Protease-activated polypeptides

Abstract

The present invention relates generally 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 wherein the linker has a length of 20 to 45 amino acids wherein the masking moiety is covalently linked to the IL-2 polypeptide through the linker, wherein the masking moiety is capable of binding to the IL-2 polypeptide, thereby reversibly concealing the IL-2 polypeptide, where the masking moiety comprises a second protease cleavage site, where the masking moiety does not conceal the IL-2 polypeptide when cleavage is at the first protease cleavage site and / or the second protease cleavage site. The present invention also relates to polynucleotides encoding such protease activatable interleukin-2 (IL-2) polypeptides and immunoconjugates, as well as vectors and host cells comprising such polynucleotides. The invention also relates to methods for producing 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 generally relates to novel protease-activated polypeptides, in particular 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 protease-activated IL-2 polypeptides or immunoconjugates, polynucleotides, vectors, and host cells comprising such vectors or polynucleotide molecules. The present invention further relates to methods for producing protease-activated IL-2 polypeptides or immunoconjugates; pharmaceutical compositions comprising protease-activated IL-2 polypeptides or immunoconjugates; and uses thereof. Background Art

[0002] In various clinical settings, it is often necessary to selectively destroy single target cells or specific target cell types. For example, a major goal of cancer therapy is to specifically destroy tumor cells while leaving healthy cells and tissues intact.

[0003] An attractive method to achieve this goal is to induce an immune response against tumors so that immune effector cells (such as natural killer (NK) cells or cytotoxic T lymphocytes (CTL)) attack and destroy tumor cells. In this regard, conjugates designed to bind to surface antigens on target cells and comprising interleukin-2 (IL-2) variants should be able to activate nearby T effector cells and NK cells. Such conjugates are combined with their targets and interleukin-2 receptors simultaneously to cause T effector cells and NK cell activation near the target (trans), or when the target is expressed on T effector cells and NK cells, the cell is activated (cis) when combined.

[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 production, survival and homeostasis. It has a length of 133 amino acids and consists of four antiparallel amphipathic α-helices that form a quaternary structure essential for its function (Smith, Science 240, 1169-76 (1988); Bazan, Science 257, 410-413 (1992)). Sequences of IL-2 from different species are found under NCBI RefSeq No. 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 is composed of up to three individual subunits, the different associations of which can produce receptor forms with different affinities for IL-2. c , CD132) subunits produce high-affinity trimeric IL-2 receptors. The dimeric IL-2 receptor composed of β subunits and γ subunits is called the medium-affinity IL-2R receptor. The α subunit forms a low-affinity monomeric IL-2 receptor. Although the medium-affinity dimeric IL-2 receptor binds IL-2 with an affinity about 100 times lower than the high-affinity trimeric receptor, both dimeric and trimeric IL-2 receptor variants are able to transmit signals when IL-2 binds (Minami et al., Annu Rev Immunol 11, 245-268 (1993)). Therefore, α-subunit CD25 is not essential for IL-2 signal transduction. The α-subunit confers high-affinity binding to its receptor, while the β subunit CD122 and γ subunits are essential for signal transduction (Krieg et al., Proc Natl Acad Sci 107, 11906-11 (2010)). The trimeric IL-2 receptor containing CD25 is expressed by (resting) CD4 + Fork frame P3 (FoxP3) + Regulatory T(T reg They are also transiently induced on conventionally activated T cells, whereas in the resting state, these cells express only dimeric IL-2 receptors. reg Cells consistently express maximal levels of CD25 in vivo (Fontenot et al., Nature Immunol 6, 1142-51 (2005)).

[0006] IL-2 is mainly produced by activated T cells, especially by CD4 +Helps T cell synthesis. It stimulates the proliferation and differentiation of T cells, induces the production of cytotoxic T lymphocytes (CTL) and the differentiation of peripheral blood lymphocytes into cytotoxic cells and lymphokine-activated killer (LAK) cells, promotes the expression of cytokines and cytolytic molecules of T cells, helps the proliferation and differentiation of B cells and the immunoglobulin synthesis of B cells, and stimulates the production, 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] Its ability to expand lymphocyte populations in vivo and increase the effector functions of these cells confers IL-2 antitumor effects, making IL-2 immunotherapy an attractive treatment option for certain metastatic cancers. As a result, high-dose IL-2 therapy has been approved for patients with metastatic renal cell carcinoma and malignant melanoma.

[0008] However, IL-2 has a dual function in immune responses as it not only mediates the expansion and activity of effector cells but also plays a key role in maintaining peripheral immune tolerance.

[0009] The main mechanism of peripheral self-tolerance is activation-induced cell death (AICD) induced by IL-2 inducing T cells. AICD is a process in which fully activated T cells undergo programmed cell death by engaging death receptors (such as CD95 (also known as Fas)) or TNF receptors expressed on the cell surface. When antigen-activated T cells expressing high-affinity IL-2 receptors (after previous exposure to IL-2) during proliferation are stimulated by antigen again via T cell receptor (T cellreceptor, TCR) / CD3 complexes, the expression of Fas ligand (Fas ligand, FasL) and / or tumor necrosis factor (tumor necrosis factor, TNF) is induced, making cells sensitive to Fas-mediated apoptosis. This process is IL-2 dependent (Lenardo, Nature 353, 858-61 (1991)) and mediated by STAT5. Through the process of AICD in T lymphocytes, tolerance can be established not only to self-antigens, but also to persistent antigens that are clearly not part of the host, such as tumor antigens.

[0010] In addition, IL-2 is also involved in maintaining peripheral CD4 + CD25 + Regulatory 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 called suppressor T cells. They inhibit the destruction of their (self) targets by effector T cells by cell-to-cell contact with the help and activation of suppressor T cells or by releasing immunosuppressive cytokines such as IL-10 or TGF-β. T reg Depletion of IL-2 cells was shown to enhance IL-2-induced anti-tumor immunity (Imai et al., Cancer Sci 98, 416-23 (2007)).

[0011] Therefore, IL-2 is not optimal for inhibiting tumor growth because in the presence of IL-2, the generated CTLs may recognize the tumor as self and undergo AICD, or the immune response may be blocked by IL-2-dependent T reg Cell inhibition.

[0012] Another problem relevant to IL-2 immunotherapy is the side effect produced by recombinant human IL-2 treatment. Patients receiving high-dose IL-2 treatment often experience serious cardiovascular, lung, kidney, liver, gastrointestinal, neurological, skin, blood and systemic adverse events, which need to be closely monitored and hospitalized. Most of these side effects can be explained by the development of so-called vascular (or capillary) leak syndrome (vascular leak syndrome, VLS), which is that the pathological increase of vascular permeability causes fluid extravasation (causing, for example, lung and skin edema and hepatocyte damage) and intravascular fluid depletion (causing blood pressure to drop and heart rate compensatory increase) in multiple organs. Except eliminating IL-2, VLS has no other treatment methods. Low-dose IL-2 schemes have been tested in patients to avoid VLS, but the cost is suboptimal treatment results. VLS is thought to be caused by the release of proinflammatory cytokines such as tumor necrosis factor (TNF)-α from IL-2-activated NK cells, however, IL-2-induced pulmonary edema has recently been shown to be caused by direct binding of IL-2 to lung endothelial cells, which 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 methods have been taken to overcome these problems associated with IL-2 immunotherapy. For example, it has been found that the combination of IL-2 and certain anti-IL-2 monoclonal antibodies enhances the in vivo therapeutic effect of IL-2 (Kamimura et al., J Immunol 177, 306-14 (2006); Boyman et al., Science 311, 1924-27 (2006)). In an alternative method, IL-2 has been mutated in various ways to reduce its toxicity and / or enhance its efficacy. Hu et al. (Blood 101, 4853-4861 (2003), U.S. Patent Publication No. 2003 / 0124678) have replaced the arginine residue at position 38 of IL-2 with tryptophan to eliminate the vascular permeability activity of IL-2. Shanafelt et al. (Nature Biotechnol 18, 1197-1202 (2000)) have mutated asparagine 88 to arginine to enhance the selectivity of T cells over that of NK cells. Heaton et al. (Cancer Res 53, 2597-602 (1993); U.S. Pat. No. 5,229,109) have introduced two mutations, Arg38Ala and Phe42Lys, to reduce proinflammatory cytokine secretion by NK cells. Gillies et al. (U.S. Pat. Publication No. 2007 / 0036752) have replaced three residues (Asp20Thr, Asn88Arg and Gln126Asp) of IL-2 that contribute to the affinity for the medium-affinity IL-2 receptor to reduce VLS. Gillies et al. (WO 2008 / 0034473) have also mutated the interface of IL-2 with CD25 by amino acid substitutions of Arg38Trp and Phe42Lys to reduce interaction with CD25 and T cells. reg Activation of cells, thereby enhancing efficacy. For the same purpose, Wittrup et al. (WO 2009 / 061853) have produced IL-2 mutants that have enhanced CD25 affinity but do not activate the receptor, thus acting as antagonists. The introduced mutations are intended to disrupt the interaction with the β subunit and / or γ subunit of the receptor.

[0014] WO 2012 / 107417 describes specific mutant IL-2 polypeptides designed to overcome the above-mentioned problems associated with IL-2 immunotherapy (toxicity induced by VLS, tumor tolerance induced by AICD, and immunosuppression caused by Treg cell activation). The phenylalanine residue at position 42 of IL-2 is substituted with alanine, the tyrosine residue at position 45 is substituted with alanine, and the leucine residue at position 72 is substituted with glycine, thereby substantially eliminating the binding of the mutant IL-2 polypeptide to the α subunit of the IL-2 receptor (CD25).

[0015] However, none of the known IL-2 mutants has been shown to overcome all of the above-mentioned problems associated with IL-2 immunotherapy, namely, toxicity induced by VLS, tumor tolerance induced by AICD, and tumor resistance induced by T cells. reg Immunosuppression caused by cell activation.

[0016] In addition, for the above methods, IL-2 immunotherapy can be improved by selectively targeting IL-2 to tumors, for example in the form of an immunoconjugate comprising an antibody that binds to an antigen expressed on tumor cells or binds to effector cells 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 A1).

[0017] Given the clinical success and unprecedented efficacy of PD-1 / PD-L1 checkpoint inhibitors, there remains a significant medical need to further increase the response rate and duration of response in patients with pre-existing T cell immunity. Recent reports indicate that PD-1 antibodies target two tumor-specific CD8 T cell populations: exhausted TILs and newly described TCF1+ precursor TResource cells with stem cell-like properties. Of the two, the latter are associated with favorable disease prognosis and response to anti-PD-1 therapy. Cytokines, such as interleukin-2, have also been described to induce proliferation / differentiation of TResource cells toward functional effector T cells.

[0018] IL-2 is the first effective cancer immunotherapy for the treatment of metastatic melanoma and renal cell carcinoma. Unfortunately, high concentrations of IL-2 are toxic by inducing vascular leak syndrome (VLS), and harmfully amplify regulatory T cells and induce activation-induced cell death due to binding with CD25. In order to overcome these limitations of wild-type IL2 / aldesleukin (Proleukin), IL-2v variants with elimination of CD25 binding have been described. However, due to the mechanism of signal transduction by the medium affinity IL-2Rbg complex of IL-2 via heterodimerization, IL2v and other IL2 variants will automatically activate IL-2R signal transduction when encountering IL-2R, and therefore mediate nonspecific and peripheral immune cell activation outside tumors in blood, vascular system and lymphoid tissue, thereby causing dose-limiting toxicity. Therefore, it is impossible to administer IL-2 or IL2v to patients for obtaining the required amount of maximum therapeutic benefit.

[0019] In summary, by cis-targeting PD1-IL2v to PD-1+ T cells, a stronger therapeutic effect of PD1-IL2v can be achieved. In fact, cis-targeting PD1-IL2v to appropriate antigen-specific T cell subsets, together with PD-1 / -L1 inhibition, is a better way to therapeutically exploit endogenous immunity and is one of the strongest immunomodulatory pathways known to release endogenous immunity for cancer immunotherapy. However, since the IL2v portion may trigger IL-2R signaling in the periphery, the maximum required dose cannot be administered for PD1-IL2v due to peripheral non-tumor-specific IL-2R activation. Therefore, the therapeutic index is considered to be still narrow, and the expected MTD has a flat dose of >10-30 mg in humans, which may limit the potential for utilizing the complete pathway. CD8 T cells can also be targeted, as well as other T cell targets.

[0020] Therefore, it is critical to generate next-generation IL-2 molecules that are cis-targeted to T cells undergoing antigenic stimulation but 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 cancer types (Duffy, MJ 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) are overexpressed in, for example, breast cancer and ovarian cancer (McGowan, PM & Duffy, MJ 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 the ascites of patients with cervical cancer, breast cancer, ovarian cancer, and epithelial ovarian cancer (EOC), but not in the serum of these patients (Demeter, A. et al. Molecular prognostic markers in recurrent and in non-recurrent epithelial ovarian cancer. Anticancer Res. 25, 2885–2889 (2005)). Although matriptase can be detected in normal epithelial cells, matriptase activity is mainly detected in cancer (LeBeau, AM et al. Imaging a functional tumorigenic biomarker in the transformed epithelium. Proc. Natl. Acad. Sci. USA 110, 93–98 (2013)).

[0022] Although current immunotherapies targeting the PD1 / PDL1 axis have demonstrated unprecedented efficacy in a variety of cancer indications, a significant proportion of patients remain unresponsive or relapse to treatment, while other tumor types remain largely refractory to these therapies. Therefore, there is a clear, high unmet need for a sizable population of cancer patients with some type of pre-existing T cell immune response. Examples of indications where PD1 antagonism results in objective responses are, for example, 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 sufficient CRC, and hematological malignancies such as DLBCL and PMBCL (after autologous stem cell transplantation) and HL (Editiorial: 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 generating IL-2 variants and conjugates suitable for treatment presents several technical challenges related to efficacy, toxicity, applicability and manufacturability that must be met. In the case where the conjugate targets an antigen on a target cell (e.g., a cancer cell) that is also expressed in a non-target tissue, toxicity may occur. Therefore, there remains a need in the art to further enhance the therapeutic usefulness 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 tissues and that a masking therapeutic agent, preferably protease-activatable interleukin-2, has reduced or abolished systemic activity and, when activated by a protease, is fully active in the tumor environment.

[0025] Thus, a first aspect of the 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, wherein the masking moiety is covalently attached to the IL-2 polypeptide via a linker, wherein the masking moiety is capable of binding to the IL-2 polypeptide, thereby reversibly concealing the IL-2 polypeptide, wherein the masking moiety comprises a second protease cleavage site, wherein when cleaved at the first protease cleavage site and / or the second protease cleavage site, the masking moiety does not conceal the IL-2 polypeptide. 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 the linker has a sequence according to SEQ ID NO: 64 or SEQ ID NO: 66. In one embodiment, the linker has a length of 38 amino acids, preferably the linker has a sequence according to SEQ ID NO: 63 or SEQ ID NO: 65. In one embodiment, the masking part is covalently linked to the amino terminal or carboxyl terminal of the interleukin-2 polypeptide through a linker. In one embodiment, the masking part is an IL-2 antagonist. In one embodiment, the masking part 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 part is an antibody derived from MT204, preferably MT204. MT204 antibody is disclosed in, for example, Volkland et al., Molecular Immunology 44 (2007) 1743–1753, and PCT publication WO 2006 / 128690 A1. More preferably, the masking part is a deimmunized MT204-derived conjugate. In one embodiment, the Fab molecule is a single-chain Fab molecule. In one embodiment, the second protease cleavage site is located between the heavy chain variable domain (VH) and the light chain variable domain (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. 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 YAARKGGI according to SEQ ID NO:60 and / or PQARK according to SEQ ID NO:61.

[0026] In one embodiment, the IL-2 polypeptide is a wild-type IL-2, preferably a human IL-2 according to 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 according to 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 according to 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 according to 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 carboxyl-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 carboxyl-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 as 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-terminal peptide bond or a carboxyl-terminal peptide bond with the antigen-binding portion or the effector cell-binding portion. In one embodiment, the immunoconjugate comprises a first antigen-binding portion and a second antigen-binding portion, or a first effector cell antigen-binding portion 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 carboxyl-terminal peptide bond with the first antigen-binding portion, and the second antigen-binding portion shares an amino-terminal peptide bond or a carboxyl-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-terminal peptide bond or a carboxyl-terminal peptide bond with the first effector cell-binding portion, and the second effector cell-binding portion shares an amino-terminal peptide bond with either a) the protease-activatable IL-2 polypeptide or b) the first effector cell-binding portion or carboxyl-terminal peptide bond; (iii) the protease-activatable IL-2 polypeptide shares an amino-terminal peptide bond or carboxyl-terminal peptide bond with the antigen-binding portion, and the effector cell-binding portion shares an amino-terminal peptide bond or carboxyl-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-terminal peptide bond or carboxyl-terminal peptide bond with the effector cell-binding portion, and the antigen-binding portion shares an amino-terminal peptide bond or carboxyl-terminal peptide bond with either 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 contained 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 a 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 in a tumor cell or a tumor cell environment, and / or wherein the effector cell binding portion is directed against an effector cell present in a tumor cell environment to achieve cis targeting.

[0029] In one embodiment, (i) 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; (ii) 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. ID NO:24; (iii) 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; or (iv) 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. 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 immunoconjugate comprises the amino acid sequence according to SEQ ID NO:5, the amino acid sequence according to SEQ ID NO:22, and the amino acid sequence according to SEQ ID NO:23; (ii) the immunoconjugate comprises the amino acid sequence according to SEQ ID NO:5, the amino acid sequence according to SEQ ID NO:22, and the amino acid sequence according to SEQ ID NO:24; (iii) the immunoconjugate comprises the amino acid sequence according to SEQ ID NO:5, the amino acid sequence according to SEQ ID NO:22, and the amino acid sequence according to SEQ ID NO:25; or (iv) the immunoconjugate comprises the amino acid sequence according to SEQ ID NO:5, the amino acid sequence according to SEQ ID NO:22, and the amino acid sequence according to SEQ ID NO:26.

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

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

[0033] Also provided is a protease-activatable IL-2 polypeptide or immunoconjugate as described herein, produced by the methods described herein. Also provided is a pharmaceutical composition comprising a protease-activatable IL-2 polypeptide or immunoconjugate disclosed herein and a pharmaceutically acceptable carrier. In particular, the present invention encompasses a protease-activatable IL-2 polypeptide or immunoconjugate as described herein for use in treating a disease in an individual in need thereof. In a specific embodiment, the disease is cancer. In a specific embodiment, the individual is a human.

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

[0035] Also provided is a method of stimulating the immune system of an individual, the method 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 DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 AI. IgG antibodies were generated to evaluate deimmunized MT204 mask and anti-PD1 binder variants by SPR along with corresponding antigen and parental controls. Figure 1 A (P1AH2050-P1AH2052) deimmunized MT204 mask in human IgG PG LALA format; Figure 1 B (P1AH4157-P1AH4161) The first set of deimmunized anti-PD1 binder variants in human IgG PG LALA format; Figure 1 C (P1AI0356-P1AI0360) a second set of deimmunized anti-PD1 binder variants in human IgG PG LALA format; Figure 1 D(P1AI1648-P1AI1652) a third group of deimmunized anti-PD1 binder variants in the form of human IgG PG LALA; Figure 1 E(P1AF7506) one-armed parental MT204 in human IgG PG LALA format; Figure 1 F(P1AA6888) parental anti-PD1 binder in the form of human IgG PGLALA; Figure 1 G (P1AD9704) Fc fusion of human PD1 antigen with C-terminal biotinylated avi tag and His tag; Figure 1 H (P1AG0879) IL2v cytokine with a C-terminal avi tag and a His tag; Figure 1 I(P1AA9690) Parental MT204 in human IgG PG LALA format.

