Immunoconjugates

Abstract

The present invention generally relates to immunoconjugates, particularly immunoconjugates comprising a mutant interleukin-2 polypeptide and an antibody that binds to PD-1. In addition, the present invention relates to polynucleotide molecules encoding the immunoconjugates, as well as vectors and host cells comprising such polynucleotide molecules. The present invention further relates to methods for producing the mutant immunoconjugates, pharmaceutical compositions comprising them, and uses thereof.

Description

[Technical Field]

[0001] The present invention generally relates to immunoconjugates, particularly immunoconjugates comprising a mutant interleukin-2 polypeptide and an antibody that binds to PD-1. In addition, the present invention relates to polynucleotide molecules encoding the immunoconjugates, as well as vectors and host cells comprising such polynucleotide molecules. The present invention further relates to methods for producing the mutant immunoconjugates, pharmaceutical compositions comprising them, and uses thereof. [Background technology]

[0002] Interleukin-2 (IL-2), also known as T-cell growth factor (TCGF), is a 15.5 kDa globular glycoprotein that plays a central role in lymphocyte development, survival, and homeostasis. IL-2 is 133 amino acids long and consists of four nonparallel amphipathic α-helices that form a quaternary structure essential for its function (Smith, Science 240, 1169-76 (1988); Bazan, Science 257, 410-413 (1992)). IL-2 sequences from different species are found in NCBI RefSeq numbers NP000577 (human), NP032392 (mouse), NP446288 (rat), or NP517425 (chimpanzee).

[0003] IL-2 mediates its effects by binding to the IL-2 receptor (IL-2R), which consists of three individual subunits, different associations of which can generate receptor forms with different affinities for IL-2: α (CD25), β (CD122), and γ (γ cThe association of the CD25 and CD132 subunits results in a trimeric high-affinity receptor for IL-2. The dimeric IL-2 receptor, consisting of the β and γ subunits, is called the intermediate-affinity IL-2R. The α subunit forms a monomeric low-affinity IL-2 receptor. The dimeric intermediate-affinity IL-2 receptor binds IL-2 with approximately 100-fold lower affinity than the trimeric high-affinity receptor, but both the dimeric and trimeric IL-2 receptor variants can transmit signals upon binding to IL-2 (Minami et al., Annu Rev Immunol 11, 245-268 (1993)). Therefore, the α-subunit CD25 is not essential for IL-2 signaling. The α-subunit confers high affinity binding to the receptor, while the β-subunit CD122 and γ-subunit are important for signal transduction (Krieg et al., Proc Natl Acad Sci 107, 11906-11 (2010)). The trimeric IL-2 receptor, which contains CD25, binds to (resting) CD4 + Forkhead Box P3 (FoxP3) + Controllability T(T reg ) cells. They are also transiently induced in conventionally activated T cells, whereas in the resting state, these cells express only the dimeric IL-2 receptor. reg The cells consistently express the highest levels of CD25 in vivo (Fontenot et al., Nature Immunol 6, 1142-51 (2005)).

[0004] IL-2 stimulates activated T cells, especially CD4 +It is mainly synthesized by helper T cells. IL-2 stimulates the proliferation and differentiation of T cells, induces the production of cytotoxic T lymphocytes (CTLs) and the differentiation of peripheral blood lymphocytes into cytotoxic cells and lymphokine-activated killer (LAK) cells, promotes the expression of cytokines and cytolytic molecules by T cells, facilitates the proliferation and differentiation of B cells and the synthesis of immunoglobulins by B cells, and stimulates the generation, proliferation, and activation of natural killer (NK) cells (e.g., Waldmann, Nat Rev Immunol 6, 595-601 (2009); Olejniczak and Kasprzak, Med Sci Monit 14, RA179-89 (2008); Malek, Annu Rev Immunol 26, 453-79 (2008)).

[0005] The ability of IL-2 to expand lymphocyte populations in vivo and increase the effector functions of these cells confers antitumor effects to IL-2 and makes IL-2 immunotherapy an attractive treatment option for certain metastatic cancers. Consequently, high-dose IL-2 treatment has been approved for use in patients with metastatic renal carcinoma and malignant melanoma.

[0006] However, IL-2 has a dual function in the immune response in that it not only mediates the expansion and activity of effector cells but is also critically involved in the maintenance of peripheral immune tolerance.

[0007] The primary mechanism underlying peripheral self-tolerance is activation-induced cell death (AICD) in T cells induced by IL-2. AICD is a process in which fully activated T cells undergo programmed cell death through the engagement of cell surface death receptors, such as CD95 (also known as Fas) or TNF receptors. When antigen-activated T cells (after previous exposure to IL-2), which express high-affinity IL-2 receptors during proliferation, are restimulated with antigen via the T cell receptor (TCR) / CD3 complex, expression of Fas ligand (FasL) and / or tumor necrosis factor (TNF) is induced, rendering the cells susceptible to Fas-mediated apoptosis. This process is IL-2-dependent (Lenardo, Nature 353, 858-61 (1991)) and mediated by STAT5. The process of AICD in T lymphocytes allows tolerance to be established not only to self-antigens but also to persistent antigens that are not clearly part of the host genome, such as tumor antigens.

[0008] Furthermore, IL-2 stimulates peripheral CD4 T cells, also known as suppressor T cells. + CD25 + Controllability T(T reg ) cells (Fontenot et al., Nature Immunol 6, 1142-51 (2005); D'Cruz and Klein, Nature Immunol 6, 1152-59 (2005); Maloy and Powrie, Nature Immunol 6, 1171-72 (2005)). These cells suppress effector T cells from destroying their (self) targets through cell-cell contact by inhibiting T cell help and activation, or through the release of immunosuppressive cytokines such as IL-10 or TGF-β. reg Cell depletion has been shown to enhance IL-2-induced anti-tumor immunity (Imai et al., Cancer Sci 98, 416-23 (2007)).

[0009] Thus, in the presence of IL-2, the generated CTLs either recognize the tumor as self and undergo AICD, or the immune response evolves into an IL-2-dependent T cell line. reg IL-2 is not optimal for inhibiting tumor growth because it is inhibited by the cells.

