Protease-activating polypeptides

Abstract

The present invention generally relates to novel protease-activatable interleukin-2 (IL-2) polypeptides and immunoconjugates, comprising (i) an IL-2 polypeptide, (ii) a masking moiety, and (iii) a linker comprising a first protease cleavage site, the linker having a length of 20 to 45 amino acids, the masking moiety being covalently attached to the IL-2 polypeptide via the linker, the masking moiety being capable of binding to the IL-2 polypeptide and thereby reversibly masking the IL-2 polypeptide, and the masking moiety comprising a second protease cleavage site, the masking moiety not masking the IL-2 polypeptide upon cleavage at the first and / or second protease cleavage sites. The present invention also relates to polynucleotides encoding such protease-activatable interleukin-2 (IL-2) polypeptides and immunoconjugates, and vectors and host cells comprising such polynucleotides. The present invention further relates to methods of making the protease-activatable interleukin-2 (IL-2) polypeptides and immunoconjugates of the invention, and methods of using these protease-activatable interleukin-2 (IL-2) polypeptides and immunoconjugates in the treatment of disease.

Description

[Technical Field]

[0001] The present invention relates generally to novel protease-activated polypeptides, and in particular to interleukin-2 (IL-2) polypeptides. More specifically, the present invention relates to protease-activated IL-2 polypeptides that exhibit improved properties for use as immunotherapeutic agents. In addition, the present invention relates to immunoconjugates, polynucleotides, vectors, and host cells comprising protease-activated IL-2 polypeptides, or such vectors or polynucleotide molecules. The present invention further relates to methods for producing protease-activated IL-2 polypeptides or immunoconjugates, pharmaceutical compositions containing same, and uses thereof. [Background technology]

[0002] The selective destruction of individual target cells or specific target cell types is often desirable in various clinical settings. For example, a primary goal of cancer therapy is the specific destruction of tumor cells while leaving healthy cells and tissues intact.

[0003] An attractive way to achieve this is to induce an immune response against tumors, inducing immune effector cells, such as natural killer (NK) cells or cytotoxic T lymphocytes (CTLs), to attack and destroy tumor cells. In this regard, conjugates containing interleukin-2 (IL-2) variants designed to bind to surface antigens on target cells are thought to activate nearby T effector cells and NK cells. Simultaneous binding of such conjugates to their target and the interleukin-2 receptor either leads to activation of T effector cells and NK cells near the target (in trans), or, when the target is expressed on T effector cells and NK cells, activates these cells upon binding (in cis).

[0004] Interleukin-2 (IL-2), also known as T-cell growth factor (TCGF), is a 15.5 kDa globular glycoprotein that plays a central role in lymphocyte development, survival, and homeostasis. IL-2 is 133 amino acids long and consists of four nonparallel amphipathic α-helices that form a quaternary structure essential for its function (Smith, Science 240, 1169-76 (1988); Bazan, Science 257, 410-413 (1992)). IL-2 sequences from different species are found in NCBI RefSeq numbers NP000577 (human), NP032392 (mouse), NP446288 (rat), or NP517425 (chimpanzee).

[0005] IL-2 mediates its effects by binding to the IL-2 receptor (IL-2R), which consists of three individual subunits, different associations of which can generate receptor forms with different affinities for IL-2: α (CD25), β (CD122), and γ (γ c The association of the CD25 and CD132 subunits results in a trimeric high-affinity receptor for IL-2. The dimeric IL-2 receptor, consisting of the β and γ subunits, is called the intermediate-affinity IL-2R. The α subunit forms the monomeric low-affinity IL-2 receptor. The dimeric intermediate-affinity IL-2 receptor binds IL-2 with approximately 100-fold lower affinity than the trimeric high-affinity receptor, but both the dimeric and trimeric IL-2 receptor variants can transduce signals upon binding to IL-2 (Minami et al., Annu Rev Immunol 11, 245-268 (1993)). Therefore, the α-subunit CD25 is not essential for IL-2 signaling. The α-subunit confers high affinity binding to the receptor, while the β-subunit CD122 and γ-subunit are important for signal transduction (Krieg et al., Proc Natl Acad Sci 107, 11906-11 (2010)). The trimeric IL-2 receptor, which includes CD25, binds to (resting) CD4 + Forkhead Box P3 (FoxP3) +Controllability T(T reg ) cells. They are also transiently induced in conventionally activated T cells, whereas in the resting state, these cells express only the dimeric IL-2 receptor. reg The cells consistently express the highest levels of CD25 in vivo (Fontenot et al., Nature Immunol 6, 1142-51 (2005)).

[0006] IL-2 stimulates activated T cells, especially CD4 + It is mainly synthesized by helper T cells. IL-2 stimulates the proliferation and differentiation of T cells, induces the production 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 (e.g., Waldmann, Nat Rev Immunol 6, 595-601 (2009); Olejniczak and Kasprzak, Med Sci Monit 14, RA179-89 (2008); Malek, Annu Rev Immunol 26, 453-79 (2008)).

[0007] The ability of IL-2 to expand lymphocyte populations in vivo and increase the effector functions of these cells confers antitumor effects to IL-2 and makes IL-2 immunotherapy an attractive treatment option for certain metastatic cancers. Consequently, high-dose IL-2 treatment has been approved for use in patients with metastatic renal carcinoma and malignant melanoma.

[0008] However, IL-2 has a dual function in the immune response in that it not only mediates the expansion and activity of effector cells but is also critically involved in the maintenance of peripheral immune tolerance.

[0009] The primary mechanism underlying peripheral self-tolerance is activation-induced cell death (AICD) in T cells triggered by IL-2. AICD is a process in which fully activated T cells undergo programmed cell death through the engagement of cell surface death receptors, such as CD95 (also known as Fas) or TNF receptors. When antigen-activated T cells (after previous exposure to IL-2) that express high-affinity IL-2 receptors during proliferation are restimulated with antigen via the T cell receptor (TCR) / CD3 complex, expression of Fas ligand (FasL) and / or tumor necrosis factor (TNF) is induced, rendering the cells susceptible to Fas-mediated apoptosis. This process is IL-2-dependent (Lenardo, Nature 353, 858-61 (1991)) and mediated by STAT5. The process of AICD in T lymphocyte tolerance allows tolerance to be established not only against self-antigens but also against persistent antigens that are not clearly part of the host genome, such as tumor antigens.

[0010] Furthermore, IL-2 inhibits peripheral CD4 + CD25 + Controllability T(T reg ) cells (Fontenot et al., Nature Immunol 6, 1142-51 (2005); D'Cruz and Klein, Nature Immunol 6, 1152-59 (2005); Maloy and Powrie, Nature Immunol 6, 1171-72 (2005)), which are also known as suppressor T cells. These cells suppress effector T cells from destroying their (self) targets through cell-cell contact by inhibiting T cell help and activation, or through the release of immunosuppressive cytokines such as IL-10 or TGF-β. T reg Cell depletion has been shown to enhance IL-2-induced anti-tumor immunity (Imai et al., Cancer Sci 98, 416-23 (2007)).

[0011] Thus, in the presence of IL-2, the generated CTLs either recognize the tumor as self and undergo AICD, or the immune response evolves into an IL-2-dependent T cell line. reg IL-2 is not optimal for inhibiting tumor growth because it is inhibited by the cells.

[0012] An additional problem associated with IL-2 immunotherapy is the side effects caused by recombinant human IL-2 treatment. Patients receiving high-dose IL-2 treatment frequently experience serious cardiovascular, pulmonary, renal, hepatic, gastrointestinal, neurological, cutaneous, hematologic, and systemic adverse events, requiring intensive monitoring and inpatient care. Most of these side effects can be explained by the progression of so-called vascular (or capillary) leak syndrome (VLS), a pathological increase in vascular permeability that causes fluid leakage in multiple organs (e.g., causing pulmonary and cutaneous edema and hepatocyte damage) and intravascular fluid depletion (causing a drop in blood pressure and a compensatory increase in heart rate). No treatment for VLS exists other than IL-2 withdrawal. Low-dose IL-2 regimens have been tested in patients to avoid VLS, but with suboptimal therapeutic outcomes. VLS was thought to be caused by the release of proinflammatory cytokines, such as tumor necrosis factor (TNF)-α, from IL-2-activated NK cells. However, it has recently been shown that IL-2-induced pulmonary edema results from direct binding of IL-2 to pulmonary endothelial cells that express low to moderate levels of functional αβγ IL-2 receptors (Krieg et al., Proc Nat Acad Sci USA 107, 11906-11 (2010)).

[0013] Several approaches have been taken to overcome these problems associated with IL-2 immunotherapy. For example, the combination of IL-2 with a specific anti-IL-2 monoclonal antibody enhances the therapeutic effect of IL-2 in vivo (Kamimura et al., J Immunol 177, 306-14 (2006); Boyman et al., Science 311, 1924-27 (2006)). In an alternative approach, IL-2 has been mutated in various ways to reduce its toxicity and / or improve its efficacy. Hu et al. (Blood 101, 4853-4861 (2003); U.S. Patent Application Publication No. 2003 / 0124678) replaced the arginine residue at position 38 of IL-2 with tryptophan to eliminate IL-2's vascular permeability activity. Shanafelt et al. (Nature Biotechnol 18, 1197-1202 (2000)) mutated asparagine 88 to arginine to enhance selectivity for T cells over NK cells. Heaton et al. (Cancer Res 53, 2597-602 (1993); U.S. Patent No. 5,229,109) introduced two mutations, Arg38Ala and Phe42Lys, to reduce secretion of inflammatory cytokines from NK cells. Gillies et al. (U.S. Patent Application Publication No. 2007 / 0036752) substituted three residues in IL-2 (Asp20Thr, Asn88Arg, and Gln126Asp) that contribute to affinity for the intermediate-affinity IL-2 receptor to reduce VLS. Gillies et al. (WO 2008 / 0034473) also mutated the interface between IL-2 and CD25 with the amino acid substitutions Arg38Trp and Phe42Lys to enhance efficacy, and demonstrated that the interaction with CD25 and T reg (WO 2009 / 061853) created IL-2 mutants that had increased affinity for CD25 but did not activate the receptor and acted as antagonists. The mutations introduced were intended to disrupt the interaction with the β- and / or γ-subunits of the receptor.

[0014] Certain mutant IL-2 polypeptides designed to overcome the above-mentioned problems associated with IL-2 immunotherapy (toxicity caused by the induction of VLS, tumor resistance caused by the induction of AICD, and immunosuppression caused by the activation of Treg cells) are described in WO 2012 / 107417. Substitution of the phenylalanine residue at position 42 with alanine, substitution of the tyrosine residue at position 45 with alanine, and substitution of the leucine residue at position 72 of IL-2 with glycine essentially abolishes binding of the mutant IL-2 polypeptide to the α-subunit of the IL-2 receptor (CD25).

[0015] However, among the known IL-2 variants, all of the above-mentioned problems associated with IL-2 immunotherapy, namely, toxicity caused by the induction of VLS, tumor resistance caused by the induction of AICD, and T reg None have been shown to overcome the immunosuppression caused by cell activation.

[0016] In addition to the above-mentioned approaches, IL-2 immunotherapy can be improved by selectively targeting IL-2 to tumors, for example, in the form of an immunoconjugate containing an antibody that binds to an antigen expressed on tumor cells or to an effector cell in the tumor environment. Several such immunoconjugates have been described (see, for example, Ko et al., J Immunother (2004) 27, 232-239; Klein et al., Oncoimmunology (2017) 6(3), e1277306; WO 2018 / 184964).

[0017] Given the clinical success and unprecedented efficacy of PD-1 / PD-L1 checkpoint inhibitors, there remains a significant medical need to increase response rates and duration in patients with pre-existing T cell immunity. Recent reports have demonstrated that two populations of tumor-specific CD8 T cells, namely exhausted TILs and their newly described TCF1+ precursors with stem-like properties, T-resource cells, can be targeted by PD-1 antibodies. Of these two, the latter correlates with favorable disease prognosis and responds to anti-PD-1 therapy. Cytokines such as interleukin-2 have also been described to induce proliferation / differentiation of T-resource cells toward functional effector T cells.

[0018] IL-2 was the first effective cancer immunotherapy used to treat metastatic melanoma and renal cell carcinoma. Unfortunately, high concentrations of IL-2 are toxic by inducing vascular leak syndrome (VLS), detrimentally expanding regulatory T cells due to binding to CD25, and inducing activation-induced cell death. To overcome these limitations of wild-type IL2 / Proleukin, IL-2v variants with abolished CD25 binding have been described. However, due to the mechanism of IL-2 signaling through the moderate affinity of the heterodimer, IL-2Rbg complex IL2v and other IL2 variants automatically activate IL-2R signaling upon encountering IL-2R, resulting in nonspecific peripheral immune cell activation outside the tumor in the blood, vasculature, and lymphatic tissues, leading to dose-limiting toxicity. As a result, it is not possible to administer the same amount of IL-2 or IL2v to patients as desired to achieve maximal therapeutic efficacy.

[0019] In summary, PD1-IL2v is expressed in cis as PD-1 +Targeting PD1-IL2v to T cells may achieve a more potent therapeutic effect. Indeed, cis-targeting of PD1-IL2v to the appropriate antigen-specific T cell subset, in conjunction with PD-1 / -L1 blockade, is a better way to therapeutically exploit endogenous immunity, one of the most powerful immunomodulatory pathways known to unlock endogenous immunity for cancer immunotherapy. However, even with PD1-IL2v, the IL2v moiety may trigger IL-2R signaling in the periphery, preventing the administration of the desired maximum dose due to activation of peripheral non-tumor-specific IL-2Rs. Therefore, the therapeutic index is likely to remain narrow, with a flat dose of >10–30 mg in humans, which may limit utilization of the full pathway potential. Alternatively, CD8 T cells could be targeted along with other T cell targets.

[0020] It is therefore important to produce next-generation IL-2 molecules that are targeted in cis to antigen-experienced T cells in amounts that have a broader therapeutic index.

[0021] Serine proteases (e.g., matriptase), cysteine ​​proteases (e.g., cathepsin S), and matrix metalloproteinases (e.g., MMP-2 and MMP-9) are overexpressed in several types of cancer (Duffy, M.J., Proteases as prognostic markers in cancer. Clin. Cancer Res. 2, 613-618 (1996)). Matriptase, matrix metalloproteinase 2 (MMP-2, gelatinase A), and matrix metalloproteinase 9 (MMP-9, gelatinase B), for example, are overexpressed in breast and ovarian cancer (McGowan, P.M., & Duffy, M.J., Matrix metalloproteinase expression and outcome in patients with breast cancer: analysis of a published database. Ann. Oncol. 19, 1566-1572 (2008)). MMP-2 and MMP-9 activity was detected in cervical, breast, and ovarian tumors and ascites of patients with epithelial ovarian cancer (EOC), but not in the serum of these patients (Demeter, A. et al. Molecular prognostic markers in recurrent and non-recurrent epithelial ovarian cancer. Anticancer Res. 25, 2885-2889 (2005)). Matriptase can be detected in normal epithelial cells, but matriptase activity is primarily detected in cancer (LeBeau, A. et al. Imaging a functional tumorigenic biomarker in the transformed epithelium. Proc. Natl. Acad. Sci. USA 110, 93-98 (2013)).

[0022] Although current immunotherapies directed at the PD1 / PDL1 axis have shown unprecedented efficacy in multiple cancer indications, there are significant numbers of patients who do not respond to treatment or who relapse, and other tumor types remain largely resistant to such therapies. Thus, there is a clear and high unmet need in the significant number of cancer patients who have some kind of pre-existing T cell immune response. Examples of indications in which PD1 antagonism has resulted in objective responses include advanced or metastatic melanoma, Merkel cell carcinoma, NSCLC, SCLC, RCC, gastric cancer, hepatocellular carcinoma, head and neck cancer, breast cancer, ovarian cancer, mismatch repair deficient and mismatch repair proficient CRC and hematologic malignancies such as DLBCL and PMBCL after autologous hematopoietic stem cell transplantation and HL (Editorial: PD-Loma: a cancer entity with a shared sensitivity to the PD-1 / PD-L1 pathway blockade, British Journal of Cancer (2019) 120: 3-5; https: / / doi.org / 10.1038 / s41416-018-0294-4).

[0023] The task of producing IL-2 variants and conjugates suitable for treatment presents multiple technical challenges that must be met, including efficacy, toxicity, applicability, and productivity. If the conjugate targets an antigen on target cells, such as cancer cells, that is also expressed in non-target tissues, toxicity may occur. Thus, there is still a need in the art to further improve the therapeutic utility of IL-2 polypeptides. Summary of the Invention

[0024] The present invention is based, in part, on the recognition that the tumor environment (TME) highly expresses proteases compared to normal tissue, and that a masked therapeutic agent, preferably a protease-activatable interleukin-2, reduces or eliminates systemic and complete activity in the tumor environment upon activation by a protease.

[0025] Thus, a first aspect of the present invention provides a protease-activatable interleukin-2 (IL-2) polypeptide comprising: (i) an IL-2 polypeptide; (ii) a masking moiety; and (iii) a linker comprising a first protease cleavage site, wherein the linker has a length of 20 to 45 amino acids, the masking moiety is covalently attached to the IL-2 polypeptide via the linker, the masking moiety is capable of binding to the IL-2 polypeptide, thereby reversibly masking the IL-2 polypeptide, and the masking moiety comprises a second protease cleavage site, and the masking moiety does not mask the IL-2 polypeptide upon cleavage at the first and / or second protease cleavage sites. In one embodiment, the linker has a length of 22 to 43 amino acids. In one embodiment, the linker has a length of 25 to 38 amino acids. In one embodiment, the linker has a length of 25 amino acids, preferably having the sequence set forth in SEQ ID NO: 64 or SEQ ID NO: 66. In one embodiment, the linker has a length of 38 amino acids, and preferably the linker has the sequence set forth in SEQ ID NO: 63 or SEQ ID NO: 65. In one embodiment, the masking moiety is covalently attached to the amino or carboxy terminus of the interleukin-2 polypeptide via a linker. In one embodiment, the masking moiety is an IL-2 antagonist. In one embodiment, the masking moiety is an IL-2 antibody or an IL-2 receptor subunit. In one embodiment, the IL-2 antibody comprises a Fab molecule. In one embodiment, the masking moiety is MT204, preferably an antibody derived from MT204. The MT204 antibody is disclosed, for example, in Volkland et al., Molecular Immunology 44 (2007) 1743-1753 and PCT Application WO 2006 / 128690. More preferably, the masking moiety is a deimmunized MT204-derived binder. In one embodiment, the Fab molecule is a single-chain Fab molecule. In one embodiment, the second protease cleavage site is located between the variable domain of the heavy chain (VH) and the variable domain of the light chain (VL) of the single-chain Fab molecule.In one embodiment, each of the first and second protease cleavage sites comprises at least one protease recognition sequence, and in one embodiment, the protease recognition sequence of the first protease cleavage site and / or the protease recognition sequence of the second protease cleavage site is either YAARKGGI set forth in SEQ ID NO:60 and / or PQARK set forth in SEQ ID NO:61.

[0026] In one embodiment, the IL-2 polypeptide is wild-type IL-2, preferably human IL-2 set forth in SEQ ID NO: 62, or a mutant IL-2 polypeptide. In one embodiment, the mutant IL-2 polypeptide comprises any amino acid substitution selected from the group T3A, F42A, Y45A, L72G, C125A of human IL-2 set forth in SEQ ID NO: 62. In one embodiment, the mutant IL-2 polypeptide comprises the amino acid substitutions F42A, Y45A, and L72G of human IL-2 set forth in SEQ ID NO: 62. In one embodiment, the mutant IL-2 polypeptide comprises the amino acid substitutions T3A, F42A, Y45A, L72G, and C125A of human IL-2 set forth in SEQ ID NO: 62. In one embodiment, the protease-activatable IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30. In one embodiment, the IL-2 polypeptide is further linked to a non-IL-2 moiety. In one embodiment, the IL-2 polypeptide shares a carboxy-terminal peptide bond with the masking moiety and an amino-terminal peptide bond with the non-IL-2 moiety, or the IL-2 polypeptide shares an amino-terminal peptide bond with the masking moiety and a carboxy-terminal peptide bond with the non-IL-2 moiety. In one embodiment, the non-IL-2 moiety is an antigen-binding moiety or an effector cell-binding moiety.

[0027] In a further aspect, the present invention provides an immunoconjugate comprising a protease-activatable IL-2 polypeptide described herein and an antigen-binding portion and / or an effector cell-binding portion. In one embodiment, the protease-activatable IL-2 polypeptide shares an amino- or carboxy-terminal peptide bond with the antigen-binding portion or effector cell-binding portion. In one embodiment, the immunoconjugate comprises a first and a second antigen-binding portion, or a first and a second effector cell antigen-binding portion, or an antigen-binding portion and an effector cell-binding portion. In one embodiment, (i) the protease-activatable IL-2 polypeptide shares an amino-terminal peptide bond or a carboxy-terminal peptide bond with the first antigen-binding portion and the second antigen-binding portion shares an amino-terminal peptide bond or a carboxy-terminal peptide bond with a) the protease-activatable IL-2 polypeptide or b) the first antigen-binding portion; or (ii) the protease-activatable IL-2 polypeptide shares an amino-terminal peptide bond or a carboxy-terminal peptide bond with the first effector cell-binding portion and the second effector cell-binding portion shares an amino-terminal peptide bond or a carboxy-terminal peptide bond with a) the protease-activatable IL-2 polypeptide or b) the first effector cell-binding portion. (iii) the protease-activatable IL-2 polypeptide shares an amino- or carboxy-terminal peptide bond with the antigen-binding portion and the effector cell-binding portion shares an amino- or carboxy-terminal peptide bond with a) the protease-activatable IL-2 polypeptide or b) the antigen-binding portion; or (iv) the protease-activatable IL-2 polypeptide shares an amino- or carboxy-terminal peptide bond with the effector cell-binding portion and the antigen-binding portion shares an amino- or carboxy-terminal peptide bond with a) the protease-activatable IL-2 polypeptide or b) the effector cell-binding portion.