[0037] Figure 2 AF. Human PD1-targeted masked IL2v constructs with PQARK or YAARKGGI matriptase sites and corresponding non-masked PD1-targeted or FAP-targeted controls. Figure 2 A (P1AI4322) bivalent human PD1-targeted human IgG PG LALA with masked IL2v (“Linear”) fused to the C-terminus of the Fc knob chain, 2 PQARK matriptase sites for mask release and a 38 amino acid linker between the IL2v cytokine and the scFv mask; Figure 2B (P1AI4323) bivalent human PD1-targeted human IgG PG LALA with masked IL2v (“linear”) fused to the C-terminus of the Fc knob chain, 2 PQARK matriptase sites for mask release and a 25 amino acid linker between the IL2v cytokine and the scFv mask; Figure 2 C (P1AI4324) bivalent human PD1-targeted human IgG PG LALA with masked IL2v (“linear”) fused to the C-terminus of the Fc knob chain, 2 YAARKGGI matriptase sites for release of the mask and a 38 amino acid linker between the IL2v cytokine and the scFv mask; Figure 2 D(P1AI4325) bivalent human PD1-targeted human IgG PG LALA with masked IL2v ("Linear") fused to the C-terminus of the Fc knob chain, 2 YAARKGGI matriptase sites for release of the mask and a 25 amino acid linker between the IL2v cytokine and the scFv mask; constructs AE contain the preferred deimmunized V domain sequences of the anti-PD1 binder and the MT204 scFv mask; Figure 2 E(P1AE4422) bivalent human PD1-targeted human IgG PG LALA with IL2v fused to the C-terminus of the Fc knob chain as a non-masking control; Figure 2 F(P1AA5355) Bivalent human FAP-targeted human IgG PG LALA with IL2v fused to the C-terminus of the Fc knob chain as a non-masking control.

[0038] Figure 3 A to Figure 3 C. Human PD1 targeting masked IL2v construct as uncleavable control and corresponding non-masking control. Figure 3 A (P1AI4646) bivalent human PD1-targeted human IgG PG LALA with masked IL2v fused to the C-terminus of the Fc knob chain (“linear”), a 38 amino acid linker between the IL2v cytokine and the scFv mask, and no matriptase release site (non-cleavable control); Figure 3 B (P1AI4647) bivalent human PD1-targeted human IgG PGLALA with masked IL2v fused to the C-terminus of the Fc knob chain (“linear”), 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 a non-masked control; constructs AC contain the preferred deimmunized V domain sequence of the anti-PD1 binder and MT204 scFv mask (constructs A and B).

[0039] Figure 4 A to Figure 4 B. Murine alternatives to the human PD1 -targeted masked IL2v construct with the YAARKGGI matriptase site and corresponding controls. Figure 4 A (P1AI4650) bivalent human PD1-targeted murine IgG DA PG with masked IL2v (“linear”) fused to the C-terminus of the FcDD-chain and 2 YAARKGGI matriptase sites for release of the mask; Figure 4 B (P1AI4651) Bivalent human PD1-targeted murine IgG DA PG with masked IL2v fused to the C-terminus of the Fc DD-chain (“Linear”) and without a matriptase release site (non-cleavable control).

[0040] Figure 5 AB. Measurement of the potency of deimmunized aPD-1 constructs to block PD1 / PD-L1 interactions. PD-1 effector cells were incubated with PD-L1aAPC / CHO-K1 in the presence of anti-PD1 antibodies. Bio-Glo reagent was added and luminescence was measured on a luminometer. Data (mean ± SEM) were analyzed using GraphPad Prism software. Data from 1 experiment. Figure 5 A shows blocking with P1AH4159, P1AH4160, and P1AH4161. Figure 5 B shows blocking by P1AH4157 and P1AH4158.

[0041] Figure 6 A to Figure 6 B. Binding of deimmunized aPD-1 to CD3 / CD28 activated CD4 T cells compared to aPD-1IgG PG LALA, PD1-IL2v and FAP-IL2v. PD1 antibody constructs were added to activated CD4 T cells at different concentrations and dose-dependent responses were measured by flow cytometry. Data (mean ± SEM) were analyzed using GraphPad Prism software. Data from 2 donors. Figure 6 A shows binding to P1AH4159, P1AH4160, and P1AH4161. Figure 6 B shows binding to P1AH4157 and P1AH4158.

[0042] Figure 7 A to Figure 7 B. Binding of deimmunized aPD-1 to CD3 / CD28 activated CD4 T cells compared to aPD-1IgG PG LALA, PD1-IL2v and FAP-IL2v. PD1 antibody constructs were added to activated CD4 T cells at different concentrations and dose-dependent responses were measured by flow cytometry. Data (mean ± SEM) were analyzed using GraphPad Prism software. Data from 2 donors. Figure 7 A shows binding to P1AH4159, P1AH4160, and P1AH4161. Figure 7 B shows binding to P1AH4157 and P1AH4158.

[0043] Figure 8 A to Figure 8 B. Minimal mixed lymphocyte reaction. The effect of several anti-PD-1 blocking antibodies on the increase of allospecific T-cell effector function was investigated by describing the frequency of granzyme B production in proliferating CD4 T cells. Data were acquired by flow cytometry and analyzed using GraphPad Prism software (mean ± SEM). Data from 1 experiment (CD4 T cells from 2 donors and mDCs from 2 donors). Figure 8 A shows the production of granzyme B exposed to P1AH4159, P1AH4160, and P1AH4161. Figure 8 B shows the production of granzyme B exposed to P1AH4157 and P1AH4158.

[0044] Fig. 9 A to Fig. 9 B. Minimal mixed lymphocyte reaction. The effect of several anti-PD-1 blocking antibodies on the increase of allospecific T cell effector function was investigated by describing the frequency of interferon-γ production in proliferating CD4 T cells. Data were acquired by flow cytometry and analyzed using GraphPad Prism software (mean ± SEM). Data from 1 experiment (CD4 T cells from 2 donors and mDCs from 2 donors). Fig. 9 A shows interferon-γ production in cells exposed to P1AH4159, P1AH4160, and P1AH4161. Fig. 9 B shows interferon-γ production in cells exposed to P1AH4157 and P1AH4158.

[0045] Fig.10Binding of deimmunized PD1-IgG to CHO cells overexpressing human PD1 was determined by flow cytometry. A fluorescently labeled anti-human Fc-specific secondary antibody was used to detect the molecule.

[0046] Fig.11 Blockade 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.

[0047] Fig.12 A to Fig.12 B. Binding of the indicated TA PD1-IL2v constructs to CHO cells overexpressing human PD1 was determined by flow cytometry. Molecules were detected with a fluorescently labeled anti-human Fc specific secondary antibody. Fig.12 A relates to a construct with a PQARK cleavage site. Fig.12 B relates to a construct with a YAARKGGI cleavage site.

[0048] Fig.13 A to Fig.13 F. Proliferation of KHYG-1 cells induced by a panel of TA PD1-IL2v constructs was measured using CellTiter Glo. Fig.13 A and Fig.13 B relates to a construct with a PQARK cleavage site. Fig.13 C and Fig.13 D relates to a construct with a YARKGGI cleavage site. Fig.13 E and Fig.13 F relates to a construct without a cleavage site.

[0049] Fig.14 Results from efficacy experiments using TA-PD1-IL2v cleavable (YAARKGGI 38-mer linker) and non-cleavable Mabs as single agents are presented. MCA205 fibrosarcoma cancer cell line was injected subcutaneously into Black 6-huPD1 tg mice to study tumor growth inhibition in a subcutaneous model. Tumor size was measured using a caliper. Tumors were observed to reach 150 mm 3 Treatment was started at 1:10 p.m. For TA-PD1-IL2v YAARKGGI 38-mer cleavable and TA-PD1-IL2v non-cleavable, the amount of antibody injected per mouse was 2 mg / kg, given twice a week. Treatment lasted for 1 week. Compared with the vehicle and non-cleavable Mab single-agent groups, TA-PD-IL2v YAARKGGI 38-mer mediated excellent efficacy in tumor growth inhibition.

[0050] Fig.15. Combination partners for in vivo efficacy studies (human FolR1-targeted T cell engagers). (P1AK1120) 2+1 human FolR1-targeted T cell engagers were used as combination partners for in vivo efficacy studies.

[0051] Fig.16 A to Fig.16 C. Murine alternatives to human PD1-targeted masked IL2v constructs with and without PQARK matriptase sites. Fig. 16A: (P1AK3638) Bivalent human PD1-targeted murine IgG DA PG with masked IL2v fused to the C-terminus of the FcDD-chain (“Linear”) and 2 PQARK matriptase sites for releasing the mask; Fig.16 B: (P1AK3649) Bivalent human PD1-targeted murine IgG DA PG with masked IL2v fused to the C-terminus of the Fc DD-chain (“Linear”) and without a matriptase release site (non-cleavable control). Fig.16 C: (P1AG7552) Bivalent human PD1 targeting murine IgG DA PG with IL2v fused to the C-terminus of the Fc DD-chain (non-masked control).

[0052] Fig.17 A to Fig.17 D. Murine surrogates of murine PD1 -targeted masked IL2v constructs with and without the PQARK matriptase site and control IgG. Fig.17 A: (P1AK3641) bivalent murine PD1-targeted murine IgG DA PG with masked IL2v (“linear”) fused to the C-terminus of the Fc DD-chain and 2 PQARK matriptase sites for release of the mask; Fig.17 B: (P1AK3640) bivalent murine PD1-targeted murine IgG DA PG with masked IL2v fused to the C-terminus of the Fc DD-chain (“linear”) and without a matriptase release site (non-cleavable control); Fig.17 C: (P1AD4006) Murine PD1 targeting murine IgG used as a non-IL2v fusion control construct. Fig.17 D: (P1AG9991) Bivalent murine PD1 targeted mouse IgG DA PG with IL2v fused to the C-terminus of the Fc DD-chain (non-masked control).

[0053] Fig.18 A to Fig.18B. HEK Blue IL2 reporter cell assay using HEK Blue IL2 cells overexpressing human PD1 was performed to test the activity of the murinized TA PD1-IL2v construct. The construct was pre-digested with recombinant human Matripatase ( Fig.18 A). Constructs were tested without predigestion ( Fig.18 B).

[0054] Fig.19 A to Fig.19 B. HEK Blue IL2 reporter cell assay was performed using HEK Blue IL2 cells overexpressing mouse PD1 to test the activity of the murinized TA PD1-IL2v construct containing a mouse-specific PD1 binder. The construct was pre-digested with recombinant human Matriptase ( Fig.19 A). Constructs were tested without predigestion ( Fig.19 B).

[0055] Fig. 20 A to Fig. 20 B. NK cells measured by CFSE dilution after treatment with TA PD1-IL2v construct digested with recombinant human Matriptase ( Fig. 20 A) and CD8 T cells ( Fig. 20 B) proliferation.

[0056] Fig.21 A to Fig.21 B. NK cells measured by CD25 upregulation after treatment with TA PD1-IL2v construct digested with recombinant human Matriptase ( Fig.21 A) and CD8 T cells ( Fig.21 B) activation.

[0057] Fig. 22 Results from the efficacy experiments of TA-PD1-IL2v cleavability (PQARK 25-mer linker), non-cleavability, and pembrolizumab Mab as a single agent are presented. MCA205 fibrosarcoma cancer cell line was injected subcutaneously into Black6-huPD1 tg mice to study tumor growth inhibition in a subcutaneous model. Tumor size was measured using a caliper. Tumors were expressed when they reached 200mm 3 Treatment was started at 1:10 pm. For TA-PD1-IL2v PQARK 25-mer cleavable and pembrolizumab, the amount of antibody injected per mouse was 1 and 3 mg / kg and 3 mg / kg, and TA-PD1-IL2v non-cleavable was given twice a week. Treatment lasted for 1 week. Compared with the vehicle, pembrolizumab and non-cleavable Mab single agent groups, TA-PD-IL2v PQARK25-mer mediated excellent efficacy in tumor growth inhibition.

[0058] Fig.23 Results from efficacy experiments using muTA-PD1-IL2v cleavable (PQARK 25-mer linker) and muPD1 mAb as single agents are presented. GL261 glioblastoma cell line was injected subcutaneously into Black 6 mice to study tumor growth inhibition in a subcutaneous model. Tumor size was measured using a caliper. Tumors were expressed when they reached 100 mm 3 Treatment was initiated at 1 hr. The amount of antibody injected per mouse was 1 and 3 mg / kg and 3 mg / kg for muTA-PD1-IL2v PQARK 25-mer cleavable and muPD1, given twice a week. Treatment lasted for 1 week. Compared with vehicle and muPD1Mab single agent groups, mu-TA-PD-IL2v PQARK 25-mer mediated superior efficacy in tumor growth inhibition.

[0059] Fig.24 .The results of the efficacy experiment evaluating the combination of TA-PD1-IL2v and FOLR1-TCB Mab are shown. BC004 human breast cancer PDX cells were subcutaneously injected into humanized NSG mice to study tumor growth inhibition in a subcutaneous xenograft model of breast cancer. The amount of antibody (mg / kg) injected per mouse is as follows: 1mg / kg TA-PD1-IL2v PQARK 25-mer cleavable linker, 0.1mg / kg PD1-IL2v non-masking, 1mg / kg pembrolizumab and 0.3mg / kg FOLR1-TCBMab. The antibody was injected intravenously once a week for 4 weeks. Compared with the FOLR1-TCB single agent group and the FOLR1-TCB+pembrolizumab combination group, significantly superior tumor growth inhibition was observed in the combination FOLR1-TCB+TA-PD1-IL2v PQARK 25-mer cleavable linker group. The combination of FOLR1-TCB+TA-PD1-IL2v PQARK cleavable linker showed similar tumor growth inhibition effect as the combination of FOLR1-TCB+PD1-IL2v non-masking group. DETAILED DESCRIPTION

[0060] definition

[0061] Unless otherwise defined below, the terms used herein are generally as used in the art.

[0062] Unless otherwise indicated, the term "interleukin-2" or "IL-2" as used herein refers to any natural IL-2 from any vertebrate source, including mammals such as primates (e.g., humans), and rodents (e.g., mice and rats). The term includes unprocessed IL-2 and any form of IL-2 produced by processing in cells. The term also encompasses naturally occurring IL-2 variants, such as splice variants or allelic variants. The amino acid sequence of an exemplary human IL-2 is shown in SEQ ID NO: 62.

[0063] As used herein, the term "IL-2 mutant" or "mutant IL-2 polypeptide" is intended to cover any mutant form of the various forms of IL-2 molecules, including full-length IL-2, truncated forms of IL-2, and forms of IL-2 connected to another molecule such as by fusion or chemical conjugation. When used in relation to IL-2, "full length" is intended to mean a mature, native length IL-2 molecule. For example, full-length human IL-2 refers to a molecule having 133 amino acids (see, e.g., SEQ ID NO: 62). Various forms of IL-2 mutants are characterized by having at least one amino acid mutation that affects the interaction of IL-2 with CD25. The mutation may involve substitution, deletion, truncation or modification of a wild-type amino acid residue normally located at that position. Mutants obtained by amino acid substitution are preferred. Unless otherwise indicated, IL-2 mutants may be referred to herein as IL-2 mutant peptide sequences, IL-2 mutant polypeptides, IL-2 mutant proteins or IL-2 mutant analogs.

[0064] Various forms of IL-2 are named herein with respect to the sequence shown in SEQ ID NO: 62. Various names may be used herein to indicate the same mutation. For example, a mutation from phenylalanine to alanine at position 42 may be represented as 42A, A42, A 42 , F42A or Phe42Ala.

[0065] As used herein, a "wild-type" form of IL-2 is a form of IL-2 that is otherwise identical to a mutant IL-2 polypeptide, except that the wild-type form has a wild-type amino acid at each amino acid position of the mutant IL-2 polypeptide. For example, if the IL-2 mutant is a full-length IL-2 (i.e., IL-2 is not fused or conjugated to any other molecule), the wild-type form of the mutant is a full-length native IL-2. If the IL-2 mutant is a fusion between IL-2 and another polypeptide (e.g., an antibody chain) encoded downstream of IL-2, the wild-type form of the IL-2 mutant is an IL-2 fused to the same downstream polypeptide and having a wild-type amino acid sequence. In addition, if the IL-2 mutant is a truncated form of IL-2 (a mutation or modified sequence within the non-truncated portion of IL-2), the wild-type form of the IL-2 mutant is a similarly truncated IL-2 with a wild-type sequence. For the purpose of comparing the IL-2 receptor binding affinity or biological activity of various forms of IL-2 mutants 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 that do not affect IL-2 receptor binding compared to naturally occurring native IL-2, such as replacing alanine with cysteine ​​at a position corresponding to residue 125 of human IL-2. In some embodiments, the wild-type IL-2 for the purposes of the present invention comprises the amino acid substitution C125A. In certain embodiments according to the present invention, the wild-type IL-2 polypeptide compared to the mutant IL-2 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 62.

[0066] Unless otherwise indicated, the term "CD25" or "alpha subunit of the IL-2 receptor" as used herein refers to any native CD25 from any vertebrate source, including mammals such as primates (e.g., humans), and rodents (e.g., mice and rats). The term includes "full-length," unprocessed CD25, as well as any form of CD25 produced by processing in cells. The term also encompasses naturally occurring CD25 variants, such as splice variants or allelic variants. In certain embodiments, CD25 is human CD25.

[0067] As used herein, the term "high affinity IL-2 receptor" refers to the heterotrimeric form of the IL-2 receptor, which consists of the receptor gamma subunit (also known as the common cytokine receptor gamma subunit, gamma cOr CD132), receptor β subunit (also known as CD122 or p70) and 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 γ subunits and β subunits, but not α subunits (for review, see, e.g., Olejniczak and Kasprzak, Med Sci Monit 14, RA179-189 (2008)).

[0068] "Regulatory T cells" or "T reg "Cells" are special types of CD4 T cells that can suppress the response of other T cells. + T cells. reg The cells are characterized by expressing 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)), and play a key role in the induction and maintenance of peripheral self-tolerance to antigens, including those expressed by tumors. reg Cells require IL-2 to carry out their function and development and to induce their suppressive characteristics.

[0069] As used herein, the term "effector cell" refers to a lymphocyte population that mediates the cytotoxic effects of IL-2. Effector cells include effector T cells, such as CD8 + Cytotoxic T cells, NK cells, lymphokine-activated killer (LAK) cells and macrophages / monocytes.

[0070] As used herein, the term "antigen binding molecule" in its broadest sense refers to a molecule that specifically binds to an antigenic determinant. Examples of antigen binding molecules are immunoglobulins and their derivatives, such as fragments thereof.

[0071] The term "bispecific" refers to an antigen binding molecule that is able to specifically bind to at least two different antigenic determinants. Typically, a bispecific antigen binding molecule comprises two antigen binding sites, each of which is specific for different antigenic determinants. In certain embodiments, a bispecific antigen binding molecule is able to simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two unique cells.