[0010] An additional problem associated with IL-2 immunotherapy is the side effects caused by recombinant human IL-2 treatment. Patients receiving high-dose IL-2 treatment frequently experience serious cardiovascular, pulmonary, renal, hepatic, gastrointestinal, neurological, cutaneous, hematologic, and systemic adverse events, requiring intensive monitoring and inpatient care. Most of these side effects can be explained by the progression of so-called vascular (or capillary) leak syndrome (VLS), a pathological increase in vascular permeability that causes fluid leakage in multiple organs (e.g., causing pulmonary and cutaneous edema and hepatocyte damage) and intravascular fluid depletion (causing a drop in blood pressure and a compensatory increase in heart rate). There is no treatment for VLS other than IL-2 withdrawal. Low-dose IL-2 regimens have been tested in patients to avoid VLS, but with suboptimal therapeutic outcomes. VLS was thought to be caused by the release of proinflammatory cytokines, such as tumor necrosis factor (TNF)-α, from IL-2-activated NK cells. However, it has recently been shown that IL-2-induced pulmonary edema results from direct binding of IL-2 to pulmonary endothelial cells that express low to moderate levels of functional αβγ IL-2 receptors (Krieg et al., Proc Nat Acad Sci USA 107, 11906-11 (2010)).

[0011] Several approaches have been taken to overcome these problems associated with IL-2 immunotherapy. For example, the combination of IL-2 with a specific anti-IL-2 monoclonal antibody enhances the therapeutic effect of IL-2 in vivo (Kamimura et al., J Immunol 177, 306-14 (2006); Boyman et al., Science 311, 1924-27 (2006)). In an alternative approach, IL-2 has been mutated in various ways to reduce its toxicity and / or improve its efficacy. Hu et al. (Blood 101, 4853-4861 (2003); U.S. Patent Application Publication No. 2003 / 0124678) replaced the arginine residue at position 38 of IL-2 with tryptophan to eliminate IL-2's vascular permeability activity. Shanafelt et al. (Nature Biotechnol 18, 1197-1202 (2000)) mutated asparagine 88 to arginine to enhance selectivity for T cells over NK cells. Heaton et al. (Cancer Res 53, 2597-602 (1993); U.S. Patent No. 5,229,109) introduced two mutations, Arg38Ala and Phe42Lys, to reduce secretion of inflammatory cytokines from NK cells. Gillies et al. (U.S. Patent Application Publication No. 2007 / 0036752) substituted three residues in IL-2 (Asp20Thr, Asn88Arg, and Gln126Asp) that contribute to affinity for the intermediate-affinity IL-2 receptor to reduce VLS. Gillies et al. (WO 2008 / 0034473) also mutated the interface between IL-2 and CD25 with the amino acid substitutions Arg38Trp and Phe42Lys to enhance efficacy, and demonstrated that the interaction with CD25 and T reg (WO 2009 / 061853) created IL-2 mutants that increased affinity for CD25 but did not activate the receptor, acting as antagonists. The mutations introduced were intended to disrupt the interaction with the β- and / or γ-subunits of the receptor.

[0012] The aforementioned problems associated with IL-2 immunotherapy (toxicity caused by the induction of VLS, tumor resistance caused by the induction of AICD, and T reg Certain mutant IL-2 polypeptides designed to overcome immune suppression caused by cell activation are described in WO 2012 / 107417. Substitution of the phenylalanine residue at position 42 with alanine, the tyrosine residue at position 45 with alanine, and the leucine residue at position 72 of IL-2 with glycine essentially abolishes binding of the mutant IL-2 polypeptide to the α-subunit of the IL-2 receptor (CD25). Liu et al. describe engineered interleukin antagonists (Liu et al., J Immunother. 2009;32(9):887-894).

[0013] In addition to the above-mentioned approaches, IL-2 immunotherapy can be improved by selectively targeting IL-2 to tumors, for example, in the form of immunoconjugates containing antibodies that bind to antigens expressed on tumor cells. 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).

[0014] However, tumors can escape such targeting by reducing, mutating, or downregulating the antibody's target antigen. Furthermore, tumor-targeted IL-2 may not have optimal contact with effector cells, such as cytotoxic T lymphocytes (CTLs), in a tumor microenvironment that actively excludes lymphocytes.

[0015] Therefore, there remains a need for further improvements in IL-2 immunotherapy. An approach that may circumvent the problem of tumor targeting is to target IL-2 directly to effector cells, particularly CTLs.

[0016] Ghasemi et al. describe a fusion protein of IL-2 with an NKG2D-binding protein to target IL-2 to NKG2D-bearing cells, such as natural killer (NK) cells (Ghashemi et al., Nat Comm (2016) 7, 12878).

[0017] Programmed cell death protein 1 (PD-1 or CD279) is an inhibitory member of the CD28 family of receptors, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is a cell surface receptor expressed on activated B cells, T cells, and myeloid cells (Okazaki et al. (2002) Curr. Opin. Immunol. 14:391779-82; Bennett et al. (2003) J Immunol 170:711-8). PD-1 is a monomeric type 1 transmembrane protein consisting of a single immunoglobulin variable-like extracellular domain and a cytoplasmic domain containing immunoreceptor inhibitory motifs (ITIMs) and immunoreceptor tyrosine-based switch motifs (ITSMs). Two PD-1 ligands, PD-L1 and PD-L2, have been identified that have been shown to downregulate T cell activation upon binding to PD-1 (Freeman et al (2000) J Exp Med 192:1027-34; Latchman et al (2001) Nat Immunol 2:261-8; Carter et al (2002) Eur J Immunol 32:634-43). Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1 but do not bind to other CD28 family members. One PD-1 ligand, PD-L1, is abundant in a variety of human cancers (Dong et al (2002) Nat. Med 8:787-9). The interaction between PD-1 and PD-L1 reduces tumor-infiltrating lymphocytes, reduces T cell receptor-mediated proliferation, and leads to immune evasion by cancerous cells (Dong et al. (2003) J. MoI. Med. 81:281-7; Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314; Konishi et al. (2004) Clin. Cancer Res. 10:5094-100).Immune suppression can be reversed by inhibiting the local interaction between PD-1 and PD-L1, and the effect is additive when the interaction between PD-1 and PD-L2 is also blocked (Iwai et al. (2002) Proc. Nat 7. Acad. ScL USA 99:12293-7; Brown et al. (2003) J. Immunol. 170:1257-66).

[0018] Antibodies that bind to PD-1 are described, for example, in WO 2017 / 055443. Immunoconjugates that bind to PD-1 are described, for example, in WO 2018 / 184964. Summary of the Invention

[0019] The present invention provides a novel approach to targeting mutant forms of IL-2 with advantageous properties for immunotherapy directed at immune effector cells, e.g., cytotoxic T lymphocytes, rather than tumor cells. Targeting to immune effector cells is achieved by conjugation of the mutant IL-2 molecule to an antibody that binds to PD-1.

[0020] The IL-2 mutants used in the present invention address the problems associated with IL-2 immunotherapy, in particular the toxicity caused by the induction of VLS, tumor resistance caused by the induction of AICD, and T reg In addition to avoiding tumor escape from tumor targeting as described above, targeting IL-2 mutants to immune effector cells has been designed to overcome the immune suppression caused by activation of immunosuppressive T cells. reg The use of antibodies that bind to PD-1 can further reverse the suppression of T cell activity induced by the interaction of PD-1 with its ligand PD-L1, thereby further enhancing the immune response.