[0028] In one embodiment, the antigen-binding portion or effector cell-binding portion comprised in the protease-activatable IL-2 polypeptide disclosed herein or the immunoconjugate disclosed herein is an antibody or antibody fragment. In one embodiment, the antigen-binding portion and / or the effector cell-binding portion is a Fab molecule or an scFv molecule. In one embodiment, the antigen-binding portion and / or the effector cell-binding portion is an immunoglobulin molecule, particularly an IgG molecule. In one embodiment, the antigen-binding portion is directed against an antigen presented on or in the tumor cell environment, and / or the effector cell-binding portion is directed against an effector cell present in the tumor cell environment to achieve cis-targeting.

[0029] In one embodiment, (i) the immune complex comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:5, an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:22, and an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:23; or (ii) the immune complex comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:5, an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:22, and an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:24. or (iii) the immune complex comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:5, an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:22, and an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:25; or (iv) the immune complex comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:5, an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:22, and an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:26.

[0030] In one embodiment, (i) the immune complex comprises the amino acid sequence set forth in SEQ ID NO: 5, the amino acid sequence set forth in SEQ ID NO: 22, and the amino acid sequence set forth in SEQ ID NO: 23; (ii) the immune complex comprises the amino acid sequence set forth in SEQ ID NO: 5, the amino acid sequence set forth in SEQ ID NO: 22, and the amino acid sequence set forth in SEQ ID NO: 24; (iii) the immune complex comprises the amino acid sequence set forth in SEQ ID NO: 5, the amino acid sequence set forth in SEQ ID NO: 22, and the amino acid sequence set forth in SEQ ID NO: 25; or (iv) the immune complex comprises the amino acid sequence set forth in SEQ ID NO: 5, the amino acid sequence set forth in SEQ ID NO: 22, and the amino acid sequence set forth in SEQ ID NO: 26.

[0031] The present invention further provides one or more isolated polynucleotides encoding the protease-activatable IL-2 polypeptides described herein or the immunoconjugates described herein, one or more expression vectors comprising the polynucleotides described herein, and one or more host cells comprising the polynucleotides described herein or the expression vectors described herein.

[0032] Further provided is a method of producing a protease-activatable IL-2 polypeptide or immunoconjugate described herein, the method comprising culturing a host cell described herein under conditions suitable for expression of the protease-activatable IL-2 polypeptide or immunoconjugate.

[0033] Further provided are the protease-activatable IL-2 polypeptides or immunoconjugates described herein produced by the methods described herein. Furthermore, provided are pharmaceutical compositions comprising the protease-activatable IL-2 polypeptides or immunoconjugates disclosed herein and a pharmaceutically acceptable carrier. In particular, the present invention encompasses the protease-activatable IL-2 polypeptides or immunoconjugates described herein for use in treating a disease in an individual in need thereof. In certain embodiments, the disease is cancer. In certain embodiments, the individual is a human.

[0034] Further included in the present invention is the use of a protease-activatable IL-2 polypeptide or immunoconjugate described herein for the manufacture of a medicament for treating a disease in an individual in need thereof. Additionally, provided is a method of treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of a composition comprising a protease-activatable IL-2 polypeptide or immunoconjugate described herein in a pharmaceutically acceptable form. The disease is preferably cancer.

[0035] Also provided are methods of stimulating the immune system of an individual comprising administering to the individual an effective amount of a composition comprising a protease-activatable IL-2 polypeptide or immunoconjugate disclosed herein in a pharmaceutically acceptable form. [Brief explanation of the drawings]

[0036] [Figures 1A-1I]IgG antibodies generated to evaluate deimmunized MT204 mask and anti-PD1 binder variants with SPR and their respective antigen and parental controls. Figure 1A (P1AH2050-P1AH2052) Deimmunized MT204 mask in human IgG PG LALA format; Figure 1B (P1AH4157-P1AH4161) First set of deimmunized anti-PD1 binder variants in human IgG PG LALA format; Figure 1C (P1AI0356-P1AI0360) Second set of deimmunized anti-PD1 binder variants in human IgG PG LALA format; Figure 1D (P1AI1648-P1AI1652) Third set of deimmunized anti-PD1 binder variants in human IgG PG LALA format; Figure 1E (P1AF7506) One-arm parent MT204 in human IgG PG LALA format; Figure 1F (P1AA6888) Human IgG PG Parental anti-PD1 binders in LALA format; Figure 1G (P1AD9704) human PD1 antigen as an Fc fusion with C-terminal biotinylated avi tag and His tag; Figure 1H (P1AG0879) IL2v cytokine with C-terminal avi tag and His tag; Figure 1I (P1AA9690) human IgG PG parental MT204 in LALA format. [Figures 2A-2F]Human PD1-targeted masked IL2v constructs with PQARK or YAARKGGI matriptase sites and the respective unmasked PD1-targeted or FAP-targeted controls. Figure 2A (P1AI4322) Bivalent human PD1-targeting human IgG PG-LALA with masked IL2v fused to the C-terminus of the Fc knob chain ("in-line"), two PQARK matriptase sites for release of the mask, and a 38-amino acid linker between the IL2v cytokine and the scFv mask; Figure 2B (P1AI4323) Bivalent human PD1-targeting human IgG PG-LALA with masked IL2v fused to the C-terminus of the Fc knob chain ("in-line"), two PQARK matriptase sites for release of the mask, and a 25-amino acid linker between the IL2v cytokine and the scFv mask; Figure 2C (P1AI4324) Bivalent human PD1-targeting human IgG PG with masked IL2v fused to the C-terminus of the Fc knob chain ("in-line") LALA, two YAARKGGI matriptase sites for release of the mask, and a 38 amino acid linker between the IL2v cytokine and the scFv mask; Figure 2D (P1AI4325) bivalent human PD1-targeting human IgG PG-LALA with masked IL2v (inline) fused to the C-terminus of the Fc knob chain, two YAARKGGI matriptase sites for release of the mask, and a 25 amino acid linker between the IL2v cytokine and the scFv mask; constructs A-E contain the preferred deimmunized V-domain sequences of an anti-PD1 binder and the MT204 scFv mask; Figure 2E (P1AE4422) bivalent human PD1-targeting human IgG PG-LALA with IL2v fused to the C-terminus of the Fc knob chain as an unmasked control; Figure 2F (P1AA5355) bivalent human FAP-targeting human IgG PG-LALA with IL2v fused to the C-terminus of the Fc knob chain as an unmasked control. [Figure 3A-3C]A non-cleavable control and a human PD1-targeted masked IL2v construct as the respective unmasked control. Figure 3A (P1AI4646) Bivalent human PD1-targeting human IgG PG-LALA with masked IL2v fused to the C-terminus of the Fc knob chain ("in-line"), a 38 amino acid linker between the IL2v cytokine and the scFv mask, and no matriptase release site (non-cleavable control); Figure 3B (P1AI4647) Bivalent human PD1-targeting human IgG PG-LALA with masked IL2v fused to the C-terminus of the Fc knob chain ("in-line"), a 25 amino acid linker between the IL2v cytokine and the scFv mask, and no matriptase release site (non-cleavable control); Figure 3C (P1AI4648) Bivalent human PD1-targeting human IgG PG-LALA with IL2v fused to the C-terminus of the Fc knob chain as an unmasked control; Constructs A-C contain the preferred deimmunized V-domain sequences of an anti-PD1 binder and the MT204 scFv mask (constructs A and B). [Figure 4A-4B] Human PD1-targeted masked IL2v constructs with YAARKGGI matriptase sites and their respective control murine surrogates. Figure 4A (P1AI4650) Bivalent human PD1-targeted murine IgG DA PG with masked IL2v ("in-line") fused to the C-terminus of the Fc DD-chain and two YAARKGGI matriptase sites for mask release; Figure 4B (P1AI4651) Bivalent human PD1-targeted murine IgG DA PG with masked IL2v ("in-line") fused to the C-terminus of the Fc DD-chain and no matriptase release sites (non-cleavable control). [Figure 5A-5B]Determining the potency of deimmunized aPD-1 constructs to inhibit PD1 / PD-L1 interaction. PD-1 effector cells were incubated with PD-L1 aAPC / CHO-K1 in the presence of anti-PD1 antibody. Bio-Glo reagent was added and luminescence was measured using a luminometer. Data were analyzed using GraphPad Prism software (mean ± SEM). Data from one experiment. Figure 5A shows inhibition by P1AH4159, P1AH4160, and P1AH4161. Figure 5B shows inhibition by P1AH4157 and P1AH4158. [Figures 6A-6B] Figure 6 shows binding of deimmunized aPD-1 to CD3 / CD28-activated CD4 T cells compared to PD-1 IgG PG LALA, PD1-IL2v, and FAP-IL2v. PD1 antibody constructs were added to activated CD4 T cells at various concentrations, and dose-dependent responses were measured by flow cytometry. Data were analyzed using GraphPad Prism software (mean ± SEM). Data from two donors. Figure 6A shows binding with P1AH4159, P1AH4160, and P1AH4161. Figure 6B shows binding with P1AH4157 and P1AH4158. [Figures 7A-7B] Figure 7 shows binding of deimmunized aPD-1 to CD3 / CD28-activated CD4 T cells compared to aPD-1 IgG PG LALA, PD1-IL2v, and FAP-IL2v. PD1 antibody constructs were added to activated CD4 T cells at various concentrations, and dose-dependent responses were measured by flow cytometry. Data were analyzed using GraphPad Prism software (mean ± SEM). Data from two donors. Figure 7A shows binding with P1AH4159, P1AH4160, and P1AH4161. Figure 7B shows binding with P1AH4157 and P1AH4158. [Figure 8A-8B]Figure 8A shows granzyme B production upon exposure to P1AH4159, P1AH4160, and P1AH4161. Figure 8B shows granzyme B production upon exposure to P1AH4157 and P1AH4158. [Figure 9A-9B] Figure 9A shows interferon-γ production upon exposure to P1AH4159, P1AH4160, and P1AH4161. Figure 9B shows interferon-γ production upon exposure to P1AH4157 and P1AH4158. [Figure 10] Binding of deimmunized PD1-IgG to human PD1-overexpressing CHO cells was measured by flow cytometry, and the molecules were detected using a fluorescently labeled anti-human Fc-specific secondary antibody. [Figure 11] Inhibition of IL2v activity by the deimmunized MT204 mask compared to the parental mask was determined by measuring proliferation of human NK92 cells using CellTiter Glo. [Figures 12A-12B]Binding of the indicated TA PD1-IL2v constructs to human PD1-overexpressing CHO cells was measured by flow cytometry. Molecules were detected using a fluorescently labeled anti-human Fc-specific secondary antibody. Figure 12A relates to constructs with a PQARK cleavage site. Figure 12B relates to constructs with a YAARKGGI cleavage site. [Figures 13A-13F] The proliferation of KHYG-1 cells induced by a set of TA PD1-IL2v constructs was measured using CellTiter Glo. Figures 13A and 13B relate to constructs with a PQARK cleavage site. Figures 13C and 13D relate to constructs with a YARKGGI cleavage site. Figures 13E and 13F relate to constructs without a cleavage site. [Figure 14] These figures show the results of efficacy experiments using the TA-PD1-IL2v cleavable (YAARKGGI 38-mer linker) and non-cleavable Mabs as single agents. The MCA205 fibrosarcoma carcinoma cell line was subcutaneously injected into Black6-huPD1 tg mice to study tumor growth inhibition in a subcutaneous model. Tumor size was measured using a caliper. Treatment began when tumors reached 150 mm3. The amount of antibody injected per mouse was 2 mg / kg for the TA-PD1-IL2v YAARKGGI 38-mer cleavable and TA-PD1-IL2v non-cleavable antibodies, administered twice weekly. Treatment continued for one week. The TA-PD-IL2v YAARKGGI 38-mer demonstrated superior efficacy in tumor growth inhibition compared with the vehicle and non-cleavable Mab single-agent groups. [Figure 15] Combination partner (human FolR1-targeting T cell engager) for in vivo efficacy studies. (P1AK1120) A 2+1 human FolR1-targeting T cell engager used as a combination partner in in vivo efficacy studies. [Figures 16A-16C]Figure 16A shows mouse surrogates of human PD1-targeted masked IL2v constructs with and without PQARK matriptase sites. Figure 16A: (P1AK3638) Bivalent human PD1-targeted mouse IgG DA PG with masked IL2v fused to the C-terminus of the FcDD chain and two PQARK matriptase sites ("in-line") for mask release; Figure 16B: (P1AK3649) Bivalent human PD1-targeted mouse IgG DA PG with masked IL2v fused to the C-terminus of the FcDD chain ("in-line") and without a matriptase release site (non-cleavable control). Figure 16C: (P1AG7552) Bivalent human PD1-targeted mouse IgG DA PG with IL2v fused to the C-terminus of the FcDD chain (unmasked control). [Figures 17A-17D] Figure 17A shows mouse surrogates of the mouse PD1-targeted masked IL2v construct with or without a PQARK matriptase site and a control IgG. Figure 17A: (P1AK3641) Bivalent mouse PD1-targeted mouse IgG DA PG with masked IL2v fused to the C-terminus of the FcDD-chain and two PQARK matriptase sites ("in-line") for mask release; Figure 17B: (P1AK3640) Bivalent mouse PD1-targeted mouse IgG DA PG with masked IL2v fused to the C-terminus of the FcDD-chain ("in-line") and no matriptase release site (non-cleavable control); Figure 17C: (P1AD4006) Mouse PD1-targeted mouse IgG used as a non-IL2v-fused control construct. Figure 17D: (P1AG9991) Bivalent mouse PD1-targeted mouse IgG DAPG with IL2v fused to the C-terminus of the FcDD-chain (unmasked control). [Figures 18A-18B] HEK Blue IL2 reporter cell assay using HEK Blue IL2 cells overexpressing human PD1 to test the activity of the murine TA PD1-IL2v construct. The construct was predigested with recombinant human matripatase (Figure 18A). The construct was tested without predigestion (Figure 18B). [Figures 19A-19B]HEK Blue IL2 reporter cell assay using HEK Blue IL2 cells overexpressing mouse PD1 to test the activity of the murine TA PD1-IL2v construct containing the mouse-specific PD1 binder. The construct was predigested with recombinant human matriptase (FIG. 19A). The construct was tested without predigestion (FIG. 19B). [Figures 20A-20B] Proliferation of NK cells (FIG. 20A) and CD8 T cells (FIG. 20B) measured by CFSE dilution upon treatment with recombinant human matriptase-digested TA PD1-IL2v construct. [Figures 21A-21B] Activation of NK cells (FIG. 21A) and CD8 T cells (FIG. 21B) as measured by CD25 upregulation upon treatment with recombinant human matriptase-digested TAPD1-IL2v construct. [Figure 22] These results show the results of efficacy experiments using TA-PD1-IL2v cleavable (PQARK 25mer linker), non-cleavable, and pembrolizumab Mabs as single agents. The MCA205 fibrosarcoma carcinoma cell line was subcutaneously injected into Black6-huPD1 tg mice to study tumor growth inhibition in a subcutaneous model. Tumor size was measured using calipers. Treatment began when tumors reached 200 mm3. The amount of antibody injected per mouse was 1 and 3 mg / kg for the TA-PD1-IL2v PQARK 25mer cleavable, and 3 mg / kg for pembrolizumab and TA-PD1-IL2v non-cleavable, administered twice weekly. Treatment continued for one week. The TA-PD-IL2v PQARK 25mer demonstrated superior efficacy in tumor growth inhibition compared with vehicle, pembrolizumab, and non-cleavable Mab single-agent groups. [Figure 23]Results of efficacy experiments using the cleavable muTA-PD1-IL2v (PQARK 25mer linker) and muPD1 Mab as single agents are shown. The GL261 glioblastoma cell line was subcutaneously injected into Black6 mice to study tumor growth inhibition in a subcutaneous model. Tumor size was measured using calipers. Treatment began when tumors reached 100 mm3. The amount of antibody injected per mouse was 1 and 3 mg / kg for the muTA-PD1-IL2v PQARK 25mer cleavable, administered twice weekly, and 3 mg / kg for muPD1. Treatment continued for one week. The mu-TA-PD-IL2v PQARK 25mer demonstrated superior efficacy in tumor growth inhibition compared with the vehicle and muPD1 Mab single-agent groups. [Figure 24] Results from an efficacy study evaluating the combination of TA-PD1-IL2v and FOLR1-TCBMab are shown. BC004 human breast cancer PDX cells were subcutaneously injected into humanized NSG mice to test tumor growth inhibition in a subcutaneous breast xenograft model. The amounts of antibodies (mg / kg) injected per mouse were as follows: 1 mg / kg TA-PD1-IL2v PQARK 25mer cleavable linker, 0.1 mg / kg unmasked PD1-IL2v, 1 mg / kg pembrolizumab, and 0.3 mg / kg FOLR1-TCB Mab. Antibodies were intravenously injected once weekly for 4 weeks. Significantly superior tumor growth inhibition was observed with the FOLR1-TCB + TA-PD1-IL2v PQARK 25mer cleavable linker combination compared to the FOLR1-TCB monotherapy and FOLR1-TCB + pembrolizumab combination groups. The combination of FOLR1-TCB + TA-PD1-IL2v PQARK cleavable linker showed similar tumor growth inhibition as the combination of FOLR1-TCB + PD1-IL2v unmasked group. DETAILED DESCRIPTION OF THE INVENTION

[0037] definition Unless otherwise defined below, terms are used herein as commonly used in the art.

[0038] The term "interleukin-2" or "IL-2," as used herein, unless otherwise indicated, refers to any native IL-2 from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses unprocessed IL-2 and any form of IL-2 resulting from processing of cells. The term also encompasses naturally occurring variants of IL-2, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human IL-2 is set forth in SEQ ID NO:62.

[0039] As used herein, the term "IL-2 variant" or "variant IL-2 polypeptide" is intended to encompass all variants of the various forms of the IL-2 molecule, including full-length IL-2, truncated forms of IL-2, and forms in which IL-2 is linked to another molecule by fusion, chemical conjugation, or the like. "Full-length," when used in reference to IL-2, is intended to mean the mature, native-length IL-2 molecule. For example, full-length human IL-2 refers to a molecule containing 133 amino acids (e.g., SEQ ID NO: 62). Various forms of IL-2 variants are characterized by having at least one amino acid mutation that affects the interaction of IL-2 with CD25. This mutation may include substitution, deletion, truncation, or modification of the wild-type amino acid residue normally located at that position. Variants obtained by amino acid substitution are preferred. Unless otherwise indicated, IL-2 variants may be referred to herein as IL-2 variant peptide sequences, IL-2 variant polypeptides, IL-2 variant proteins, or IL-2 variant analogs.

[0040] The nomenclature of various forms of IL-2 is given herein with reference to the sequence shown in SEQ ID NO: 62. Various names may be used herein to denote the same mutation. For example, a mutation from phenylalanine to alanine at position 42 is designated 42A, A42, A42 , F42A or Phe42Ala.

[0041] As used herein, a "wild-type" form of IL-2 is a form of IL-2 that is the same as the mutant IL-2 polypeptide, except that the wild-type form has the wild-type amino acid at each amino acid position of the mutant IL-2 polypeptide. For example, if the IL-2 variant is full-length IL-2 (i.e., IL-2 is not fused or conjugated to any other molecule), the wild-type form of this isomer is full-length native human IL-2. If the IL-2 variant is a fusion of IL-2 with another polypeptide (e.g., an antibody chain) encoded downstream of IL-2, the wild-type form of this IL-2 isomer is IL-2 with the wild-type amino acid sequence fused to the same downstream polypeptide. Furthermore, if the IL-2 variant is a truncated form of IL-2 (a mutated or modified form of the non-truncated portion of IL-2), the wild-type form of this IL-2 isomer is similarly truncated IL-2 with the wild-type sequence. For purposes of comparing the IL-2 receptor binding affinity or bioactivity of various forms of IL-2 variants with the corresponding wild-type form of IL-2, the term wild-type encompasses forms of IL-2 that contain one or more amino acid mutations (e.g., a substitution of alanine for cysteine ​​at a position corresponding to residue 125 in human IL-2) relative to naturally occurring native IL-2 that do not affect IL-2 receptor binding. In some embodiments, wild-type IL-2 for purposes of the present invention contains the amino acid substitution C125A. In a specific embodiment of the present invention, the wild-type IL-2 polypeptide that is compared to the variant IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO:62.

[0042] The term "CD25" or "α-subunit of the IL-2 receptor," as used herein, unless otherwise indicated, refers to any native CD25 from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed CD25 and any form of CD25 resulting from processing of cells. The term also encompasses naturally occurring variants of CD25, such as splice variants or allelic variants. In a specific embodiment, the CD25 is human CD25.

[0043] As used herein, the term "high affinity IL-2 receptor" refers to the receptor γ subunit (common cytokine receptor γ subunit, γ c , or CD132), a receptor β subunit (also known as CD122 or p70), and a receptor α subunit (also known as CD25 or p55). In contrast, the term "intermediate affinity IL-2 receptor" refers to an IL-2 receptor that contains only the γ- and β-subunits, but not the α-subunit (for a review, see, e.g., Olejniczak and Kasprzak, Med Sci Monit 14, RA179-189 (2008)).

[0044] "Regulatory T cells" or "T reg "T cells" are a special type of CD4 T cell that can suppress the responses of other T cells. + It stands for T cells. reg T cells are characterized by expression of the α-subunit of the IL-2 receptor (CD25) and the transcription factor forkhead box P3 (FOXP3) (Sakaguchi, Annu Rev Immunol 22, 531-62 (2004)), which play an important role in the induction and maintenance of peripheral self-tolerance to antigens, including those expressed by tumors. reg Cells require IL-2 for their function and growth, as well as for the induction of their suppressive properties.

[0045] As used herein, the term "effector cells" refers to a population of lymphocytes that mediate the cytotoxic effects of IL-2. Effector cells include effector T cells, e.g., CD8 + These include cytotoxic T cells, NK cells, lymphokine-activated killer (LAK) cells and macrophages / monocytes.