[0072] The term "valency" as used herein means that there is a specified number of antigen binding sites in an antigen binding molecule. Thus, the term "monovalently binds to an antigen" means that there is one (and no more than one) antigen binding site specific for an antigen in an antigen binding molecule.

[0073] "Antigen binding site" refers to the site of an antigen binding molecule that provides interaction with an antigen, i.e., one or more amino acid residues. For example, the antigen binding site of an antibody comprises amino acid residues from the complementarity determining region (CDR). Natural immunoglobulin molecules typically have two antigen binding sites, and Fab molecules typically have a single antigen binding site.

[0074] As used herein, the term "antigen binding portion" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, the antigen binding portion can guide the entity to which it is attached (e.g., a second antigen binding portion) to a target site, such as a tumor cell or tumor stroma of a specific type with an antigenic determinant. In another embodiment, the antigen binding portion can activate signal transduction by its target antigen (e.g., a T cell receptor complex antigen). The antigen binding portion includes antibodies and fragments thereof as further defined herein. A specific antigen binding portion includes an antigen binding domain of an antibody, which includes an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, the antigen binding portion may include an antibody constant region as further defined herein and known in the art. Available heavy chain constant regions include any of the following five isotypes: α, δ, ε, γ, or μ. Available light chain constant regions include any of the following two isotypes: κ and λ.

[0075] 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 continuous amino acid or a conformational configuration consisting of different regions of non-continuous amino acids), to which an antigen binding portion binds, thereby forming an antigen binding portion-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, in free matter in serum and / or in the extracellular matrix (ECM). Unless otherwise indicated, the protein referred to as an antigen herein can be any natural form of protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). In a specific embodiment, the antigen is a human protein. When referring to a specific protein herein, the term encompasses "full length", unprocessed protein, and any form of protein produced by intracellular processing. The term also encompasses naturally occurring protein variants, such as splice variants or allelic variants. The ability of an antigen binding portion to bind 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 (SPR) technology (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding of an antigen binding portion to an unrelated protein is less than about 10% of the extent of binding of the antigen binding portion to the antigen, as measured, for example, by SPR. In certain embodiments, an antigen binding portion that binds to an antigen, or an antigen binding molecule comprising the antigen binding portion, has a dissociation constant (K) of D ): ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM or ≤0.001 nM (e.g., 10 -8 M or smaller, for example, from 10 -8 M to 10 -13 M, for example, from 10 -9 M to 10 -13 M).

[0076] "Affinity" refers to the strength of the sum 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, as used herein, "binding affinity" refers to intrinsic binding affinity, which 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 can generally be expressed in terms of the dissociation constant (K D ) indicates that the dissociation constant is the sum of the dissociation rate constant and the association rate constant (k off and k on ). 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).

[0077] "Decreased binding" (e.g., reduced binding to an Fc receptor) refers to a decrease in affinity for the corresponding interaction, as measured, for example, by SPR. For clarity, the term also includes a decrease in affinity to zero (or below the detection limit of the analytical method), i.e., complete elimination of the interaction. Conversely, "increased binding" refers to an increase in binding affinity for the corresponding interaction.

[0078] 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.

[0079] As used herein, "target cell antigen" refers to an antigenic determinant present on the surface of a target cell, for example, a cell in a tumor (such as a cancer cell or a cell of a tumor stroma).

[0080] As used herein, the terms "first" and "second" with respect to antigen binding moieties and the like are used for convenience in distinguishing when there is more than one of each type of moiety. Unless explicitly stated, the use of these terms is not intended to confer a particular order or orientation on the protease-activatable IL-2 polypeptides or immunoconjugates.

[0081] "Fab molecule" refers to a protein composed of the VH and CH1 domains of the heavy chain ("Fab heavy chain") and the VL and CL domains of the light chain ("Fab light chain") of an immunoglobulin.

[0082] "TA" stands for tumor activated. "OA" stands for both arms.

[0083] "Fusion" means that the components (eg, Fab molecule and Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0084] 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 a Fab light chain and a Fab heavy chain are connected by a peptide linker to form a single peptide chain. Another term is a single-chain variable fragment (scFv). In a specific embodiment of this type, the C-terminus of the Fab light chain in the single-chain Fab molecule is connected to the N-terminus of the Fab heavy chain.

[0085] "Cross" Fab molecules (also referred to as "Crossfab") refer to a Fab molecule in which the variable regions or constant regions of the Fab heavy and light chains are exchanged, i.e., the cross-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 cross-Fab molecule in which the variable regions of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain constant region is referred to herein as the "heavy chain" of the cross-Fab molecule. Conversely, in a cross-Fab molecule in which the constant regions of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain variable region is referred to herein as the "heavy chain" of the cross-Fab molecule.

[0086] In contrast, a "conventional" Fab molecule refers to a Fab molecule in its native form, ie, comprising a heavy chain consisting of a heavy chain variable region and a constant region (VH-CH1), and a light chain consisting of a light chain variable region and a constant region (VL-CL).

[0087] The term "immunoglobulin molecule" refers to a protein with the structure of a naturally occurring antibody. For example, the IgG class immunoglobulin is a heterotetrameric glycoprotein of about 150,000 daltons, which is composed of two light chains and two heavy chains bonded by disulfide bonds. From the N-terminal to the C-terminal, each heavy chain has a variable region (VH) (also referred to as a variable heavy chain domain or a heavy chain variable domain), followed by three constant domains (CH1, CH2 and CH3) (also referred to as a heavy chain constant region). Similarly, from the N-terminal to the C-terminal, each light chain has a variable region (VL) (also referred to as a variable light chain domain or a light chain variable domain), followed by a constant light chain (CL) domain (also referred to as a light chain constant region). The heavy chains of immunoglobulins can be assigned to one of five types: called α (IgA), δ (IgD), ε (IgE), γ (IgG) or μ (IgM), some of which can be further divided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chains of immunoglobulins can be assigned to one of two types based on the amino acid sequence of their constant domains: called kappa (κ) and lambda (λ). Immunoglobulins are essentially composed of two Fab molecules and an Fc domain connected by the immunoglobulin hinge region.

[0088] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0089] "Antibody fragment" refers to a molecule other than a complete antibody, which comprises a portion of a complete antibody that binds to the antigen to which the complete 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 certain antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, for example, Plückthun in The harmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab fragments and F(ab')2 fragments that contain salvage receptor binding epitope residues and have extended in vivo half-lives, see U.S. Pat. No. 5,869,046. Diabodies are antibody fragments with two antigen binding sites that can be bivalent or bispecific. See, e.g., EP 404,097; WO 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 certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516B1). Antibody fragments can be prepared by various techniques, as described herein, including but not limited to proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage).

[0090] The term "antigen binding domain" refers to a portion of an antibody that includes a region that specifically binds to and is complementary to part or all of an antigen. The antigen binding domain can be provided, for example, by one or more antibody variable domains (also referred to as antibody variable regions). In particular, the antigen binding domain includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0091] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that participates in the binding of an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6th edition, WH Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity.

[0092] As used herein, the term "hypervariable region" or "HVR" refers to any of the regions of the antibody variable domain that are hypervariable in sequence and / or form structurally defined loops ("hypervariable loops"). Typically, a natural four-chain antibody comprises six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and / or from complementary determining regions (CDRs), which have the highest sequence variability and / or are involved in antigen recognition. In addition to CDR1 in VH, CDRs typically comprise amino acid residues that form hypervariable loops. Hypervariable regions (HVRs) are also referred to as "complementarity determining regions" (CDRs), and these terms are used interchangeably herein to refer to the variable region portions that form the antigen binding region. This specific region has been described by Kabat et al., USDept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and Chothia et al., J Mol Biol 196:901-917 (1987), wherein the definition includes overlap or subsets of amino acid residues when compared to each other. However, using any of the two definitions to refer to the CDR of an antibody or its variants should be within the scope of the term as defined and used herein. As a comparison, the corresponding amino acid residues comprising the CDR defined by each of the references cited above are listed in Table 1 below. The exact number of residues comprising a specific CDR will vary according to the sequence and size of the CDR. In the case of the variable region amino acid sequence of a given antibody, those skilled in the art can routinely determine which residues comprise a specific CDR.

[0093] Table 1. CDR definitions 1

[0094] CDR Kabat Chothia <![CDATA[AbM 2 ]]> <![CDATA[V H CDR1]]> 31-35 26-32 26-35 <![CDATA[V H CDR2]]> 50-65 52-58 50-58 <![CDATA[V H CDR3]]> 95-102 95-102 95-102 <![CDATA[V L CDR1]]> 24-34 26-32 24-34 <![CDATA[V L CDR2]]> 50-56 50-52 50-56 <![CDATA[V L CDR3]]> 89-97 91-96 89-97

[0095] 1 The numbering of all CDR definitions in Table 1 is according to the numbering convention proposed by Kabat et al. (see below).

[0096] 2 "AbM" with a lowercase "b" as used in Table 1 refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

[0097] Kabat et al. also defined a numbering system applicable to the variable region sequence of any antibody. One of ordinary skill in the art can clearly assign the "Kabat numbering" system to any variable region sequence, independent of any experimental data outside the sequence itself. As used herein, "Kabat numbering" refers to the numbering system proposed by Kabat et al., US Pat. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise stated, reference to the numbering of specific amino acid residue positions in the variable region of an antibody is based on the Kabat numbering system.

[0098] The polypeptide sequences of the sequence listing are not numbered according to the Kabat numbering system. However, it is entirely within the capabilities of those of ordinary skill in the art to convert the sequence numbers of the sequence listing into Kabat numbers.

[0099] "Framework" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. The FR of the variable domain is usually composed of the following four FR domains: FR1, FR2, FR3 and FR4. Therefore, HVR and FR sequences usually appear in the following sequence in VH (or VL): FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.

[0100] The "class" of an antibody or immunoglobulin refers to the type of constant domain or region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these antibodies can be further divided into subclasses (isotypes), such as 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.

[0101] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one aspect, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the antibody produced by the host cell can undergo post-translational cleavage of one or more (particularly one or two) amino acids from the C-terminus of the heavy chain. Therefore, the antibody produced by the host cell by expressing a specific nucleic acid molecule encoding a full-length heavy chain can include a full-length heavy chain, or the antibody can include a cleaved variant of the full-length heavy chain. This may be a situation where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU numbering system). Therefore, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (Lys447) in the Fc region may be present or may not be present. If not otherwise specified, the amino acid sequence of the heavy chain comprising the Fc region is represented herein as having no C-terminal glycine-lysine dipeptide. In one aspect, the heavy chain comprising the Fc region as specified herein is included in an antibody according to the present invention, the heavy chain comprising an additional C-terminal glycine-lysine dipeptide (G446 and K447, EU numbering system). In one aspect, the heavy chain comprising the Fc region as specified herein is included in an antibody according to the present invention, the heavy chain comprising an additional C-terminal glycine residue (G446, numbered according to the EU index). Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is carried out according to the EU numbering system (also referred to as the EU index), as described by Kabat et al. (Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991) (see also above). As used herein, the "subunit" of the Fc domain refers to one of the two polypeptides forming the dimeric Fc domain, i.e., a polypeptide comprising the C-terminal constant region of an immunoglobulin heavy chain, which polypeptide is capable of stable self-association. For example, the subunit of an IgG Fc domain comprises the IgG CH2 and IgG CH3 constant domains.

[0102] By "fused", it is meant that the components (eg, Fab molecule and Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0103] "Modifications that promote the association of the first and second subunits of the Fc domain" are manipulations of the peptide backbone or post-translational modifications of the Fc domain subunits that reduce or prevent the association of a polypeptide comprising the Fc domain subunit with the same polypeptide to form a homodimer. As used herein, modifications that promote association particularly include separate modifications to each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) that are desired to associate, wherein the modifications complement each other to promote the association of the two Fc domain subunits. For example, modifications that promote association may change the structure or charge of one or both of the Fc domain subunits so as to make their association sterically or electrostatically favorable, respectively. Thus, (heterologous) dimerization occurs between a polypeptide comprising the first Fc domain subunit and a polypeptide comprising the second Fc domain subunit, which may be different in the sense that the additional components (e.g., antigen binding moieties) fused to each subunit are inconsistent. In some embodiments, modifications that promote association include amino acid mutations, particularly amino acid substitutions, in the Fc domain. In a specific embodiment, the modifications promoting association comprise individual amino acid mutations, in particular amino acid substitutions, of each of the two subunits of the Fc domain.

[0104] The term "effector function" refers to those biological activities attributable to the Fc region of an antibody that vary with 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.

[0105] As used herein, the terms "engineering, engineered, engineered" are considered to include any manipulation of the peptide backbone, or post-translational modification of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modifications to the amino acid sequence, glycosylation pattern, or side chain groups of individual amino acids, as well as combinations of these methods.

[0106] As used herein, the term "immunoconjugate" refers to a polypeptide molecule comprising at least one IL-2 portion and at least one antigen binding portion or effector cell binding portion. In certain embodiments, the immunoconjugate comprises at least one IL-2 portion and at least two antigen binding portions or at least two effector cell binding portions. The specific immunoconjugate according to the present invention is essentially composed of an IL-2 portion and two antigen binding portions joined by one or more linker sequences. The antigen binding portion can be joined to the IL-2 portion by various interactions and in various configurations as described herein. The specific immunoconjugate according to the present invention is essentially composed of an IL-2 portion and two effector cell binding portions joined by one or more linker sequences. The effector cell binding portion can be joined to the IL-2 portion by various interactions and in various configurations as described herein.

[0107] As used herein, the term "amino acid mutation" means that amino acid substitution, deletion, insertion and modification are covered. Any combination of substitution, deletion, insertion and modification can be performed to obtain the final construct, provided that the final construct has the desired characteristics, such as reduced binding to Fc receptors, or increased association with another peptide. Amino acid sequence deletions and insertions include amino terminal and / or carboxyl terminal deletions and insertions of amino acids. Specific amino acid mutations are amino acid substitutions. For the purpose of changing the binding characteristics of, for example, Fc regions, non-conservative amino acid substitutions, i.e., replacing one amino acid with another amino acid having different structures and / or chemical properties, are particularly preferred. Amino acid substitutions include substitutions with non-natural amino acids or with amino acid derivatives of twenty naturally occurring standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be produced using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. It is also useful to envision methods for changing amino acid side chain groups by methods other than genetic engineering (such as chemical modification). Various names can be used herein to indicate the same amino acid mutation. For example, substitution of proline at position 329 of the Fc domain with glycine can be represented as 329G, G329, G 329 , P329G or Pro329Gly.

[0108] As used herein, the term "polypeptide" refers to a molecule consisting of monomers (amino acids) linearly connected by amide bonds (also referred to as peptide bonds). The term "polypeptide" refers to any chain with two or more amino acids, rather than the specific length of the product. Therefore, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains" or any other terms used to refer to chains with two or more amino acids are included in the definition of "polypeptide", and the term "polypeptide" can be used instead of any of these terms, or can be used interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the post-expression modification product of the polypeptide, which includes but is not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization with known protection / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. The polypeptide can be derived from a natural biological source or produced by recombinant technology, and is not necessarily translated from a specified nucleic acid sequence. It can be generated in any way, including by chemical synthesis. The size of a polypeptide of the invention can 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, 1,000 or more, or 2,000 or more amino acids. Polypeptides can have a defined three-dimensional structure, but they do not necessarily have such a structure. Polypeptides with a defined three-dimensional structure are referred to as folded; and polypeptides that do not have a defined three-dimensional structure, but can adopt a large number of different conformations, are referred to as unfolded.

[0109] An "isolated" polypeptide or variant or derivative thereof means a polypeptide that is not in its natural environment. No particular level of purification is required. For example, an isolated polypeptide can be removed from the polypeptide's 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.

[0110] " Amino acid sequence identity percentage (%) " relative to a reference polypeptide sequence is defined as after comparing a candidate sequence with a reference polypeptide sequence and introducing a vacancy (if necessary) to achieve the maximum sequence identity percentage, and without considering any conservative substitution as a component of sequence identity, the percentage of the amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence. The comparison for determining the amino acid sequence identity percentage can be implemented in various ways within the technical scope 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 the appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment on the full length of the compared sequence. However, for the purposes of this article, the sequence comparison computer program ALIGN-2 is used to generate amino acid sequence identity values%. The ALIGN-2 sequence comparison computer program is written by Genentech, Inc., and the source code has been submitted to US Copyright Office, Washington DC, 20559, with user documentation, where it is registered with 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 source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged. In the case of using ALIGN-2 for amino acid sequence comparison, the amino acid sequence identity % (which can be alternatively expressed as a given amino acid sequence A having or comprising a certain amino acid sequence identity % with a given amino acid sequence B) of a given amino acid sequence A and a given amino acid sequence B is calculated as follows:

[0111] Multiply 100 by the fraction X / Y

[0112] Where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be understood that where 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 be equal to the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0113] The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), virally derived RNA, or plasmid DNA (pDNA). A polynucleotide may contain conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, such as those present in peptide nucleic acids (PNA)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.

[0114] "Isolated" nucleic acid molecules or polynucleotides mean nucleic acid molecules, DNA or RNA, that have been taken out of their natural environment. For example, recombinant polynucleotides encoding polypeptides contained in vectors are considered to be isolated for the purposes of the present invention. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially purified) polynucleotides in solution. Isolated polynucleotides include polynucleotide molecules that are contained in cells that usually contain polynucleotide molecules, but the polynucleotide molecules are present outside the chromosome or at a chromosomal position different from their natural chromosomal position. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present invention, as well as positive and negative strand forms and double-stranded forms. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced by synthesis. In addition, polynucleotides or nucleic acids may be or may include regulatory elements, such as promoters, ribosome binding sites or transcription terminators.

[0115] With respect to a nucleic acid or polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence of the invention, it is meant that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These changes in the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, or may be interspersed individually among the residues of the reference sequence, or may be interspersed in one or more consecutive groups within the reference sequence. As a practical matter, known computer programs, such as those discussed below for polypeptides (e.g., ALIGN-2), can be used to routinely determine whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the invention.

[0116] The term "expression cassette" refers to a polynucleotide produced by recombination or synthesis, which has a series of specific nucleic acid elements that allow a specific nucleic acid to be transcribed in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus or nucleic acid fragment. Typically, the recombinant expression cassette portion of the expression vector includes, in addition to other sequences, a nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassette of the present invention comprises a polynucleotide sequence encoding a bispecific antigen binding molecule of the present invention or a fragment thereof.

[0117] The term "vector" or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule used to introduce a specific gene into a target cell operably associated therewith and to direct the expression of the gene. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of a host cell into which it has been introduced. The expression vector of the present invention comprises an expression cassette. The expression vector allows transcription of a large amount of stable mRNA. Once the expression vector is inside the target cell, a ribonucleic acid molecule or protein encoded by the gene is produced by cellular transcription and / or translation mechanisms. In one embodiment, the expression vector of the present invention comprises an expression cassette comprising a polynucleotide sequence encoding a bispecific antigen binding molecule of the present invention or a fragment thereof.