[0021] An IL-2 fusion protein containing the anti-PD-L1 antibody atezolizumab has been described by Chen et al. (Chen et al., Biochem Biophys Res Comm (2016) 480, 160-165).

[0022] Notably, immunoconjugates of the invention comprising antibodies that bind to PD-1 exhibit significantly superior anti-tumor efficacy in vivo compared to similar immunoconjugates that target PD-L1 (see Example 4 herein below).

[0023] In a first aspect, the present invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to PD-1, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising the amino acid substitutions F42A, Y45A, L72G, and Q126T (numbering relative to SEQ ID NO: 90 of the human IL-2 sequence).

[0024] In a further aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to PD-1, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising amino acid substitutions F42A, Y45A, L72G, and Q126T (numbering relative to SEQ ID NO: 90 of the human IL-2 sequence); and the antibody comprises (a) a heavy chain variable region (VH) comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 74, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 75, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 76, and (b) a light chain variable region (VL) comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 77, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 78, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 79.

[0025] In another aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to PD-1, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising amino acid substitutions F42A, Y45A, L72G, and Q126T (numbering relative to SEQ ID NO:90 of the human IL-2 sequence); and the antibody comprises (a) a heavy chain variable region (VH) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:80, and (b) a light chain variable region (VL) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:81.

[0026] In some embodiments of the immunoconjugates described herein, the mutant IL-2 polypeptide further comprises the amino acid substitution T3A and / or the amino acid substitution C125A. In some embodiments, the mutant IL-2 polypeptide comprises the sequence of SEQ ID NO: 92. In some embodiments, the immunoconjugate comprises no more than one mutant IL-2 polypeptide. In some embodiments, the antibody comprises an Fc domain composed of a first subunit and a second subunit. In some such embodiments, the Fc domain is an Fc domain of the IgG class, particularly the IgG1 subclass, and / or the Fc domain is a human Fc domain. In some embodiments, the antibody is an immunoglobulin of the IgG class, particularly the IgG1 subclass.

[0027] In some embodiments, the immunoconjugate comprises an Fc domain, wherein the Fc domain comprises a modification that promotes association of a first subunit with a second subunit of the Fc domain. In some embodiments, in the CH3 domain of a first subunit of the Fc domain, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit that can reposition within a cavity in the CH3 domain of the second subunit, and in the CH3 domain of a second subunit of the Fc domain, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit into which the protrusion in the CH3 domain of the first subunit can reposition. In some embodiments, the first subunit of the Fc domain has a threonine residue at position 366 replaced with a tryptophan residue (T366W), and the second subunit of the Fc domain has a tyrosine residue at position 407 replaced with a valine residue (Y407V), optionally with a serine residue at position 366 (T366S), and a leucine residue at position 368 replaced with an alanine residue (L368A) (Kabat EU index numbering). In some such embodiments, the first subunit of the Fc domain further has a serine residue at position 354 replaced with a cysteine ​​residue (S354C) or a glutamic acid residue at position 356 replaced with a cysteine ​​residue (E356C), and the second subunit of the Fc domain further has a tyrosine residue at position 349 replaced with a cysteine ​​residue (Y349C) (Kabat EU index numbering). In some embodiments, the mutant IL-2 polypeptide is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the subunits of the Fc domain, particularly the first subunit of the Fc domain, optionally via a linker peptide. In some such embodiments, the linker peptide has the amino acid sequence of SEQ ID NO:93.

[0028] In some embodiments, the immunoconjugate comprises an Fc domain, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor, particularly an Fcγ receptor, and / or effector function, particularly antibody-dependent cell-mediated cytotoxicity (ADCC). In some such embodiments, the one or more amino acid substitutions are at one or more positions selected from the group of L234, L235, and P329 (Kabat EU index numbering). In some embodiments, each subunit of the Fc domain comprises amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbering).

[0029] In some embodiments, an immunoconjugate according to the invention comprises a polypeptide comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 21, a polypeptide comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 23 or SEQ ID NO: 22, and a polypeptide comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 35. In some embodiments, the immunoconjugate consists essentially of a mutant IL-2 polypeptide and an IgG1 immunoglobulin molecule linked by a linker sequence.

[0030] The present invention further provides one or more isolated polynucleotides encoding the immunoconjugates of the invention, one or more vectors (particularly expression vectors) comprising said polynucleotides, and host cells comprising said polynucleotides or said vectors.

[0031] Also provided is a method for producing an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds PD-1, the method comprising (a) culturing a host cell of the invention under conditions suitable for expression of the immunoconjugate, and optionally (b) recovering the immunoconjugate. Also provided by the invention is an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds PD-1 produced by the method.

[0032] The present invention further provides pharmaceutical compositions comprising the immunoconjugates of the invention and a pharmaceutically acceptable carrier, as well as methods of using the immunoconjugates of the invention.

[0033] In particular, the present invention encompasses immunoconjugates according to the present invention for use as pharmaceuticals and for use in the treatment of disease. In particular embodiments, the disease is cancer.

[0034] The present invention also encompasses the use of an immunoconjugate according to the present invention in the manufacture of a medicament for the treatment of a disease. In certain embodiments, the disease is cancer.

[0035] Further provided is a method of treating a disease in an individual, comprising administering to said individual a therapeutically effective amount of a composition comprising an immunoconjugate described in the present invention in a pharmaceutically acceptable form. In certain embodiments, the disease is cancer.

[0036] Also provided is a method of stimulating the immune system of an individual, comprising administering to said individual an effective amount of a composition comprising an immunoconjugate according to the invention in a pharmaceutically acceptable form. DETAILED DESCRIPTION OF THE INVENTION

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

[0038] The term "interleukin-2" or "IL-2," as used herein, unless otherwise indicated, refers to any native IL-2 from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses unprocessed IL-2 and any form of IL-2 resulting from processing of cells. The term also encompasses naturally occurring variants of IL-2, such as splice variants or allelic variants. The amino acid sequence of an exemplary human IL-2 is set forth in SEQ ID NO:90. Unprocessed human IL-2 further includes an N-terminal 20-amino acid signal peptide having the sequence of SEQ ID NO:94, which is not present in the mature IL-2 molecule.

[0039] The term "IL-2 mutant" or "mutant IL-2 polypeptide," as used herein, is intended to encompass all variant forms of the various forms of the IL-2 molecule, including full-length IL-2, truncated forms of IL-2, and forms in which IL-2 is linked to another molecule by fusion, chemical conjugation, or the like. "Full-length," when used in reference to IL-2, is intended to mean the mature, native-length IL-2 molecule. For example, full-length human IL-2 refers to a molecule containing 133 amino acids (e.g., SEQ ID NO: 90). 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. This mutation may include substitution, deletion, truncation, or modification of the 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 mutant IL-2 peptide sequences, mutant IL-2 polypeptides, mutant IL-2 proteins, or mutant IL-2 analogs.

