK4, CEA, CTLA4, Cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE ​​protein (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, mesoserine, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX 3. The tumor-associated antigen ("TAA") selected from 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, and uroplakin-3. It can be joined, The IL2 proprotein according to claim 1.

14. The IL2 proprotein according to claim 1, wherein the Fc region is a homodimer.

15. The IL2 proprotein according to claim 1, wherein the Fc region is a heterodimer.

16. The IL2 proprotein according to claim 1, having the configuration depicted in Figure 1A.

17. IL2 proprotein, wherein the IL2 proprotein is optionally the IL2 proprotein described in claim 1, and the IL2 proprotein is (a) In order from the N-terminus to the C-terminus, (i) A first amino acid sequence having at least about 95% sequence identity with any one of the amino acid sequences of sequence numbers 346, 347, 348, 349, 350, and 351, (ii) A second amino acid sequence comprising or consisting of a non-cleavable linker sequence, (iii) A third amino acid sequence having at least about 95% sequence identity with any one of the amino acid sequences of sequence numbers 3, 4, and 5, (iv) A fourth amino acid sequence comprising one or more amino acid sequences described in Table B or Table D, and (v) A first polypeptide chain comprising a fifth amino acid sequence having at least approximately 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 2, (b) In order from the N-terminus to the C-terminus, (i) A sixth amino acid sequence having at least approximately 95% sequence identity with any one of sequence numbers 346, 347, 348, 349, 350, and 351, (ii) A seventh amino acid sequence comprising or consisting of a non-cleavable linker sequence, (iii) An eighth amino acid sequence having at least approximately 95% sequence identity with any one of sequence numbers 3, 4, and 5, (iv) A ninth amino acid sequence comprising one or more amino acid sequences listed in Table B or Table D, and (v) IL2 proprotein comprising a second polypeptide chain having a tenth amino acid sequence having at least about 95% sequence identity with respect to the amino acid sequence of SEQ ID NO:

2.

18. (a) The first polypeptide comprises at the N-terminus of the first amino acid sequence an eleventh amino acid sequence having at least about 95%, at least about 98%, or 100% sequence identity with any one of SEQ ID NOs: 352, 353, 354, and 355, and / or (b) The second polypeptide comprises a twelfth amino acid sequence having at least about 95%, at least about 98%, or 100% sequence identity with any one of SEQ ID NOs: 352, 353, 354, and 355 at the N-terminus of the first amino acid sequence. The IL2 proprotein according to claim 17.

19. The IL2 proprotein according to claim 17, wherein the first amino acid sequence has at least about 98% or 100% sequence identity with any one of SEQ ID NOs: 346, 347, 348, 349, 350, and 351.

20. The IL2 proprotein according to claim 17, wherein the sixth amino acid sequence has at least about 98% or 100% sequence identity with any one of sequence numbers 346, 347, 348, 349, 350, and 351.

21. (a) The second amino acid sequence is the amino acid sequence listed in Table E, and / or (b) The seventh amino acid sequence is the amino acid sequence listed in Table E. The IL2 proprotein according to claim 17.

22. (a) The third amino acid sequence has at least about 98% or 100% sequence identity with any one of sequence numbers 3, 4, and 5, and / or (b) The eighth amino acid sequence has at least about 98% or 100% sequence identity with any one of sequence numbers 3, 4, and 5. The IL2 proprotein according to claim 17.

23. (a) The fourth amino acid sequence includes (a) one or more amino acid sequences listed in Table B, or (b) an amino acid sequence listed in Table D, and / or (b) The ninth amino acid sequence includes one or more amino acid sequences listed in (a) Table B, or (b) an amino acid sequence listed in Table D. The IL2 proprotein according to claim 17.

24. (a) The fifth amino acid sequence has at least about 98% or 100% sequence identity with respect to SEQ ID NO: 2, and / or (b) The 10th amino acid sequence has at least about 98% or 100% sequence identity with respect to SEQ ID NO:

2. The IL2 proprotein according to claim 17.

25. (a) the first polypeptide chain lacks an additional sequence between the first amino acid sequence and the second amino acid sequence, (b) an additional sequence between the second amino acid sequence and the third amino acid sequence, (c) an additional sequence between the third amino acid sequence and the fourth amino acid sequence, and / or (d) an additional sequence between the fourth amino acid sequence and the fifth amino acid sequence, and / or (b) The second polypeptide chain (a) lacks an additional sequence between the sixth amino acid sequence and the seventh amino acid sequence, (b) lacks an additional sequence between the seventh amino acid sequence and the eighth amino acid sequence, (c) lacks an additional sequence between the eighth amino acid sequence and the ninth amino acid sequence, and / or (d) lacks an additional sequence between the ninth amino acid sequence and the tenth amino acid sequence. The IL2 proprotein according to claim 17.

26. A nucleic acid or a plurality of nucleic acids encoding the IL2 proprotein according to any one of claims 1 to 25.

27. A host cell manipulated to express the IL2 proprotein according to any one of claims 1 to 25.

28. A method for producing the IL2 proprotein according to any one of claims 1 to 25, comprising culturing the host cell according to claim 27 and recovering the IL2 proprotein expressed thereby.

29. A pharmaceutical composition comprising the IL2 proprotein according to any one of claims 1 to 25 and an excipient.

30. A pharmaceutical composition according to claim 29 for the treatment of cancer.

31. A pharmaceutical composition according to claim 29 for a method of local delivery of IL-2 protein, a method of treating cancer with IL-2 protein selectively activated in cancer tissue, a method of administering IL-2 therapy with reduced systemic exposure and / or reduced systemic toxicity, a method of targeted delivery of activated IL-2 protein to cancer tissue, or a method of locally inducing an immune response in target tissue, The method includes administering the pharmaceutical composition to the target, A pharmaceutical composition comprising one or more protease-cleavable linkers, each of which contains one or more substrates for one or more proteases expressed by a tissue (e.g., cancer tissue) to which the IL2 protein is locally delivered.

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