[0118] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived from the primary transformed cells, regardless of the number of passages. Progeny may not be completely consistent with the nucleic acid content of the parent cell, but may contain mutations. This article includes mutant progeny with the same function or biological activity as screened or selected in the original transformed cells. Host cells are any type of cell system that can be used to generate the bispecific antigen binding molecules of the present invention. Host cells include cultured cells, for example, cultured mammalian cells, such as CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells and plant cells, as well as cells included in transgenic animals, transgenic plants or cultured plants or animal tissues.

[0119] An "activating Fc receptor" is an Fc receptor that, upon engagement by the Fc domain of an antibody, initiates a signaling event that stimulates cells bearing the receptor to perform effector functions. Human activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0120] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that causes immune effector cells to lyse antibody-coated target cells. Target cells are cells that specifically bind to antibodies or derivatives thereof comprising Fc regions, and the specific binding is usually through the protein portion of the N-terminus of the Fc region. As used herein, the term "reduced ADCC" is defined as a reduction in the number of target cells lysed by the ADCC mechanism defined above at a given antibody concentration in the culture medium surrounding the target cells within a given time, and / or an increase in the antibody concentration necessary to achieve lysis of a given number of target cells by the ADCC mechanism in the culture medium surrounding the target cells within a given time. ADCC reduction is relative to ADCC mediated by the same antibody produced by the same type of host cells but not yet engineered using the same standard production, purification, formulation and storage methods (such methods are known to those skilled in the art). For example, the reduction of ADCC mediated by an antibody comprising an amino acid substitution that reduces ADCC in the Fc domain is relative to ADCC mediated by the same antibody without the amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, e.g., PCT Publication No. WO 2006 / 082515 or PCT Publication No. WO 2012 / 130831).

[0121] An "effective amount" of an agent is that amount required to produce a physiological change in the cell or tissue to which it is administered.

[0122] 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 preventive result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes or prevents the adverse effects of a disease.

[0123] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, 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.

[0124] The term "pharmaceutical composition" refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered.

[0125] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical composition other than the active ingredient that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0126] As used herein, "treatment" (and grammatical variants such as treat or treating) refers to an attempt to alter the natural course of a disease in the individual being treated, and may be performed for prevention or may be performed during clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, diminishing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the protease-activatable IL-2 polypeptides or immunoconjugates of the invention are used to delay the development of a disease or slow the progression of a disease.

[0127] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0128] As used herein, "idiotype-specific polypeptide" refers to a polypeptide that recognizes the idiotype of an antigen binding moiety, for example, an antigen binding moiety that is specific for CD3. The idiotype-specific polypeptide is capable of specifically binding to the variable region of the antigen binding moiety, thereby reducing or preventing the specific binding of the antigen binding moiety to its cognate antigen. When bound to a molecule comprising an antigen binding moiety, the idiotype-specific polypeptide can act as a masking portion of the molecule. Specifically disclosed herein are anti-idiotype antibodies or anti-idiotype binding antibody fragments that are specific for the idiotype of an anti-CD3 binding molecule.

[0129] As used herein, "protease" or "proteolytic enzyme" refers to any proteolytic enzyme that cleaves a linker at a recognition site and is expressed by a target cell. Such proteases may be secreted by the target cell or remain associated with the target cell, for example, on the surface of the target cell. Examples of proteases include, but are not limited to, metalloproteinases, such as matrix metalloproteinases 1-28 and disintegrin and metalloproteinases (ADAM) 2, 7-12, 15, 17-23, 28-30 and 33, serine proteases, such as urokinase-type plasminogen activator and matriptase, cysteine ​​proteases, aspartic proteases, and members of the cathepsin family.

[0130] As used herein, "protease-activatable", with respect to an interleukin-2 polypeptide, means that the interleukin-2 polypeptide has a reduced or eliminated ability to bind to an interleukin-2 receptor due to a masking moiety, which reduces or eliminates the ability of the interleukin-2 polypeptide to bind to the interleukin-2 receptor. When the masking moiety is degraded by proteolytic cleavage (e.g., by proteolytic cleavage of a linker that connects the masking moiety to the interleukin-2 polypeptide and / or within the masking moiety), binding to the interleukin-2 receptor is restored, thereby activating the interleukin-2 polypeptide.

[0131] As used herein, "reversibly hiding" refers to the binding of a masking moiety to an interleukin-2 polypeptide, such as to prevent the interleukin-2 polypeptide from binding to its receptor. Such hiding is reversible because the masking moiety can be released from the interleukin-2 polypeptide, for example, by protease cleavage, and thereby free the interleukin-2 polypeptide to bind to its receptor.

[0132] Embodiments of the present disclosure

[0133] 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, wherein the masking moiety is covalently attached to the IL-2 polypeptide via the linker, wherein the masking moiety is capable of binding to the IL-2 polypeptide, thereby reversibly concealing the IL-2 polypeptide, wherein the masking moiety comprises a second protease cleavage site, wherein when cleaved at the first protease cleavage site and / or the second protease cleavage site, the masking moiety does not conceal the IL-2 polypeptide. 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 terminus or carboxyl terminus of the interleukin-2 polypeptide via the 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 an antibody derived from MT204, preferably MT204. The MT204 antibody is disclosed in, for example, Volkland et al., Molecular Immunology 44 (2007) 1743–1753, and PCT publication WO 2006 / 128690 A1. In a preferred embodiment, the masking moiety is a deimmunized MT204-derived conjugate. In a preferred embodiment, the masking moiety derived from MT204 comprises a VL domain according to SEQ ID NO: 55 and a VH domain according to 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 heavy chain variable domain (VH) and the light chain variable domain (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.

[0134] 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 YAARKGGI according to SEQ ID NO: 60 and / or PQARK according to SEQ ID NO: 61. In one embodiment, the protease recognition sequence of the first protease cleavage site is YAARKGGI according to SEQ ID NO: 60 or PQARK according to SEQ ID NO: 61. In one embodiment, the protease recognition sequence of the second protease cleavage site is YAARKGGI according to SEQ ID NO: 60 or PQARK according to SEQ ID NO: 61. In one embodiment, the protease recognition sequence of the first protease cleavage site is YAARKGGI according to SEQ ID NO: 60 and the protease recognition sequence of the second protease cleavage site is PQARK according to SEQ ID NO: 61. In one embodiment, the protease recognition sequence of the first protease cleavage site is PQARK according to SEQ ID NO: 61 and the protease recognition sequence of the second protease cleavage site is YAARKGGI according to SEQ ID NO: 60.

[0135] In one embodiment, the IL-2 polypeptide is a wild-type IL-2, preferably a human IL-2 according to 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 according to 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 according to 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 according to SEQ ID NO: 62.

[0136] In a specific embodiment, the protease-activatable IL-2 polypeptide 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: 27. In a specific embodiment, the protease-activatable IL-2 polypeptide 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: 28. In a specific embodiment, the protease-activatable IL-2 polypeptide 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: 29. In a specific embodiment, the protease-activatable IL-2 polypeptide 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: 30.

[0137] In a specific embodiment, the protease-activatable IL-2 polypeptide comprises the amino acid sequence according to SEQ ID NO: 27. In a specific embodiment, the protease-activatable IL-2 polypeptide comprises the amino acid sequence according to SEQ ID NO: 28. In a specific embodiment, the protease-activatable IL-2 polypeptide comprises the amino acid sequence according to SEQ ID NO: 29. In a specific embodiment, the protease-activatable IL-2 polypeptide comprises the amino acid sequence according to SEQ ID NO: 30.

[0138] 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 carboxyl-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 carboxyl-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.

[0139] Immunoconjugates

[0140] In one embodiment, the present invention provides an immunoconjugate comprising a protease-activatable IL-2 polypeptide as described herein and an antigen binding portion and an effector cell binding portion. In one embodiment, the present invention provides an immunoconjugate comprising a protease-activatable IL-2 polypeptide as described herein and an antigen binding portion. In one embodiment, the present invention provides an immunoconjugate comprising a protease-activatable IL-2 polypeptide as described herein and an antigen binding portion. In one embodiment, the present invention provides an immunoconjugate comprising a protease-activatable IL-2 polypeptide as described herein and an effector cell binding portion.

[0141] In one embodiment, the protease-activatable IL-2 polypeptide shares an amino-terminal peptide bond or a carboxyl-terminal peptide bond with the antigen-binding portion or the effector cell-binding portion. In one embodiment, the immunoconjugate comprises a first antigen-binding portion and a second antigen-binding portion, or a first effector cell antigen-binding portion 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 carboxyl-terminal peptide bond with the first antigen-binding portion, and the second antigen-binding portion shares an amino-terminal peptide bond or a carboxyl-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-terminal peptide bond or a carboxyl-terminal peptide bond with the first effector cell-binding portion, and the second effector cell-binding portion shares an amino-terminal peptide bond with either a) the protease-activatable IL-2 polypeptide or b) the first effector cell-binding portion or carboxyl-terminal peptide bond; (iii) the protease-activatable IL-2 polypeptide shares an amino-terminal peptide bond or carboxyl-terminal peptide bond with the antigen-binding portion, and the effector cell-binding portion shares an amino-terminal peptide bond or carboxyl-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-terminal peptide bond or carboxyl-terminal peptide bond with the effector cell-binding portion, and the antigen-binding portion shares an amino-terminal peptide bond or carboxyl-terminal peptide bond with either a) the protease-activatable IL-2 polypeptide or b) the effector cell-binding portion.

[0142] In one embodiment, the antigen binding portion or effector cell binding portion contained 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 Fab molecules and scFv molecules. 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 in a tumor cell or a tumor cell environment, and / or wherein the effector cell binding portion is directed against an effector cell present in a tumor cell environment to achieve cis targeting.

[0143] In specific 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 specific 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 specific 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 specific 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.

[0144] In a specific embodiment, the immunoconjugate comprises an amino acid sequence according to SEQ ID NO: 23. In a specific embodiment, the immunoconjugate comprises an amino acid sequence according to SEQ ID NO: 24. In a specific embodiment, the immunoconjugate comprises an amino acid sequence according to SEQ ID NO: 25. In a specific embodiment, the immunoconjugate comprises an amino acid sequence according to SEQ ID NO: 26.

[0145] In one embodiment, the immunoconjugate comprises an amino acid sequence according to SEQ ID NO: 5, an amino acid sequence according to SEQ ID NO: 22, and an amino acid sequence according to SEQ ID NO: 23. In a specific embodiment, the immunoconjugate comprises an amino acid sequence according to SEQ ID NO: 5, an amino acid sequence according to SEQ ID NO: 22, and an amino acid sequence according to SEQ ID NO: 24. In a specific embodiment, the immunoconjugate comprises an amino acid sequence according to SEQ ID NO: 5, an amino acid sequence according to SEQ ID NO: 22, and an amino acid sequence according to SEQ ID NO: 25. In a specific embodiment, the immunoconjugate comprises an amino acid sequence according to SEQ ID NO: 5, an amino acid sequence according to SEQ ID NO: 22, and an amino acid sequence according to SEQ ID NO: 26.

[0146] Masking part

[0147] 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 sequence of SEQ ID NO: 55 and SEQ ID NO: 56. In one embodiment, the masking moiety comprises the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3 in the VH domain according to SEQ ID NO: 56, and the light chain CDR1, light chain CDR2, and light chain CDR3 in the VL domain according to SEQ ID NO: 55. In a specific embodiment, the masking moiety masking the IL-2 polypeptide comprises a VL domain according to SEQ ID NO: 55 and a VH domain according to SEQ ID NO: 56, wherein the masking moiety is a single chain Fab molecule.

[0148] Connectors

[0149] In one embodiment, the protease-activatable IL-2 polypeptide or immunoconjugate comprises a linker having a protease recognition site comprising a polypeptide sequence that is 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 specific embodiment, the protease recognition site comprises the polypeptide sequence of SEQ ID NO: 63, 64, 65 or 66.

[0150] Polynucleotide

[0151] The present invention further provides an isolated polynucleotide encoding a protease-activatable IL-2 polypeptide or immunoconjugate or fragment thereof as described herein.

[0152] The polynucleotide encoding the protease-activatable IL-2 polypeptide or immunoconjugate of the present invention can be expressed as a single polynucleotide encoding the entire protease-activatable IL-2 polypeptide or immunoconjugate, or as a plurality of (e.g., two or more) polynucleotides that are co-expressed. The polypeptides encoded by the co-expressed polynucleotides can be associated, for example, by disulfide bonds or other means to form functional protease-activatable IL-2 polypeptides or immunoconjugates. For example, in the context of an immunoconjugate, the light chain portion of the antigen binding portion can be encoded by a polynucleotide separate from the polynucleotide encoding the heavy chain, Fc domain subunit, and optionally another antigen binding portion (a portion) of the immunoconjugate. When co-expressed, the heavy chain polypeptide will associate with the light chain polypeptide to form an antigen binding portion. In another example, the portion of the immunoconjugate comprising one of the two Fc domain subunits and optionally one or more antigen binding portions (a portion) can be encoded by a polynucleotide separate from the portion of the immunoconjugate comprising another of the two Fc domain subunits and an optional antigen binding portion (a portion). When co-expressed, the Fc domain subunits will associate to form an Fc domain.

[0153] In some embodiments, the isolated polynucleotide encodes a complete immunoconjugate according to the invention as described herein. In other embodiments, the isolated polynucleotide encodes a polypeptide comprised in an immunoconjugate according to the invention as described herein.

[0154] In another embodiment, the invention relates to an isolated polynucleotide encoding a protease-activatable IL-2 polypeptide or immunoconjugate of the invention or a fragment thereof. In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide of the invention is RNA, for example in the form of messenger RNA (mRNA). The RNA of the invention may be single-stranded or double-stranded.

[0155] Recombination methods

[0156] 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. For recombinant production, one or more polynucleotides encoding a protease-activatable IL-2 polypeptide or immunoconjugate, such as described above, are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such polynucleotides can be easily isolated and sequenced using conventional methods. In one embodiment, a vector, preferably an expression vector, is provided, which contains one or more polynucleotides of the polynucleotides of the present invention. Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of the IL-2 polypeptide or immunoconjugate and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, synthetic techniques, and in vivo recombination / genetic recombination. See, for example, the techniques described in the following documents: 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). The expression vector may be part of a plasmid, a virus, or may be a nucleic acid fragment. The expression vector includes an expression cassette into which a polynucleotide encoding a protease-activatable IL-2 polypeptide or an immunoconjugate (i.e., a coding region) is cloned in operable 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 that consists of codons that are translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into amino acids, it (if present) may be considered to be part of the coding region, while any flanking 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 may be present in a single polynucleotide construct (e.g., on a single vector), or in separate polynucleotide constructs (e.g., on separate (different) vectors). In addition, any vector may contain a single coding region, or may contain two or more coding regions, for example, the vector of the invention may encode one or more polypeptides that are separated into final proteins by proteolytic cleavage after or during translation. In addition, the vector, polynucleotide or nucleic acid of the invention may encode a heterologous coding region, which may or may not be fused to a polynucleotide encoding a protease-activatable IL-2 polypeptide or immunoconjugate of the 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 is when the coding region of a gene product (e.g., a polypeptide) is associated with one or more regulatory sequences in a certain manner so that the expression of the gene product is under the influence or control of the regulatory sequences. If the induction of promoter function leads to the transcription of mRNA encoding the desired gene product, and if the nature of the bond between the two DNA fragments does not interfere with the ability of the expression regulatory sequence to direct the expression of the gene product or the ability of the DNA template to be transcribed, then the two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated". Therefore, if the promoter is able to affect the transcription of the nucleic acid, the promoter region will be operably associated with the nucleic acid encoding the polypeptide. The promoter can be a cell-specific promoter that directs substantial transcription of DNA only in a predetermined cell. In addition to the promoter, other transcription control elements, such as enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein. A variety of transcription control regions are known to those skilled in the art. These include, but are not limited to, those that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (e.g., the immediate early promoter in conjunction with intron-A), simian virus 40 (e.g., the early promoter), and retroviruses (such as, for example, Rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes (such as actin, heat shock protein, bovine growth hormone, and rabbit. The expression cassette may also include other features, such as an origin of replication, and / or chromosomal integration elements, such as retroviral long terminal repeats (LTRs), or adeno-associated virus (AAV) inverted terminal repeats (ITRs).

[0157] The polynucleotides and nucleic acid coding regions of the present invention may be associated with additional coding regions encoding secretory or signal peptides that direct the secretion of the polypeptides encoded by the polynucleotides of the present invention. For example, if secretion of an IL-2 polypeptide or immunoconjugate is desired, DNA encoding a signal sequence may be placed upstream of the nucleic acid of the 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 have a signal peptide or secretory leader sequence that is cleaved from the mature protein once the growing protein chain has been initiated to export across the rough endoplasmic reticulum. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells typically have a signal peptide fused to the N-terminus of the polypeptide that is cleaved from the translated polypeptide to produce a 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. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof may be used. For example, the wild-type leader sequence can be replaced by the leader sequence of human tissue plasminogen activator (TPA) or mouse beta-glucuronidase.

[0158] DNA encoding a short protein sequence that can be used to facilitate subsequent purification (e.g., a histidine tag) or to aid in labeling the protease-activatable IL-2 polypeptide or immunoconjugate may be included internally or at the end of the polynucleotide encoding the protease-activatable IL-2 polypeptide or immunoconjugate.

[0159] In another embodiment, a host cell comprising one or more polynucleotides of the present invention is provided. In certain embodiments, a host cell comprising one or more vectors of the present invention is provided. Polynucleotides and vectors may be incorporated into any of the features described herein for polynucleotides and vectors, respectively, either alone or in combination. In one such embodiment, the host cell comprises a vector (e.g., one or more vectors have been transformed or transfected), the vector comprising a polynucleotide encoding a protease-activatable IL-2 polypeptide or immunoconjugate (a portion thereof) of the present invention. As used herein, the term "host cell" refers to any type of cell system 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 replication and support for expression of protease-activatable IL-2 polypeptides or immunoconjugates are well known in the art. Such cells can be appropriately transfected or transduced with specific expression vectors, and large quantities of cells containing the vectors can be grown for inoculation of large-scale fermenters to obtain sufficient amounts of IL-2 polypeptides or immunoconjugates for clinical applications. Suitable host cells include prokaryotic microorganisms, such as Escherichia coli, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, and the like. For example, polypeptides can be produced in bacteria, particularly when glycosylation is not required. The polypeptides can be separated from bacterial cell paste in a soluble fraction after expression, and can be further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding polypeptides, including fungi and yeast strains whose glycosylation pathways have been "humanized", thereby resulting in the production of polypeptides with partial or complete 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 expressing (glycosylated) polypeptides also come from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains that can be used with insect cells have been identified, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES for producing antibodies in transgenic plants). TMTechniques). Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells, as described, for example, in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells, as described, for example, 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 (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (as described, for example, in Mather et al., Annals NY Acad Sci 383, 44-68 (1982)), MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr - 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 certain mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, Humana Press, Totowa, NJ), pp. 255-268 (2003). Host cells include cultured cells, such as mammalian cultured cells, yeast cells, insect cells, bacterial cells and plant cells, to name just a few examples, and also include 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 lymphocyte (e.g., Y0, NS0, Sp20 cell).

[0160] Standard techniques for expressing foreign genes in these systems are known in the art. Cells expressing polypeptides comprising antigen binding domains, such as heavy or light chains of antibodies, can be engineered to also express another antibody chain so that the expressed product is an antibody having heavy and light chains.