[0040] The nomenclature of various forms of IL-2 is given herein with reference to the sequence shown in SEQ ID NO: 90. Various names may be used herein to denote the same mutation. For example, a mutation from phenylalanine to alanine at position 42 is designated 42A, A42, A 42 , F42A or Phe42Ala.

[0041] "Human IL-2 molecule," as used herein, means an IL-2 molecule comprising an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, or at least about 96% identical to the human IL-2 sequence of SEQ ID NO: 90. In particular, the sequence identity is at least about 95%, more particularly at least about 96%. In certain embodiments, the human IL-2 molecule is a full-length IL-2 molecule.

[0042] The term "amino acid mutation" as used herein is intended to encompass amino acid substitution, deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be performed to arrive at a final construct, as long as the final construct possesses the desired characteristics, e.g., reduced binding to CD25. Deletions and insertions in amino acid sequences include deletions and insertions of amino and / or carboxy terminal amino acids. An example of a terminal deletion is the deletion of the alanine residue at position 1 of full-length human IL-2. Preferred amino acid mutations are amino acid substitutions. For example, for the purpose of altering the binding characteristics of an IL-2 polypeptide, non-conservative amino acid substitutions, i.e., replacing one amino acid with another amino acid with different structural and / or chemical properties, are particularly preferred. Preferred amino acid substitutions include replacing a hydrophobic amino acid with a hydrophilic amino acid. Amino acid substitutions include substitutions with non-naturally occurring amino acids or naturally occurring amino acid derivatives of the 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. It is contemplated that methods of modifying the side chain groups of amino acids by methods other than genetic engineering, such as chemical modification, may also be useful.

[0043] As used herein, a "wild-type" form of IL-2 is a form of IL-2 that is the same as a mutant IL-2 polypeptide, except that the wild-type form has the wild-type amino acid at each amino acid position of the mutant IL-2 polypeptide. For example, if an IL-2 mutant is 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 full-length native IL-2. If an IL-2 mutant is a fusion of IL-2 with another polypeptide (e.g., an antibody chain) encoded downstream of IL-2, the wild-type form of the IL-2 mutant is IL-2 with the wild-type amino acid sequence fused to the same downstream polypeptide. Furthermore, if an IL-2 mutant is a truncated form of IL-2 (a mutated or modified sequence within the uncleaved portion of IL-2), the wild-type form of the IL-2 mutant is similarly truncated IL-2 with the wild-type sequence. For purposes 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, relative to naturally occurring native IL-2, one or more amino acid mutations that do not affect IL-2 receptor binding, such as a substitution of alanine for cysteine ​​at the position corresponding to residue 125 in human IL-2. In some embodiments, wild-type IL-2 for purposes of the present invention contains the amino acid substitution C125A. In certain embodiments described herein, the wild-type IL-2 polypeptide that is compared to a mutant IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO:90. In other embodiments, the wild-type IL-2 polypeptide that is compared to a mutant IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO:95.

[0044] The term "CD25" or "α-subunit of the IL-2 receptor," as used herein, unless otherwise indicated, refers to any native CD25 from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed CD25 and any form of CD25 resulting from intracellular processing. The term also encompasses naturally occurring variants of CD25, such as splice variants or allelic variants. In certain embodiments, the CD25 is human CD25. The amino acid sequence of human CD25 can be found, for example, in UniProt entry number P01589 (version 185).

[0045] The term "high affinity IL-2 receptor" as used herein refers to the receptor γ-subunit (common cytokine receptor γ-subunit, γ c (also known as CD132, see UniProt entry number P14784 (version 192)), receptor β-subunit (also known as CD122 or p70, see UniProt entry number P31785 (version 197)), and receptor α-subunit (also known as CD25 or p55, see UniProt entry number P01589 (version 185)). In contrast, the term "intermediate affinity IL-2 receptor" refers to an IL-2 receptor that contains only the γ- and β-subunits, but not the α-subunit (for a review, see, e.g., Olejniczak and Kasprzak, Med Sci Monit 14, RA179-189 (2008)).

[0046] "Affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antigen-binding moiety and an antigen, or a receptor and its ligand). The affinity of molecule X for binding partner Y is typically measured by the dissociation constant (K D ), and the dissociation rate constant and association rate constant (k off and k on ) is the ratio of the rate constants. Thus, equivalent affinities can involve 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).

[0047] The affinity of mutant or wild-type IL-2 polypeptides for various forms of the IL-2 receptor can be determined by surface plasmon resonance (SPR) using standard equipment such as a BIAcore instrument (Cytiva) and receptor subunits such as those obtainable by recombinant expression, according to the methods described in WO 2012 / 107417 (see, e.g., Shanafelt et al., Nature Biotechnol 18, 1197-1202 (2000)). Alternatively, the binding affinity of IL-2 mutants for different forms of the IL-2 receptor can be assessed using cell lines known to express one or other such forms of the receptor. Specific illustrative and exemplary embodiments for measuring binding affinity are described herein below.

[0048] "Regulatory T cells" or "T reg "T cells" are a special type of CD4 T cell that can suppress the responses of other T cells. + It stands for T cells. regT cells are characterized by expression of the α-subunit of the IL-2 receptor (CD25) and the transcription factor forkhead box P3 (FOXP3) (Sakaguchi, Annu Rev Immunol 22, 531-62 (2004)), which play an important role in the induction and maintenance of peripheral self-tolerance to antigens, including those expressed by tumors. reg Cells require IL-2 for their function and growth, as well as for the induction of their suppressive properties.

[0049] As used herein, the term "effector cells" refers to a population of lymphocytes that mediate the cytotoxic effects of IL-2. Effector cells include effector T cells, e.g., CD8 + These include cytotoxic T cells, NK cells, lymphokine-activated killer (LAK) cells and macrophages / monocytes.