[0046] As used herein, the term "antigen-binding molecule" refers in the broadest sense to a molecule that specifically binds to an antigenic determinant. Examples of antigen-binding molecules are immunoglobulins and derivatives, such as fragments thereof.

[0047] The term "bispecific" means that an antigen-binding molecule can specifically bind to at least two distinct antigenic determinants. Typically, a bispecific antigen-binding molecule contains two antigen-binding sites, each specific for a different antigenic determinant. In certain embodiments, a bispecific antigen-binding molecule can simultaneously bind to two antigenic determinants (particularly, two antigenic determinants expressed on two distinct cells).

[0048] As used herein, the term "valency" indicates that a specific number of antigen-binding sites is present in an antigen-binding molecule. Thus, the term "monovalent binding to an antigen" indicates that one (and not more than one) antigen-binding site specific for that antigen is present in the antigen-binding molecule.

[0049] "Antigen-binding site" refers to the site (i.e., one or more amino acid residues) of an antigen-binding molecule that interacts with an antigen. For example, the antigen-binding site of an antibody comprises amino acid residues from the complementarity-determining regions (CDRs). A native immunoglobulin molecule typically contains two antigen-binding sites, and a Fab molecule typically has one antigen-binding site.

[0050] As used herein, the term "antigen-binding moiety" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, an antigen-binding moiety can direct the entity to which it binds (e.g., a second antigen-binding moiety) to a target site, such as a particular type of tumor cell or tumor stroma bearing the antigenic determinant. In another embodiment, an antigen-binding moiety can activate signaling through its target antigen, e.g., a T-cell receptor complex antigen. Antigen-binding moieties include antibodies and fragments thereof as further defined herein. Particular antigen-binding moieties comprise the antigen-binding domain of an antibody, comprising an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, an antigen-binding moiety can comprise an antibody constant region as further defined herein and known in the art. Useful heavy chain constant regions include any of the five isotypes: α, δ, ε, γ, or μ. Useful light chain constant regions include any of the two isotypes: κ and λ.

[0051] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site on a polypeptide macromolecule (e.g., a three-dimensional structure composed of a contiguous stretch of amino acids or distinct regions of non-contiguous amino acids) to which an antigen-binding moiety binds to form an antigen-binding moiety-antigen complex. Useful antigenic determinants may be found, for example, on the surface of tumor cells, on the surface of virally infected cells, on the surface of other diseased cells, on the surface of immune cells, free in serum, and / or in the extracellular matrix (ECM). Unless otherwise indicated, proteins referred to herein as antigens are proteins found in mammals, including primates (e.g., humans) and rodents (e.g., mice and rats). The antigen may be in its native form from any vertebrate source, including any mammalian species. In certain embodiments, the antigen is a human protein. When a particular protein is referred to herein, the term encompasses not only the "full-length," unprocessed protein, but also any form of the protein resulting from processing within the cell. The term also encompasses naturally occurring variants of the protein, such as splice variants or allelic variants. The binding ability of an antigen-binding moiety to a particular antigenic determinant can be determined by enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art, such as surface plasmon resonance (SPR) analysis (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and classical binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the degree of binding of the antigen-binding moiety to an unrelated protein is less than about 10% of the binding of the antigen-binding moiety to the antigen as measured, for example, by SPR. In certain embodiments, the antigen-binding moiety or antigen-binding molecule comprising the antigen-binding moiety binds to the antigen with a specific affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., ≦10 -8 M or less, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 Dissociation constant (K D )

[0052] "Affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antigen-binding moiety and an antigen, or a receptor and its ligand). The affinity of a molecule X for its partner Y is usually expressed by a dissociation constant, and this dissociation constant (K D ) are the dissociation rate constant and the association rate constant (k off and k on ) is the ratio of the rate constants. Thus, equivalent affinities can include different rate constants as long as the ratio of the rate constants remains the same. Affinity can be measured by well-established methods known in the art, including those described herein. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0053] "Decreased binding," e.g., decreased binding to an Fc receptor, refers to a decrease in affinity for the respective interaction, as measured, for example, by SPR. For clarity, the term also includes a reduction in affinity to zero (or below the detection limit of the analytical method), i.e., a complete loss of interaction. Conversely, "increased binding" refers to an increase in binding affinity for the respective interaction.

[0054] As used herein, "T cell activation" refers to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers.

[0055] As used herein, "target cell antigen" refers to an antigenic determinant displayed on the surface of a target cell, e.g., a cell within a tumor, such as a cancer cell or a cell of the tumor stroma.

[0056] As used herein, the terms "first" and "second" with respect to antigen-binding moieties, etc., are used for convenience to distinguish when there is more than one of each type of moiety. The use of these terms is not intended to confer a particular order or orientation of the protease-activatable IL-2 polypeptide or immune complex unless explicitly stated.

[0057] A "Fab molecule" refers to a protein consisting of the VH and CH1 domains of an immunoglobulin heavy chain (a "Fab heavy chain") and the VL and CL domains of a light chain (a "Fab light chain").

[0058] "TA" stands for tumor activatable. "OA" stands for 2-arm.

[0059] By "fused" is meant that the components (eg, a Fab molecule and an Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0060] As used herein, the term "single chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In certain embodiments, one of the antigen-binding moieties is a single-chain Fab molecule, i.e., a Fab molecule in which the Fab light chain and the Fab heavy chain are linked by a peptide linker to form a single peptide chain. Another term is single-chain variable fragment (scFv). In certain such embodiments, the C-terminus of the Fab light chain in the single-chain Fab molecule is linked to the N-terminus of the Fab heavy chain.

[0061] A "crossover" Fab molecule (also referred to as "Crossfab") refers to a Fab molecule in which either the variable or constant regions of the Fab heavy and light chains have been exchanged; i.e., the crossover Fab molecule comprises a peptide chain consisting of a light chain variable region and a heavy chain constant region, and a peptide chain consisting of a heavy chain variable region and a light chain constant region. For clarity, in a crossover Fab molecule in which the variable regions of the Fab light chain and the Fab heavy chain have been exchanged, the peptide chain comprising the heavy chain constant region is referred to herein as the "heavy chain" of the crossover Fab molecule. Conversely, in a crossover Fab molecule in which the constant regions of the Fab light chain and the Fab heavy chain have been exchanged, the peptide chain comprising the heavy chain variable region is referred to herein as the "heavy chain" of the crossover Fab molecule.

[0062] In contrast, a "conventional" Fab molecule refers to a Fab molecule in its native format, i.e., a Fab molecule comprising a heavy chain composed of a heavy chain variable and constant region (VH-CH1) and a light chain composed of a light chain variable and constant region (VL-CL).

[0063] The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, IgG class immunoglobulins are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two disulfide-bonded light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH) (also called a variable heavy domain or a heavy chain variable domain) followed by three constant domains (CH1, CH2, and CH3) (also called a heavy chain constant region). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL) (also called a variable light domain or a light chain variable domain), followed by a constant light (CL) domain (also called a light chain constant region). Immunoglobulin heavy chains can be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), and several of these can be divided into subtypes, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Immunoglobulin light chains may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. Immunoglobulins essentially consist of two Fab molecules and an Fc domain, connected via an immunoglobulin hinge region.

[0064] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0065] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and single-domain antibodies. For a review of specific antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); and WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. See U.S. Patent No. 5,869,046 for a description of Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and having extended in vivo half-lives. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, European Patent No. 404,097, International Publication No. 1993 / 01161, Hudson et al., Nat Med 9, 129-134 (2003), and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In some embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516). Antibody fragments may be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies, as well as production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0066] The term "antigen-binding domain" refers to a part of an antibody that specifically binds to and is complementary to part or all of an antigen. An antigen-binding domain may be provided, for example, by one or more antibody variable domains (also called antibody variable regions). In particular, an antigen-binding domain comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0067] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, and each domain contains four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6 th ed., WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0068] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops ("hypervariable loops"). Native four-chain antibodies generally contain six HVRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs generally contain amino acid residues from the hypervariable loops and / or from the complementarity-determining regions (CDRs), the latter of which exhibit the highest sequence variability and / or are involved in antigen recognition. With the exception of CDR1 in VH, CDRs generally contain amino acid residues that form the hypervariable loops. Hypervariable regions (HVRs) are also referred to as "complementarity-determining regions" (CDRs), and these terms are used interchangeably herein with respect to the portions of the variable domain that form the antigen-binding region. This particular region is described in Kabat et al., US Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and Chothia et al., J Mol Biol 196:901-917 (1987), and the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to the CDR of an antibody or its variants is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues that encompass the CDRs defined by each of the above cited documents are set forth in Table 1 below for comparison. The actual residue numbers that encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the amino acid sequence of the variable region of an antibody. [Table 1]

[0069] Kabat et al. also defined a numbering system for variable region sequences that is applicable to any antibody. One of skill in the art can unambiguously assign this system of "Kabat numbering" to any variable region sequence without reliance on experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described in Kabat et al., U.S. Department of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise specified, references to the numbering of specific amino acid residue positions in antibody variable regions follow the Kabat numbering system.

[0070] The polypeptide sequences in the sequence listing are not numbered according to the Kabat numbering system, however, it is well within the ordinary skill of one in the art to convert the numbering of the sequences in the sequence listing to Kabat numbering.

[0071] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0072] The "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by the antibody or immunoglobulin heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0073] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational truncation of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, antibodies produced by host cells by expression of a particular nucleic acid molecule encoding a full-length heavy chain may contain a full-length heavy chain or a truncated variant of the full-length heavy chain. This may be the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU numbering system). Thus, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present. The amino acid sequence of a heavy chain comprising an Fc region is shown herein without the C-terminal glycine-lysine dipeptide unless otherwise indicated. In one embodiment, a heavy chain comprising an Fc region as designated herein and comprised in an antibody according to the invention comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, EU numbering system). In one embodiment, a heavy chain comprising an Fc region as designated herein and comprised in an antibody according to the invention comprises an additional C-terminal glycine residue (G446, EU index numbering). Unless otherwise specified herein, the numbering of amino acid residues in an Fc region or constant region is according to the EU numbering system (also referred to as the EU index), as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.As used herein, a "subunit" of an Fc domain refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide comprising the C-terminal constant region of an immunoglobulin heavy chain capable of stable self-association. For example, a subunit of an IgG Fc domain comprises the IgG CH2 and IgG CH3 constant domains.

[0074] By "fused" is meant that the components (eg, a Fab molecule and an Fc domain subunit) are linked by a peptide bond, either directly or via one or more peptide linkers.

[0075] A "modification that promotes association of a first subunit and a second subunit of an Fc domain" refers to manipulation of the peptide backbone or post-translational modification of an Fc domain subunit that reduces or prevents the association of a polypeptide containing the Fc domain subunit with an identical polypeptide to form a homodimer. As used herein, a modification that promotes association specifically includes a separate modification for each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) that are desired to associate, where the modifications are complementary to each other so as to promote the association of the two Fc domain subunits. For example, a modification that promotes association may alter the structure or charge of one or both of the Fc domain subunits to sterically or electrostatically favor their association, respectively. Thus, (hetero)dimerization occurs between a polypeptide containing a first Fc domain subunit and a polypeptide containing a second Fc domain subunit, and these polypeptides may not be identical in the sense that the additional components (e.g., antigen-binding moieties) fused to each of the subunits are not the same. In some embodiments, the modification that promotes association comprises an amino acid mutation, particularly an amino acid substitution, within the Fc domain, hi certain embodiments, the modification that promotes association comprises a distinct amino acid mutation, particularly an amino acid substitution, in each of the two subunits of the Fc domain.

[0076] The term "effector function" refers to the biological activity attributable to the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependent cellular phagocytosis (ADCP); cytokine secretion; immune complex-mediated antigen uptake by antigen-presenting cells; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0077] As used herein, the terms "engineer, engineered, engineering" are intended to include any manipulation of the peptide backbone or post-translational modification of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modification of the amino acid sequence, modification of the glycosylation pattern or modification of the side groups of individual amino acids, as well as combinations of these techniques.

[0078] As used herein, the term "immunoconjugate" refers to a polypeptide molecule comprising at least one IL-2 moiety and at least one antigen-binding moiety or effector cell-binding moiety. In some embodiments, the immunoconjugate comprises at least one IL-2 moiety and at least two antigen-binding moieties or at least two effector cell-binding moieties. Particular immunoconjugates according to the invention consist essentially of an IL-2 moiety and two antigen-binding moieties joined by one or more linker sequences. The antigen-binding moieties may be joined to the IL-2 moiety by various interactions and in various configurations described herein. Particular immunoconjugates according to the invention consist essentially of an IL-2 moiety and two effector cell-binding moieties joined by one or more linker sequences. The effector cell-binding moieties may be joined to the IL-2 moiety by various interactions and in various configurations described herein.

[0079] As used herein, the term "amino acid mutation" is intended to encompass amino acid substitution, deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be performed to arrive at the final construct, provided that the final construct possesses the desired characteristics (e.g., decreased binding to Fc receptors or increased association with another peptide). Deletion and insertion of amino acid sequences include deletion and insertion of amino and / or carboxy terminal amino acids. A particular amino acid mutation is an amino acid substitution. For example, to alter the binding characteristics of the Fc region, non-conservative amino acid substitutions, i.e., replacing one amino acid with another amino acid having different structural and / or chemical properties, are particularly preferred. Amino acid substitutions include substitutions with non-naturally occurring amino acids or substitutions with naturally occurring amino acid derivatives of the 20 common amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. It is contemplated that methods other than genetic engineering, such as chemical modification, for modifying the side chain group of an amino acid may also be useful. Various names may be used herein to refer to the same amino acid mutation. For example, a proline to glycine substitution at position 329 of the Fc domain is referred to as 329G, G329, G 329 , P329G or Pro329Gly.

[0080] As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The term "polypeptide" refers to a chain of two or more amino acids, not a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain of two or more amino acids is included within the definition of "polypeptide," and the term "polypeptide" may be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. Polypeptides may be derived from natural biological sources or produced by recombinant technology, but are not necessarily translated from a specified nucleic acid sequence. Polypeptides may be generated by any method, including chemical synthesis. Polypeptides of the invention may be about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1000 or more, or 2000 or more amino acids in size. Polypeptides may have a well-defined three-dimensional structure, but do not necessarily have such a structure. Polypeptides that have a defined three-dimensional structure are referred to as folded, while polypeptides that do not have a defined three-dimensional structure but rather can adopt a number of different conformations are referred to as unfolded.

[0081] By "isolated" polypeptide or a variant or derivative thereof is intended a polypeptide that is not in its natural environment. A particular level of purification is not required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purposes of the present invention, as are native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0082] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California), or can be compiled from its source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is used for amino acid sequence comparison, the percent amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B, with amino acid sequence B, or with amino acid sequence B (alternatively, it can be written as a given amino acid sequence A having or containing a certain percent amino acid sequence identity with or opposite amino acid sequence B to a given amino acid sequence B) is calculated as follows: 100 x fraction X / Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically indicated otherwise, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0083] The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), viral RNA, or plasmid DNA (pDNA). Polynucleotides can contain conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, as found in peptide nucleic acids (PNAs)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.

[0084] By "isolated" nucleic acid molecule or polynucleotide is intended a nucleic acid molecule, DNA, or RNA, that has been removed from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for purposes of the present invention. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) in solution. Isolated polynucleotides include polynucleotide molecules that are originally contained in a cell that contains the polynucleotide molecule, but where the polynucleotide molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present invention, positive- and negative-stranded forms, and double-stranded forms. Furthermore, isolated polynucleotides or nucleic acids of the present invention include such molecules produced synthetically. In addition, polynucleotides or nucleic acids may be or include regulatory elements, such as a promoter, ribosome binding site, or transcription terminator.

[0085] A nucleic acid or polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention is intended to be identical to the reference sequence, except that the nucleotide sequence of the polynucleotide may contain up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with other nucleotides, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These changes to the reference sequence may occur at the 5' or 3' terminal position of the reference nucleotide sequence, or anywhere between these terminal positions, and may be interspersed individually among the residues in the reference sequence, or may be interspersed among one or more consecutive groups in the reference sequence. In practical terms, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be conventionally determined using known computer programs, such as those described above for polypeptides (e.g., ALIGN-2).

[0086] The term "expression cassette" refers to a recombinantly or synthetically produced polynucleotide that contains a specific set of nucleic acid elements capable of transcribing a specific nucleic acid in a target cell. Recombinant expression cassettes can be incorporated into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, the recombinant expression cassette portion of an expression vector contains, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In certain embodiments, expression cassettes of the invention contain a polynucleotide sequence encoding a bispecific antigen-binding molecule of the invention, or a fragment thereof.

[0087] The term "vector" or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule used to introduce and direct the expression of a specific gene with which it is operably associated in a cell. This term encompasses vectors that integrate into the genome of a host cell into which they are introduced, as well as vectors as self-replicating nucleic acid structures. The expression vector of the present invention comprises an expression cassette. The expression vector allows for the stable transcription of large amounts of mRNA. Once the expression vector is inside the target cell, the protein encoded by the ribonucleic acid molecule or gene is produced by the cell's transcriptional and / or translational machinery. In one embodiment, the expression vector of the present invention comprises an expression cassette comprising a polynucleotide sequence encoding a bispecific antibody of the present invention, or a fragment thereof.

[0088] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," and include the primary transformed cell and its progeny, regardless of the number of passages. The progeny may not have exactly the same nucleic acid content as the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. Host cells are any type of cell line that can be used to produce the bispecific antibodies of the present invention. Host cells include cultured cells, e.g., cultured mammalian cells such as CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, and PER.C6 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, to name a few, as well as cells contained within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0089] An "activating Fc receptor" is an Fc receptor that, following binding of the Fc domain of an antibody, triggers signaling events that stimulate the receptor-bearing cell to carry out an effector function. Human activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0090] Antibody-dependent cellular cytotoxicity (ADCC) is an immune mechanism that causes immune effector cells to lyse antibody-coated target cells. Target cells are cells to which an antibody or its derivative, including an Fc region, specifically binds via a protein portion generally N-terminal to the Fc region. As used herein, the term "reduced ADCC" is defined as either a reduction in the number of target cells lysed in a given time period with a given concentration of antibody in the medium surrounding the target cells, via the ADCC mechanism defined above, and / or an increase in the concentration of antibody in the medium surrounding the target cells required to achieve lysis of a given number of target cells in a given time period via the ADCC mechanism. Reduced ADCC is compared to unengineered ADCC mediated by the same antibody produced by the same type of host cell using the same standard production, purification, formulation, and storage methods (known to those skilled in the art). For example, an amino acid substitution that reduces ADCC mediated by an antibody containing its Fc domain is relative to ADCC mediated by the same antibody without this amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, for example, PCT Application Nos. WO 2006 / 082515 or WO 2012 / 130831).

[0091] An "effective amount" of a drug is the amount necessary to effect a physiological change in the cells or tissue to which it is administered.

[0092] A "therapeutically effective amount" of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents the side effects of a disease.

[0093] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, livestock animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.

[0094] The term "pharmaceutical composition" refers to a formulation in a form that allows for the biological activity of the active ingredient contained therein to be effective and does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0095] "A 'pharmaceutically acceptable carrier' refers to an ingredient in a pharmaceutical composition, other than an active ingredient, that is not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0096] As used herein, "treatment" (and grammatical variants thereof, e.g., "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of disease in the treated individual and may be performed prophylactically or during the course of clinical pathology. Desired effects of treatment include preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, remission or palliation of disease symptoms, and improving or improving prognosis. In some embodiments, the protease-activatable IL-2 polypeptides or immunoconjugates of the invention are used to delay the onset of disease or slow the progression of disease.

[0097] The term "package insert" is used to refer to instructions customarily included in commercial packaging of therapeutic products that contain information about the indications, usage, dosage, administration, concomitant therapy, contraindications and / or warnings for the use of such therapeutic product.

[0098] As used herein, "idiotype-specific polypeptide" refers to a polypeptide that recognizes the idiotype of an antigen-binding portion, such as an antigen-binding portion for CD3. An idiotype-specific polypeptide specifically binds to the variable region of the antigen-binding portion, thereby reducing or preventing the specific binding of the antigen-binding portion to its cognate antigen. When associated with a molecule containing an antigen-binding portion, the idiotype-specific polypeptide can function as a masking portion of the molecule. Specifically disclosed herein are anti-idiotype antibodies or anti-idiotype-binding antibody fragments specific for the idiotype of an anti-CD3 binding molecule.

[0099] As used herein, "protease" or "protease" refers to any proteolytic enzyme expressed by a target cell that cleaves a linker at a recognition site. Such proteases may be secreted by the target cell or may remain associated with the target cell, for example, on the target cell surface. Examples of proteases include, but are not limited to, metalloproteinases, such as matrix metalloproteinases 1-28 and a disintegrin and adenosine phosphoproteinases (ADAM) 2, 7-12, 15, 17-23, 28-30, and 33; serine proteases, such as urokinase-type plasminogen activator and matriptase; cysteine ​​proteases; aspartic acid proteases; and members of the cathepsin family.

[0100] "Protease-activatable," as used herein with respect to an interleukin-2 polypeptide, refers to an interleukin-2 polypeptide that has reduced or eliminated ability to bind to an interleukin-2 receptor due to a masking moiety that reduces or eliminates the ability of the interleukin-2 polypeptide to bind to the interleukin-2 receptor. Upon release of the masking moiety by proteolytic cleavage, e.g., by proteolytic cleavage of a linker connecting the masking moiety to the interleukin-2 polypeptide and / or within the masking moiety, binding to the interleukin-2 receptor is restored and the interleukin-2 polypeptide is thereby activated.

[0101] As used herein, "reversibly masking" refers to the binding of a masking moiety to an interleukin-2 polypeptide such that the interleukin-2 polypeptide is prevented from binding to its receptor. Such masking is reversible in that the masking moiety can be released from the interleukin-2 polypeptide, for example, by protease cleavage, thereby freeing the interleukin-2 polypeptide to bind to its receptor.