[0161] In one embodiment, a method for producing a protease IL-2 polypeptide or immunoconjugate according to the present invention is provided, wherein the method comprises culturing a host cell comprising a polynucleotide encoding a protease activatable IL-2 polypeptide or immunoconjugate as provided herein under conditions suitable for expression of the protease activatable IL-2 polypeptide or immunoconjugate, and optionally recovering the protease activatable IL-2 polypeptide or immunoconjugate from the host cell (or host cell culture medium).

[0162] 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 directly fused to each other or indirectly fused through a linker sequence. The composition and length of the linker can be determined according to methods well known in the art, and the efficacy of the linker can be tested. Examples of linker sequences between different components of the protease-activatable IL-2 polypeptide or immunoconjugate are found in the sequences provided herein. If desired, additional sequences (e.g., endopeptidase recognition sequences) may also be included to incorporate cleavage sites to separate the fused components.

[0163] In certain embodiments, one or more antigen binding moieties of the immunoconjugate at least comprise an antibody variable region capable of binding an antigenic determinant. The variable region may form a part of and be derived from a naturally or non-naturally occurring antibody and its fragment. Methods for producing polyclonal antibodies 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, may be recombinantly produced (e.g., as described in U.S. Patent No. 4,186,567), or may be obtained, for example, by screening a combinatorial library comprising a variable heavy chain and a variable light chain (see, e.g., U.S. Patent No. 5,969,108 of McCafferty).

[0164] 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 that can be used in the present invention can be of murine, primate or human origin. If the protease-activatable IL-2 polypeptide or immunoconjugate is intended for human use, a chimeric form of the antibody can be used, wherein the constant region of the antibody is from a human. Humanized or fully human forms of antibodies can also be prepared according to methods well known in the art (see, e.g., U.S. Pat. No. 5,565,332 to Winter). Humanization can be achieved by various methods, including but not limited to (a) grafting non-human (e.g., donor antibody) CDRs onto human (e.g., acceptor antibody) frameworks and constant regions with or without retaining key framework residues (e.g., key framework residues important for maintaining good antigen binding affinity or antibody function), (b) grafting only non-human specificity determining regions (SDRs or a-CDRs; residues that are critical for antibody-antigen interactions) onto human frameworks and constant regions, or (c) grafting entire non-human variable domains but "hiding" them with human-like segments by replacing surface residues.Humanized antibodies and methods for their preparation are reviewed, e.g., in Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), and further 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. Pat. 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 the "guided selection" method for FR shuffling). Various techniques known in the art can be used to produce human antibodies and human variable regions. 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). Human variable regions can form a part of a human monoclonal antibody prepared by a hybridoma method and be derived from the antibody (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 a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions that respond to antigenic 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 phage display libraries of human origin (see, e.g., Hoogenboom et al. Methods in Molecular Biology 178, 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001); and McCafferty et al., Nature 348, 552-554; Clackson et al., Nature 352, 624-628 (1991)). Phages typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments.

[0165] In certain embodiments, antigen binding moieties useful in the present invention are engineered to have enhanced binding affinity according to the methods disclosed, for example, in U.S. Patent Application Publication No. 2004 / 0132066, which is incorporated herein by reference in its entirety. The ability of the immunoconjugates of the present invention to bind 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 (analyzed on a BIACORE T100 system) (Liljeblad et al., Glyco J 17, 323-329 (2000)) and traditional 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) as the reference antibody. Detailed exemplary methods for mapping epitopes bound by antibodies are provided in Morris (1996) "Epitope Mapping in Methods in Molecular Biology Vol. 66 (Humana Press, Totowa, NJ). The protease-activatable IL-2 polypeptide or immunoconjugate 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, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and will be apparent to those skilled in the art. For affinity chromatography purification, an antibody, ligand, receptor, or antigen that binds to the protease-activatable IL-2 polypeptide or immunoconjugate can be used. For example, for affinity chromatography purification of the protease-activatable IL-2 polypeptide and immunoconjugate of the present invention, a matrix with protein A or protein G can be used. Sequential protein A or G affinity chromatography and size exclusion chromatography can be used to separate the protease-activatable IL-2 polypeptide or immunoconjugate, essentially as described in the Examples. The purity of the protease-activatable IL-2 polypeptide or immunoconjugate can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high pressure liquid chromatography, and the like.

[0166] Determination

[0167] The physical / chemical properties and / or biological activities of the protease-activatable IL-2 polypeptides and immunoconjugates provided herein can be identified, screened or characterized by various assays known in the art.

[0168] Affinity determination

[0169] The affinity of the immunoconjugate for an Fc receptor or target antigen can be determined by surface plasmon resonance (SPR) using standard instruments such as a BIAcore instrument (GE Healthcare) and receptors or target proteins such as those obtainable by recombinant expression according to the methods described in the Examples. Alternatively, the binding of protease-activatable IL-2 polypeptides and immunoconjugates to different receptors or target antigens can be assessed using cell lines expressing specific receptors or target antigens, for example, by flow cytometry (FACS). Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0170] According to one embodiment, the K was measured by surface plasmon resonance using a T100 instrument (GE Healthcare). D .

[0171] To analyze the interaction between the Fc part and the Fc receptor, His-tagged recombinant Fc receptors were captured by anti-pentahistidine antibodies (Qiagen) immobilized on a CM5 chip, and the bispecific constructs were used as analytes. Briefly, carboxymethylated dextran biosensor chips (CM5, GE Healthcare) were activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The anti-pentahistidine antibody was diluted to 40 μg / ml with 10 mM sodium acetate pH 5.0 and then injected at a flow rate of 5 μl / min to obtain approximately 6500 response units (RU) of coupled protein. After the ligand was injected, 1 M ethanolamine was injected to block unreacted groups. The Fc receptor was then captured at 4 or 10 nM for 60 s. For kinetic measurements, four-fold serial dilutions of the bispecific constructs (ranging between 500 nM and 4000 nM) were injected into HBS-EP (GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, pH 7.4) at 25°C at a flow rate of 30 μl / min for 120 sec.

[0172] To determine the affinity to the target antigen, the bispecific construct was captured by an anti-human Fab specific antibody (GE Healthcare) immobilized on the surface of an activated CM5 sensor chip, as described for the anti-pentahistidine antibody. The final amount of coupled protein was approximately 12000RU. The bispecific construct was captured at 300nM for 90s. The target antigen was passed through the flow cell at a concentration range of 250 to 1000nM at a flow rate of 30μl / min for 180s. Dissociation was monitored for 180s.

[0173] Bulk refractive index differences were corrected by subtracting the response obtained in a reference flow cell. The steady-state response was used to derive the dissociation constant, K, by nonlinear curve fitting of the Langmuir binding isotherm. D Using a simple one-to-one Langmuir binding model ( T100Evaluation Software version 1.1.1) was used to calculate the association rate (k) by simultaneously fitting the association and dissociation sensorgrams. on ) and dissociation rate (k off ). Equilibrium dissociation constant (K D ) is calculated as the ratio k off / k on See, e.g., Chen et al., J Mol Biol 293, 865-881 (1999).

[0174] Activity assay

[0175] The biological activity of the protease-activatable IL-2 polypeptide or immunoconjugate of the invention can be measured by various assays as described in the Examples. The biological activity can, for example, include induction of T cell proliferation, induction of signaling in T cells, induction of activation marker expression in T cells, induction of T cell cytokine secretion, induction of target cell (e.g., tumor cell) lysis, and induction of tumor regression and / or improved survival.

[0176] Composition, formulation and route of administration

[0177] In a further aspect, the present invention provides a pharmaceutical composition comprising any of the protease-activatable IL-2 polypeptides or immunoconjugates provided herein, for example, for use in any of the following treatment methods. 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, for example, as described below.

[0178] Further provided is a method for producing a protease-activatable IL-2 polypeptide or immunoconjugate of the present invention in a form suitable for in vivo administration, the method comprising (a) obtaining a protease-activatable IL-2 polypeptide or immunoconjugate according to the present invention, and (b) formulating the protease-activatable IL-2 polypeptide or immunoconjugate with at least one pharmaceutically acceptable carrier, thereby formulating the protease-activatable IL-2 polypeptide or immunoconjugate preparation for in vivo administration.

[0179] The 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 pharmaceutical carrier. The phrase "pharmaceutically 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 to animals (such as, for example, humans) as appropriate. The preparation of pharmaceutical compositions containing at least one protease-activatable IL-2 polypeptide or immunoconjugate and optional additional active ingredients will be known to those skilled in the art in view of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, which is incorporated herein by reference. In addition, for animal (e.g., human) administration, it should be understood that the preparation should meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Office of Biological Standards or other relevant authorities in other countries. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, similar substances and combinations thereof, as would be known to one of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Edition, Mack Printing Company, 1990, pp. 1289-1329, which is incorporated herein by reference). Except in the case where any conventional carrier is incompatible with the active ingredient, the use of the carrier in the therapeutic or pharmaceutical composition is contemplated.

[0180] The composition may 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 administration routes such as injection. The protease-activatable IL-2 polypeptides or immunoconjugates of the invention (and any additional therapeutic agent) can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrasplenicly, intrarenally, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intracapsularly, intramucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, topically, by inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, local infusion directly into target cells, via a catheter, via lavage, in the form of a cream, in the form of a lipid composition (e.g., liposomes), or by other methods known to one of ordinary skill in the art, or any combination of the foregoing (see, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Printing Company, 1990, which is incorporated herein by reference). Parenteral administration, particularly intravenous injection, is most commonly used to administer polypeptide molecules, such as protease-activatable IL-2 polypeptides or immunoconjugates of the invention.

[0181] Parenteral compositions include those designed for 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 an aqueous solution, preferably in a physiologically compatible buffer (such as Hanks solution, Ringer's solution or physiological saline buffer). The solution may contain a formulation agent (formulatoryagent), such as a suspending agent, a stabilizer and / or a dispersant. Alternatively, the protease-activatable IL-2 polypeptide or immunoconjugate may be in powder form for reconstitution with a suitable solvent (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 desired amount together with various other ingredients listed below in an appropriate solvent as needed. For example, sterility can be easily achieved by filtration through a sterile filtration membrane. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile solvent containing a basic dispersion medium and / or other components. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsions, preferred preparation methods are vacuum drying or freeze drying techniques, which produce a powder of the active ingredient from a previously sterile filtered liquid medium plus any additional required ingredients. If necessary, the liquid medium should be properly buffered, and sufficient saline or glucose should first be used to make the liquid diluent isotonic before injection. The composition must be stable under manufacturing and storage conditions, and is preserved as a contamination effect against microorganisms such as bacteria and fungi. It should be understood that endotoxin contamination should be kept to a minimum, for example, at a safety level below 0.5 ng / mg protein. Suitable pharmaceutical carriers include, but are not limited to, buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as Serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinyl pyrrolidone; 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., zinc protein complexes); and / or nonionic 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 a suitable stabilizer or an agent that increases the solubility of the compound to allow the preparation of a highly concentrated solution. In addition, the suspension of the active compound may be prepared as an appropriate oily injection suspension. 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.

[0182] The active ingredient can be embedded in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared, for example, by coacervation techniques or by interfacial polymerization; embedded in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules); or embedded in coarse emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th edition, Mack Printing Company, 1990). Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing polypeptides, which are in the form of shaped articles such as films or microcapsules. In particular embodiments, prolonged absorption of injectable compositions can be achieved by using an agent that delays absorption (such as, for example, aluminum monostearate, gelatin, or a combination thereof) in the composition.

[0183] In addition to the compositions described previously, the 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 implantation) or by intramuscular injection. Thus, for example, the protease-activatable IL-2 polypeptide or immunoconjugate can be formulated with a suitable polymeric or hydrophobic material (e.g., as an emulsion in an acceptable oil) or with an ion exchange resin, or as a slightly soluble derivative, such as a slightly soluble salt.

[0184] Pharmaceutical compositions comprising the protease-activatable IL-2 polypeptide or immunoconjugate of the invention can be manufactured by conventional mixing, dissolving, emulsifying, encapsulating, embedding or lyophilizing processes. Pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or adjuvants that facilitate processing of the protein into pharmaceutically usable preparations. Appropriate formulations depend on the chosen route of administration.

[0185] The protease-activatable IL-2 polypeptide or immunoconjugate can be formulated into the composition in the form of a free acid or base, neutral or salt. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or free base. These pharmaceutically acceptable salts include acid addition salts, such as acid addition salts formed with free amino groups of the protein composition, or acid addition salts formed with inorganic acids (such as hydrochloric acid or phosphoric acid) or 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 hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide; or organic bases such as isopropylamine, trimethylamine, histidine or procaine. Pharmaceutically acceptable salts tend to be more soluble in aqueous and other protic solvents than the corresponding free base forms.

[0186] Methods of treatment and compositions

[0187] 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, for the treatment of cancer.

[0188] For use in therapeutic methods, the protease-activatable IL-2 polypeptides or immunoconjugates of the invention will be formulated, dosed and administered in a manner consistent with good medical practice. Factors to be considered in this context include the specific disorder being treated, the specific 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 timing of administration, and other factors known to practitioners.

[0189] In one aspect, a protease-activatable IL-2 polypeptide or immunoconjugate of the invention for use as a medicament is provided. In a further aspect, a protease-activatable IL-2 polypeptide or immunoconjugate of the invention for use in treating a disease is provided. In certain embodiments, a protease-activatable IL-2 polypeptide or immunoconjugate of the invention for use in a method of treatment is provided. In one embodiment, the invention provides a protease-activatable IL-2 polypeptide or immunoconjugate as described herein for use in treating a disease in an individual in need thereof. In certain embodiments, the invention provides a protease-activatable IL-2 polypeptide or immunoconjugate for use in a method of treating an individual suffering from a disease, the method comprising administering to the individual a therapeutically effective amount of a protease-activatable IL-2 polypeptide or immunoconjugate. In certain embodiments, the disease to be treated is a proliferative disorder. In a specific embodiment, 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, such as an anticancer agent if the disease to be treated is cancer. In a further embodiment, the invention provides a protease-activatable IL-2 polypeptide or immunoconjugate as described herein for 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, the method comprising administering to the individual an effective amount of a protease-activatable IL-2 polypeptide or immunoconjugate to induce lysis of the target cells. According to any of the above embodiments, the "individual" is a mammal, preferably a human.

[0190] In a further aspect, the present invention provides the use of a protease-activatable IL-2 polypeptide or immunoconjugate of the present invention in the manufacture or preparation of a medicament. In one embodiment, the medicament is used to treat a disease in an individual in need thereof. In another embodiment, the medicament is used in a method for treating a disease, the method comprising administering a therapeutically effective amount of the medicament to an individual having the disease. In certain embodiments, the disease to be treated is a proliferative disorder. In a specific embodiment, the disease is cancer. In one embodiment, the method further comprises administering a therapeutically effective amount of at least one additional therapeutic agent to the individual, for example, if the disease to be treated is cancer, an anticancer agent is used. In a further embodiment, the medicament is used to induce lysis of target cells, particularly tumor cells. In yet another embodiment, the medicament is used in a method for inducing lysis of target cells, particularly tumor cells, in an individual, comprising administering an effective amount of the medicament to the individual to induce lysis of the target cells. The "individual" according to any of the above embodiments may be a mammal, preferably a human.

[0191] In a further aspect, the present invention provides a method for treating a disease. In one embodiment, the method comprises administering a therapeutically effective amount of a protease-activatable IL-2 polypeptide or immunoconjugate of the present invention to an individual suffering from such a disease. In one embodiment, a composition is administered to the individual, the composition comprising a protease-activatable IL-2 polypeptide or immunoconjugate of the present invention in a pharmaceutical form. In certain embodiments, the disease to be treated is a proliferative disorder. In a specific embodiment, the disease is cancer. In certain embodiments, the method further comprises administering a therapeutically effective amount of at least one additional therapeutic agent to the individual, for example, if the disease to be treated is cancer, an anticancer agent is used. According to any of the above embodiments, the "individual" can be a mammal, preferably a human.

[0192] In another aspect, the present invention provides a method for inducing lysis of a target cell, particularly a tumor cell.

[0193] 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, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other cell proliferative disorders that can be treated using the protease-activatable IL-2 polypeptide or immunoconjugate of the present invention include, but are not limited to, tumors located in the following parts: abdomen, bones, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary glands, testicles, ovaries, thymus, thyroid gland), eyes, head and neck, nervous system (central and peripheral nervous system), lymphatic system, pelvis, skin, soft tissue, spleen, chest, and urogenital system. Precancerous conditions or lesions and cancer metastasis 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, head and neck cancer. The skilled person will readily recognize that in many cases, a protease-activatable IL-2 polypeptide or immunoconjugate may not provide a cure, but may only provide a partial benefit. In some embodiments, physiological changes with some benefit are also considered therapeutically beneficial. Therefore, in some embodiments, the amount of a 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, more particularly a human.

[0194] In some embodiments, an effective amount of a protease-activatable IL-2 polypeptide or immunoconjugate of the invention is administered to a cell. In 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.

[0195] For the prevention or treatment of disease, the appropriate dosage of the protease-activatable IL-2 polypeptide or immunoconjugate of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be 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 protease-activatable IL-2 polypeptide or immunoconjugate is administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's clinical history and response to the protease-activatable IL-2 polypeptide or immunoconjugate, and the judgment of the attending physician. In any case, the practitioner responsible for administration will determine the concentration and appropriate dosage of the active ingredient in the composition for an individual subject. Various dosing schedules are contemplated herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulse infusions.

[0196] The 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 methods in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine the LD 50 (the dose that causes 50% of the population to die) and ED 50 (the dose that is therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD 50 / ED 50 . Protease-activatable IL-2 polypeptides or immunoconjugates that exhibit large therapeutic indices are preferred. In one embodiment, a protease-activatable IL-2 polypeptide or immunoconjugate according to the present invention exhibits a high therapeutic index. Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages suitable for use in humans. Doses are preferably within a range that includes ED with little or no toxicity. 50 The dosage may vary within this range depending on a variety of factors, such as the dosage form employed, the route of administration utilized, the condition of the subject, etc. The exact formulation, route of administration, and dosage may be selected by an individual physician based on the patient's condition (see, e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Chapter 1, page 1, the entire contents of which are incorporated herein by reference).

[0197] The attending physician of a patient treated with a protease-activatable IL-2 polypeptide or immunoconjugate of the invention should know how and when to terminate, interrupt or adjust administration due to toxicity, organ dysfunction, etc. Conversely, if the clinical response is inadequate (excluding toxicity), the attending physician will also know to adjust the treatment to a higher level. The size of the dose administered in the management of the target disorder will vary with the severity of the condition to be treated, the route of administration, etc. For example, the severity of the condition can be assessed in part by standard prognostic assessment methods. In addition, the dose and possible dose frequency will also vary according to the age, weight and response of the individual patient.

[0198] Other medicines and treatments

[0199] The protease-activatable IL-2 polypeptide or immunoconjugate according to the present invention can be administered in combination with one or more other agents in therapy. For example, the protease-activatable IL-2 polypeptide or immunoconjugate of the present invention can be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" includes any agent that is administered to treat the symptoms or diseases of an individual in need of such treatment. Such additional therapeutic agents may include any active ingredients suitable for the specific indications being treated, preferably active ingredients with complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is an immunomodulator, a cell growth inhibitor, a cell adhesion inhibitor, a cytotoxic agent, an apoptosis activator, or an agent that increases the sensitivity of cells to apoptosis-inducing agents. In a specific embodiment, the additional therapeutic agent is an anticancer agent, such as a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormone therapy, a kinase inhibitor, a receptor antagonist, a tumor cell apoptosis activator, or an anti-angiogenic agent.