[0050] As used herein, "PD1," "human PD1," "PD-1," or "human PD-1" (also known as programmed cell death protein 1, or Programmed Death 1) refers to the human protein PD1 (SEQ ID NO: 96, protein without signal sequence) / (SEQ ID NO: 97, protein with signal sequence). See also UniProt entry number Q15116 (version 156). As used herein, "binding to PD-1," "specifically binding to PD-1," "antibody that binds to PD-1," or "anti-PD-1 antibody" refers to an antibody capable of binding to PD-1, particularly cell surface-expressed PD-1 polypeptides, with sufficient affinity so that the antibody is useful as a diagnostic and / or therapeutic agent in targeting PD-1. In one embodiment, the binding of an anti-PD-1 antibody to an unrelated, non-PD-1 protein is less than about 10% of the binding of the antibody to PD-1, as measured, for example, by radioimmunoassay (RIA) or flow cytometry (FACS), or by surface plasmon resonance assays using a biosensor system such as a Biacore® system. In certain embodiments, an antibody that binds to PD-1 has a binding affinity KD value for binding to human PD-1 of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., ≦10 -8 M or less, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 In one embodiment, the KD value of the binding affinity is determined in a surface plasmon resonance assay using the extracellular domain (ECD) of human PD-1 (PD-1-ECD, see SEQ ID NO: 43) as the antigen.

[0051] "Specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or nonspecific interactions. The ability of an antibody to bind to a specific antigen (e.g., PD-1) 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 (e.g., 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 degree of binding of the antibody to an unrelated protein is less than about 10% of the binding of the antibody to the antigen, as measured, for example, by SPR. The antibody contained in the immunoconjugate described herein specifically binds to PD-1.

[0052] As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The term "polypeptide" refers to a chain of two or more amino acids, not a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other term used to refer to a chain of two or more amino acids are included within the definition of "polypeptide," and the term "polypeptide" may be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modifications with non-naturally occurring amino acids. Polypeptides may be derived from natural biological sources or produced by recombinant technology, but are not necessarily translated from a specified nucleic acid sequence. Polypeptides may be generated by any method, including chemical synthesis. A polypeptide may, but does not necessarily, have a defined three-dimensional structure. Polypeptides that have a defined three-dimensional structure are said to be folded, and polypeptides that do not have a defined three-dimensional structure but rather can adopt a number of different conformations are said to be unfolded.

[0053] By "isolated" polypeptide or a variant or derivative thereof is intended a polypeptide that is not in its natural environment. A particular level of purification is not required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purposes of the present invention, as are native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0054] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA program package. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the ggsearch program in the FASTA package version 36.3.8c, or subsequently using the BLOSUM50 comparison matrix. The FASTA program package was written by W.R. Pearson and D.J. Lipman (1988), "Improved Tools for Biological Sequence Analysis," PNAS 85:2444-2448; W.R. Pearson (1996) "Effective protein sequence comparison," Meth. Enzymol. 266:227-258; and Pearson et al. (1997) Genomics 46:24-36, and is publicly available at http: / / fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml.Alternatively, sequences can be compared using the public server accessible at http: / / fasta.bioch.virginia.edu / fasta_www2 / index.cgi using the ggsearch(globalprotein:protein) program and default options (BLOSUM50; open: -10; ext: -2; Ktup=2), ensuring a global rather than local alignment. The percent amino acid identity is shown in the output alignment header.

[0055] The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), viral RNA, or plasmid DNA (pDNA). A polynucleotide may contain conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNAs)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.

[0056] By "isolated" nucleic acid molecule or polynucleotide is intended a nucleic acid molecule, DNA, or RNA, removed from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for purposes of the present invention. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) in solution. Isolated polynucleotides include polynucleotide molecules originally contained in cells that contain the polynucleotide molecule, but where the polynucleotide molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present invention, positive- and negative-stranded forms, and double-stranded forms. Furthermore, isolated polynucleotides or nucleic acids of the present invention include such molecules produced synthetically. In addition, polynucleotides or nucleic acids may be or include regulatory elements, such as promoters, ribosome binding sites, or transcription terminators.

[0057] An "isolated polynucleotide (or nucleic acid) encoding [e.g., an immunoconjugate of the invention]" refers to one or more polynucleotide molecules encoding an antibody heavy and light chain and / or an IL-2 polypeptide (or fragment thereof), and includes such polynucleotide molecules in a single vector or separate vectors, and such nucleic acid molecules present in one or more locations in a host cell.

[0058] The term "expression cassette" refers to a recombinantly or synthetically produced polynucleotide that contains a specific set of nucleic acid elements capable of transcription of a specific nucleic acid in a target cell. Recombinant expression cassettes can be incorporated into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, the recombinant expression cassette portion of an expression vector contains, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassette contains a polynucleotide sequence encoding an immunoconjugate of the invention, or a fragment thereof.

[0059] The term "vector" or "expression vector" refers to a DNA molecule used to introduce and direct the expression of a particular gene with which it is operably associated into a cell. This term includes vectors as self-replicating nucleic acid structures as well as vectors that integrate into the genome of the host cell into which they are introduced. The expression vector of the present invention comprises an expression cassette. The expression vector allows for the transcription of large amounts of stable mRNA. Once the expression vector is inside the cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the intracellular transcription and / or translation machinery. In one embodiment, the expression vector of the present invention comprises an expression cassette comprising a polynucleotide sequence encoding an immunoconjugate of the present invention, or a fragment thereof.

[0060] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny, regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. Host cells are any type of cell line that can be used to produce the immunoconjugates of the invention. Host cells include cultured cells, e.g., mammalian cultured cells, such as HEK cells, CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells, to name just a few, yeast cells, insect cells, and plant cells, but also cells contained in transgenic animals, transgenic plants, or cultured plant or animal tissue.

[0061] The term "antibody" exhibits antigen-binding activity and encompasses a variety of antibody structures that exhibit antigen-binding activity, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments.

[0062] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., each individual antibody comprising the population is identical and / or binds to the same epitope (with the exception of possible variant antibodies that contain, for example, naturally occurring mutations or that arise during production of the monoclonal antibody preparation; such variants are generally present in minor amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies in accordance with the invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods, as well as other exemplary methods for producing monoclonal antibodies, are described herein.

[0063] An "isolated" antibody is one that has been separated from a component of its natural environment (i.e., is not in its native environment). No particular level of purification is required. For example, an isolated antibody can be removed from its native or natural environment. Recombinantly produced antibodies expressed in host cells are considered isolated for purposes of the present invention, as are native or recombinant antibodies that have been separated, fractionated, or partially or substantially purified by any suitable technique. Thus, the immunoconjugates of the present invention are isolated. In some embodiments, antibodies are purified to greater than 95% or greater than 99% purity as determined by electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse-phase HPLC) methods. For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0064] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to the structure of a native antibody.

[0065] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAbs), and multispecific antibodies formed from antibody fragments.

[0066] The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, IgG class immunoglobulins are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two disulfide-bonded light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy chain domain or heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3), also called a heavy chain constant region. Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called a variable light chain domain or light chain variable region, followed by a constant light chain (CL) domain, also called a light chain constant region. Immunoglobulin heavy chains can be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further divided into subtypes, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Immunoglobulin light chains may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. Immunoglobulins essentially consist of two Fab molecules and an Fc domain connected via an immunoglobulin hinge region.