[0102] Embodiments of the present disclosure In one embodiment, a protease-activatable interleukin-2 (IL-2) polypeptide is provided, comprising: (i) an IL-2 polypeptide; (ii) a masking moiety; and (iii) a linker comprising a first protease cleavage site, wherein the linker has a length of 20 to 45 amino acids; the masking moiety is covalently attached to the IL-2 polypeptide via the linker; the masking moiety is capable of binding to the IL-2 polypeptide, thereby reversibly masking the IL-2 polypeptide; and the masking moiety comprises a second protease cleavage site, wherein the masking moiety does not mask the IL-2 polypeptide upon cleavage at the first and / or second protease cleavage sites. In a preferred embodiment, the linker has a length of 22 to 43 amino acids. In a preferred embodiment, the linker has a length of 25 to 38 amino acids. In a preferred embodiment, the linker has a length of 25. In another preferred embodiment, the linker has a length of 38 amino acids. In one embodiment, the masking moiety is covalently attached to the amino or carboxy terminus of the interleukin-2 polypeptide via a linker. In one embodiment, the masking moiety is an IL-2 antagonist. In one embodiment, the masking moiety is an IL-2 antibody or an IL-2 receptor subunit. In one embodiment, the IL-2 antibody comprises a Fab molecule. In a preferred embodiment, the masking moiety is MT204, preferably an antibody derived from MT204. The MT204 antibody is disclosed, for example, in Volkland et al., Molecular Immunology 44 (2007) 1743-1753, and PCT Application WO 2006 / 128690. In a preferred embodiment, the masking moiety is a deimmunized MT204-derived binder. In a preferred embodiment, the MT204-derived masking moiety comprises a VL domain set forth in SEQ ID NO: 55 and a VH domain set forth in SEQ ID NO: 56. In one embodiment, the Fab molecule is a single-chain Fab molecule. In one embodiment, the second protease cleavage site is located between the variable domain of the heavy chain (VH) and the variable domain of the light chain (VL) of the single-chain Fab molecule.In one embodiment, the first protease cleavage site and the second protease cleavage site each comprise at least one protease recognition sequence.

[0103] In one embodiment, the protease recognition sequence of the first protease cleavage site and / or the protease recognition sequence of the second protease cleavage site is either YAARKGGI set forth in SEQ ID NO: 60 and / or PQARK set forth in SEQ ID NO: 61. In one embodiment, the protease recognition sequence of the first protease cleavage site is YAARKGGI set forth in SEQ ID NO: 60 or PQARK set forth in SEQ ID NO: 61. In one embodiment, the protease recognition sequence of the second protease cleavage site is YAARKGGI set forth in SEQ ID NO: 60 or PQARK set forth in SEQ ID NO: 61. In one embodiment, the protease recognition sequence of the first protease cleavage site is YAARKGGI set forth in SEQ ID NO: 60 and the protease recognition sequence of the second protease cleavage site is PQARK set forth in SEQ ID NO: 61. In one embodiment, the protease recognition sequence of the first protease cleavage site is PQARK set forth in SEQ ID NO:61, and the protease recognition sequence of the second protease cleavage site is YAARKGGI set forth in SEQ ID NO:60.

[0104] In one embodiment, the IL-2 polypeptide is wild-type IL-2, preferably human IL-2 as set forth in SEQ ID NO: 62, or a mutant IL-2 polypeptide. In one embodiment, the mutant IL-2 polypeptide comprises any amino acid substitution selected from the group T3A, F42A, Y45A, L72G, C125A of human IL-2 as set forth in SEQ ID NO: 62. In one embodiment, the mutant IL-2 polypeptide comprises the amino acid substitutions F42A, Y45A, and L72G of human IL-2 as set forth in SEQ ID NO: 62. In one embodiment, the mutant IL-2 polypeptide comprises the amino acid substitutions T3A, F42A, Y45A, L72G, and C125A of human IL-2 as set forth in SEQ ID NO: 62.

[0105] In one specific embodiment, the protease-activatable IL-2 polypeptide comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 27. In one specific embodiment, the protease-activatable IL-2 polypeptide comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 28. In one specific embodiment, the protease-activatable IL-2 polypeptide comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 29. In one specific embodiment, the protease-activatable IL-2 polypeptide comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 30.

[0106] In one particular embodiment, the protease-activatable IL-2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 27. In one particular embodiment, the protease-activatable IL-2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 28. In one particular embodiment, the protease-activatable IL-2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 29. In one particular embodiment, the protease-activatable IL-2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 30.

[0107] In one embodiment, the IL-2 polypeptide is further linked to a non-IL-2 moiety. In one embodiment, the IL-2 polypeptide shares a carboxy-terminal peptide bond with the masking moiety and an amino-terminal peptide bond with the non-IL-2 moiety. In one embodiment, the IL-2 polypeptide shares an amino-terminal peptide bond with the masking moiety and a carboxy-terminal peptide bond with the non-IL-2 moiety. In one embodiment, the non-IL-2 moiety is an antigen-binding moiety or an effector cell-binding moiety.

[0108] immune complex In one embodiment, the invention provides an immunoconjugate comprising a protease-activatable IL-2 polypeptide described herein and an antigen-binding portion and an effector cell-binding portion. In one embodiment, the invention provides an immunoconjugate comprising a protease-activatable IL-2 polypeptide described herein and an antigen-binding portion. In one embodiment, the invention provides an immunoconjugate comprising a protease-activatable IL-2 polypeptide described herein and an effector cell-binding portion.

[0109] In one embodiment, the protease-activatable IL-2 polypeptide shares an amino- or carboxy-terminal peptide bond with the antigen-binding portion or the effector cell-binding portion. In one embodiment, the immune complex comprises a first and a second antigen-binding portion, or a first and a second effector cell antigen-binding portion, or an antigen-binding portion and an effector cell-binding portion. In one embodiment, (i) the protease-activatable IL-2 polypeptide shares an amino- or carboxy-terminal peptide bond with the first antigen-binding portion, and the second antigen-binding portion shares an amino- or carboxy-terminal peptide bond with a) the protease-activatable IL-2 polypeptide or b) the first antigen-binding portion; or (ii) the protease-activatable IL-2 polypeptide shares an amino- or carboxy-terminal peptide bond with the first effector cell-binding portion, and the second effector cell-binding portion shares an amino- or carboxy-terminal peptide bond with a) the protease-activatable IL-2 polypeptide or b) the first effector cell-binding portion. (iii) the protease-activatable IL-2 polypeptide shares an amino- or carboxy-terminal peptide bond with the antigen-binding portion and the effector cell-binding portion shares an amino- or carboxy-terminal peptide bond with either a) the protease-activatable IL-2 polypeptide or b) the antigen-binding portion; or (iv) the protease-activatable IL-2 polypeptide shares an amino- or carboxy-terminal peptide bond with the effector cell-binding portion and the antigen-binding portion shares an amino- or carboxy-terminal peptide bond with either a) the protease-activatable IL-2 polypeptide or b) the effector cell-binding portion.

[0110] In one embodiment, the antigen-binding portion or effector cell-binding portion comprised in the protease-activatable IL-2 polypeptide disclosed herein or the immunoconjugate disclosed herein is an antibody or antibody fragment. In one embodiment, the antigen-binding portion and / or the effector cell-binding portion is selected from a Fab molecule and an scFv molecule. In one embodiment, the antigen-binding portion and / or the effector cell-binding portion is an immunoglobulin molecule, particularly an IgG molecule. In one embodiment, the antigen-binding portion is directed against an antigen presented on or in the tumor cell environment, and / or the effector cell-binding portion is directed against an effector cell present in the tumor cell environment to achieve stimulatory targeting.

[0111] In certain embodiments, the immunoconjugate comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 5, an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 22, and an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 23. In certain embodiments, the immunoconjugate comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 5, an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 22, and an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 24. In certain embodiments, the immunoconjugate comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 5, an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 22, and an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 25. In certain embodiments, the immunoconjugate comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 5, an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 22, and an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 26.

[0112] In one particular embodiment, the immune complex comprises the amino acid sequence set forth in SEQ ID NO: 23. In one particular embodiment, the immune complex comprises the amino acid sequence set forth in SEQ ID NO: 24. In one particular embodiment, the immune complex comprises the amino acid sequence set forth in SEQ ID NO: 25. In one particular embodiment, the immune complex comprises the amino acid sequence set forth in SEQ ID NO: 26.

[0113] In one embodiment, the immune complex comprises the amino acid sequence set forth in SEQ ID NO: 5, the amino acid sequence set forth in SEQ ID NO: 22, and the amino acid sequence set forth in SEQ ID NO: 23. In a particular embodiment, the immune complex comprises the amino acid sequence set forth in SEQ ID NO: 5, the amino acid sequence set forth in SEQ ID NO: 22, and the amino acid sequence set forth in SEQ ID NO: 24. In a particular embodiment, the immune complex comprises the amino acid sequence set forth in SEQ ID NO: 5, the amino acid sequence set forth in SEQ ID NO: 22, and the amino acid sequence set forth in SEQ ID NO: 25. In a particular embodiment, the immune complex comprises the amino acid sequence set forth in SEQ ID NO: 5, the amino acid sequence set forth in SEQ ID NO: 22, and the amino acid sequence set forth in SEQ ID NO: 26.

[0114] Masking part The protease-activatable IL-2 polypeptide of the present invention comprises at least one masking moiety. In one embodiment, the masking moiety masks the IL-2 polypeptide and comprises at least one of the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 of the MT204-derived Fab antibody. In a specific embodiment, the masking moiety masks the IL-2 polypeptide and comprises at least one of the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 of the MT204-derived Fab antibody having the amino acid sequences of SEQ ID NO:55 and SEQ ID NO:56. In one embodiment, the masking moiety comprises the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of the VH domain set forth in SEQ ID NO:56, and the light chain CDR1, light chain CDR2, and light chain CDR3 of the VL domain set forth in SEQ ID NO:55. In a specific embodiment, the masking moiety that masks the IL-2 polypeptide comprises a VL domain set forth in SEQ ID NO: 55 and a VH domain set forth in SEQ ID NO: 56, and the masking moiety is a single chain Fab molecule.

[0115] Linker In one embodiment, the protease-activatable IL-2 polypeptide or immunoconjugate comprises a linker having a protease recognition site comprising a polypeptide sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 63, 64, 65 or 66. In a particular embodiment, the protease recognition site comprises the polypeptide sequence of SEQ ID NO: 63, 64, 65 or 66.

[0116] Polynucleotides The present invention further provides isolated polynucleotides encoding the protease-activatable IL-2 polypeptides or immunoconjugates or fragments thereof described herein.

[0117] Polynucleotides encoding the protease-activatable IL-2 polypeptides or immunoconjugates of the present invention can be expressed as a single polynucleotide encoding the entire protease-activatable IL-2 polypeptide or immunoconjugate, or as multiple (e.g., two or more) co-expressed polynucleotides. The polypeptides encoded by the co-expressed polynucleotides can associate, for example, by disulfide bonds or other means, to form a functional protease-activatable IL-2 polypeptide or immunoconjugate. For example, in the case of an immunoconjugate, the light chain portion of the antigen-binding moiety can be encoded by a separate polynucleotide from the polynucleotide encoding the heavy chain, Fc domain subunit, and optionally another (portion of) antigen-binding moiety of the immunoconjugate. When co-expressed, the heavy chain polypeptide combines with the light chain polypeptide to form the antigen-binding moiety. In another example, a partial immune complex comprising one of two Fc domain subunits and, optionally, one or more (portions of) antigen-binding moieties can be encoded by a separate polynucleotide from the portion of the immune complex comprising the other of the two Fc domain subunits and, optionally, one or more (portions of) antigen-binding moieties. When co-expressed, the Fc domain subunits associate to form the Fc domain.

[0118] In some embodiments, the isolated polynucleotide encodes the entire immunoconjugate of the invention described herein, hi other embodiments, the isolated polynucleotide encodes a polypeptide included in an immunoconjugate of the invention described herein.

[0119] In another embodiment, the present invention relates to an isolated polynucleotide encoding a protease-activatable IL-2 polypeptide or immunoconjugate of the present invention, or a fragment thereof. In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide of the present invention is RNA, e.g., RNA in the form of messenger RNA (mRNA). The RNA of the present invention may be single-stranded or double-stranded.

[0120] Recombinant methods The IL-2 polypeptides or immunoconjugates of the present invention can be obtained, for example, by solid-state peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant production. In the case of recombinant production, for example, one or more polynucleotides encoding the protease-activatable IL-2 polypeptides or immunoconjugates described above are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such polynucleotides can be readily isolated and sequenced using conventional procedures. In one embodiment, vectors, preferably expression vectors, containing one or more of the polynucleotides of the present invention are provided. Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of an IL-2 polypeptide or immunoconjugate along with appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination. See, for example, the techniques described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY (1989). An expression vector may be part of a plasmid, a virus, or a nucleic acid fragment. Expression vectors include expression cassettes into which a polynucleotide encoding a protease-activatable IL-2 polypeptide or immune complex (i.e., a coding region) is cloned in operative association with a promoter and / or other transcriptional or translational control elements. As used herein, a "coding region" is a portion of a nucleic acid consisting of codons that are translated into amino acids. "Stop codons" (TAG, TGA, or TAA) are not translated into amino acids but are considered part of the coding region (if present). However, any adjacent sequences, such as promoters, ribosome binding sites, transcription terminators, introns, 5' and 3' untranslated regions, etc., are not part of the coding region.Two or more coding regions can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate vectors. Furthermore, any vector can contain a single coding region or two or more coding regions; for example, vectors of the present invention can encode one or more polypeptides that are separated into final proteins post- or co-translationally via proteolytic cleavage. Additionally, vectors, polynucleotides, or nucleic acids of the present invention can encode heterologous coding regions, either fused or unfused to the polynucleotide encoding the protease-activatable IL-2 polypeptide or immunoconjugate of the present invention, or a variant or derivative thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as secretory signal peptides or heterologous functional domains. Operable association occurs when the coding region for a gene product, such as a polypeptide, is associated with one or more regulatory sequences in a manner that places expression of the gene product under the influence or control of the one or more regulatory sequences. Two DNA fragments (such as a polypeptide coding region and its associated promoter) are "operably linked" if induction of promoter function results in transcription of mRNA encoding the desired gene product, and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression control sequences to direct expression of the gene product or the ability of the DNA template to be transcribed. Thus, a promoter region is said to be operably linked to a nucleic acid encoding a polypeptide if the promoter is capable of effecting transcription of that nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in a predetermined cell. In addition to the promoter, other transcriptional regulatory elements, such as enhancers, operators, repressors, and transcription termination signals, may be operably linked to the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcriptional control regions are disclosed herein. A variety of transcriptional regulatory regions are known to those skilled in the art.These include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (e.g., the immediate-early promoter linked to intron A), Simian Virus 40 (e.g., the early promoter), and retroviruses (e.g., Rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes, such as actin, heat shock proteins, bovine growth hormone, and rabbit α-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Further suitable transcriptional control regions include tissue-specific promoters and enhancers, and inducible promoters (e.g., tetracycline-inducible promoters). Similarly, various translational control elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly internal ribosome entry sites, or IRES, also known as CITE sequences). The expression cassette may also include other features, such as an origin of replication and / or chromosomal integration elements, such as the long terminal repeats (LTRs) of retroviruses or the inverted terminal repeats (ITRs) of adeno-associated viruses (AAV).

[0121] Polynucleotide and nucleic acid coding regions of the present invention can be associated with additional coding regions encoding secretory or signal peptides that direct the secretion of a polypeptide encoded by a polynucleotide of the present invention. For example, if secretion of an IL-2 polypeptide or immunoconjugate is desired, DNA encoding a signal sequence can be located upstream of the nucleic acid encoding a protease-activatable IL-2 polypeptide or immunoconjugate of the present invention, or a fragment thereof. According to the signal hypothesis, proteins secreted by mammalian cells possess a signal peptide or secretory leader sequence that is cleaved from the mature protein upon initiation of transport of the growing protein chain across the rough endoplasmic reticulum. Those skilled in the art will recognize that polypeptides secreted by vertebrate cells typically possess a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to generate the secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide, e.g., an immunoglobulin heavy or light chain signal peptide, is used, or a functional derivative of that sequence that retains the ability to direct the secretion of a polypeptide operably associated therewith is used. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof can be used. For example, the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

[0122] DNA encoding a short protein sequence that can be used to facilitate subsequent purification (e.g., a histidine tag) or that can aid in labeling the protease-activatable IL-2 polypeptide or immunoconjugate may be included within or at the end of the polynucleotide encoding the protease-activatable IL-2 polypeptide or immunoconjugate.

[0123] In further embodiments, host cells are provided that comprise one or more polynucleotides of the present invention. In certain embodiments, host cells are provided that comprise one or more vectors of the present invention. The polynucleotides and vectors may incorporate any of the features described herein in connection with the polynucleotides and vectors, respectively, alone or in combination. In one such embodiment, the host cell comprises a vector (e.g., is transformed or transfected with such a vector) that comprises a polynucleotide encoding (a portion of) a protease-activatable IL-2 polypeptide or immunoconjugate of the present invention. As used herein, the term "host cell" refers to any type of cell line that can be engineered to produce a protease-activatable IL-2 polypeptide or immunoconjugate of the present invention, or a fragment thereof. Host cells suitable for replicating and supporting expression of a protease-activatable IL-2 polypeptide or immunoconjugate are well known in the art. Such cells can be appropriately transfected or transduced with a particular expression vector, and large quantities of the vector-containing cells can be grown to inoculate large-scale fermenters to obtain sufficient quantities of the IL-2 polypeptide or immunoconjugate for clinical use. Suitable host cells include prokaryotic microorganisms (e.g., Escherichia coli) or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, and the like. For example, polypeptides may be produced in bacteria, particularly if glycosylation is not required. After expression, the polypeptide may be isolated from the bacterial cell paste in a soluble fraction and further purified. In addition to prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeast, are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized" to produce polypeptides with partially or fully human glycosylation patterns. See Gerngross, Nat Biotech 22, 1409-1414 (2004) and Li et al., Nat Biotech 24, 210-215 (2006).Suitable host cells for the expression of (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified and can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be utilized as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (PLANTIBODIES FOR PRODUCING ANTIBODIES IN TRANSGENIC PLANTS). (商標) (See, for example, the description of the technique. Vertebrate cells can also be used as hosts. For example, mammalian cell lines that have been adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines include SV40 (COS-7) transformed monkey kidney CV1; human embryonic kidney lines (e.g., 293 or 293T cells as described in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human hepatocytes (HepG2), mouse mammary tumor cells (MMT060562), TRI cells (e.g., Mather et al., Annals of NY Acad Sci 383, 44-68 (1982), MRC5 cells, and FS4 cells. Other useful mammalian host cell lines include dhfr -Examples include Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)), and myeloma cell lines such as YO, NS0, P3X63, and Sp2 / 0. For a review of specific mammalian host cells suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). Host cells include cultured cells, such as cultured mammalian cells, yeast cells, insect cells, bacterial cells, and plant cells, to name just a few, but also cells contained in transgenic animals, transgenic plants, or cultured plants or animal tissues. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a lymphocytic cell (e.g., a Y0, NS0, Sp20 cell).

[0124] Standard techniques for expressing foreign genes in these systems are known in the art. Cells that express a polypeptide containing either the heavy or light chain of an antigen-binding domain, such as an antibody, may also be engineered to express the other antibody chain, such that the expression product is an antibody having both a heavy and a light chain.

[0125] In one embodiment, a method for producing a protease-IL-2 polypeptide or immunoconjugate according to the invention is provided, the method comprising culturing a host cell comprising a polynucleotide encoding a protease-activatable IL-2 polypeptide or immunoconjugate provided herein under conditions suitable for expression of the protease-activatable IL-2 polypeptide or immunoconjugate, and recovering the protease-activatable IL-2 polypeptide or immunoconjugate from the host cell (or host cell medium).

[0126] The components of the protease-activatable IL-2 polypeptide or immunoconjugate are genetically fused to each other. The protease-activatable IL-2 polypeptide or immunoconjugate can be designed so that its components are fused to each other directly or indirectly through a linker sequence. The composition and length of the linker can be determined according to methods well known in the art and tested for effectiveness. Examples of linker sequences between different components of the protease-activatable IL-2 polypeptide or immunoconjugate can be found in the sequences provided herein. Additional sequences, such as endopeptidase recognition sequences, can be included as needed to incorporate cleavage sites for separating the individual components of the fusion.

[0127] In certain other embodiments, one or more antigen-binding portions of the immune complex comprise at least an antibody variable region capable of binding to an antigenic determinant. The variable region may form part of, and be derived from, naturally occurring or non-naturally occurring antibodies and fragments thereof. Methods for producing polyclonal and monoclonal antibodies are well known in the art (see, e.g., Harlow and Lane, "Antibodies, a laboratory manual," Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodies may be constructed using solid-phase peptide synthesis, produced recombinantly (e.g., as described in U.S. Pat. No. 4,186,567), or obtained, for example, by screening combinatorial libraries containing variable heavy and light chains (see, e.g., U.S. Pat. No. 5,969,108 to McCafferty).