[0200] Such other agents are suitably present in combination in amounts effective for the intended purpose. The effective amount of such other agents depends on the amount of protease-activatable IL-2 polypeptide or immunoconjugate used, the type of disorder or treatment, and other factors discussed above. The protease-activatable IL-2 polypeptide or immunoconjugate is generally used in the same dosages and administration routes as described herein, or in about 1% to 99% of the dosages described herein, or in any dosage and any route determined empirically / clinically to be appropriate.

[0201] Such combination therapies as indicated above encompass both combined administration (where two or more therapeutic agents are included in the same or different compositions) and separate administration, in which case the administration of the protease-activatable IL-2 polypeptide or immunoconjugate of the invention may be performed before, simultaneously with, and / or after the administration of the additional therapeutic agent and / or adjuvant. The protease-activatable IL-2 polypeptide or immunoconjugate of the invention may also be used in combination with radiation therapy.

[0202] Products

[0203] In another aspect of the present invention, an article of manufacture is provided, which contains substances useful for treating, preventing and / or diagnosing the above-mentioned diseases. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous (IV) solution bags, etc. The container can be formed of a variety of materials such as glass or plastic. The container holds a composition that can be effectively used to treat, prevent and / or diagnose the disease itself or in combination with another composition, and the container can have a sterile access port (for example, the container can be an IV solution bag or vial with a stopper that can be pierced 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 to treat the selected disease. In addition, the article of manufacture may include (a) a first container containing a composition, wherein the composition comprises a protease-activatable IL-2 polypeptide or immunoconjugate of the present invention; and (b) a second container containing a composition, wherein the composition comprises an additional cytotoxic agent or other therapeutic agent. The article of manufacture in this embodiment of the invention may further include a package insert indicating that these compositions can be used to treat specific conditions. Alternatively or additionally, the article of manufacture may further include a second (or third) container comprising a pharmaceutical buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may further include other substances desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0204] sequence

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240] Further aspects of the present disclosure

[0241] In another aspect, the present disclosure provides a deimmunized PD-1 binder comprising a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 71, HCDR 2 of SEQ ID NO: 72, and HCDR 3 of SEQ ID NO: 73, and a light chain variable region (VL) comprising a light chain complementary determining region (LCDR) 1 of SEQ ID NO: 68, LCDR 2 of SEQ ID NO: 69, and LCDR 3 of SEQ ID NO: 70. In a specific aspect, the present disclosure provides a deimmunized PD1 binder 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: 53 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: 54. In another aspect, the deimmunized PD-1 binder comprises an amino acid sequence according to SEQ ID NO: 53 and an amino acid sequence according to SEQ ID NO: 54. In another aspect, the present disclosure provides a use of a deimmunized PD-1 binder as 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.

[0242] In another aspect, the present 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, HCDR 2 of SEQ ID NO: 78, and HCDR 3 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, LCDR 2 of SEQ ID NO: 75, and LCDR 3 of SEQ ID NO: 76. In a specific aspect, the present disclosure provides a deimmunized MT204-derived binder 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: 55 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: 56. In another aspect, the deimmunized MT204-derived conjugate comprises an amino acid sequence according to SEQ ID NO:55 and an amino acid sequence according to SEQ ID NO:56. In another aspect, the deimmunized MT204-derived conjugate is a single-chain Fab. In another aspect, the deimmunized MT204-derived conjugate is a single-chain Fv. In another aspect, the disclosure provides the use of a deimmunized MT204-derived conjugate as disclosed herein, wherein the deimmunized MT204-derived conjugate is present in a therapeutic agent. Preferably, the therapeutic agent is an isolated polypeptide. More preferably, the therapeutic agent is a cancer treatment.

[0243] Examples

[0244] The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced given the general description provided above.

[0245] Example 1

[0246] Design of conjugates with reduced immunogenicity potential

[0247] To reduce potential immunogenicity issues, we plan to increase the germline content of two binders that bind to either PD1 or interleukin 2 (IL2).

[0248] PD1 binders (P1AA0927, SEQ ID NOs 51 and 52) were aligned with human germline sequences IGHV3-23-01 (Accession No.: M99660) for the heavy chain and IGKV4-1-01 (Accession No.: Z00023) for the light chain, respectively. In addition, we analyzed protein sequences using commercial software to predict MHC class 2 binding peptides (software: ISPRI; supplier: Epivax).

[0249] In particular, those sequence stretches with predicted MHC class 2 binding should be addressed either by increasing the germline content or by introducing mutations that reduce the MHC binding score, while maintaining the affinity and stability of the antibody. In particular, the sequences around framework 2 and CDR2 of the light chain showed three overlapping peptides with predicted MHC binding. Since germlining would reduce binding properties, we manually selected mutations that reduced the predicted binding score, and we subsequently experimentally verified the maintenance of the desired biochemical properties, as described below.

[0250] For the heavy chain, we focused more on germlining. The leucine at Kabat position 5 was replaced by valine, which is more common at this position. Therefore, a greater number of 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.

[0251] IL2 binder MT204 is treated in a similar manner. The light chain is compared with human germline IGKV1-16-01 or trastuzumab VL (CAS No. 180288-69-1) sequences, which are considered to have low immunogenicity. For the heavy chain, the sequence IGHV3-23-01 (accession number: M99660) is also used as a comparator. Subsequently, the optimized sequence has a higher degree of human germline content. In addition, variant MT204_VL1a has a higher homology (at Kabat position 54) with the trastuzumab framework. The deimmunized MT204 binder used in the following examples comprises a VH sequence of SEQ ID NO:56 and a VL sequence of SEQ ID NO:55.

[0252] Production and purification of deimmunized MT204 masked and deimmunized anti-PD1 binder variants as IgG

[0253] Deimmunized sequences of anti-IL2v masks (P1AH2050-P1AH2052) and anti-PD1 binders (P1AH4157-P1AH4161, P1AI0356-P1AI0360, and P1AI1648-P1AI1652) were cloned into human IgG PG LALA for initial characterization ( Figure 1 A to Figure 1 D). The corresponding cDNA was synthesized and cloned into the vector system of evitria using conventional (non-PCR-based) cloning techniques. Plasmid DNA was prepared under low endotoxin conditions based on anion exchange chromatography. DNA concentration was determined by measuring the absorbance at a wavelength of 260 nm. The correctness of the sequence was verified by Sanger sequencing (two sequencing reactions were performed for each plasmid).

[0254] Suspension-adjusted CHO K1 cells (originally received from ATCC and adapted to serum-free growth in suspension culture at evitria) 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 after transfection cells were grown in eviMake2, an animal component-free, serum-free medium. Supernatants were collected by centrifugation and subsequent filtration (0.2 μm filter).

[0255] Using MabSelect TM SuRe TM IgG was purified with Dulbecco's PBS (Lonza BE17-512Q) as wash buffer, 0.1 M glycine pH 3.5 as elution buffer, and 1 M Tris HCl as neutralization buffer (pH 9). Subsequent size exclusion chromatography was performed on a HiLoad Superdex 200 pg column using the final buffer as running buffer. A Pierce Slide-A-Lyzer with a 2K molecular weight cutoff was used. TM G2 dialysis cassettes were used for dialysis (if necessary). Ultracentrifugal filters are used for antibody concentration (if required).

[0256] The concentration was determined by measuring the absorbance at a wavelength of 280 nm. The extinction coefficient was calculated using a proprietary algorithm from evitria. Purity was determined by analytical size exclusion chromatography using an Agilent AdvanceBio SEC column (300A 2.7um 7.8x300mm) with DPBS as running buffer at a flow rate of 0.8 ml / min. Endotoxin content was measured using a Charles River Endosafe PTS system.

[0257] Design, production and purification of complex PD1-targeted masked IL2v formats

[0258] Four complex PD1-targeted masked IL2v formats were cloned using a deimmunized anti-IL2v mask sequence (derived from P1AH2051, i.e. hu IgG1GL MT204_VL1a – combined VH2 & VH6 PG LALA) and a deimmunized anti-PD1 binder sequence (derived from P1AH4157, i.e. PD1-376_VL2_VH4) Figure 2 A to Figure 2 D). They were cloned as bivalent human IgG1 PG LALA antibodies with two heavy chains heterodimerized in a knob-in-hole structure. The masked IL2v comprising the IL2v cytokine and the C-terminally fused scFv mask was fused to the C-terminus of the knob heavy chain. In addition, the glycine-serine linker sequence between the IL2v cytokine and the scFv mask and the glycine-serine linker between the VH and VL domains of the scFv mask comprised a protease recognition site 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 is PQARK( Figure 2 A to Figure 2 B), the protease recognition site used in P1AI4324 and P1AI4325 is YAARKGGI ( Figure 2 C to Figure 2 D). In addition, the four constructs differed in the length of the linker containing the protease recognition site between the IL2v cytokine and the scFv mask, which was 38 amino acids in P1AI4322 and P1AI4324 and 25 amino acids in P1AI4323 and P1AI4325. As control constructs, 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) lacking the protease release site were generated, as well as the non-masked construct P1AI4648 ( Figure 3 A to Figure 3 C).

[0259] These complex PD1-targeted masked IL2v forms (P1AI4322, P1AI4323, P1AI4324 and P1AI4325) were produced and purified by WuXi Biologics. Briefly, they were transiently transfected into HEK293 and purified by MabSelectSuRe LX protein A affinity chromatography and Superdex200 size exclusion chromatography. The purification of P1AI4324 involved two additional HiTrap SP HP-1 and SP HP-2 cation exchange chromatography purification steps between MabSelectSuRe LX protein A affinity chromatography and Superdex200 size exclusion chromatography.

[0260] To facilitate in vivo efficacy studies in human PD1 transgenic mice, cleavable (with YAARKGGI site) and noncleavable human PD1-targeted masked IL2v constructs were generated, P1AI4650 and P1AI4651 ( Figure 4 A to Figure 4 B). These murine surrogates have the same format as the human constructs but contain murine sequences to avoid immunogenicity. The V domain of the PD1 binder corresponds to the non-humanized predecessor, while all constant antibody domain sequences are murine. The only unavoidable human sequences in these surrogates are the human IL2v and the masked V domains. The murine surrogates were also produced and purified by WuXi Biologics.

[0261] Surface plasmon resonance (determination of masked affinity to human IL2v)

[0262] Deimmunized MT204 variants (P1AH2050-P1AH2052, Figure 1 A) Affinity for human IL2v (SEQ ID NO: 67). 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).

[0263] Anti-PGLALA antibody (M-1.7.24muIgG2b) was directly immobilized on 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 0.4M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 0.1M N-hydroxysuccinimide (NHS), 10ug / ml anti-PGLALA (diluted in 10mM acetate pH 5.0) was injected at a flow rate of 10μl / min for 100 seconds. After blocking with 1M ethanolamine-HCl pH 8.5, the coupling process produced an anti-PG LALA surface density of approximately 5400RU.

[0264] The MT204 variants of deimmunization were captured at a flow rate of 10 μl / min and a concentration of 50 nM for 60 seconds. Human IL2v G4S avi his was injected at different concentrations (800–0.391 nM, 1:2 dilution) and passed through the flow cell at a flow rate of 30 μl / min. Association and dissociation were monitored for 240 seconds and 1000 seconds, respectively. After each cycle, the chip surface was regenerated by injecting once (60 seconds) 10 mM glycine pH 2.0. The bulk refractive index difference was corrected by subtracting the response obtained in the reference flow cell. The curve was fitted using a 1:1 Langmuir interaction model using BiacoreInsight evaluation software 3.0 (Cytiva, Freiburg / Germany), and the results are summarized in Table 1.1.

[0265] Table 1.1

[0266]

[0267]

[0268] Two of the three deimmunized variants of the anti-IL2v mask, P1AH2050 and P1AH2051, were comparable to the parental anti-IL2v mask P1AF7506 ( Figure 1 E) were comparable, while the third deimmunized variant, P1AH2052, showed the fastest off-rate of all tested variants and also had the lowest affinity, 995 pM. Variant P1AH2051, hu IgG1 GL MT204_VL1a - combined VH2 & VH6 PG LALA, showed the highest affinity of 101 pM and was selected for cloning the complex masked IL2v format.

[0269] Surface plasmon resonance determines affinity to human PD1

[0270] The affinity of deimmunized α-PD1 binders to human PD1-Fc was assessed by surface plasmon resonance (SPR). SPR experiments were performed on Biacore T200 (for P1AH4157-P1AH4161 and P1AI0356-P1AI0360) or on Biacore 8K+ (for P1AI1648-P1AI1652). Figure 1 BC) were characterized on a Biacore T200 at 25°C using HBS-EP run 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).

[0271] Anti-PGLALA antibody (M-1.7.24muIgG2b) was directly immobilized on 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 0.4M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 0.1M N-hydroxysuccinimide (NHS), 20 μg / ml anti-PGLALA (diluted in 10 mM acetate pH 5.0) was injected at a flow rate of 5 μl / min for 900 seconds. After blocking with 1M ethanolamine-HCl pH 8.5, the coupling process produced an anti-PG LALA surface density of more than 12000 RU.

[0272] The deimmunized α-PD1 conjugate was captured at a flow rate of 10 μl / min and a concentration of 10 nM for 60 seconds. Recombinant huPD1-ECD_Fc-knob / mortise structure (internal IDP1AD9704) was injected through the flow cell at different concentrations (300-9,4 nM, 1: 1 dilution) and a flow rate of 50 μl / min. Association and dissociation were monitored for 120 seconds and 600 seconds, respectively. After each cycle, the chip surface was regenerated by injecting 20 mM NaOH (35 seconds each time). The bulk refractive index difference was corrected by subtracting the response obtained in the reference flow cell. The curve was 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.

[0273] P1AI1648-P1AI1652( Figure 1D) Characterized on a Biacore 8K+ 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) at 25°C.

[0274] Anti-PGLALA antibody (M-1.7.24muIgG2b) was directly immobilized on a C1 chip using a standard amine coupling kit (Cytiva, Freiburg / Germany) at pH 5.0. After activating the sensor surface with a 1:1 mixture of 0.4M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and 0.1M N-hydroxysuccinimide (NHS), 20 μg / ml 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 1M ethanolamine-HCl pH 8.5, the coupling process produced an anti-PG LALA surface density of approximately 1000 RU.

[0275] The deimmunized α-PD1 conjugate was captured at a flow rate of 10 μl / min and a concentration of 5 nM for 80 seconds. The recombinant huPD1-ECD_Fc-knob / mortise structure was injected through the flow cell at a flow rate of 30 μl / min at different concentrations (200-0.27 nM, 1:3 dilution). Association and dissociation were monitored for 240 seconds and 800 seconds, respectively. After each cycle, the chip surface was regenerated by injecting twice (60 seconds each) 10 mM glycine pH 2. The bulk refractive index difference was corrected by subtracting the response obtained in the reference flow cell. The 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.

[0276] Table 1.2. Binding properties

[0277]

[0278] Table 1.3. Binding properties

[0279]

[0280] Table 1.4. Binding properties

[0281]

[0282] A total of three groups of five deimmunized anti-PD1 variants were generated, each for evaluation as a human IgG1 PGLALA antibody: Group 1 (P1AH4157-P1AH4161; Figure 1 B), group 2 (P1AI0356-P1AI0360; Figure 1 C) and group 3 (P1AI1648-P1AI1652; Figure 1 D). Groups 1 and 2 are independent groups, while Group 3 is based on P1AH4157 (PD1-376_VL2_VH4) with additional mutations in CDR H2. The kinetic rate constants and affinity of PD1-376_VL2_VH4 to human PD1 are very similar to those of the parental humanized antibody P1AA6888 (PD1-0103), with ka 6.40E+05, kd 1.30E-04 and KD 200pM, respectively, for ka 6.30E+05, kd 1.50E-04 and KD 200pM. Based on binding properties 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 to clone the complex masked IL2v form.

[0283] Example 2

[0284] Relative luminescence units of PD1 / PD-L1 reporter Jurkat cell lines after PD-1 / PD-L1 blockade with parental (bivalent vs. monovalent) or deimmunized anti-PD1 antibodies

[0285] The PD-1 / PD-L1 Blockade Reporter Assay from Promega (Catalog #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 involves a co-culture of two genetically engineered cell lines: PD-1 T cells as effector cells, and PD-L1 APC / CHO-K1 cells as target cells.

[0286] PD-1 effector T cells are Jurkat T cells expressing human PD-1 and a luciferase reporter gene driven by the NFAT response element (NFAT-RE). PD-L1 APC / CHO-K1 target cells are CHO-K1 cells expressing human PD-L1 and engineered cell surface proteins designed to activate the cognate TCR in an antigen-independent manner. When co-cultured, the PD-1 / PD-L1 interaction inhibits TCR signaling and downregulates NFAT-RE-mediated luminescence in effector T cells. Blocking the PD-1 / PD-L1 interaction with anti-PD-1 (or anti-PD-L1) antibodies releases inhibitory signals and leads to TCR activation that NFAT-RE-mediated luminescence.

[0287] This assay was used to evaluate the ability of deimmunized PD-1 binders to block the PD-1 / PD-L1 interaction. To this end, eight 1:10 dilution steps (maximum concentration of 66 nM) of PD1 IgG PG LALA PD1-IL2v or deimmunized anti-PD-1 were added to cells expressing PD-L1-CHO, and the two cell lines were immediately co-cultured for 5 hours at 37°C. After 5 hours of incubation, substrate (BIO-Glo reagent) was added and samples were measured on a photometer (PerkinElmer Reader).

[0288] Figure 5 A and 5B show that PD1-376_VL2_VH1_2_3_4IgG PG LALA, PD1-376_VL2_7_VH4 IgG PG LALA and PD1-376_VL2_VH4 IgG PG LALA are roughly as effective as parental anti-PD1 in blocking PD-1 / PD-L1 interactions and overcoming downstream inhibitory signals. On the other hand, mutations on PD1-376_VL7_VH1_2_4IgG PG LALA and PD1-376_VL7_VH4 IgG PG LALA result in a 4-5-fold reduction in potency (Table 2.1).

[0289] Table 2.1. EC50 of blocking efficacy of deimmunized PD-1 on PD-1 effector cells in dose response in relative luminescence units.

[0290]

[0291]

[0292] PD1-IL2v (as disclosed in WO 2018 / 184964 A1) and FAP IL2v (as disclosed in WO 2012 / 107417 A1; INN: Simlukafusp alfa) were used as controls, and they showed that the lack of aPD-1 in FAP-IL2v prevented the PD-1 / PD-L1 inhibition of TCR signaling and the resulting luminescence. On the other hand, NFAT-RE-mediated luminescence was active because the interaction was blocked by aPD-1 on the PD1-IL2v construct.