[0067] The term "antigen-binding domain" refers to a portion of an antibody that specifically binds to and is complementary to part or all of an antigen. An antigen-binding domain may be provided, for example, by one or more antibody variable domains (also called antibody variable regions). In particular, an antigen-binding domain comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0068] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, and each domain contains four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6 th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. As used herein with reference to variable region sequences, "Kabat numbering" refers to the numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0069] As used herein, the amino acid positions of all constant regions and domains of the heavy and light chains are numbered according to the Kabat numbering system as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), and is referred to herein as "Kabat numbering" or "Kabat numbering." Specifically, the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991) pp. 647-660) is used for the light chain constant domains CL of the kappa and lambda isotypes, and the Kabat EU index numbering system (see pp. 661-723) is used for the heavy chain constant domains (CH1, hinge, CH2 and CH3), which is further clarified herein by referring to "numbering according to the Kabat EU index" in this case.

[0070] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence and determines antigen-binding specificity, e.g., the "complementarity-determining region" (CDR).

[0071] Generally, antibodies contain six CDRs, three in the VH (CDR-H1, CDR-H2, CDR-H3) and three in the VL (CDR-L1, CDR-L2, CDR-L3). CDRs are defined by various methods / systems by those skilled in the art. These systems and / or definitions have been developed and refined over the years and include Kabat, Chothia, IMGT, AbM, and Contact. The Kabat definition is based on sequence variability and is generally the most commonly used. The Chothia definition is based on the location of structural loop regions. The IMGT system is based on sequence variability and location within the structure of the variable domain. The AbM definition is a compromise between Kabat and Chothia. The Contact definition is based on analysis of available antibody crystal structures. Software programs (eg, abYsis: http: / / www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi) are available and known to those of skill in the art for analyzing antibody sequences and determining CDRs.

[0072] Exemplary CDRs herein include (amino acid residue numbering according to the cited reference, i.e., Chothia numbering for the Chothia and Contact definitions, Kabat numbering for the Kabat definitions, and IMGT numbering for the IMGT definitions), (a) Hypervariable loops present at amino acid residues 26–32 (L1), 50–52 (L2), 91–96 (L3), 26–32 (H1), 53–55 (H2), and 96–101 (H3) (according to Chothia and Lesk, J. Mol. Biol. 196:901–917 (1987) (“Chotia definition”)); (b) CDRs located at amino acid residues 24–34 (L1), 50–56 (L2), 89–97 (L3), 31–35b (H1), 50–65 (H2), and 95–102 (H3) (according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991) (“Kabat definition”)); (c) antigenic contacts (according to MacCallum et al. J. Mol. Biol. 262:732-745 (1996) ("Contact definition") present at amino acid residues 30-36 (L1), 46-55 (L2), 89-96 (L3), 30-35 (H1), 47-58 (H2), and 93-101 (H3), and (d) CDRs located at amino acid residues 27-38 (L1), 56-65 (L2), 105-117 (L3), 27-38 (H1), 56-65 (H2), and 105-117 (H3) (according to Lefranc et al. Dev. Comp. Immunol. 27:55-77 (2003) ("IMGT definition")).

[0073] Unless otherwise indicated, CDRs are determined according to Kabat et al. (supra). Those skilled in the art will understand that CDR designations can also be determined according to Chothia, supra, MacCallum, supra, Lefranc, supra, or any other scientifically accepted definition / system.

[0074] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences are usually presented in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0075] A "humanized" antibody refers to an antibody that contains amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody contains substantially all of at least one, and typically two, variable domains, with all or substantially all of the CDRs corresponding to those of a non-human antibody and all or substantially all of the FRs corresponding to those of a human antibody. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0076] A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or an antibody derived from the human antibody repertoire or other non-human source that utilizes human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0077] The "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by the antibody or immunoglobulin heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0078] The term "Fc domain" or "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226 or from Pro230 to the carboxy terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, upon expression of a particular nucleic acid molecule encoding a full-length heavy chain, antibodies produced by host cells may contain a full-length heavy chain or a truncated variant of the full-length heavy chain (also referred to herein as a "truncated variant heavy chain"). This is the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, according to the Kabat EU index). Thus, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and lysine (K447) of the Fc region may or may not be present. The amino acid sequence of a heavy chain comprising an Fc domain (or a subunit of an Fc domain as defined herein) is shown herein without the C-terminal glycine-lysine dipeptide, unless otherwise indicated. In one embodiment of the invention, a heavy chain comprising a subunit of an Fc domain as specified herein comprised in an immunoconjugate described in the invention comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the EU index of Kabat). In one embodiment of the invention, a heavy chain comprising a subunit of an Fc domain as specified herein comprised in an immunoconjugate described in the invention comprises an additional C-terminal glycine residue (G446, numbered according to the EU index of Kabat). Compositions of the invention, e.g., pharmaceutical compositions described herein, comprise a population of immunoconjugates of the invention. The population of immunoconjugates may include molecules containing full-length heavy chains and molecules containing truncated variant heavy chains.The population of immunoconjugates may consist of a mixture of molecules having full-length heavy chains and molecules having cleaved variant heavy chains, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the immunoconjugates have cleaved variant heavy chains. In one embodiment of the invention, a composition comprising a population of immunoconjugates of the invention comprises immunoconjugates comprising heavy chains comprising subunits of an Fc domain as specified herein, which comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, as numbered by the EU index of Kabat). In one embodiment of the invention, a composition comprising a population of immunoconjugates of the invention comprises immunoconjugates comprising heavy chains comprising subunits of an Fc domain as specified herein, which comprises an additional C-terminal glycine residue (G446, as numbered by the EU index of Kabat). In one embodiment of the present invention, such compositions comprise a population of immunoconjugates comprised of molecules comprising a heavy chain comprising a subunit of an Fc domain as specified herein, molecules comprising a heavy chain comprising a subunit of an Fc domain as specified herein with an additional C-terminal glycine residue (G446, numbering according to the EU index of Kabat), and molecules comprising a heavy chain comprising a subunit of an Fc domain as specified herein with an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to the EU index of Kabat). Unless otherwise specified herein, the numbering of amino acid residues within the Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also supra). As used herein, a "subunit" of an Fc domain refers to one of the two polypeptides that form a dimeric Fc domain, i.e., the polypeptide comprising the C-terminal constant region of an immunoglobulin heavy chain capable of stable self-association.For example, the subunits of the IgG Fc domain include the IgG CH2 and IgG CH3 constant domains.