[0128] Antibodies, antibody fragments, antigen-binding domains, or variable regions of any animal species can be used in the immunoconjugates of the present invention. Non-limiting antibodies, antibody fragments, antigen-binding domains, or variable regions useful in the present invention can be of murine, primate, or human origin. If the protease-activatable IL-2 polypeptide or immunoconjugate is intended for use in humans, chimeric forms of antibodies in which the antibody constant region is derived from humans can be used. "Humanized" or fully human forms of antibodies can also be prepared according to methods well known in the art (see, for example, U.S. Patent No. 5,565,332 to Winter). Humanization can be achieved by various methods, including, but not limited to, (a) grafting CDRs of a non-human (e.g., donor antibody) into the framework and constant regions of a human (e.g., recipient antibody) with or without retaining critical framework residues (e.g., those important for maintaining good antigen-binding affinity or antibody function); (b) grafting only non-human specificity-determining regions (SDRs or a-CDRs; residues important for antibody-antigen interactions) into human framework and constant regions; or (c) grafting entire non-human variable domains but "cloaking" them with human-like sections by replacing surface residues.Humanized antibodies and methods for their production are reviewed, e.g., by Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), and are described, e.g., in Riechmann et al., Nature 332, 323-329 (1988); Queen et al., Proc Natl Acad Sci USA 86, 10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Jones et al., Nature 321, 522-525 (1986); Morrison et al., Proc Natl Acad Sci 81, 6851-6855 (1984); Morrison and Oi, Adv Immunol 44, 65-92 (1988); Verhoeyen et al., Science 239, 1534-1536 (1988); Padlan, Molec Immun 31(3), 169-217 (1994); Kashmiri et al., Methods 36, 25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol Immunol 28, 489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36, 43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36, 61-68 (2005) and Klimka et al., Br J Cancer 83, 252-260 (2000) (describing a "guided selection" approach to FR shuffling). Human antibodies and human variable regions can be produced using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr Opin Pharmacol. 5, 368-74 (2001) and Lonberg, Curr Opin Immunol 20, 450-459 (2008).The human variable region may form part of, and be derived from, a human monoclonal antibody produced by the hybridoma method (see, e.g., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). Human antibodies and human variable regions can also be prepared by administering an immunogen to transgenic animals that have been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge (see, e.g., Lonberg, Nat Biotech 23, 1117-1125 (2005)). Human antibodies and human variable regions can also be produced by isolating Fv clone variable region sequences selected from human-derived phage display libraries (see, e.g., Hoogenboom et al. in Methods in Molecular Biology 178, 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001); and McCafferty et al., Nature 348, 552-554; Clackson et al., Nature 352, 624-628 (1991)). Phage typically display antibody fragments as single-chain Fv (scFv) fragments or as Fab fragments.

[0129] In certain embodiments, antigen-binding moieties useful in the present invention are engineered to have enhanced binding affinity, for example, according to the methods disclosed in U.S. Patent Application Publication No. 2004 / 0132066, the entire contents of which are incorporated herein by reference. The binding ability of the immune complexes of the present invention to a specific antigenic determinant can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance technology (analyzed on a BIACORE T 100 system) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). Competition assays can be used to identify antibodies, antibody fragments, antigen-binding domains, or variable domains that compete with a reference antibody for binding to a specific antigen. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) bound by the reference antibody. Detailed exemplary methods for mapping antibody-binding epitopes are provided in Morris (1996) "Epitope Mapping Protocols" in Methods in Molecular Biology, vol. 66 (Humana Press, Totowa, NJ). Protease-activatable IL-2 polypeptides or immune complexes prepared as described herein can be purified by techniques known in the art, such as high-performance liquid chromatography, ion-exchange chromatography, gel electrophoresis, affinity chromatography, and size-exclusion chromatography. The actual conditions used to purify a particular protein will depend, in part, on factors such as net charge, hydrophobicity, and hydrophilicity, and will be apparent to those skilled in the art. For affinity chromatography purification, antibodies, ligands, receptors, or antigens to which the protease-activatable IL-2 polypeptide or immune complex binds can be used. For example, for affinity chromatography purification of the protease-activatable IL-2 polypeptide or immune complex of the present invention, matrices containing protein A or protein G can be used.Sequential protein A or G affinity chromatography and size exclusion chromatography can be used to isolate protease-activatable IL-2 polypeptides or immune complexes. The purity of the protease-activatable IL-2 polypeptides or immune complexes can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high pressure liquid chromatography, and the like.

[0130] Assay The protease-activatable IL-2 polypeptides or immunoconjugates provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by a variety of assays known in the art.

[0131] Affinity assay The affinity of immune complexes for Fc receptors or target antigens can be determined by surface plasmon resonance (SPR) using standard equipment, such as a BIAcore instrument (GE Healthcare), and the receptor or target protein as obtained by recombinant expression, according to the methods described in the Examples. Alternatively, binding of protease-activatable IL-2 polypeptides or immune complexes to different receptors or target antigens can be assessed, for example, by flow cytometry (FACS), using cell lines expressing the particular receptor or target antigen. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0132] According to one embodiment, K D is measured by surface plasmon resonance using a BIACORE® T100 machine (GE Healthcare) at 25°C.

[0133] To analyze the interaction between the Fc moiety and Fc receptors, His-tagged recombinant Fc receptors were captured by anti-Penta His antibodies (Qiagen) immobilized on a CM5 chip, and the bispecific constructs were used as analytes. Briefly, a carboxymethylated dextran biosensor chip (CM5, GE Healthcare) was activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The anti-Penta His antibody was diluted to 40 μg / ml in 10 mM sodium acetate (pH 5.0) and then injected at a flow rate of 5 μl / min, yielding approximately 6500 response units (RU) of bound protein. After the ligand injection, 1 M ethanolamine was injected to block unreacted groups. The Fc receptors were then captured at 4 nM or 10 nM for 60 s. For kinetic measurements, four-fold serial dilutions of the bispecific constructs (ranging between 500 nM and 4000 nM) are injected for 120 seconds in HBS-EP (GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20, pH 7.4) at 25°C and a flow rate of 30 μl / min.

[0134] To determine affinity for the target antigen, the bispecific construct is captured by an anti-human Fab specific antibody (GE Healthcare) immobilized on an activated CM5 sensor chip surface, as described for the anti-Penta-His antibody. The final amount of bound protein is approximately 12,000 RU. The bispecific construct is captured at 300 nM for 90 seconds. The target antigen is passed through the flow cell for 180 seconds at a flow rate of 30 μl / min, ranging in concentration from 250 nM to 1,000 nM. Dissociation is monitored for 180 seconds.

[0135] Bulk refractive index differences are corrected for by subtracting the response obtained with a reference flow cell. The steady-state response is used to determine the dissociation constant, K, by nonlinear curve fitting of the Langmuir binding isotherm. D The association rate (k on ) and dissociation rate (k offThe equilibrium dissociation constant (K) is calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® T100 Evaluation Software Version 1.1.1). D ) is k off / k on It is calculated as a ratio. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999).

[0136] Activity assay The biological activity of the protease-activatable IL-2 polypeptides or immunoconjugates of the invention can be measured by various assays described in the Examples. Biological activity includes, for example, inducing T cell proliferation, inducing signal transduction in T cells, inducing expression of activation markers in T cells, inducing cytokine secretion by T cells, inducing lysis of target cells such as tumor cells, and inducing tumor regression and / or improving survival.

[0137] Compositions, Formulations and Routes of Administration In a further aspect, the present invention provides pharmaceutical compositions comprising any of the protease-activatable IL-2 polypeptides or immunoconjugates provided herein, e.g., for use in any of the following methods of treatment. In one embodiment, the pharmaceutical composition comprises any of the protease-activatable IL-2 polypeptides or immunoconjugates provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises any of the protease-activatable IL-2 polypeptides or immunoconjugates provided herein and at least one additional therapeutic agent, e.g., as described below.

[0138] Further provided is a method for producing a protease-activatable IL-2 polypeptide or immunoconjugate of the invention in a form suitable for in vivo administration, comprising: (a) obtaining a protease-activatable IL-2 polypeptide or immunoconjugate according to the invention; and (b) formulating the protease-activatable IL-2 polypeptide or immunoconjugate with at least one pharmaceutically acceptable carrier, thereby formulating a preparation of the protease-activatable IL-2 polypeptide or immunoconjugate for in vivo administration.

[0139] Pharmaceutical compositions of the present invention comprise a therapeutically effective amount of one or more protease-activatable IL-2 polypeptides or immunoconjugates dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically acceptable or pharmacologically acceptable" refers to molecular entities and compositions that are generally nontoxic to recipients at the dosages and concentrations employed, i.e., do not produce adverse, allergic, or other untoward reactions when administered as needed to animals, e.g., humans. The preparation of pharmaceutical compositions containing at least one protease-activatable IL-2 polypeptide or immunoconjugate, and optionally additional active ingredients, will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed., Mack Printing Company, 1990, incorporated herein by reference. Furthermore, it will be understood that for animal (e.g., human) administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Office of Biological Standards or other national equivalents. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceutically acceptable carriers" are known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference), and include any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonicity agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, such materials, and combinations thereof. Except insofar as a conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0140] The composition can contain different types of carriers depending on whether it is to be administered in solid, liquid, or aerosol form, and whether it needs to be sterile for the route of administration, such as injection. The protease-activatable IL-2 polypeptides or immunoconjugates of the invention (and any additional therapeutic agents) may be administered intravenously, intradermally, intra-arterially, intraperitoneally, intralesionally, intracranially, intra-articularly, intraprostatically, intrasplenicly, intrarenally, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesicularly, transmucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, by local perfusion directly bathing target cells, via a catheter, via a lavage solution, in a cream, in a lipid composition (e.g., liposomes), or by other methods or any combination of the above that would be known to one of skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference). Parenteral administration, particularly intravenous infusion, is most commonly used to administer polypeptide molecules such as the protease-activatable IL-2 polypeptides and immunoconjugates of the present invention.

[0141] Parenteral compositions include those designed for administration by injection, e.g., subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal, or intraperitoneal injection. For injection, the protease-activatable IL-2 polypeptide or immunoconjugate of the present invention can be formulated in aqueous solution, preferably in a physiologically compatible buffer, such as Hank's solution, Ringer's solution, or physiological saline buffer. The solution may contain formulating agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the protease-activatable IL-2 polypeptide and immunoconjugate may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. Sterile injectable solutions are prepared by incorporating the protease-activatable IL-2 polypeptide or immunoconjugate of the present invention in the required amount in an appropriate solvent, with various other ingredients, as listed below, as needed. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and / or other ingredients. In the case of sterile powders for preparing sterile injectable solutions, suspensions, or emulsions, the preferred preparation method is vacuum drying or freeze-drying, which yields a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered liquid medium. The liquid medium should be appropriately buffered, if necessary, and the liquid diluent should first be rendered isotonic with sufficient saline or glucose prior to injection. The composition must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. It will be understood that endotoxin contamination should be kept to a safe level, e.g., less than 0.5 ng / mg protein, to a minimum.Suitable pharmaceutically acceptable carriers include, but are not limited to, buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; Proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, dextran, etc. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound, allowing for the preparation of highly concentrated solutions. Additionally, suspensions of the active compound may be prepared as appropriate oil injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.

[0142] The active ingredient can also be incorporated into colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or into macroemulsions, for example, by coacervation techniques or microcapsules prepared by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained-release preparations may also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, such as films or microcapsules. In certain embodiments, sustained absorption of injectable compositions can be achieved by using agents that delay absorption (e.g., aluminum monostearate, gelatin, or a combination thereof) in the composition.

[0143] In addition to the above-mentioned compositions, protease-activatable IL-2 polypeptide or immunoconjugate can also be formulated as a depot preparation. Such long-acting preparations can be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Thus, for example, protease-activatable IL-2 polypeptide or immunoconjugate can be formulated with a suitable polymer or hydrophobic material (e.g., as an emulsion in an acceptable oil) or ion exchange resin, or as a sparingly soluble derivative, for example, as a sparingly soluble salt.

[0144] Pharmaceutical compositions containing the protease-activatable IL-2 polypeptides or immunoconjugates of the present invention can be produced by conventional mixing, dissolving, emulsifying, encapsulating, entrapping, or lyophilizing processes. Pharmaceutical compositions may be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, additives, or adjuvants that facilitate processing of the protein into a pharmaceutically usable preparation. The appropriate formulation depends on the chosen route of administration.

[0145] The protease-activatable IL-2 polypeptide or immunoconjugate can be formulated into a composition in a free acid or free base, neutral, or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or free base. These include acid addition salts, such as those formed with the free amino groups of the protein composition, or with inorganic acids such as hydrochloric acid or phosphoric acid, or with organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide; or organic bases such as isopropylamine, trimethylamine, histidine, or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than the corresponding free base forms.

[0146] Therapeutic methods and compositions Any of the protease-activatable IL-2 polypeptides or immunoconjugates provided herein can be used in therapeutic methods. The protease-activatable IL-2 polypeptides or immunoconjugates of the invention can be used as immunotherapeutic agents, for example, in the treatment of cancer.

[0147] For use in therapeutic methods, the protease-activatable IL-2 polypeptides or immunoconjugates of the invention will be formulated, administered, and administered in a manner consistent with good medical practice. Factors to consider in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the administration schedule, and other factors known to medical practitioners.

[0148] In one aspect, a protease-activatable IL-2 polypeptide or immunoconjugate of the present invention is provided for use as a pharmaceutical. In a further aspect, a protease-activatable IL-2 polypeptide or immunoconjugate of the present invention is provided for use in treating a disease. In certain embodiments, a protease-activatable IL-2 polypeptide or immunoconjugate of the present invention is provided for use in a method of treatment. In one embodiment, the present invention provides a protease-activatable IL-2 polypeptide or immunoconjugate described herein for use in treating a disease in an individual in need thereof. In certain embodiments, the present invention provides a protease-activatable IL-2 polypeptide or immunoconjugate for use in a method of treating an individual having a disease, the method comprising administering to the individual a therapeutically effective amount of the protease-activatable IL-2 polypeptide or immunoconjugate. In certain embodiments, the disease being treated is a proliferative disorder. In certain embodiments, the disease is cancer. In certain embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease being treated is cancer. In further embodiments, the present invention provides a protease-activatable IL-2 polypeptide or immunoconjugate described herein for use in inducing lysis of target cells, particularly tumor cells. In certain embodiments, the present invention provides a protease-activatable IL-2 polypeptide or immunoconjugate for use in a method of inducing lysis of target cells, particularly tumor cells, in an individual, comprising administering to the individual an effective amount of a protease-activatable IL-2 polypeptide or immunoconjugate to induce lysis of the target cells. An "individual" according to any of the above embodiments is a mammal, preferably a human.

[0149] In a further aspect, the present invention provides use of a protease-activatable IL-2 polypeptide or immunoconjugate of the invention in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of a disease in an individual in need thereof. In a further embodiment, the medicament is for use in a method of treating a disease, comprising administering a therapeutically effective amount of the medicament to an individual having the disease. In a particular embodiment, the disease being treated is a proliferative disorder. In a particular embodiment, the disease is cancer. In one embodiment, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease being treated is cancer. In a further embodiment, the medicament is for inducing lysis of target cells, particularly tumor cells. In yet a further embodiment, the medicament is for use in a method of inducing lysis of target cells, particularly tumor cells, in an individual, the method comprising administering to the individual an effective amount of the medicament to induce lysis of the target cells. The "individual" according to any of the above embodiments may be a mammal, preferably a human.

[0150] In a further aspect, the present invention provides a method for treating a disease. In one embodiment, the method comprises administering to an individual having such a disease a therapeutically effective amount of a protease-activatable IL-2 polypeptide or immunoconjugate of the present invention. In one embodiment, a composition comprising a protease-activatable IL-2 polypeptide or immunoconjugate of the present invention in a pharmaceutically acceptable form is administered to the individual. In certain embodiments, the disease being treated is a proliferative disorder. In certain embodiments, the disease is cancer. In certain embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease being treated is cancer. An "individual" according to any of the above embodiments may be a mammal, preferably a human.

[0151] In a further aspect, the present invention provides a method for inducing lysis of target cells, particularly tumor cells.

[0152] In certain embodiments, the disease to be treated is a proliferative disorder, particularly cancer. Non-limiting examples of cancer include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, stomach cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other cell proliferative diseases that can be treated using the protease-activatable IL-2 polypeptides or immunoconjugates of the present invention include, but are not limited to, neoplasms located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary gland, testes, ovaries, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thoracic region, and genitourinary system. Precancerous conditions or lesions and cancer metastases are also included. In certain embodiments, the cancer is selected from the group consisting of renal cell carcinoma, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, and head and neck cancer. Those skilled in the art will readily recognize that a protease-activatable IL-2 polypeptide or immunoconjugate may often only provide a partial benefit without providing a cure. In some embodiments, a physiological change that has some benefit is also considered therapeutically beneficial. Thus, in some embodiments, the amount of protease-activatable IL-2 polypeptide or immunoconjugate that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount." The subject, patient, or individual in need of treatment is typically a mammal, and more particularly, a human.

[0153] In some embodiments, an effective amount of a protease-activatable IL-2 polypeptide or immunoconjugate of the invention is administered to a cell, hi other embodiments, a therapeutically effective amount of a protease-activatable IL-2 polypeptide or immunoconjugate of the invention is administered to an individual to treat a disease.

[0154] The appropriate dosage of a protease-activatable IL-2 polypeptide or immunoconjugate of the present invention (when used alone or in combination with one or more other additional therapeutic agents) for the prevention or treatment of disease will depend on the type of disease being treated, the route of administration, the patient's weight, the type of IL-2 polypeptide or immunoconjugate, the severity and course of the disease, whether the IL-2 polypeptide or immunoconjugate is administered for prophylactic or therapeutic purposes, previous or current therapeutic interventions, the patient's medical history and response to the protease-activatable IL-2 polypeptide or immunoconjugate, and the judgment of the attending physician. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in the composition and appropriate dose for the individual subject. Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations over various time periods, bolus administration, and pulse infusion.

[0155] A therapeutically effective dose of a protease-activatable IL-2 polypeptide or immunoconjugate described herein will generally provide therapeutic benefit without causing substantial toxicity. The toxicity and therapeutic efficacy of a protease-activatable IL-2 polypeptide or immunoconjugate can be determined by standard pharmaceutical procedures in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine the LD 50 (the dose that is lethal to 50% of the population) and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index, which is the LD 50 / ED 50 The therapeutic index may be expressed as a ratio of 1:1. Protease-activatable IL-2 polypeptides or immunoconjugates that exhibit large therapeutic indices are preferred. In one embodiment, the protease-activatable IL-2 polypeptides or immunoconjugates according to the present invention exhibit a high therapeutic index. Data obtained from cell culture assays and animal studies can be used in formulating a dosage range appropriate for human use. Dosages preferably exceed the ED 100 with little or no toxicity. 50The blood concentration range includes: (a) a concentration of 100 mg / kg / day or more ...

[0156] The attending physician of a patient being treated with a protease-activatable IL-2 polypeptide or immunoconjugate of the present invention will know how and when to discontinue, interrupt, or adjust administration due to toxicity, organ failure, and the like. Conversely, the attending physician will also know to adjust treatment to higher levels if the clinical response is inadequate (without causing toxicity). The magnitude of an administered dose in the management of a target disorder will vary depending on the severity of the condition being treated, the route of administration, and the like. The severity of the condition can, for example, be assessed, in part, by standard prognostic evaluation methods. Furthermore, the dose, and perhaps the frequency of administration, will also vary according to the age, weight, and response of the individual patient.

[0157] Other drugs and treatments The protease-activatable IL-2 polypeptides or immunoconjugates of the present invention may be administered in combination with one or more other agents in a therapeutic regimen. For example, the protease-activatable IL-2 polypeptides or immunoconjugates of the present invention may be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any agent administered to treat a condition or disease in an individual in need of such treatment. Such additional therapeutic agents may include any active ingredients appropriate for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is an immunomodulatory agent, a cytostatic agent, an inhibitor of cell adhesion, a cytotoxic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of cells to apoptosis-inducing factors. In certain embodiments, the additional therapeutic agent is an anti-cancer agent, such as a microtubule-disrupting agent, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, hormone therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an anti-angiogenic agent.

[0158] Such other agents are suitably present in combination in amounts effective for the intended purpose. The effective amount of such other agents will depend on the amount of protease-activatable IL-2 polypeptide or immunoconjugate used, the type of disorder or treatment, and other factors discussed above. Protease-activatable IL-2 polypeptides and immunoconjugates are typically used in the same dosages and using the same routes of administration as described herein, or at about 1-99% of the dosages described herein, or at any dosage and by any route empirically / clinically determined to be appropriate.

[0159] Such combination therapy, as described above, encompasses combined administration (wherein the two or more therapeutic agents are contained in the same or separate compositions) and separate administration, where administration of a protease-activatable IL-2 polypeptide or immunoconjugate of the invention may occur before, simultaneously with, and / or after administration of an additional therapeutic agent and / or adjuvant. A protease-activatable IL-2 polypeptide or immunoconjugate of the invention may be used in combination with radiation therapy.

[0160] manufactured goods In another aspect of the present invention, an article of manufacture containing materials useful for the treatment, prevention, and / or diagnosis of the aforementioned disorders is provided. The article of manufacture comprises a container and a label or package insert affixed to or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container holds the composition by itself or in combination with another composition effective for treating, preventing, and / or diagnosing a condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). At least one active agent in the composition is a protease-activatable IL-2 polypeptide or immunoconjugate of the present invention. The label or package insert indicates that the composition is used for treating the condition of choice. Additionally, the article of manufacture includes (a) a first container containing a composition comprising a protease-activatable IL-2 polypeptide or immunoconjugate of the present invention; and (b) a second container containing the composition, the second container containing an additional cytotoxic or other therapeutic agent. In this embodiment of the invention, the article of manufacture may further comprise a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0161] TIFF2025534285000002.tif226170TIFF2025534285000003.tif246170TIFF2025534285000004.tif209170TIFF2025534285000005.tif209170TIFF2025534285000006.tif209170TIFF2025534285000007.tif209170TIFF2025534285000008.tif209170TIFF2025534285000009.tif209170TIFF2025534285000010.tif209170TIFF2025534285000011.tif239170TIFF2025534285000012.tif239170TIFF2025534285000013.tif239170TIFF2025534285000014.tif197170TIFF2025534285000015.tif197170TIFF2025534285000016.tif240170TIFF2025534285000017.tif234170TIFF2025534285000018.tif172170TIFF2025534285000019.tif197170TIFF2025534285000020.tif197170TIFF2025534285000021.tif239170TIFF2025534285000022.tif239170TIFF2025534285000023.tif197170TIFF2025534285000024.tif239170TIFF2025534285000025.tif246170TIFF2025534285000026.tif239170TIFF2025534285000027.tif238170TIFF2025534285000028.tif246170TIFF2025534285000029.tif233170TIFF2025534285000030.tif233170TIFF2025534285000031.tif233170TIFF2025534285000032.tif233170TIFF2025534285000033.tif197170TIFF2025534285000034.tif197170TIFF2025534285000035.tif69170.