[0293] Binding of deimmunized PD-1 to activated CD4 T cells

[0294] In the following experiments, deimmunized PD-1 antibodies were compared side by side in binding assays to assess whether deimmunization affects binding affinity / avidity to PD-1 on T cells. For this purpose, CD4 T cells were isolated from healthy donor PBMCs with CD4 beads (#130-045-101, Miltenyi) and activated for 3 days in the presence of 1 μg / ml plate-bound anti-CD3 (pre-coated overnight, 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 three days, cells were harvested and washed to remove endogenous IL-2. The cells were then seeded into V-bottom plates and stained with increasing concentrations of the treated antibodies for 30 min at 4°C: 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_4IgG PG LALA, PD1-376_VL2_VH1_2_3_4IgGPG LALA, parental anti-PD-1PD1-IL2v and FAP IL2v (50 μl, 1:10 dilution step, highest concentration of 66 nM).

[0295] The cells were washed with PBS to remove unbound molecules. Then, 50 μl of diluted AF647 anti-PGLALA antibody and Fixable Viability Dye eFluor TM 780 (eBioscience) was added to the cells, followed by a wash step after incubation at 4°C for 30 minutes. Finally, the cells were fixed with BD cell fixative (70ul, #340181, BD Biosciences) and collected 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.

[0296] like Figure 6As shown in A 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_4IgG PG LALA and the corresponding parental anti-PD1- and PD1-IL2v bind to CD4 T cells with similar potency. PD1-376_VL7_VH4 IgG PG LALA and PD1-376_VL7_VH1_2_4IgG PG LALA show reduced binding to CD4 T cells. FAP-IL2v was used as a non-targeting control to compare the effect of a single IL2v-based immunocytokine that does not target PD-1.

[0297] Tables 2.2 and 2.3 show the frequency and EC50 of MFI for dose-responsive binding of various antibodies to activated CD4 T cells obtained from 2 donors.

[0298] Table 2.2. Selected deimmunized PD-1 in PD-1 from healthy donors + EC50 of binding on dose-response frequency of CD4 T cells.

[0299] molecular EC50 PD-1IgG PF LALA 80.62 PD1-IL2v (P1AE4422-18309) 45.49 PD1-376_VL2_VH1_2_3_4IgG PG LALA 47.92 PD1-376_VL7_VH1_2_4IgG PG LALA 747.6 PD1-376_VL2_7_VH4 IgG PG LALA 74.42 PD1-376_VL7_VH4 IgG PG LALA 948.4 PD1-376_VL2_VH4 IgG PG LALA 88.64.4 FAP IL2v(P1AA5355-004) 9.679.8

[0300] Table 2.3. Selected deimmunized PD-1 in PD-1 from healthy donors + EC50 of binding for dose-response MFI on CD4 T cells.

[0301] molecular EC50 PD-1IgG PF LALA 234.2 PD1-IL2v (P1AE4422-18309) 153.6 PD1-376_VL2_VH1_2_3_4IgG PG LALA 121.1 PD1-376_VL7_VH1_2_4IgG PG LALA 1655 PD1-376_VL2_7_VH4 IgG PG LALA 206 PD1-376_VL7_VH4 IgG PG LALA 2052 PD1-376_VL2_VH4 IgG PG LALA 226.5 FAP IL2v(P1AA5355-004) 708

[0302] Effects of different anti-PD-1 antibodies on cytotoxic granzyme B and IFN-γ secretion by allogeneic specific human CD4 T cells co-cultured with allogeneic mature dendritic cells

[0303] To evaluate deimmunized PD-1 antibodies in functional assays, CD4 T cells were exposed to allogeneic mature DCs to induce the generation of allogeneic specific T cells with different antigen specificities and TCR affinities. Importantly, allogeneic specific T cells express immune checkpoints such as PD-1 and can therefore be used to measure the functional efficacy of anti-PD1 antibodies because they can release T cell effector functions. To screen the function and efficacy of PD-1 blocking antibodies in an allogeneic setting, freshly purified CD4 T cells were co-cultured in the presence of monocyte-derived allogeneic mature dendritic cells (mDC) for 5 days. Monocytes were isolated from fresh PBMCs using CD14 beads (130-050-201, Miltenyi) one week ago. Immature DCs were then generated from monocytes by culturing them in medium containing GM-CSF (50 ng / ml) and IL-4 (100 ng / ml) for 5 days. To induce iDC maturation, TNF-α, IL-1β, and IL-6 (50 ng / ml each) were added to the culture medium and cultured for another 2 days.

[0304] On the day of minimal mixed lymphocyte reaction (mMLR), CD4 T cells were enriched from PBMC obtained from unrelated donors via a microbead kit (Miltenyi). Before culture, CD4 T cells were labeled with 5 μM Cell Trace Violet (CTV, #C34557, ThermoFisher). Then 105 CD4 T cells were plated with mature allogeneic DC (10:1 ratio) in flat-bottom 96-well plates in the presence of a range of concentrations of the following purified anti-PD1 monoclonal antibodies at 37°C, 5% CO2 for 5 days: PD1-376_VL2_VH4 IgG PG LALA, PD1-376_VL7_VH4IgG PG LALA, PD1-376_VL2_7_VH4 IgG PG LALA, PD1-376_VL7_VH1_2_4IgG PG LALA, PD1-376_VL2_VH1_2_3_4IgG PGLALA, parental anti-PD1 and PD1-IL2v (50 μl, 1:10 dilution step, highest concentration of 66 nM). No antibody or FAP IL2v was used as a negative control.

[0305] After five days, the cells were placed in the presence of Golgi Plug (Brefeldin A) and Golgi Stop (Monensin) at 37°C for another 5 hours, then washed and stained with anti-human CD4 antibody and the fixable viability dye eFluor TM780 (eBioscience) was stained on the surface and fixed / permeabilized with Fix / Perm Buffer (BD Bioscience). The cells were then stained intracellularly 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).

[0306] Anti-PD1 monoclonal antibodies promoted the expression of GrzB ( Figure 8 A and 8B) and IFN-γ ( Fig. 9 A and 9B) T cell secretion. It was found that all anti-PD-1 variants enhanced 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) of GrzB and IFNγ secretion by CD4 T cells in dose-dependent response to allogeneic stimulation and anti-PD-1 antibody treatment.

[0307] Table 2.4. EC50 and area under the curve for dose-response granzyme B secretion of selected deimmunized PD-1 on CD4 T cells from healthy donors.

[0308]

[0309]

[0310] Table 2.5. EC50 and area under the curve for dose-response IFN-γ secretion of selected deimmunized PD-1 on CD4 T cells from healthy donors.

[0311]

[0312] The parental anti-PD-1 obtained a lower EC50 for cytokine production, followed by PD1-376_VL2_VH4 IgG PGLALA. The highest AUC was provided by the deimmunized variant PD1-376_VL2_VH4 IgG PG LALA and the control PD1-IL2v.

[0313] Example 3

[0314] Binding of deimmunized PD1 binders to CHO-huPD1 cells

[0315] We evaluated the binding of five different deimmunized PD1-IgGs to CHO cells overexpressing human PD1 and compared them with the respective parental PD1-IgG molecules and the TA PD1-IL2v construct containing the parental PD1 binder (Table 3.1).

[0316] Table 3.1. Constructs tested

[0317] Constructs ID PD1-376_VL2_VH4 IgG PG LALA P1AH4157-001 PD1-376_VL7_VH4 PG LALA P1AH4158-001 PD1-376_VL2_7_VH4 PG LALA P1AH4159-001 PD1-376_VL7_VH1_2_4PG LALA P1AH4160-001 PD1-376_VL2_VH1_2_3_4PG LALA P1AH4161-001 PD1 IgG-IL2v aIL2 MT204 scFv-PQARK (parental) P1AG9606-008 PD1-IgG P1AD4476-002

[0318] 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, 5mM EDTA, 0.025% NaN3). Then, 100'000 cells were seeded into each well of a round bottom plate. The cells were stained in FACS buffer at 4 ° C for 30 min with 30 μl of PD1-IgG, parent PD1-IgG and TA PD1-IL2v constructs containing parent PD1-IgG at a specified concentration. After staining, the cells were washed twice with FACS buffer to remove unbound molecules. Then 30 μl of diluted PE anti-human Fc specific secondary antibodies (1:50 dilution, 109-116-170, Jackson ImmunoResearch) were added to the cells. After incubation at 4 ° C for 30 min, the cells were washed twice with FACS buffer. Finally, cells were resuspended in 150 μl FACS buffer and measured using a BD Fortessa.

[0319] All five deimmunized PD1-IgGs bound similarly to human PD1 on CHO cells compared to their respective parental PD1-IgGs, with the PD1-IgG containing 2 VL7s having slightly reduced binding compared to the other deimmunized PD1-IgGs ( Fig.10 ).

[0320] Blocking IL2v activity using deimmunized MT204 mask

[0321] Next, we tested the blocking of FAP-IL2v activity by three different deimmunized MT204 masks compared to the parental MT204 mask (Table 3.2).

[0322] Table 3.2. Constructs tested

[0323] Constructs ID hu IgG1 GL MT204_VL1-combination VH2&VH6 PG LALA P1AH2050-001 hu IgG1 GL MT204_VL1a-combination VH2&VH6 PG LALA P1AH2051-001 hu IgG1 GL MT204_VL1b-combination VH2&VH6 PG LALA P1AH2052-002 Anti-IL2 MM7 MM8 PG LALA, Anti-MT204 P1AA9690-001 FAP-IL2v P1AA5355-004

[0324] NK92 cells were harvested, counted and their viability was assessed. The cells were washed three times with PBS to remove residual IL2. The washed NK92 cells were resuspended in fresh culture medium (advanced RPMI1640, 2% FCS, 1% glutamine) without IL2, and 50 μl of cell suspension containing 10'000 cells was transferred to a 96-well cell culture treated flat plate. First, 25 μl of 0.5 nM FAP-IL2v antibody was added to each well. Then, 25 μl of MT204 antibody was added to each well to reach a final volume of 100 μl per well. The plate was incubated in an incubator for 3 days. After 3 days, CellTiter-Glo (G7571, Promega) reagent and cell culture plates were balanced to room temperature. CellTiter-Glo solution was prepared as described in the manufacturer's instructions, and 100 μl of solution was added to each well. After 10 min incubation, the 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 with a Tecan Spark 10M multimode reader.

[0325] MT204_VL1 and MT204_VL1a blocked IL2v activity comparable to the parental MT204 mask. MT204_VL1b showed lower blocking efficiency on IL2v activity compared to the parental MT204 mask ( Fig.11 ).

[0326] Binding of TA PD1-IL2v construct to CHO-hu PD1 cells

[0327] 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.

[0328] Table 3.3. Constructs tested

[0329]

[0330] 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, 5mM EDTA, 0.025% NaN3). 100'000 cells per well were seeded in a round bottom plate. The cells were stained with TA PD1-IL2v constructs in 30 μl FACS buffer at 4 ° C for 30 minutes. After staining, the cells were washed twice with FACS buffer to remove unbound molecules. Then 30 μl of diluted APC anti-human Fc specific secondary antibodies (1:50 dilution, 109-136-098, Jackson ImmunoResearch) were added to the cells. After incubation at 4 ° C for 30 min, the cells were washed twice with FACS buffer. Finally, the cells were resuspended in 150 μl FACS buffer and measured using BD Fortessa.

[0331] All four tested constructs contained deimmunized VL2_VH4 PD1 binders and MT204 VL1a deimmunized masks. We tested their binding to CHO cells overexpressing human PD1 and compared them to the respective uncleavable, non-masked and parental TA PD1-IL2v molecules. All tested TA PD1-IL2v constructs bound comparably to human PD1 on CHO cells ( Fig.12 ).

[0332] TA PD1-IL2v construct induces KHYG-1 proliferation

[0333] Next, we tested the induction of KHYG-1 cell proliferation by four different TA PD1-IL2v constructs containing either the PQARK cleavage site or the YAARKGGI cleavage site and either a 38-mer linker or a 25-mer linker (see Table 3.3).

[0334] The NK cell line KHYG-1 was used to test the proliferation induction of the TA PD1-IL2v construct. The cells were harvested, counted and assessed for viability. The cells were washed three times with PBS to remove residual IL2. The washed KHYG-1 cells were resuspended in fresh medium (advanced RPMI1640, 2% FCS, 1% glutamine) without IL2, and 12.5 μl of the cell suspension containing 2'000 cells was transferred to a 384-well cell culture treated flat bottom plate. 10 μg of TA PD1-IL2v construct was digested with 2 μl Matriptase (Enzo ~ 2.5U / μl, ALX-201-246-U25, batch number 08102104, or no Matriptase was used as an undigested control) at 37 ° C for 2 hours in 20 μl Matriptase buffer (50mM Tris, 50mM NaCl, 0.01% Tween 20, pH 9.0), and 12.5 μl of antibody was added to each well to reach a final volume of 25 μl per well. The plate was incubated in an incubator for 3 days. After 3 days, CellTiter-Glo (G7571, Promega) reagent and cell culture plates were equilibrated to room temperature. CellTiter-Glo solution was prepared as described in the manufacturer's instructions, and 25 μl of solution was added to each well. After 10 min incubation, the remaining aggregates were resuspended by pipetting and 40 μl of the mixture was transferred to a white flat bottom plate.Luminescence was measured with a Tecan Spark 10M multimode reader.

[0335] All four constructs tested contained the deimmunized VL2_VH4 PD1 binder and the MT204 VL1a deimmunized mask. As controls, two respective non-cleavable molecules and non-masked PD1-IL2v were included. All molecules were tested undigested and after digestion with recombinant matriptase. After digestion with matriptase, TA PD1-IL2v with PQARK and YAARKGGI linkers regained activity. There was a slight decrease in activity compared to the non-masked control, most likely due to incomplete cleavage. The activity of the TA PD-IL2v construct containing the 38-mer linker was comparable to that of the 25-mer linker ( Fig.13 A, 13C). No undigested TA PD1-IL2v induced proliferation ( Fig.13 A, 13D, 13F). Non-cleavable TA PD1-IL2v digested with matriptase did not induce proliferation ( Fig.13 E). In contrast to the uncleavable construct, the activity of the non-masked PD1-IL2v was not affected by matriptase cleavage ( Fig.13 E, 13F).

[0336] Example 4

[0337] Syngeneic model of murine tumor cell lines using murine TA-PD1-IL2v immunoconjugate

[0338] In vivo efficacy in (MCA205 subcutaneous syngeneic model)

[0339] The murinized TA-PD1-IL2v immunoconjugate was tested in the mouse fibrosarcoma cell line MCA205 injected subcutaneously into Black 6-huPD1 transgenic mice.

[0340] MCA205 fibrosarcoma cancer cells were originally obtained from Sigma Aldrich (Catalog No. SCC173) and stored 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 11 was used for transplantation with a survival rate of 95.3%. Using a 1 ml tuberculin syringe (BD Biosciences, Germany), 1x10 per animal was used. 6 The cells were injected subcutaneously into the flank of mice in 100 μl of RPMI cell culture medium (Gibco).

[0341] Female Black 6-huPD1 mice (bred at Charles Rivers, Lyon, France), 10-11 weeks old at the start of the experiment, were maintained under specific pathogen-free conditions with a 12 h light / 12 h dark daily cycle according to regulatory guidelines (GV-Solas; Felasa; TierschG). The experimental study protocol was reviewed and approved by the local authorities (P 184 / 2020). After arrival, the animals were maintained for one week to acclimate to the new environment and were observed. Continuous health status monitoring was performed regularly.

[0342] On day 0 of the study, mice were injected subcutaneously with 1x10 6 MCA205 cells were randomly divided into groups and weighed. Ten days after tumor cell injection (tumor volume > 150 mm 3 ), mice were injected iv with TA-PD1-IL-2v YAARKGGI 38-mer cleavable linker or TA-PD1-IL-2v 38-mer non-cleavable linker twice a week for one week. All mice were injected iv with 200 μl of the appropriate solution. Mice in the vehicle group were injected with histidine buffer. In order to obtain the appropriate amount of immunoconjugate per 200 μl, the stock solution was diluted with histidine buffer as necessary (Table 4).

[0343] Table 4.

[0344]

[0345] Fig.14 It was shown that TA-PD-IL2vYAARKGGI 38-mer mediated superior efficacy in tumor growth inhibition compared with vehicle and non-cleavable Mab single-agent groups.

[0346] Example 5

[0347] Design of a murine alternative to a complex PD1-targeted, masked IL2v immunoconjugate with a scFv mask

[0348] To facilitate in vivo tolerability and efficacy studies in non-tumor bearing mice or mouse models of cancer, mouse surrogates of PD1-targeted masked IL2v immunoconjugates targeting human (P1AK3638 and P1AK3649) or mouse (P1AK3641 and P1AK3640) PD1 were generated. To reduce immunogenicity, all constant antibody domains in these constructs correspond to mouse sequences. The mouse surrogates target human PD1 for use in humanized mice or human PD1 transgenic mice; or target mouse PD1 for use in syngeneic mouse models with immunocompetent mice. Human IL2v has been used in all constructs due to the cross-reactivity of human IL2v with the mouse IL2 receptor and the lack of cross-reactivity of the scFv mask with mouse IL2v.

[0349] These murine surrogate constructs bind bivalently to human or murine PD1 via N-terminal Fab arms on the Fc DD- and Fc KK+ chains, which also carry a masked (matriptase cleavable or matriptase non-cleavable) C-terminal IL2v. The C-terminus of the scFv mask is "linearly" fused to IL2v on the same Fc DD-chain. Heterodimerization is achieved by applying complementary charges in the murine IgG1 CH3 domain (Fc DD- and Fc KK+ chains), and binding to activating Fcγ receptors and complement component C1q can be abolished by introducing a DAP G mutation in the murine IgG1 CH2Fc domain of the antibody. The matriptase cleavable C-terminally masked IL2v constructs P1AK3638 and P1AK3641 each carry two PQARK matriptase recognition sites, one of which is located in the linker between the VH and VL domains of the scFv mask and the other is located in the linker between the scFv mask and IL2v. In addition, corresponding matriptase non-cleavable control constructs (lacking the PQARK matriptase recognition site) P1AK3649 and P1AK3640 and a non-masked control construct (lacking the scFv mask) were also generated. These masked constructs are schematically depicted in Fig.16 AC and 17A-B, D. For comparison, to test only the in vivo efficacy of checkpoint inhibition, a mouse IgG2a (P1AD4006) with anti-mouse PD1 specificity was included in some studies and Fig.17 Depicted in C.

[0350] Production and purification of PD1-targeted masked IL2v immunoconjugates and control constructs

[0351] Murine surrogate PD1 -targeted masked IL2v immunoconjugates (P1AK3638 and P1AK3649 and P1AK3641 and P1AK3640, respectively) were produced and purified by WuXi Biologics. They were transiently expressed in HEK293 and purified by a 2-column DSP process: 1. MabSelectSuRe LX affinity chromatography (equilibration and 1st wash: 25 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, pH 7.5; 2nd wash: 25 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 0.1% Triton 100 / 114, pH 7.5; elution: 50 mM sodium citrate-citric acid, 150 mM NaCl, pH 3.0; neutralization: 1 M arginine, 0.4 M succinic acid, pH 9.0; and 2. Superdex200 size exclusion chromatography (equilibration and formulation buffer: 20 mM histidine-HCl, 140 mM NaCl, pH 6.0). Purity has been confirmed by SEC-HPLC and reducing and non-reducing 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).