[0079] A "modification that promotes association of a first subunit and a second subunit of an Fc domain" refers to a manipulation of the peptide backbone or a post-translational modification of an Fc domain subunit that reduces or prevents the association of a polypeptide containing the Fc domain subunit with an identical polypeptide to form a homodimer. As used herein, a modification that promotes association specifically includes separate modifications made to each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) that are desired to associate, which are complementary to each other to promote the association of the two Fc domain subunits. For example, a modification that promotes association can alter the structure or charge of one or both of the Fc domain subunits to sterically or electrostatically favor their association, respectively. Thus, (hetero)dimerization occurs between a polypeptide containing a first Fc domain subunit and a polypeptide containing a second Fc domain subunit, which may not be identical in the sense that the additional components (e.g., antigen-binding moieties) fused to each of the subunits are not the same. In some embodiments, a modification that promotes association includes an amino acid mutation, specifically an amino acid substitution, within the Fc domain. In a specific embodiment, the association-promoting modifications comprise distinct amino acid mutations, particularly amino acid substitutions, in each of the two subunits of the Fc domain.

[0080] The term "effector function," when used in reference to an antibody, refers to a biological activity attributable to the Fc region of an antibody and varies 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, immunoconjugate-mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0081] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism by which immune effector cells lyse antibody-coated target cells. Target cells are cells to which an antibody or its derivative containing an Fc region specifically binds, typically via a protein portion at the N-terminus of the Fc region. As used herein, the term "reduced ADCC" is defined as either a decrease in the number of target cells lysed in a given time period at a given concentration of antibody in the medium surrounding the target cells by the ADCC mechanism defined above, and / or an increase in the concentration of antibody in the medium surrounding the target cells required to achieve lysis of a given number of target cells in a given time period by the ADCC mechanism. The reduced ADCC is compared to the ADCC mediated by the same antibody produced by the same type of host cell and using the same standard production, purification, formulation, and storage methods (known to those skilled in the art), but without genetic engineering. For example, an amino acid substitution that reduces ADCC mediated by an antibody containing its Fc domain is relative to the ADCC mediated by the same antibody without this amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, for example, PCT Publication No. WO 2006 / 082515 or PCT Publication No. WO 2012 / 130831).

[0082] An "activating Fc receptor" is an Fc receptor that, following binding of the Fc domain of an antibody, triggers signaling events that stimulate the receptor-bearing cell to carry out an effector function. Human activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0083] As used herein, the terms "engineer, engineered, manipulating" are intended to include any manipulation or post-translational modification of the peptide backbone of a naturally occurring or recombinant polypeptide or fragment thereof. Manipulation includes modifying the amino acid sequence, modifying the glycosylation pattern, or modifying the side groups of individual amino acids, and combinations of these techniques.

[0084] "Decreased binding," for example, decreased binding to an Fc receptor or CD25, refers to a decrease in affinity for the respective interaction, as measured, for example, by SPR. For clarity, this term also includes reducing affinity to zero (or below the detection limit of the analytical method), i.e., complete loss of the interaction. Conversely, "increased binding" refers to an increase in binding affinity for the respective interaction.

[0085] As used herein, the term "immunoconjugate" refers to a polypeptide molecule comprising at least one IL-2 molecule and at least one antibody. The IL-2 molecule can be linked to the antibody by a variety of interactions and in a variety of configurations, as described herein. In certain embodiments, the IL-2 molecule is fused to the antibody via a peptide linker. Certain immunoconjugates described herein consist essentially of one IL-2 molecule and an antibody linked by one or more linker sequences.

[0086] By "fused" is meant that the components (eg, an antibody and an IL-2 molecule) are linked by a peptide bond either directly or via one or more peptide linkers.

[0087] As used herein, the terms "first" and "second," with respect to Fc domain subunits and the like, are used for convenience in distinguishing when more than one of each type of moiety is present. The use of these terms is not intended to confer a particular order or orientation of the immunoconjugate unless explicitly indicated as such.

[0088] An "effective amount" of a drug refers to the amount needed to produce a physiological change in the cells or tissue to which it is administered.

[0089] A "therapeutically effective amount" of a drug, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of a drug, for example, eliminates, reduces, delays, minimizes, or prevents the side effects of a disease.

[0090] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.

[0091] The term "pharmaceutical composition" refers to a preparation in a form that is suitable for the biological activity of the active ingredient contained therein, and that does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered.

[0092] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0093] As used herein, "treatment" (and grammatical variations thereof, e.g., "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of disease in the individual being treated, and can be carried out prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing the direct or indirect pathological consequences of disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating disease symptoms, and achieving remission or improved prognosis. In some embodiments, the immunoconjugates of the invention are used to delay the onset of disease or slow the progression of disease.

[0094] Mutant IL-2 polypeptides The immunoconjugates described herein comprise mutant IL-2 polypeptides with advantageous properties for immunotherapy. In particular, pharmacological properties of IL-2 that contribute to toxicity but are not essential for IL-2 efficacy are eliminated in the mutant IL-2 polypeptides. Such mutant IL-2 polypeptides are described in detail in WO 2012 / 107417, the entire contents of which are incorporated herein by reference. As noted above, different forms of the IL-2 receptor are composed of different subunits and exhibit different affinities for IL-2. The intermediate-affinity IL-2 receptor is composed of β and γ receptor subunits, is expressed on resting effector cells, and is sufficient for IL-2 signaling. The high-affinity IL-2 receptor further comprises the α-subunit of the receptor and regulates the regulatory T (T reg ) cells and activated effector cells, which, upon engagement with IL-2, activate T regThese interactions can promote cell-mediated immune suppression or activation-induced cell death (AICD). Therefore, without wishing to be bound by theory, reduced or absent affinity of IL-2 for the α-subunit of the IL-2 receptor should reduce IL-2-induced downregulation of effector cell function by regulatory T cells and the progression of tumor resistance through the AICD process. On the other hand, maintaining affinity for the intermediate-affinity IL-2 receptor should preserve IL-2 induction of proliferation and activation of effector cells, such as NK cells and T cells.

[0095] The mutant interleukin-2 (IL-2) polypeptides included in the immunoconjugates described herein contain at least one amino acid mutation that eliminates or reduces the affinity of the mutant IL-2 polypeptide for the α-subunit of the IL-2 receptor, compared to the respective wild-type IL-2 polypeptide, and preserves the affinity of the mutant IL-2 polypeptide for the intermediate affinity IL-2 receptor.