[0162] Further aspects of the present disclosure In a further aspect, the disclosure provides a deimmunized PD-1 binder comprising a heavy chain variable region (VH) comprising heavy chain complementarity determining region (HCDR)1 of SEQ ID NO: 71, HCDR2 of SEQ ID NO: 72, and HCDR3 of SEQ ID NO: 73, and a light chain variable region (VL) comprising light chain complementarity determining region (LCDR)1 of SEQ ID NO: 68, LCDR2 of SEQ ID NO: 69, and LCDR3 of SEQ ID NO: 70. In a specific aspect, the disclosure provides a deimmunized PD-1 binder comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 53 and an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 54. In another aspect, the deimmunized PD-1 binder comprises the amino acid sequence set forth in SEQ ID NO: 53 and the amino acid sequence set forth in SEQ ID NO: 54. In another aspect, the present disclosure provides a use of a deimmunized PD-1 binder disclosed herein, wherein the deimmunized PD-1 binder is present in a therapeutic agent. Preferably, the therapeutic agent is an isolated polypeptide. More preferably, the therapeutic agent is a cancer treatment agent.

[0163] In a further aspect, the disclosure provides a deimmunized MT204-derived binder comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR)1 of SEQ ID NO: 77, an HCDR2 of SEQ ID NO: 78, and an HCDR3 of SEQ ID NO: 79, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR)1 of SEQ ID NO: 74, an LCDR2 of SEQ ID NO: 75, and an LCDR3 of SEQ ID NO: 76. In a particular aspect, the disclosure provides a deimmunized MT204-derived binder comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 55 and an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 56. In another aspect, the deimmunized MT204-derived binder comprises the amino acid sequence set forth in SEQ ID NO: 55 and the amino acid sequence set forth in SEQ ID NO: 56. In a further embodiment, the deimmunized MT204-derived binder is a single-chain Fab. In a further embodiment, the deimmunized MT204-derived binder is a single-chain Fv. In another embodiment, the present disclosure provides a use of the deimmunized MT204-derived binder disclosed herein, wherein the deimmunized MT204-derived binder is present in a therapeutic agent. Preferably, the therapeutic agent is an isolated polypeptide. More preferably, the therapeutic agent is a cancer treatment agent. [Example]

[0164] The following are examples of methods and compositions of the present invention. Given the general description provided above, it is understood that various embodiments may be practiced.

[0165] Example 1 Designing binders with low immunogenic potential To reduce potential immunogenicity issues, we aimed to increase the germline content of two binders that bind to PD1 or interleukin 2 (IL2).

[0166] The PD1 binder (P1AA0927, SEQ ID NOs: 51 and 52) was aligned to the human germline sequence IGHV3-23-01 (Acc. No.: M99660) for the heavy chain and to IGKV4-1-01 (Acc. No.: Z00023) for the light chain. Furthermore, we analyzed the protein sequence using commercially available software for predicting MHC class II binding peptides (software: ISPRI; provided by Epivax).

[0167] In particular, these sequence stretches with predicted MHC class 2 binding should be addressed by increasing germline content or by introducing mutations that reduce the MHC binding score while maintaining antibody affinity and stability. In particular, the sequences around framework 2 and CDR2 of the light chain showed three overlapping peptides with predicted MHC binding. Because germline content reduces binding properties, we manually selected mutations that reduce the predicted binding score and then experimentally verified the maintenance of the desired biochemical properties, as described below.

[0168] For the heavy chain, we focused more on germline. Leucine at position 5 according to the Kabat method was substituted with valine, which is more frequent at this position. Thus, more human germline residues were introduced. The deimmunized PD1 binder used in the following examples comprises the VH sequence of SEQ ID NO: 54 and the VL sequence of SEQ ID NO: 53.

[0169] The IL2 binder MT204 was treated in a similar manner. The light chain was compared with either the human germline IGKV1-16-01 or the trastuzumab VL (CAS No. 180288-69-1) sequence, which is considered to be less immunogenic. For the heavy chain, the sequence IGHV3-23-01 (Acc No.: M99660) was also used as a comparator. The optimized sequence would then have a higher degree of human germline content. Furthermore, the variant MT204_VL1a would have a higher homology to the trastuzumab backbone (position 54 according to the Kabat system). The deimmunized MT204 used in the following examples comprises the VH sequence of SEQ ID NO: 56 and the VL sequence of SEQ ID NO: 55.

[0170] Production and purification of deimmunized MT204 masked and deimmunized anti-PD1 binder variants as IgG Deimmunized sequences of anti-IL2v (P1AH2050-P1AH2052) and anti-PD1 binders (P1AH4157-P1AH4161, P1AI0356-P1AI0360, and P1AI1648-P1AI1652) were cloned as human IgG PGLALAs for initial characterization (Figure 1A-D). The corresponding cDNAs were gene synthesized and cloned into the evitoria vector system using conventional (non-PCR-based) cloning techniques. Plasmid DNA was prepared under low-endotoxin conditions based on anion-exchange chromatography. DNA concentration was measured by measuring absorbance at 260 nm. Sequence accuracy was verified by Sanger sequencing (two sequencing reactions per plasmid).

[0171] Suspension-adapted CHO K1 cells (received from ATCC and adapted at evitria for serum-free growth in suspension culture) were used for production. Seeds were grown in eviGrow medium, a chemically defined, animal-component-free, serum-free medium. Cells were transfected with eviFect (evitria's custom-made, proprietary transfection reagent) and, post-transfection, grown in eviMake2, an animal-component-free, serum-free medium. Supernatants were collected by centrifugation and subsequent filtration (0.2 μm filter).

[0172] IgG was purified using MabSelect™ SuRe™ with Dulbecco's PBS (LonzaBE17-512Q) as the wash buffer, 0.1 M glycine pH 3.5 as the elution buffer, and 1 M Tris-HCl as the neutralization buffer (pH 9). Subsequent size-exclusion chromatography was performed on a HiLoad Superdex 200 pg column using the final buffer as the running buffer. Dialysis (if required) was performed using Pierce Slide-A-Lyzer™ G2 Dialysis Cassettes with a 2K molecular weight cutoff. Antibody concentration (if required) was measured using Amicon® Ultra Centrifugal Filters with a 30 kDa molecular weight cutoff.

[0173] Concentration was determined by measuring absorbance at 280 nm. Extinction coefficients were calculated using a proprietary algorithm in evitria. Purity was determined by analytical size exclusion chromatography using an Agilent AdvanceBio SEC column (300A 2.7 um 7.8 x 300 mm) and DPBS as the running buffer at 0.8 ml / min. Endotoxin content was measured using a Charles River Endosafe PTS system.

[0174] Design, production and purification of a complex PD1-targeted masked IL2v format Four composite PD1-targeting masked IL2v formats were cloned using one deimmunized anti-IL2v mask sequence (derived from P1AH2051, i.e., hu IgG1 GL MT204_VL1 a-combo VH2&VH6 PG LALA) and one deimmunized anti-PD1 binder sequence (derived from P1AH4157, i.e., PD1-376_VL2_VH4) (Figures 2A-2D). These were cloned as bivalent human IgG1 PG LALA antibodies using knob-into-hole heterodimerization of the two heavy chains. The masked IL2v, containing the IL2v cytokine and a C-terminally fused scFv mask, was fused to the C-terminus of the knob heavy chain. Furthermore, the glycine-serine linker sequence between the IL2v cytokine and the scFv mask, as well as the glycine-serine linker between the VH and VL domains of the scFv mask, contain protease recognition sites for specific unmasking and activation of IL2v by proteases in the tumor microenvironment, such as matriptase. The protease recognition site used in P1AI4322 and P1AI4323 was PQARK (Figures 2A-2B), while the protease recognition site used in P1AI4324 and P1AI4325 was YAARKGGI (Figures 2C-2D). Furthermore, the four constructs differ in the length of the protease recognition site, including the linker between the IL2v cytokine and the scFv mask: 38 amino acids for P1AI4322 and P1AI4324, and 25 amino acids for P1AI4323 and P1AI4325. As control constructs, we generated the non-cleavable constructs P1AI4646 (with a 38-amino acid linker between the IL2v cytokine and the scFv mask) and P1AI4647 (with a 25-amino acid linker between the IL2v cytokine and the scFv mask) that lack the protease release site, as well as the unmasked construct P1AI4648 (Figures 3A-C).

[0175] These composite PD1-targeting masked IL2v formats (P1AI4322, P1AI4323, P1AI4324, and P1AI4325) were generated and purified by WuXi Biologics. Briefly, they were transiently transfected into HEK293 cells and purified by MabSelectSuRe LX Protein A affinity chromatography and Superdex200 size-exclusion chromatography. Purification of P1AI4324 included two additional HiTrap SP HP-1 and SP HP-2 cation-exchange chromatography steps between MabSelectSuRe LX Protein A affinity chromatography and Superdex200 size-exclusion chromatography.

[0176] To facilitate in vivo efficacy testing in human PD1 transgenic mice, we generated cleavable (containing the YAARKGGI site) and non-cleavable human PD1-targeted masked IL2v constructs, P1AI4650 and P1AI4651, respectively (Figures 4A-4B). These murine surrogates share the same format as the human constructs but contain murine sequences to avoid immunogenicity. The V domain of the PD1 binder corresponds to its nonhumanized predecessor, but all constant antibody domain sequences are murine. The only human sequences in these surrogates that could not be avoided are the V domain of human IL2v and the mask. Murine surrogates were also generated and purified by WuXi Biologics.

[0177] Surface plasmon resonance (measurement of masking affinity for human IL-2v) The affinity of deimmunized MT204 variants (P1AH2050-P1AH2052, FIG. 1A) for human IL2v (SEQ ID NO: 67) was assessed by surface plasmon resonance (SPR). SPR experiments were performed on a Biacore 8K+ at 25°C using HBS-EP running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% surfactant P20, Cytiva, Freiburg, Germany).

[0178] Anti-PGLALA antibody (M-1.7.24 mu IgG2b) was directly immobilized onto a CM5 chip at pH 5.0 using a standard amine coupling kit (Cytiva, Freiburg, Germany). After activating the sensor surface with a 1:1 mixture of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 0.1 M N-hydroxysuccinimide (NHS), 10 μg / ml of anti-PGLALA (diluted in 10 mM acetate, pH 5.0) was injected at a flow rate of 10 μl / min for 100 s. After blocking with 1 M ethanolamine-HCl, pH 8.5, the coupling procedure resulted in an anti-PGLALA surface density of approximately 5400 RU.

[0179] Deimmunized MT204 variants were captured at a concentration of 50 nM for 60 seconds at a flow rate of 10 μl / min. Human IL2v G4S avihis was injected at various concentrations (800–0.391 nM, 1:2 dilution) through the flow cell at a flow rate of 30 μl / min. Association and dissociation were monitored for 240 and 1000 seconds, respectively. The chip surface was regenerated after each cycle using a single injection (60 seconds) of 10 mM glycine pH 2.0. Bulk refractive index differences were corrected by subtracting the response obtained with a reference flow cell. Curves were fitted using a 1:1 Langmuir interaction model using Biacore Insight Evaluation Software 3.0 (Cytiva, Freiburg, Germany), and the results are summarized in Table 1.1. [Table 1.1]

[0180] Two of the three deimmunized variants of anti-IL2v mask, P1AH2050 and P1AH2051, were comparable in kinetics and affinity to the parent anti-IL2v mask, P1AF7506 (Figure 1E), whereas the third deimmunized variant, P1AH2052, exhibited the fastest dissociation rate of all tested variants and the lowest affinity of 995 pM. The variant P1AH2051, exhibiting the highest affinity of 101 pM, i.e., hu IgG1 GL MT204_VL1 a-combo VH2 and VH6 PG LALA, was selected for cloning into the complex-masked IL2v format.

[0181] Surface plasmon resonance to measure affinity for human PD1 The affinity of deimmunized a-PD1 binders for human PD1-Fc was assessed by surface plasmon resonance (SPR). SPR experiments were performed on either a Biacore T200 (for P1AH4157-P1AH4161 and P1AI0356-P1AI0360) or a Biacore 8K+ (for P1AI1648-P1AI1652). P1AH4157-P1AH4161 and P1AI0356-P1AI0360 (Figure 1B-C)) were characterized at 25 °C on a Biacore T200 using HBS-EP running buffer and sample dilution buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% surfactant P20, Cytiva, Freiburg, Germany).

[0182] Anti-PGLALA antibody (M-1.7.24 mu IgG2b) was directly immobilized onto a CM5 chip at pH 5.0 using a standard amine coupling kit (Cytiva, Freiburg, Germany). After activating the sensor surface with a 1:1 mixture of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 0.1 M N-hydroxysuccinimide (NHS), 20 μg / ml of anti-PGLALA (diluted in 10 mM acetate, pH 5.0) was injected at a flow rate of 5 μl / min for 900 s. After blocking with 1 M ethanolamine-HCl, pH 8.5, the coupling procedure resulted in an anti-PGLALA surface density of over 12,000 RU.

[0183] Deimmunized a-PD1 binder was captured at a concentration of 10 nM for 60 s at a flow rate of 10 μl / min. Recombinant huPD1-ECD_Fc-knob / hole (internal ID P1AD9704) was injected at various concentrations (300–9, 4 nM, 1:1 dilution) through the flow cell at a flow rate of 50 μl / min. Association and dissociation were monitored for 120 s and 600 s, respectively. The chip surface was regenerated after each cycle by using 20 mM NaOH injections (35 s each). Bulk refractive index differences were corrected by subtracting the response obtained with a reference flow cell. Curves were fitted using a 1:1 Langmuir interaction model using Biacore T200 Evaluation Software 3.1 (GE Healthcare Bio-Sciences), and the results are summarized in the table below.

[0184] P1AI1648-P1AI1652 (Figure 1D) were characterized on a Biacore 8K+ at 25°C using HBS-EP running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% surfactant P20, Cytiva, Freiburg / Germany).

[0185] Anti-PGLALA antibody (M-1.7.24 mu IgG2b) was directly immobilized on a C1 chip at pH 5.0 using a standard amine coupling kit (Cytiva, Freiburg, Germany). After activating the sensor surface with a 1:1 mixture of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 0.1 M N-hydroxysuccinimide (NHS), 20 μg / ml of anti-PGLALA (diluted in 10 mM acetate, pH 5.0) was injected at a flow rate of 10 μl / min for 10 min. After blocking with 1 M ethanolamine-HCl, pH 8.5, the coupling procedure resulted in an anti-PGLALA surface density of approximately 1,000 RU.

[0186] Deimmunized a-PD1 binders were captured at a concentration of 5 nM at a flow rate of 10 μl / min for 80 s. Recombinant huPD1-ECD_Fc-knob / hole was injected at various concentrations (200–0.27 nM, 1:3 dilution) through the flow cell at a flow rate of 30 μl / min. Association and dissociation were monitored for 240 s and 800 s, respectively. The chip surface was regenerated after each cycle using two injections (60 s each) of 10 mM glycine pH 2. Differences in bulk refractive index were corrected by subtracting the response obtained with a reference flow cell. Curves were fitted using a 1:1 Langmuir interaction model using Biacore Insight Evaluation Software 3.0 (Cytiva, Freiburg, Germany), and the results are summarized in Tables 1.2, 1.3, and 1.4. [Table 1.2] [Table 1.3] [Table 1.4]

[0187] In total, three sets of five deimmunized anti-PD1 variants were generated for evaluation as human IgG1 PG-LALA antibodies: Set 1 (P1AH4157-P1AH4161; Figure 1B), Set 2 (P1AI0356-P1AI0360; Figure 1C), and Set 3 (P1AI1648-P1AI1652; Figure 1D). Sets 1 and 2 were independent sets, while Set 3 was based on P1AH4157 (PD1-376_VL2_VH4) with additional mutations in CDRH2. The kinetic constants and affinity of PD1-376_VL2_VH4 for human PD1 were very similar to those of the parent humanized antibody P1AA6888 (PD1-0103), with ka 6.40E+05, kd 1.30E-04, and KD 200pM, respectively. Based on binding characteristics and cell-based in vitro evaluation, P1AH4157 (PD1-376_VL2_VH4) was selected as the preferred deimmunized anti-PD1 binder, and its V-domain sequence was used for cloning of the complex masked IL2v format.

[0188] Example 2 Relative luminescence units of the PD1 / PD-L1 reporter Jurkat cell line upon PD-1 / PD-L1 blockade with either parental (bivalent vs. monovalent) or deimmunized anti-PD1 antibodies The PD-1 / PD-L1 Blockade Reporter Assay from Promega (Cat. Nos. J1250, J1255) is a bioluminescent cell-based assay that can be used to measure the potency of antibodies designed to block the PD-1 / PD-L1 interaction. The assay relies on the co-culture of two genetically engineered cell lines: PD-1 T cells as effectors and PD-L1 APC / CHO-K1 cells as target cells.

[0189] PD-1 effector T cells are Jurkat T cells expressing human PD-1 and a luciferase reporter gene controlled by the NFAT response element (NFAT-RE). PD-L1 APC / CHO-K1 target cells are CHO-K1 cells expressing human PD-L1 and an engineered cell surface protein designed to activate their cognate TCR in an antigen-independent manner. When cocultured, PD-1 / PD-L1 interaction inhibits TCR signaling and downregulates NFAT-RE-mediated luminescence in effector T cells. Inhibition of PD-1 / PD-L1 interaction with anti-PD-1 (or anti-PD-L1) antibodies releases an inhibitory signal, leading to TCR activation of NFAT-RE-mediated luminescence.

[0190] This assay was used to evaluate the ability of deimmunized PD-1 binders to inhibit PD-1 / PD-L1 interaction. To this end, eight 1:10 dilutions of either PD1 IgG PG LALA PD1-IL2v or deimmunized anti-PD-1, with a top concentration of 66 nM, were added to PD-L1-CHO-expressing cells immediately before co-culture of the two cell lines at 37°C for 5 hours. After the 5-hour incubation, substrate (BIO-Glo Reagent) was added, and samples were measured using a luminometer (PerkinElmer Reader).

[0191] Figures 5A and 5B show that PD1-376_VL2_VH1_2_3_4 IgG PG LALA, PD1-376_VL2_7_VH4 IgG PG LALA, and PD1-376_VL2_VH4 IgG PG LALA were nearly as potent as the parental anti-PD1 in blocking PD-1 / PD-L1 interaction and overcoming downstream inhibitory signals, whereas mutations in PD1-376_VL7_VH1_2_4 IgG PG LALA and PD1-376_VL7_VH4 IgG PG LALA resulted in a 4- to 5-fold decrease in potency (Table 2.1). [Table 2.1]

[0192] PD1-IL2v (as disclosed in WO 2018 / 184964) and FAP IL2v (as disclosed in WO 2012 / 107417; INN:Simlukafusp alpha) were used as controls and showed that lack of aPD-1 in FAP-IL2v prevented PD-1 / PD-L1 inhibition of TCR signaling and the resulting luminescence, whereas inhibition of the interaction by aPD-1 on the PD1-IL2v construct activated NFAT-RE-mediated luminescence.

[0193] Binding of deimmunized PD-1 to activated CD4 T cells In the following experiment, we compared deimmunized PD-1 antibodies side-by-side in a binding assay to assess whether deimmunization affects the binding affinity / avidity of PD-1 on T cells. To this end, CD4 T cells were isolated from healthy donor PBMCs using CD4 beads (#130-045-101, Miltenyi) and activated for 3 days in the presence of 1 μg / ml plate-bound anti-CD3 (overnight precoated, clone OKT3, #317326, BioLegend) and 1 μg / ml soluble anti-CD28 (clone CD28.2, #302934, BioLegend) antibodies to induce T cell activation and PD-1 expression. After 3 days, cells were harvested and washed to remove endogenous IL-2. Cells were then seeded into V-bottom plates and stained for 30 minutes at 4°C with increasing concentrations of the following treatment antibodies: PD1-376_VL2_VH4 IgG PG LALA, PD1-376_VL7_VH4 IgG PG LALA, PD1-376_VL2_7_VH4 IgG PG LALA, PD1-376_VL7_VH1_2_4 IgG PG LALA, PD1-376_VL2_VH1_2_3_4 IgG PG LALA, parental anti-PD-1 PD1-IL2v, and FAP IL2v (50 μl, 1:10 dilution steps with a top concentration of 66 nM).

[0194] Cells were washed with PBS to remove unbound molecules. Then, 50 μl of diluted AF647 anti-PGLALA antibody and Fixable Viability Dye eFluor™ 780 (eBioscience) were added to the cells, followed by a 30-minute incubation at 4°C and a washing step. Finally, cells were fixed with BD cell fix (70 μl, #340181, BD Biosciences) and acquired on a FACSymphony A3 Cell Analyzer (BD Bioscience). The frequency and MFI of positive cells were determined using FlowJo (V10) and plotted using GraphPad Prism.

[0195] As shown in Figures 6A and 6B and 7A and 7B, PD1-376_VL2_VH4 IgG PG LALA, PD1-376_VL2_7_VH4 IgG PG LALA, PD1-376_VL2_VH1_2_3_4 IgG PG LALA, and the corresponding parental anti-PD1- and PD1-IL2v antibodies bind to CD4 T cells with similar potency. PD1-376_VL7_VH4 IgG PG LALA and PD1-376_VL7_VH1_2_4 IgG PG LALA show reduced binding to CD4 T cells. FAP-IL2v served as a non-targeting control to compare the effects of IL2v-based immunocytokines alone, which do not target PD-1.