[0352] Murine IgG2a P1AD4006 with anti-murine PD1 specificity was produced and purified by evitria AG. Suspension-adjusted CHO K1 cells (originally received from ATCC and adapted to serum-free growth in suspension culture at evitria) were used for production. Seeds were grown in eviGrow medium, a chemically defined, animal component-free, serum-free medium. Cells were transfected with eviFect, a custom, proprietary transfection reagent from evitria, and after transfection cells were grown in eviMake2, an animal component-free, serum-free medium. The supernatant was collected by centrifugation and subsequent filtration (0.2 μm filter). The supernatant was purified using MabSelect TM SuRe TM IgG was purified with Dulbecco's PBS (Lonza BE17-512Q) as wash buffer, 0.1 M glycine pH 3.5 as elution buffer, and 1 M Tris HCl as neutralization buffer (pH 9). Subsequent size exclusion chromatography was performed on a HiLoad Superdex 200 pg column using the final buffer as running buffer. A Pierce Slide-A-Lyzer with a 2K molecular weight cutoff was used. TM G2 dialysis cassettes were used for dialysis (if necessary). Ultracentrifugal filters were used for antibody concentration (if required). Concentration was determined by measuring the absorbance at a wavelength of 280 nm. Extinction coefficients were calculated using evitria's proprietary algorithm. Purity was determined by analytical size exclusion chromatography using an Agilent AdvanceBio SEC column (300A 2.7um 7.8x300mm) with DPBS as running buffer at a flow rate of 0.8ml / min. Endotoxin content was measured using a Charles River Endosafe PTS system.

[0353] The human FolR1-targeted T cell engager P1AK1120 ( Fig.15 ) has been produced by evitria AG as described above and purified at Roche by a 3-column DSP process: (1. Protein A MabSelectSure, 2. Butyl HP hydrophobic interaction chromatography (HIC), and 3. Superdex200 HiLoad16 / 6000 size exclusion chromatography (SEC)) and formulated into 20 mM histidine, 140 mM NaCl, pH 6.0. Purity has been confirmed by SEC-HPLC and reducing and non-reducing 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).

[0354] Example 6

[0355] HEK Blue IL2 reporter cell assay using HEK Blue IL2 cells overexpressing human PD1,

[0356] To test the activity of the murinized TA PD1-IL2v construct

[0357] We tested the activity of murinized cleavable and noncleavable TA PD1-IL2v molecules containing human-specific PD1 binders in a HEK Blue IL2 reporter cell assay using HEK Blue IL2 cells expressing human PD1.

[0358] The 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). The corresponding amount of undigested control was also filled to 20 μl with Matriptase buffer and then treated in the same manner as the sample to which Matriptase was added. Digestion was performed at 37°C for 2 hours. After digestion, the sample was filled with DMEM+10% FBS+1% GlutaMax (assay medium) to have a final starting concentration of 50 nM.

[0359] HEK-blue huPD1 IL-2 cells (HEK-blue-IL2_hPDCD1_clone 4) were detached using cell dissociation buffer and resuspended in DMEM+10% FBS+1% GlutaMax (assay medium) at 0.33 Mio cells / ml. 150 μl HEK-blue IL-2 cells (comprising 50'000 cells) were subsequently inoculated into 96-well flat-bottom plates. 50 μl titrated surrogate IL2v molecules were then added to each well to reach a final volume of 200 μl per well. The plate was then incubated at 37°C for 24 hours. 180 μl Quanti-blue solution (rep-qbs2, InvivoGen) and 20 μl cell supernatant were then added to each well of a 96-well flat-bottom plate and incubated at 37°C for 60 minutes. Then, the optical density (OD) was measured at 620 nm using a Tecan Spark Reader.

[0360] The activity of the molecules was compared to the respective non-masked PD1-IL2v constructs. All molecules were tested after cleavage with recombinant Matriptase or without digestion. After cleavage, the activity of the cleavable murinized TA PD1-IL2v construct was comparable to naked PD1-IL2v, while the non-cleavable TA PD1-IL2v had only very limited activity in the reporter cell assay ( Fig.18 A).

[0361] In the absence of digestion with recombinant Matriptase, both cleavable and uncleavable TA PD1-IL2v constructs had only very limited activity compared to naked PD1-IL2v ( Fig.18 B).

[0362] Example 7

[0363] A HEK Blue IL2 reporter cell assay was performed using HEK Blue IL2 cells overexpressing mouse PD1 to test the activity of the murinized TA PD1-IL2v construct containing a mouse-specific PD1 binder.

[0364] We tested the activity of murinized cleavable and noncleavable TA PD1-IL2v molecules containing mouse-specific PD1 binders in a HEK Blue IL2 reporter cell assay using HEK Blue IL2 cells expressing mouse PD1.

[0365] The 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). The corresponding amount of undigested control was also filled to 20 μl with Matriptase buffer and then treated in the same manner as the sample to which Matriptase was added. Digestion was performed at 37°C for 2 hours. After digestion, the sample was filled with DMEM+10% FBS+1% GlutaMax (assay medium) to have a final starting concentration of 50 nM.

[0366] 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 HEK-blue IL-2 cells (comprising 50'000 cells) were subsequently inoculated into 96-well flat-bottom plates. 50 μl titrated surrogate IL2v molecules were then added to each well to reach a final volume of 200 μl per well. The plate was then incubated at 37°C for 24 hours. 180 μl Quanti-blue solution (rep-qbs2, InvivoGen) and 20 μl cell supernatant were then added to each well of a 96-well flat-bottom plate and incubated at 37°C for 60 minutes. Then, the optical density (OD) was measured at 620 nm using a Tecan Spark Reader.

[0367] The activity of the molecules was compared to the respective non-masked PD1-IL2v constructs. All molecules were tested after cleavage with recombinant Matriptase or without digestion. After cleavage, the activity of the cleavable murinized TA PD1-IL2v construct was comparable to naked PD1-IL2v, while the non-cleavable TA PD1-IL2v had only very limited activity in the reporter cell assay ( Fig.19 A).

[0368] In the absence of pre-digestion with recombinant Matriptase, both cleavable and non-cleavable TA PD1-IL2v constructs had only very limited activity compared to naked PD1-IL2v. The TA PD1-IL2v cleavable construct was slightly more active than the corresponding non-cleavable construct, which may indicate that minor cleavage may occur during the incubation time ( Fig.19 B).

[0369] Example 8

[0370] The TA PD1-IL2v construct induced proliferation and activation of CD8 T cells and NK cells in vitro.

[0371] The TA PD1-IL2v construct was tested for its ability to induce proliferation of NK cells and CD8 T cells in PBMCs after digestion with recombinant human Matriptase.

[0372] The calculated amount of each molecule was digested with 3 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). Digestion was performed at 37°C for 2 hours. After digestion, the sample was filled with RPMI+10% FBS+1% GlutaMax (assay medium) to have a final starting concentration of 100 nM.

[0373] Frozen PBMC (Biomex) were thawed and immediately resuspended in preheated assay medium. The cells were then centrifuged at 350g for 5 min and washed once with preheated PBS. The CFSE stock solution was diluted 1:20 with preheated PBS to obtain a working solution with a concentration of 100 μM. 30 Mio cells were resuspended in 30 ml of preheated 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 min. Then, preheated assay medium was added to terminate the reaction. The cells were centrifuged at 400g for 10 min, resuspended in assay medium, and incubated at 37°C for 30 min. After incubation, the cells were washed once with preheated assay medium, counted and resuspended in assay medium at 2 Mio cells / ml.

[0374] CFSE-labeled PBMCs are inoculated into 96-well round-bottom plates (0.1 Mio cells per well), and IL2 antibodies are added and all wells are filled to obtain a final volume of 200 μl. After 6 days of incubation at 37°C, PBMCs are harvested. Cells are centrifuged at 400g for 4min, and washed once with PBS. Live / dead staining agents are added to 25 μl PBS (diluted at 1:1000 in PBS), and incubated at RT for 20min. Then 150 μl FACS buffers are added and the plate is centrifuged at 400g for 4min. Supernatant is removed, and cells are washed again with 150 μl FACS buffers. Then 25 μl antibody mixtures (BUV395 anti-human CD3, PE anti-human CD4, APC anti-human CD8, PE / Cy7 anti-human CD25, BV421 anti-human CD56) per well are added to cells. Cells are incubated in a refrigerator for 30min. The cells were then washed twice with FACS buffer and resuspended in 150 μl FACS buffer. Analysis was performed using BD LSR Fortessa. NK cells were defined as CD3 negative and CD56 positive, and CD8 T cells were defined as CD3 CD8 double positive.

[0375] CFSE-labeled PBMCs were treated with TA PD1-IL2v molecules containing either the PQARK cleavage site or the YAARKGGI cleavage site and either a 38-mer or 25-mer linker. The corresponding non-cleavable TA PD1-IL2v molecules were included as negative controls. The corresponding non-masked PD1-IL2v molecules were included as positive controls. After 6 days of incubation, PBMCs were analyzed by flow cytometry for upregulation of activation markers and CFSE dilution was used as a proliferation indicator.

[0376] All pre-digested TA PD1-IL2v constructs containing the cleavage site were able to induce NK cell and CD8 T cell proliferation ( Fig. 20 AB) and activation ( Fig.21 AB). In this assay, no differences were observed between the two tested linker lengths and the two cleavage sites, and the pre-cleaved TA PD1-IL2v molecules had comparable activity to the corresponding non-masked PD1-IL2v molecules. The two TA PD1-IL2vs that did not contain the protease cleavage site did not induce any proliferation or activation of NK cells and CD8 T cells, indicating that TA PD1-IL2v was active only after protease cleavage.

[0377] Example 9

[0378] Syngeneic model of murine tumor cell line with murine TA-PD1-IL2v immunoconjugate

[0379] In vivo efficacy of MCA205 in a subcutaneous syngeneic model

[0380] The murinized TA-PD1-IL2v immunoconjugate was tested in the mouse fibrosarcoma cell line MCA205 injected subcutaneously into Black 6-huPD1 transgenic mice.

[0381] MCA205 fibrosarcoma cancer cells were originally obtained from Sigma Aldrich (Catalog No. SCC173) and stored 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 percent. Using a 1 ml tuberculin syringe (BD Biosciences, Germany), 1x10 per animal was used. 6 The cells were injected subcutaneously into the flank of mice in 100 μl of RPMI cell culture medium (Gibco).

[0382] Female Black 6-huPD1 mice (bred at Charles Rivers, Lyon, France), 8-10 weeks old at the start of the experiment, were maintained under specific pathogen-free conditions with a 12 h light / 12 h dark daily cycle according to regulatory guidelines (GV-Solas; Felasa; TierschG). The experimental study protocol was reviewed and approved by the local authorities (P 184 / 2020). After arrival, the animals were maintained for one week to acclimate to the new environment and were observed. Continuous health status monitoring was performed regularly.

[0383] On day 0 of the study, mice were injected subcutaneously with 1x10 6 MCA205 cells were randomly divided into groups and weighed. Ten days after tumor cell injection (tumor volume > 200 mm 3 ), mice were injected iv with TA-PD1-IL-2v PQARK 25-mer cleavable linker or TA-PD1-IL-2v 25-mer non-cleavable linker twice a week for one week. All mice were injected iv with 200 μl of the appropriate solution. Mice in the vehicle group were injected with histidine buffer. To obtain the appropriate amount of immunoconjugate per 200 μl, the stock solution was diluted with histidine buffer as necessary.

[0384] Fig. 22 It was shown that TA-PD-IL2vPQARK 25-mer mediated superior efficacy in tumor growth inhibition compared with vehicle, uncleavable and pembrolizumab Mab single-agent groups.

[0385] Table 5.

[0386]

[0387] Example 10

[0388] Murine alternative to TA-PD1-IL2v immunoconjugate in syngeneic models of mouse tumor cell lines

[0389] In vivo efficacy in the GL261 subcutaneous syngeneic model

[0390] The murine surrogate TA-PD1-IL2v immunoconjugate was tested in the mouse glioblastoma cell line GL261, injected subcutaneously into Black 6 mice.

[0391] GL261 glioblastoma cells were originally obtained from DSMZ (Germany) and stored 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 percent. Using a 1 ml tuberculin syringe (BD Biosciences, Germany), 1x10 cells were placed in each animal. 6 The cells were injected subcutaneously into the flank of mice in 100 μl of RPMI cell culture medium (Gibco).

[0392] Female Black6 mice (bred at Charles Rivers, Lyon, France), 9-11 weeks old at the start of the experiment, were maintained under specific pathogen-free conditions with a 12 h light / 12 h dark daily cycle according to the established guidelines (GV-Solas; Felasa; TierschG). The experimental study protocol was reviewed and approved by the local authorities (P 184 / 2020). After arrival, the animals were maintained for one week to acclimate to the new environment and were observed. Continuous health status monitoring was performed regularly.

[0393] On day 0 of the study, mice were injected subcutaneously with 1x10 6 GL261 cells were randomly divided into groups and weighed. Ten days after tumor cell injection (tumor volume > 100 mm 3 ), mice were injected intravenously with muTA-PD1-IL-2v PQARK 25-mer cleavable linker or muPD1-IgG twice a week for one week. All mice were injected iv with 200 μl of the appropriate solution. Mice in the vehicle group were injected with histidine buffer. To obtain the appropriate amount of immunoconjugate per 200 μl, the stock solution was diluted with histidine buffer when necessary.

[0394] Fig.23It was shown that the muTA-PD-IL2vPQARK 25-mer mediated superior efficacy in tumor growth inhibition at both doses tested compared to vehicle and muPD1 Mab single-agent groups.

[0395] Table 6.

[0396]

[0397] Example 11

[0398] In vivo efficacy of TA-PD1-IL2v immunoconjugate in combination with FOLR1-TCB bispecific Mab in human tumor PDX xenograft models - BC004 breast patient-derived xenograft model

[0399] The anti-tumor efficacy of the TA-PD1-IL2v immunoconjugate in combination with the FOLR1-TCB bispecific antibody was tested in a xenograft model. The human TA-PD1-IL2v immunoconjugate was tested in human breast BC004 patient-derived cells injected subcutaneously into humanized NSG mice.

[0400] BC004 PDX material (human breast cancer) was originally 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 transplanted with BC004 cells. 1x10 per animal was used using a 1 ml tuberculin syringe (BD Biosciences, Germany). 6 The cells were injected subcutaneously into the flank of mice in 100 μl of RPMI cell culture medium (Gibco, Germany).

[0401] Fully humanized NSG female mice (Roche-Glycart, Schlieren, Switzerland) were maintained under specific pathogen-free conditions with a 12 h light / 12 h dark daily cycle according to regulatory guidelines (GV-Solas; Felasa; TierschG). The experimental study protocol was reviewed and approved by the local authorities (ZH184 / 2020). Continuous health status monitoring was performed regularly.

[0402] On day 0 of the study, mice were injected subcutaneously with 1x10 6 BC004 cells were randomly divided into groups and weighed. Thirty-two days after tumor cell injection (tumor volume>150mm 3), mice were injected iv with the following immunocytokines: TA-PD1-IL2v-PQARK cleavable 25-mer 1 mg / kg, PD1-IL2v 0.1 mg / kg and pembrolizumab 1 mg / kg in combination with FolR1-TCB or vehicle once a week for four weeks. All mice were injected iv with 200 μl of the appropriate solution. Mice in the vehicle group were injected with histidine buffer, and mice in the treatment groups were injected with different constructs. In order to obtain the appropriate amount of immunoconjugate per 200 μl, the stock solution was diluted with histidine buffer when necessary. Tumor growth measurements were assessed three times a week with calipers and plotted using GrahPad Prism software, with volume units in mm 3 + / -SEM.

[0403] Fig.24 The combination of FOLR1-TCB 0.3mg / kg+TA-PD1-IL2v PQARK cleavable linker 1mg / kg Mab mediated superior efficacy in tumor growth inhibition compared to FOLR1-TCB+pembrolizumab 1mg / kg. The benefit in efficacy showed similar tumor growth inhibition to the combination of FOLR1-TCB 0.3mg / kg+PD1-IL2v non-masking 0.1mg / kg Mab.

[0404] Table 7.

[0405]

[0406] ***

[0407] Although the present invention has been previously described in considerable detail by way of illustration and example for purposes of clarity of understanding, 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. 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, wherein the masking moiety is covalently linked to the IL-2 polypeptide via the linker, wherein the masking moiety is capable of binding to the IL-2 polypeptide thereby reversibly concealing the IL-2 polypeptide, wherein the masking moiety comprises a second protease cleavage site, wherein the masking moiety does not conceal the IL-2 polypeptide when cleaved at the first protease cleavage site and / or the second protease cleavage site.

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

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

4. The protease-activatable IL-2 polypeptide according to 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 according to any one of the preceding claims, wherein the masking moiety is covalently linked to the amino terminus or the carboxyl terminus of the interleukin-2 polypeptide via the linker.

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

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

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 according to any one of the preceding claims, wherein the masking moiety is an antibody derived from antibody MT204.

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

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

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

13. The protease-activatable IL-2 polypeptide according to any one of the preceding claims, 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 according to SEQ ID NO: 60 and / or PQARK according to SEQ ID NO:

61.

14. The protease-activatable IL-2 polypeptide according to any of the preceding claims, wherein the IL-2 polypeptide is wild-type IL-2, preferably human IL-2 according to SEQ ID NO: 62, or is a mutant IL-2 polypeptide.

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

62.

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

62.

17. The protease-activatable IL-2 polypeptide according to 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 according to SEQ ID NO:

62.

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

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

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

24. The immunoconjugate of claim 22 or 23, wherein the immunoconjugate comprises a first antigen binding moiety and a second antigen binding moiety, or a first effector cell antigen binding moiety and a second effector cell antigen binding moiety, or an antigen binding moiety and an effector cell binding moiety.

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

26. The protease-activatable IL-2 polypeptide of claim 21 or the immunoconjugate 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. The protease-activatable IL-2 polypeptide of claim 21 or the immunoconjugate 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 a scFv molecule.

28. The protease-activatable IL-2 polypeptide according to claim 21 or the immunoconjugate according to 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. The protease-activatable IL-2 polypeptide of claim 21 or the immunoconjugate 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 wherein the effector cell binding portion is directed against an effector cell present in the tumor cell environment to achieve cis targeting.

30. The immunoconjugate according to any one of claims 22 to 29, (i) wherein 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; (ii) wherein 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; (iii) wherein 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; or (iv) wherein 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.

31. The immunoconjugate according to any one of claims 22 to 30, (i) wherein the immunoconjugate comprises an amino acid sequence according to SEQ ID NO: 5, an amino acid sequence according to SEQ ID NO: 22, and an amino acid sequence according to SEQ ID NO: 23; (ii) wherein the immunoconjugate comprises an amino acid sequence according to SEQ ID NO: 5, an amino acid sequence according to SEQ ID NO: 22, and an amino acid sequence according to SEQ ID NO: 24; (iii) wherein the immunoconjugate comprises an amino acid sequence according to SEQ ID NO: 5, an amino acid sequence according to SEQ ID NO: 22 and an amino acid sequence according to SEQ ID NO: 25; or (iv) wherein the immunoconjugate comprises an amino acid sequence according to SEQ ID NO:5, an amino acid sequence according to SEQ ID NO:22 and an amino acid sequence according to SEQ ID NO:

26.

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

33. An expression vector comprising the polynucleotide according to claim 32.

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

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

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

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

38. A protease-activatable IL-2 polypeptide or immunoconjugate according to any one of claims 1 to 31 or 36 for use in treating a disease in a subject in need thereof.

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

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

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

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

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

44. The invention as hereinbefore described.

Images