[0196] Tables 2.2 and 2.3 show the EC50 of dose-response binding frequency and MFI of various antibodies to activated CD4 T cells obtained from two donors. [Table 2.2] [Table 2.3]

[0197] Effects of various anti-PD-1 antibodies on cytotoxic granzyme B and IFN-γ secretion by allospecific human CD4 T cells cocultured with allogeneic mature dendritic cells To evaluate deimmunized PD-1 antibodies in functional assays, CD4 T cells were exposed to allogeneic mature DCs to induce the generation of specific T cells with various antigen specificities and TCR affinities. Importantly, allospecific T cells express immune checkpoints such as PD-1, and therefore are useful for measuring the functional efficacy of anti-PD1 antibodies, as anti-PD1 antibodies can elicit T cell effector function. To screen for the functionality and efficacy of PD-1-blocking antibodies in an allogeneic environment, freshly purified CD4 T cells were co-cultured for 5 days with allogeneic mature dendritic cells (mDCs) derived from monocytes. Monocytes were isolated from fresh PBMCs using CD14 beads (130-050-201, Miltenyi) 1 week prior. Immature DCs were generated from monocytes by culturing them for 5 days in medium containing GM-CSF (50 ng / ml) and IL-4 (100 ng / ml). To induce iDC maturation, TNF-α, IL-1β, and IL-6 (50 ng / ml each) were added to the culture medium for an additional 2 days.

[0198] On the day of the minimal mixed lymphocyte reaction (mMLR), CD4+ T cells were enriched from PBMCs obtained from unrelated donors using a microbead kit (Miltenyi). Prior to culture, CD4+ T cells were labeled with 5 μM Cell Trace Violet (CTV, #C34557, ThermoFisher). 10 CD4 T cells were then plated in flat-bottom 96-well plates with mature allogeneic DCs (10:1 ratio) for 5 days at 37°C, 5% CO2 in the presence of a range of concentrations of purified anti-PD1 monoclonal antibodies: PD1-376_VL2_VH4 IgG PG LALA, PD1-376_VL7_VH4 IgG PG LALA, PD1-376_VL2_7_VH4 IgG PG LALA, PD1-376_VL7_VH1_2_4 IgG PG LALA, PD1-376_VL2_VH1_2_3_4 IgG PG LALA, parental anti-PD1, and PD1-IL2v (50 μl, 1:10 dilution steps with a top concentration of 66 nM). Either no antibody or FAP IL2v was used as a negative control.

[0199] After 5 days, cells were incubated for an additional 5 hours at 37°C in the presence of Golgi Plug (Brefeldin A) and Golgi Stop (monensin), then washed, surface stained with anti-human CD4 antibody and Fixable Viability Dye eFluor™ 780 (eBioscience), and fixed / permeabilized with Fix / Perm Buffer (BD Bioscience). Cells were then intracellularly stained for granzyme B (BD Bioscience) and IFN-γ (eBioscience). The results are shown in Figures G / H and I / J (GrzB and IFN-γ secretion / release).

[0200] Anti-PD1 monoclonal antibodies concentration-dependently promoted T cell secretion of GrzB (Figures 8A and 8B) and IFN-γ (Figures 9A and 9B). All anti-PD-1 variants were found to enhance granzyme B and IFNγ compared to FAP-IL2v or untreated cells (negative control). Tables 2.4 and 2.5 show the EC50 and area under the curve (AUC) for the percentage of GrzB and IFNγ secretion of CD4 T cells in dose-dependent responses to allogeneic stimulation and treatment with anti-PD-1 antibodies. [Table 2.4] [Table 2.5]

[0201] Lower EC50 values ​​for cytokine production were obtained with the parental anti-PD-1, followed by PD1-376_VL2_VH4 IgG PG LALA. The highest AUCs were provided by the deimmunized variant PD1-376_VL2_VH4 IgG PG LALA and the control PD1-IL2v.

[0202] Example 3 Binding of deimmunized PD1 binders to CHO-huPD1 cells We evaluated the binding of five different deimmunized PD1-IgGs to human PD1 overexpressing CHO cells in comparison to each parental PD1-IgG molecule, including the parental PD1 binder, and the TA PD1-IL2v construct (Table 3.1). [Table 3.1]

[0203] CHO-huPD1 cells (CHO-K1_MUSMU_PDCD1_Clone_42) were harvested with trypsin / EDTA, washed with PBS, and resuspended in FACS buffer (PBS, 2% FBS, 5 mM EDTA, 0.025% NaN3). 100,000 cells were then seeded per well in a round-bottom plate. Cells were stained with 30 μl of deimmunized PD1-IgG, parental PD1-IgG, and the TA PD1-IL2v construct containing parental PD1-IgG in FACS buffer at the indicated concentrations for 30 minutes at 4°C. After staining, cells were washed twice with FACS buffer to remove unbound molecules. Then, 30 μl of diluted PE anti-human Fc-specific secondary antibody (1:50 dilution, 109-116-170, Jackson ImmunoResearch) was added to the cells. After a 30-minute incubation at 4°C, cells were washed twice with FACS buffer. Finally, cells were resuspended in 150 μl of FACS buffer and measured using a BD Fortessa.

[0204] All five deimmunized PD1-IgGs bound similarly to human PD1 on CHO cells compared to their respective parental PD1-IgGs, with the 2 VL7 containing PD1-IgGs showing slightly reduced binding compared to the other deimmunized PD1-IgGs (Figure 10).

[0205] Inhibition of IL2v activity by deimmunized MT204 masks Next, we tested the inhibition of FAP-IL2v activity by three different deimmunized MT204 masks compared to the parental MT204 mask (Table 3.2). [Table 3.2]

[0206] NK92 cells were harvested, counted, and assessed for viability. Cells were washed three times with PBS to remove residual IL2. The washed NK92 cells were resuspended in fresh IL2-free medium (advanced RPMI 1640, 2% FCS, 1% glutamine), and 50 μl of the cell suspension containing 10,000 cells was transferred to a 96-well cell culture-treated flat-bottom plate. First, 25 μl of 0.5 nM FAP-IL2v antibody was added per well. Next, 25 μl of MT204 antibody was added per well to a final volume of 100 μl per well. The plate was incubated in an incubator for 3 days. After 3 days, the CellTiter-Glo (G7571, Promega) reagent and cell culture plate were allowed to equilibrate to room temperature. CellTiter-Glo solution was prepared as described in the manufacturer's instructions, and 100 μl of the solution was added to each well. After 10 min of incubation, remaining aggregates were resuspended by pipetting and 100 μl of the mixture was transferred to a 96-well white flat-bottom plate. Luminescence was measured using a Tecan Spark 10M multimode reader.

[0207] MT204_VL1 and MT204_VL1a inhibited IL2v activity comparable to the parent MT204 mask. MT204_VL1b showed less efficient inhibition of IL2v activity compared to the parent MT204 mask (FIG. 11).

[0208] Binding of TA PD1-IL2v construct to CHO-huPD1 cells Next, four different TA PD1-IL2v constructs were tested, comparing the PQARK cleavage site, the YAARKGGI cleavage site, the 38-mer linker, and the 25-mer linker. [Table 3.3]

[0209] CHO-huPD1 cells (CHO-K1_MUSMU_PDCD1_Clone_42) were harvested with trypsin / EDTA, washed with PBS, and resuspended in FACS buffer (PBS, 2% FBS, 5 mM EDTA, 0.025% NaN3). 100,000 cells were seeded per well in a round-bottom plate. Cells were stained with 30 μl of TA PD1-IL2v construct in FACS buffer for 30 minutes at 4°C. After staining, cells were washed twice with FACS buffer to remove unbound molecules. 30 μl of diluted APC anti-human Fc-specific secondary antibody (1:50 dilution, 109-136-098, Jackson ImmunoResearch) was then added to the cells. After 30 minutes of incubation at 4°C, cells were washed twice with FACS buffer. Finally, cells were resuspended in 150 μl of FACS buffer and measured using a BD Fortessa.

[0210] All four constructs tested contain a deimmunized VL2_VH4 PD1 binder and the MT204 VL1a deimmunized mask. We tested their binding to human PD1 overexpressing CHO cells in comparison with the respective uncleavable TA PD1-IL2v, unmasked TA PD1-IL2v, and parental TA PD1-IL2v molecules. All TA PD1-IL2v constructs tested bind comparable amounts to human PD1 on CHO cells (Figure 12).

[0211] KHYG-1 proliferation induced by TA PD1-IL2v construct Next, we tested the induction of proliferation of KHYG-1 cells by four different TA PD1-IL2v constructs containing the PQARK or YAARKGGI cleavage site and a 38-mer or 25-mer linker (see Table 3.3).

[0212] The NK cell line KHYG-1 was used to test the induction of proliferation by the TA PD1-IL2v construct. Cells were harvested, counted, and assessed for viability. Cells were washed three times with PBS to remove residual IL2. Washed KHYG-1 cells were resuspended in fresh IL2-free medium (advanced RPMI 1640, 2% FCS, 1% glutamine), and 12.5 μl of the cell suspension containing 2,000 cells was transferred to a 384-well cell-culture-treated flat-bottom plate. Ten μg of the TA PD1-IL2v construct was digested with 20 μl of matriptase (Enzo approximately 2.5 U / μl, ALX-201-246-U25, lot number 08102104, or no matriptase as an undigested control) in 2 μl of matriptase buffer (50 mM Tris, 50 mM NaCl, 0.01% Tween 20, pH 9.0) for 2 hours at 37°C, and 12.5 μl of antibody was added per well to a final volume of 25 μl per well. The plates were incubated in an incubator for 3 days. After 3 days, the CellTiter-Glo (G7571, Promega) reagent and cell culture plates were allowed to equilibrate to room temperature. CellTiter-Glo solution was prepared as described in the manufacturer's instructions, and 25 μl of the solution was added to each well. After 10 min of incubation, the remaining aggregates were resuspended by pipetting and 40 μl of the mixture was plated onto a white flat-bottom plate. Luminescence was measured using a Tecan Spark 10M multimode reader.

[0213] All four constructs tested contained the deimmunized VL2_VH4 PD1 binder and the MT204 VL1a deimmunized mask. Two respective uncleavable molecules and unmasked PD1-IL2v were included as controls. All molecules were tested undigested and digested with recombinant matriptase. Following digestion with matriptase, TA PD1-IL2v with PQARK and YAARKGGI linkers regained activity. Activity was slightly reduced compared to the unmasked control, likely due to incomplete cleavage. The activity of the TA PD-IL2v construct containing a 38-mer linker was comparable to that of the 25-mer linker (Figures 13A and 13C). Undigested TA PD1-IL2v did not induce proliferation (Figures 13A, 13D, and 13F). Uncleavable TA PD1-IL2v digested with matriptase did not induce proliferation (Figure 13E). Unmasked PD1-IL2v activity is unaffected by cleavage by matriptase compared to the non-cleavable construct (Figures 13E, 13F).

[0214] Example 4 In vivo efficacy of murine TA-PD1-IL2v immunoconjugate in a syngeneic model of a mouse tumor cell line (MCA205 subcutaneous syngeneic model) The murine TA-PD1-IL2v immune complex was tested in the murine fibrosarcoma cell line MCA205 injected subcutaneously into Black6-huPD1 transgenic mice.

[0215] MCA205 fibrosarcoma carcinoma cells were originally obtained from SigmaAldrich (catalog no. SCC173) and deposited in the Roche-Glycart in-house cell bank after expansion. Tumor cell lines were routinely cultured in DMEM containing 10% FCS (Gibco) at 37°C in a water-saturated atmosphere of 5% CO2. Passage 11 was used for transplantation with a survival rate of 95.3%. 1 x 10 cells per animal were used. 6 Cells were injected subcutaneously into the flank of mice in 100 μl of RPMI cell culture medium (Gibco) using a 1 ml tuberculin syringe (BD Biosciences, Germany).

[0216] Female Black6-huPD1 mice (housed at Charles Rivers, Lyon, France), aged 10–11 weeks at the start of the experiment, were maintained under specific pathogen-free conditions with a 12-h light / 12-h dark diurnal cycle in accordance with the adopted guidelines (GV-Solas; Felasa; TierschG). The experimental study protocol was reviewed and approved by the local authorities (P184 / 2020). After arrival, animals were kept for one week to acclimate to their new environment and to be observed. Continuous health monitoring was performed regularly.

[0217] Mice were given 1 x 10 6 MCA205 cells were injected subcutaneously, randomized, and weighed. 10 days after tumor cell injection (tumor volume >150 mm 3 Mice were intravenously injected twice weekly for 1 week with the TA-PD1-IL-2vYAARKGGI 38mer cleavable linker or the TA-PD1-IL-2v 38mer non-cleavable linker. All mice were injected intravenously with 200 μl of the appropriate solution. Mice in the vehicle group were injected with histidine buffer. Stock solutions were diluted as needed with histidine buffer to obtain the appropriate amount of immune complex per 200 μl (Table 4). [Table 4]

[0218] FIG. 14 shows that the TA-PD-IL2v YAARKGGI 38mer demonstrated superior efficacy in tumor growth inhibition compared to vehicle and non-cleavable Mab single agent groups.

[0219] Example 5 Design of a murine surrogate of a composite PD1-targeted masked IL2v immunoconjugate with an scFv mask To facilitate in vivo tolerability and efficacy testing in non-tumor-bearing mice or mouse models of cancer, we generated murine surrogates of PD1-targeting masked IL2v immunoconjugates targeting either human (P1AK3638 and P1AK3649) or mouse (P1AK3641 and P1AK3640) PD1. To reduce immunogenicity, all constant antibody domains in these constructs correspond to murine sequences.

[0220] The mouse surrogates target either human PD1 for use in humanized mice, human PD1 transgenic mice, or mouse PD1 for use in immunocompetent and syngeneic mouse models. Human IL2v is used in all constructs due to the cross-reactivity of human IL2v to the mouse IL2 receptor and the lack of cross-reactivity of the scFv mask to mouse IL2v.

[0221] These murine surrogate constructs bivalently bind either human or mouse PD1 via the N-terminal Fab arms on the Fc DD- and Fc KK+ chains, but the Fc DD-chain additionally bears a masked (matriptase-cleavable or non-matriptase-cleavable) C-terminal IL2v. The scFv mask is fused "in-line" C-terminally to the same Fc DD-chain as IL2v. Heterodimerization was achieved by adding complementary charges to the murine IgG1 CH3 domains (Fc DD- and Fc KK+ chains), and binding to activating Fcγ receptors and complement component C1q was abrogated by introducing a DAPG mutation into the murine IgG1 CH2 Fc domain of the antibody. The matriptase-cleavable C-terminally masked IL2v constructs, P1AK3638 and P1AK3641, each contain two PQARK matriptase recognition sites: one PQARK matriptase recognition site located in the linker between the VH and VL domains of the scFv mask, and the other located in the linker between the scFv mask and IL2v. Additionally, non-matriptase-cleavable control constructs (lacking the PQARK matriptase recognition site), P1AK3649 and P1AK3640, as well as an unmasked control construct (lacking the scFv mask), were generated. These masked constructs are shown schematically in Figures 16A-C and 17A-B, 17D. For comparison, a murine IgG2a with anti-mouse PD1 specificity (P1AD4006) was included in some studies to test the in vivo efficacy of checkpoint inhibition alone and is shown in Figure 17C.

[0222] Generation and purification of masked PD1-targeted IL2v immune complexes and control constructs Murine surrogate PD1-targeted masked IL2v immunoconjugates (P1AK3638 and P1AK3649 and P1AK3641 and P1AK3640, respectively) have been produced and purified by WuXi Biologics. They were transiently expressed in HEK293 cells and purified using the following two-column DSP process: 1. MabSelectSuRe LX affinity chromatography (equilibration and first wash: 25 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, pH 7.5; second wash: 25 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 0.1% Triton 100 / 114, pH 7.5; elution: 50 mM sodium citrate-sodium citrate, 150 mM NaCl, pH 3.0; neutralization: 1 M arginine, 0.4 M succinic acid, pH 9.0); 2. Superdex 200 size-exclusion chromatography (equilibration and formulation buffer: 20 mM histidine-HCl, 140 mM NaCl, pH 6.0). Purity was determined by SEC-HPLC and reduced and non-reduced caliper-SDS. Purified batches were tested for low endotoxin levels and the identity of the deglycosylated mass was confirmed by liquid chromatography-mass spectrometry (LC-MS).

[0223] P1AD4006, a mouse IgG2a with anti-mouse PD1 specificity, was produced and purified by vitoria AG. Suspension-adapted CHO K1 cells (received from ATCC and adapted for serum-free growth in suspension culture at vitoria) were used for production. Seeds were grown in eviGrow medium, a chemically defined, animal-component-free, serum-free medium. Cells were transfected with eviFect (vitoria's custom-made, proprietary transfection reagent) and, post-transfection, expanded in eviMake2, an animal-component-free, serum-free medium. The supernatant was collected by centrifugation and subsequent filtration (0.2 μm filter). IgG was purified using MabSelect™ SuRe™ with Dulbecco's PBS (Lonza BE17-512Q) as the wash buffer, 0.1 M glycine pH 3.5 as the elution buffer, and 1 M Tris-HCl (pH 9) as the neutralization buffer. Subsequent size-exclusion chromatography was performed on a HiLoad Superdex 200 pg column using the final buffer as the running buffer. Dialysis (if required) was performed using Pierce Slide-A-Lyzer™ G2 Dialysis Cassettes with a 2K molecular weight cutoff. Antibody concentration (if required) was performed using Amicon® Ultra Centrifugal Filters with a 30 kDa molecular weight cutoff. Concentration was determined by measuring absorbance at 280 nm. The extinction coefficient was calculated using a proprietary algorithm in evitria. Purity was determined by analytical size-exclusion chromatography using an Agilent AdvanceBio SEC column (300A 2.7 um 7.8 x 300 mm) and DPBS as the running buffer at 0.8 ml / min. Endotoxin content was measured using a Charles River Endosafe PTS system.

[0224] The human FolR1-targeting T cell engager, P1AK1120 (Figure 15), used to deliver T cell "signal 1" in several in vivo efficacy studies, was produced by evitria AG as described above and purified at Roche using a three-column DSP process (1. Protein A MabSelectSure, 2. Butyl HP Hydrophobic Interaction Chromatography (HIC), 3. Superdex 200 HiLoad 16 / 6000 size-exclusion chromatography (SEC)) and formulated in 20 mM histidine, 140 mM NaCl, pH 6.0. Purity was determined by SEC-HPLC and reduced and non-reduced CE-SDS. Purified batches were tested for low endotoxin levels, and the identity of the deglycosylated mass was confirmed by liquid chromatography-mass spectrometry (LC-MS).

[0225] Example 6 HEK blue IL2 reporter cell assay using HEK blue IL2 cells overexpressing human PD1 to test the activity of the murine TA PD1-IL2v construct. We tested the activity of murine cleavable and non-cleavable TA PD1-IL2v molecules containing a human-specific PD1 binder in a HEK blue IL2 reporter cell assay using HEK blue IL2 cells expressing human PD1.

[0226] A calculated amount of each molecule was digested with 2 μl of recombinant human matriptase (ALX-201-246-U250, Enzo) and filled to 20 μl with matriptase buffer (50 mM Tris, 50 mM NaCl, 0.01% Tween 20, pH 9.0). A corresponding amount of undigested control was also filled to 20 μl with matriptase buffer and subsequently treated in the same manner as the matriptase-spiked samples. Digestion was carried out for 2 hours at 37°C. After digestion, samples were filled to a final starting concentration of 50 nM with DMEM + 10% FBS + 1% GlutaMax (assay medium).

[0227] HEK-Blue huPD1 IL-2 cells (HEK-Blue-IL2_hPDCD1_clone4) were detached using cell dissociation buffer and resuspended in DMEM + 10% FBS + 1% GlutaMax (assay medium) at 0.33 Mio cells / ml. 150 μl of HEK-Blue IL-2 cells (containing 50,000 cells) were then seeded into a 96-well flat-bottom plate. 50 μl of titrated surrogate IL2v molecules were then added to each well for a final volume of 200 μl per well. The plate was then incubated at 37°C for 24 hours. 180 μl of Quanti-Blue solution (rep-qbs2, InvivoGen) and 20 μl of cell supernatant were then added per well to the 96-well flat-bottom plate and incubated at 37°C for 60 minutes. Optical density (OD) was then measured at 620 nm using a Tecan Spark reader.

[0228] The activity of the molecules was compared to each unmasked PD1-IL2v construct. All molecules were tested either undigested or after cleavage with recombinant matriptase. Upon cleavage, the activity of the cleavable murine TA PD1-IL2v construct is comparable to naked PD1-IL2v, whereas the uncleavable TA PD1-IL2v has very limited activity in the reporter cell assay (Figure 18A).

[0229] In the absence of digestion with recombinant matriptase, both the cleavable and non-cleavable TA PD1-IL2v constructs have very limited activity compared to naked PD1-IL2v (Figure 18B).

[0230] Example 7 HEK blue IL2 reporter cell assay using HEK blue IL2 cells overexpressing mouse PD1 to test the activity of the murine TA PD1-IL2v construct containing a mouse-specific PD1 binder. We tested the activity of murine-cleavable and non-cleavable TA PD1-IL2v molecules containing a mouse-specific PD1 binder in a HEK blue IL2 reporter cell assay using HEK blue IL2 cells expressing mouse PD1.

[0231] A calculated amount of each molecule was digested with 2 μl of recombinant human matriptase (ALX-201-246-U250, Enzo) and filled to 20 μl with matriptase buffer (50 mM Tris, 50 mM NaCl, 0.01% Tween 20, pH 9.0). A corresponding amount of undigested control was also filled to 20 μl with matriptase buffer and subsequently treated in the same manner as the matriptase-spiked samples. Digestion was carried out for 2 hours at 37°C. After digestion, samples were filled to a final starting concentration of 50 nM with DMEM + 10% FBS + 1% GlutaMax (assay medium).

[0232] HEK-Blue huPD1 IL-2 cells (HEK-Blue IL-2 cells_muPDCD1_clone 12 cells) were detached using cell dissociation buffer and resuspended in DMEM + 10% FBS + 1% GlutaMax (assay medium) at 0.33 Mio cells / ml. 150 μl of HEK-Blue IL-2 cells (containing 50,000 cells) were then seeded into a 96-well flat-bottom plate. 50 μl of titrated surrogate IL2v molecules were then added to each well for a final volume of 200 μl per well. The plate was then incubated at 37°C for 24 hours. 180 μl of Quanti-Blue solution (rep-qbs2, InvivoGen) and 20 μl of cell supernatant were then added per well to the 96-well flat-bottom plate and incubated at 37°C for 60 minutes. The optical density (OD) was then measured at 620 nm using a Tecan Spark reader.

[0233] The activity of the molecules was compared to each unmasked PD1-IL2v construct. All molecules were tested either undigested or after cleavage with recombinant matriptase. Upon cleavage, the activity of the cleavable murine TA PD1-IL2v construct is comparable to naked PD1-IL2v, whereas the uncleavable TA PD1-IL2v has very limited activity in the reporter cell assay (Figure 19A).

[0234] Without predigestion with recombinant matriptase, both the cleavable and non-cleavable TA PD1-IL2v constructs have very limited activity compared to naked PD1-IL2v. The TA PD1-IL2v cleavable constructs have slightly higher activity than the respective non-cleavable constructs, which may indicate minor cleavage that may occur during the incubation period (Figure 19B).

[0235] Example 8 In vitro proliferation and activation of CD8 T cells and NK cells induced by TA PD1-IL2v constructs. The TA PD1-IL2v construct was tested for its ability to induce proliferation of NK cells and CD8 T cells in PBMCs upon digestion with recombinant human matriptase.

[0236] The calculated amount of each molecule was digested with 3 μl of recombinant human matriptase (ALX-201-246-U250, Enzo) and filled up to 20 μl with matriptase buffer (50 mM Tris, 50 mM NaCl, 0.01% Tween 20, pH 9.0). Digestion was carried out for 2 hours at 37°C. After digestion, the sample was filled up with RPMI + 10% FBS + 1% GlutaMax (assay medium) to a final starting concentration of 100 nM.

[0237] Frozen PBMCs (Biomex) were thawed and immediately resuspended in prewarmed assay medium. The cells were then centrifuged at 350g for 5 minutes and washed once with prewarmed PBS. CFSE stock solution was diluted 1:20 with prewarmed PBS to obtain a working solution with a concentration of 100 μM. 30 Mio cells were resuspended in 30 ml of prewarmed PBS. 30 μl of CFSE working solution was added to the cell suspension, and the cells were immediately mixed and incubated at 37°C for 15 minutes. The reaction was then stopped by adding prewarmed assay medium. The cells were detached at 400g for 10 minutes, resuspended in assay medium, and incubated at 37°C for 30 minutes. After incubation, the cells were washed once with prewarmed assay medium, counted, and resuspended in assay medium at 2 Mio cells / ml.

[0238] CFSE-labeled PBMCs were seeded into a 96-well round-bottom plate (0.1 Mio cells / well), IL2 antibody was added, and all wells were filled to a final volume of 200 μl. After 6 days of incubation at 37°C, PBMCs were harvested. Cells were centrifuged at 400g for 4 minutes and washed once with PBS. Live / dead staining solution was added in 25 μl of PBS diluted 1:1000 in PBS and incubated at room temperature for 20 minutes. 150 μl of FACS buffer was then added, and the plate was centrifuged at 400g for 4 minutes. The supernatant was removed, and the cells were washed again with 150 μl of FACS buffer. 25 μl / well of an antibody mixture (BUV395 anti-human CD3, PE anti-human CD4, APC anti-human CD8, PE / Cy7 anti-human CD25, and BV421 anti-human CD56) was then added to the cells. The cells were incubated in the refrigerator for 30 minutes. Cells were then washed twice with FACS buffer and resuspended in 150 μl of FACS buffer. Analysis was performed using a BD LSR Fortessa. NK cells were defined as CD3 negative and CD56 positive, and CD8 T cells were defined as CD3CD8 double positive.

[0239] CFSE-labeled PBMCs were treated with TA PD1-IL2v molecules containing either the PQARK or YAARKGGI cleavage site and either a 38-mer or 25-mer linker. As negative controls, each uncleavable TA PD1-IL2v molecule was included. As positive controls, each unmasked PD1-IL2v molecule was included. After 6 days of incubation, PBMCs were analyzed by flow cytometry for upregulation of activation markers, and CFSE dilution was used as an indicator of proliferation.

[0240] All predigested TA PD1-IL2v constructs containing the cleavage site were able to induce proliferation (Figures 20A-B) and activation (Figures 21A-B) of NK cells and CD8 T cells. In this assay, no differences were observed between the two linker lengths and two cleavage sites tested, demonstrating that the precleaved TA PD1-IL2v molecules possessed activity comparable to their respective unmasked PD1-IL2v molecules. The two TA PD1-IL2v constructs that did not contain a protease cleavage site did not induce proliferation or activation of NK cells or CD8 T cells, indicating that TA PD1-IL2v is active only upon protease cleavage.

[0241] Example 9 In vivo efficacy of murine TA-PD1-IL2v immunoconjugates in a syngeneic model of a mouse tumor cell line - the MCA 205 subcutaneous syngeneic model The murine TA-PD1-IL2v immune complex was tested in the murine fibrosarcoma cell line MCA205 injected subcutaneously into Black6-huPD1 transgenic mice.

[0242] MCA205 fibrosarcoma carcinoma cells were originally obtained from Sigma-Aldrich (catalog no. SCC173) and deposited in the Roche-Glycart internal cell bank after expansion. Tumor cell lines were routinely cultured in DMEM containing 10% FCS (Gibco) at 37°C in a water-saturated atmosphere of 5% CO2. Passage 9 was used for transplantation with a survival rate of 97.9%. 1 x 10 cells per animal were used. 6Cells were injected subcutaneously into the flank of mice in 100 μl of RPMI cell culture medium (Gibco) using a 1 ml tuberculin syringe (BD Biosciences, Germany).

[0243] Female Black6-huPD1 mice (housed at Charles Rivers, Lyon, France), aged 8–10 weeks at the start of the experiment, were maintained under specific pathogen-free conditions with a 12-h light / 12-h dark diurnal cycle in accordance with the adopted guidelines (GV-Solas; Felasa; TierschG). The experimental study protocol was reviewed and approved by the local government (P184 / 2020). After arrival, animals were kept for one week to acclimate to their new environment and observed. Continuous health monitoring was performed regularly.

[0244] On test day 0, mice were injected with 1x10 6 MCA205 cells were injected subcutaneously, randomized, and weighed. 10 days after tumor cell injection (tumor volume >200 mm 3 Mice were intravenously injected twice weekly for 1 week with either the TA-PD1-IL-2v PQARK 25mer cleavable linker or the TA-PD1-IL-2v 25mer noncleavable linker. All mice were intravenously injected with 200 μl of the appropriate solution. Mice in the vehicle group were injected with histidine buffer. The stock solution was diluted with histidine buffer as needed to obtain the appropriate amount of immunoconjugate per 200 μl.

[0245] Figure 22 shows that both doses of the TA-PD-IL2v PQARK 25mer tested mediated superior efficacy in terms of tumor growth inhibition compared to vehicle, non-cleavable, and pembrolizumab Mab single agent groups. [Table 5]

[0246] Example 10 In vivo efficacy of a murine surrogate of TA-PD1-IL2v immunoconjugate in a syngeneic model of a mouse tumor cell line - the GL 261 subcutaneous syngeneic model The mouse surrogate TA-PD1-IL2v immunoconjugate was tested on the murine glioblastoma cell line GL261 injected subcutaneously into Black6 mice.

[0247] GL261 glioblastoma cells were originally obtained from DSMZ (Germany) and deposited in the Roche-Glycart internal cell bank after expansion. The tumor cell line was routinely cultured in DMEM containing 10% FCS (Gibco) at 37°C in a water-saturated atmosphere of 5% CO2. Passage 9 was used for transplantation with a survival rate of 97.9%. 1 × 10 cells per animal were used. 6 Cells were injected subcutaneously into the flank of mice in 100 μl of RPMI cell culture medium (Gibco) using a 1 ml tuberculin syringe (BD Biosciences, Germany).

[0248] Female Black6 mice (housed at Charles Rivers, Lyon, France), 9–11 weeks old at the start of the experiment, were maintained under specific pathogen-free conditions with a daily cycle of 12 h light / 12 h dark according to the guidelines of the GV-Solas; Ferrassa; Tiersch. The experimental study protocol was reviewed and approved by the local government (P184 / 2020). After arrival, the animals were kept for one week to acclimate to their new environment and to be observed. Continuous health monitoring was performed regularly.

[0249] Mice were randomly selected, weighed, and injected with 1x10 GL261 cells on the day of the experiment. 6 Ten days after tumor cell injection (tumor volume >100 mm) 3), mice were intravenously injected with muTA-PD1-IL-2v PQARK 25mer cleavable linker or muPD1-IgG twice a week for 1 week. All mice were injected intravenously with 200 μl of the appropriate solution. Mice in the vehicle group were injected with histidine buffer. The stock solution was diluted with histidine buffer as needed to obtain the appropriate amount of immune complex per 200 μl.

[0250] FIG. 23 shows that the muTA-PD-IL2v PQARK 25mer at both doses tested demonstrated superior efficacy in terms of tumor growth inhibition compared to vehicle and muPD1 Mab single agent groups. [Table 6]

[0251] Example 11 In vivo efficacy of TA-PD1-IL2v immunoconjugate in a human tumor PDX xenograft model combined with the FOLR1-TCB bispecific Mab-BC 004 breast patient-derived xenograft model The TA-PD1-IL2v immunoconjugate was combined with the FOLR1-TCB bispecific antibody to test its antitumor efficacy in a xenograft model. Human TA-PD1-IL2v immunoconjugates were tested on human breast BC004 patient-derived cells injected subcutaneously into humanized NSG mice.

[0252] BC004 PDX material (human breast carcinoma) was initially obtained from OncoTest (Freiburg, Germany) and deposited in the Roche-Glycart internal cell bank after in vivo expansion. Tumor fragments were digested with collagenase D and DNase I (Roche, Switzerland), and BC004 cells were used for transplantation. 1 × 10 cells per animal were transplanted in 100 μl of RPMI cell culture medium (Gibco, Germany) using a 1 ml tuberculin syringe (BD Biosciences, Germany). 6 The cells were injected subcutaneously into the flank of mice.

[0253] Fully humanized NSG female mice (Roche-Glycart, Schlieren, Switzerland) were maintained under specific pathogen-free conditions with a daily cycle of 12 h light / 12 h dark according to established guidelines (GV-Solas; Ferrassa; Thiersch). The experimental study protocol was reviewed and approved by the local government (ZH184 / 2020). Continuous health monitoring was performed regularly.

[0254] Mice were randomly selected, weighed, and then injected into a 1x10 6 BC004 cells were injected subcutaneously. 32 days after tumor cell injection (tumor volume >150 mm 3 ), mice were injected i.v. with the following immunocytokines: TA-PD1-IL2v-PQARK 25mer cleavable 1 mg / kg in combination with FolR1-TCB or vehicle, PD1-IL2v 0.1 mg / kg, and pembrolizumab 1 mg / kg administered once a week for 4 weeks. All mice were injected intravenously with 200 μl of the appropriate solution. Mice in the vehicle group were injected with histidine buffer, while treatment groups were injected with the various constructs. Stock solutions were diluted as needed with histidine buffer to obtain the appropriate amount of immunoconjugate per 200 μl. Tumor growth measurements were assessed three times a week with calipers, and volume (mm ) was calculated using GrahPad Prism software. 3 + / - SEM).

[0255] Figure 24 shows that the combination of FOLR1-TCB 0.3 mg / kg + TA-PD1-IL2v PQARK cleavable linker 1 mg / kg Mab demonstrated superior efficacy in terms of tumor growth inhibition compared to FOLR1-TCB + pembrolizumab 1 mg / kg. The efficacy benefit was similar to the combination of FOLR1-TCB 0.3 mg / kg + PD1-IL2v unmasked 0.1 mg / kg Mab, which showed similar tumor growth inhibition. [Table 7] * * *

[0256] The foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, but these descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims

1. 1. A protease-activatable interleukin-2 (IL-2) polypeptide comprising: (i) an IL-2 polypeptide; (ii) a masking moiety; and (iii) a linker comprising a first protease cleavage site, said linker having a length of 20 to 45 amino acids, said masking moiety being covalently attached to said IL-2 polypeptide via said linker, said masking moiety being capable of binding to said IL-2 polypeptide, thereby reversibly masking said IL-2 polypeptide, said masking moiety comprising a second protease cleavage site, wherein said masking moiety does not mask said IL-2 polypeptide upon cleavage at said first and / or said second protease cleavage site.

2. The protease-activatable IL-2 polypeptide of claim 1, wherein the linker has a length of 22 to 43 amino acids.

3. The protease-activatable IL-2 polypeptide of claim 1 or 2, wherein the linker has a length of 25 to 38 amino acids.

4. The protease-activatable IL-2 polypeptide of any one of claims 1 to 3, wherein the linker has a length of 25 or 38 amino acids.

5. The protease-activatable IL-2 polypeptide of any one of claims 1 to 4, wherein the masking moiety is covalently attached to the amino-terminus or carboxy-terminus of the interleukin-2 polypeptide via the linker.

6. The protease-activatable IL-2 polypeptide of any one of claims 1 to 5, wherein the masking moiety is an IL-2 antagonist.

7. The protease-activatable IL-2 polypeptide of any one of claims 1 to 6, wherein the masking moiety is an IL-2 antibody or an IL-2 receptor subunit.

8. 8. The protease-activatable IL-2 polypeptide of claim 7, wherein the IL-2 antibody comprises a Fab molecule.

9. The protease-activatable IL-2 polypeptide of any one of claims 1 to 8, wherein the masking moiety is an antibody derived from the antibody MT204.

10. The protease-activatable IL-2 polypeptide of claim 8 or 9, wherein the Fab molecule is a single-chain Fab molecule.

11. 11. The protease-activatable IL-2 polypeptide of claim 10, wherein the second protease cleavage site is located between the variable domain of the heavy chain (VH) and the variable domain of the light chain (VL) of the single-chain Fab molecule.

12. 12. The protease-activatable IL-2 polypeptide of claim 1, wherein the first protease cleavage site and the second protease cleavage site each comprise at least one protease recognition sequence.

13. 13. The protease-activatable IL-2 polypeptide of any one of claims 1 to 12, wherein the protease recognition sequence of the first protease cleavage site and / or the protease recognition sequence of the second protease cleavage site is YAARKGGI set forth in SEQ ID NO: 60 and / or PQARK set forth in SEQ ID NO:

61.

14. 14. The protease-activatable IL-2 polypeptide of any one of claims 1 to 13, wherein the IL-2 polypeptide is wild-type IL-2, preferably human IL-2 as set forth in SEQ ID NO: 62, or a mutant IL-2 polypeptide.

15. 15. The protease-activatable IL-2 polypeptide of claim 14, wherein the mutant IL-2 polypeptide comprises any amino acid substitution selected from the group consisting of T3A, F42A, Y45A, L72G, and C125A of human IL-2 as set forth in SEQ ID NO:

62.

16. 16. The protease-activatable IL-2 polypeptide of claim 14 or 15, wherein the mutant IL-2 polypeptide comprises the amino acid substitutions F42A, Y45A and L72G of human IL-2 as set forth in SEQ ID NO:

62.

17. 17. The protease-activatable IL-2 polypeptide of any one of claims 14 to 16, wherein the mutant IL-2 polypeptide comprises the amino acid substitutions T3A, F42A, Y45A, L72G, and C125A of human IL-2 as set forth in SEQ ID NO:

62.

18. 18. The protease-activatable IL-2 polypeptide of any one of claims 14 to 17, comprising the amino acid sequence of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 or SEQ ID NO:

30.

19. The protease-activatable IL-2 polypeptide of any one of claims 1 to 18, wherein the IL-2 polypeptide is further linked to a non-IL-2 moiety.

20. 20. The protease-activatable IL-2 polypeptide of claim 19, wherein the IL-2 polypeptide shares a carboxy-terminal peptide bond with the masking moiety and an amino-terminal peptide bond with the non-IL-2 moiety, or the IL-2 polypeptide shares an amino-terminal peptide bond with the masking moiety and a carboxy-terminal peptide bond with the non-IL-2 moiety.

21. 21. The protease-activatable IL-2 polypeptide of claim 19 or 20, wherein the non-IL-2 moiety is an antigen-binding moiety or an effector cell-binding moiety.

22. An immunoconjugate comprising a protease-activatable IL-2 polypeptide according to claims 1 to 18 and an antigen-binding portion and / or an effector cell-binding portion.

23. 23. The immunoconjugate of claim 22, wherein the protease-activatable IL-2 polypeptide shares an amino- or carboxy-terminal peptide bond with the antigen-binding portion or effector cell-binding portion.

24. 24. The immunoconjugate of claim 22 or 23, comprising a first and a second antigen-binding portion, or a first and a second effector cell antigen-binding portion, or an antigen-binding portion and an effector cell-binding portion.

25. (i) the protease-activatable IL-2 polypeptide shares an amino- or carboxy-terminal peptide bond with the first antigen-binding portion, and the second antigen-binding portion shares an amino- or carboxy-terminal peptide bond with either a) the protease-activatable IL-2 polypeptide or b) the first antigen-binding portion; (ii) the protease-activatable IL-2 polypeptide shares an amino- or carboxy-terminal peptide bond with the first effector cell-binding portion, and the second effector cell-binding portion shares an amino- or carboxy-terminal peptide bond with either a) the protease-activatable IL-2 polypeptide or b) the first effector cell-binding portion; 25. The immune complex of claim 24, wherein (iii) the protease-activatable IL-2 polypeptide shares an amino- or carboxy-terminal peptide bond with the antigen-binding portion and the effector cell-binding portion shares an amino- or carboxy-terminal peptide bond with either a) the protease-activatable IL-2 polypeptide or b) the antigen-binding portion; or (iv) the protease-activatable IL-2 polypeptide shares an amino- or carboxy-terminal peptide bond with the effector cell-binding portion and the antigen-binding portion shares an amino- or carboxy-terminal peptide bond with either a) the protease-activatable IL-2 polypeptide or b) the effector cell-binding portion.

26. 26. The protease-activatable IL-2 polypeptide of claim 21 or the immune complex of any one of claims 22 to 25, wherein the antigen-binding portion or effector cell-binding portion is an antibody or antibody fragment.

27. 26. The protease-activatable IL-2 polypeptide of claim 21 or the immune complex of any one of claims 22 to 25, wherein the antigen-binding portion and / or the effector cell-binding portion is selected from a Fab molecule and an scFv molecule.

28. 26. The protease-activatable IL-2 polypeptide of claim 21 or the immune complex of any one of claims 22 to 25, wherein the antigen-binding portion and / or the effector cell-binding portion is an immunoglobulin molecule, in particular an IgG molecule.

29. 29. The protease-activatable IL-2 polypeptide of claim 21 or the immune complex of any one of claims 22 to 28, wherein the antigen-binding portion is directed against an antigen presented on a tumor cell or in the tumor cell environment, and / or the effector cell-binding portion is directed against an effector cell present in the tumor cell environment to achieve cis-targeting.

30. (i) comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:5, an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:22, and an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:23; (ii) comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:5, an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:22, and an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:24; (iii) comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:5, an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:22, and an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:25; or (iv) The immune complex of any one of claims 22 to 29, comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 5, an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 22, and an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:

26.

31. (i) comprising the amino acid sequence set forth in SEQ ID NO: 5, the amino acid sequence set forth in SEQ ID NO: 22, and the amino acid sequence set forth in SEQ ID NO: 23; (ii) comprises the amino acid sequence set forth in SEQ ID NO: 5, the amino acid sequence set forth in SEQ ID NO: 22, and the amino acid sequence set forth in SEQ ID NO: 24; (iii) comprising the amino acid sequence set forth in SEQ ID NO: 5, the amino acid sequence set forth in SEQ ID NO: 22, and the amino acid sequence set forth in SEQ ID NO: 25; or (iv) The immune complex according to any one of claims 22 to 30, comprising the amino acid sequence set forth in SEQ ID NO: 5, the amino acid sequence set forth in SEQ ID NO: 22, and the amino acid sequence set forth in SEQ ID NO:

26.

32. An isolated polynucleotide encoding the protease-activatable IL-2 polypeptide or immunoconjugate of any one of claims 1 to 27.

33. 33. An expression vector comprising the polynucleotide of claim 32.

34. 34. A host cell comprising the polynucleotide of claim 32 or the expression vector of claim 33.

35. 35. A method for producing a protease-activatable IL-2 polypeptide or immunoconjugate thereof, comprising culturing the host cell of claim 34 under conditions suitable for expression of the protease-activatable IL-2 polypeptide or immunoconjugate.

36. A protease-activatable IL-2 polypeptide or immunoconjugate produced by the method of claim 35.

37. A pharmaceutical composition comprising a protease-activatable IL-2 polypeptide or immunoconjugate according to any one of claims 1 to 31 or 36 and a pharmaceutically acceptable carrier.

38. 37. A protease-activatable IL-2 polypeptide or immunoconjugate according to any one of claims 1 to 31 or 36 for use in the treatment of disease in an individual in need thereof.

39. 39. The protease-activatable IL-2 polypeptide or immunoconjugate of claim 38, wherein the disease is cancer.

40. 40. Use of a protease-activatable IL-2 polypeptide or immunoconjugate according to any one of claims 1 to 31 or 36 for the manufacture of a medicament for treating a disease in an individual in need thereof.

41. 10. A method of treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of a composition comprising a protease-activatable IL-2 polypeptide or immunoconjugate of any one of claims 1 to 31 or 36 in a pharmaceutically acceptable form.

42. 42. The method of claim 41, wherein the disease is cancer.

43. 37. A method of stimulating the immune system of an individual, comprising administering to said individual an effective amount of a composition comprising a protease-activatable IL-2 polypeptide or immunoconjugate of any one of claims 1 to 31 or 36 in a pharmaceutically acceptable form.

44. 10. The invention as hereinbefore described.

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