[0096] Mutants of human IL-2 (hIL-2) with reduced affinity for CD25 may be made, for example, by amino acid substitutions at amino acid positions 35, 38, 42, 43, 45, or 72, or combinations thereof (numbering relative to the human IL-2 sequence of SEQ ID NO: 90). Exemplary amino acid substitutions include K35E, K35A, R38A, R38E, R38N, R38F, R38S, R38L, R38G, R38Y, R38W, F42L, F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, K43E, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R and L72K. Particular IL-2 mutants useful in the immunoconjugates of the invention comprise an amino acid mutation at an amino acid position corresponding to residue 42, 45, or 72 of human IL-2, or a combination thereof. In one embodiment, the amino acid mutation is an amino acid substitution selected from the group of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K, and more particularly, an amino acid substitution selected from the group of F42A, Y45A, and L72G. These mutants exhibit substantially similar binding affinities to the intermediate-affinity IL-2 receptor and have significantly reduced affinity for the α-subunit of the IL-2 receptor and the high-affinity IL-2 receptor compared to the wild-type forms of the IL-2 mutants.

[0097] Other characteristics of useful mutants may include the ability to induce proliferation of IL-2 receptor-containing T cells and / or NK cells, the ability to induce IL-2 signaling in IL-2 receptor-containing T cells and / or NK cells, the ability to produce interferon (IFN)-γ as a secondary cytokine by NK cells, a reduced ability to induce the production of secondary cytokines (particularly IL-10 and TNF-α) by peripheral blood mononuclear cells (PBMCs), a reduced ability to activate regulatory T cells, a reduced ability to induce apoptosis in T cells, and a reduced toxicity profile in vivo.

[0098] A particular mutant IL-2 polypeptide useful in the present invention contains three amino acid mutations that confer no or low affinity to the α-subunit of the IL-2 receptor, but preserve the affinity of the mutant IL-2 polypeptide for the moderate-affinity IL-2 receptor. In one embodiment, the three amino acid mutations are at positions corresponding to residues 42, 45, and 72 of human IL-2. In one embodiment, the three amino acid mutations are amino acid substitutions. In one embodiment, the three amino acid mutations are amino acid substitutions selected from the group of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R and L72K. In a specific embodiment, the three amino acid mutations are amino acid substitutions F42A, Y45A and L72G (numbering relative to the human IL-2 sequence of SEQ ID NO: 90).

[0099] A particular mutant IL-2 polypeptide useful in the present invention contains four amino acid mutations that confer no or low affinity to the α-subunit of the IL-2 receptor, but preserve the affinity of the mutant IL-2 polypeptide for the moderate-affinity IL-2 receptor. In one embodiment, the three amino acid mutations are at positions corresponding to residues 42, 45, 72, and 126 of human IL-2. In one embodiment, the three amino acid mutations are amino acid substitutions. In one embodiment, the three amino acid mutations are amino acid substitutions selected from the group consisting of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, L72K and Q126T. In a specific embodiment, the three amino acid mutations are amino acid substitutions F42A, Y45A, L72G and Q126T (numbering relative to the human IL-2 sequence of SEQ ID NO: 90). In a specific embodiment, the three amino acid mutations are amino acid substitutions F42A, Y45A, L72G, and N88D (numbering relative to the human IL-2 sequence of SEQ ID NO: 90). In a specific embodiment, the three amino acid mutations are amino acid substitutions F42A, Y45A, L72G, and N88Q (numbering relative to the human IL-2 sequence of SEQ ID NO: 90).

[0100] In certain embodiments, the amino acid mutations reduce the affinity of the mutant IL-2 polypeptide for the α-subunit of the IL-2 receptor by at least 5-fold, particularly at least 10-fold, and more particularly at least 25-fold. In embodiments in which more than one amino acid mutation that reduces the affinity of the mutant IL-2 polypeptide for the α-subunit of the IL-2 receptor is present, the combination of these amino acid mutations may reduce the affinity of the mutant IL-2 polypeptide for the α-subunit of the IL-2 receptor by at least 30-fold, at least 50-fold, or even at least 100-fold. In one embodiment, the amino acid mutation or combination of amino acid mutations abolishes the affinity of the mutant IL-2 polypeptide for the α-subunit of the IL-2 receptor such that binding is not detectable by surface plasmon resonance.

[0101] Substantially similar binding to the intermediate affinity receptor (i.e., preservation of the affinity of the mutant IL-2 polypeptide for said receptor) is achieved when the IL-2 mutant exhibits an affinity for the intermediate affinity IL-2 receptor that is greater than about 70% of the wild-type affinity of the IL-2 mutant. The IL-2 mutants of the invention may exhibit an affinity that is greater than about 80%, or even greater than about 90%, of such affinity.

[0102] Reducing the affinity of IL-2 for the α-subunit of the IL-2 receptor, combined with eliminating O-glycosylation of IL-2, results in an IL-2 protein with improved properties. For example, the absence of O-glycosylation sites results in a more homogeneous product when the mutant IL-2 polypeptide is expressed in mammalian cells, such as CHO or HEK cells.

[0103] Thus, in certain embodiments, the mutant IL-2 polypeptide comprises an additional amino acid mutation that eliminates the IL-2 O-glycosylation site at a position corresponding to residue 3 of human IL-2. In one embodiment, the additional amino acid mutation that eliminates the IL-2 O-glycosylation site at a position corresponding to residue 3 of human IL-2 is an amino acid substitution. Exemplary amino acid substitutions include T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P. In a specific embodiment, the additional amino acid mutation is the amino acid substitution T3A.

[0104] In certain embodiments, the mutant IL-2 polypeptide is essentially a full-length IL-2 molecule. In certain embodiments, the mutant IL-2 polypeptide is a human IL-2 molecule. In one embodiment, the mutant IL-2 polypeptide comprises the sequence of SEQ ID NO: 90 with at least one amino acid mutation that eliminates or reduces the affinity of the mutant IL-2 polypeptide for the α-subunit of the IL-2 receptor, compared to an IL-2 polypeptide comprising SEQ ID NO: 90 without the mutation, but preserves the affinity of the mutant polypeptide for the intermediate-affinity IL-2 receptor. In another embodiment, the mutant IL-2 polypeptide comprises the sequence of SEQ ID NO: 95 with at least one amino acid mutation that eliminates or reduces the affinity of the mutant IL-2 polypeptide for the α-subunit of the IL-2 receptor, compared to an IL-2 polypeptide comprising SEQ ID NO: 95 without the mutation, but preserves the affinity of the mutant polypeptide for the intermediate-affinity IL-2 receptor.

[0105] In specific embodiments, the mutant IL-2 polypeptide is capable of eliciting one or more cellular responses selected from the group consisting of activated T lymphocyte cell proliferation, activated T lymphocyte cell differentiation, cytotoxic T cell (CTL) activity, activated B cell expansion, activated B cell differentiation, natural killer (NK) cell proliferation, NK cell differentiation, cytokine secretion by activated T cells or NK cells, and NK / lymphocyte-activated killer (LAK) anti-tumor cytotoxicity.

[0106] In one embodiment, the mutant IL-2 polypeptide has a reduced ability to induce IL-2 signaling in regulatory T cells compared to the wild-type IL-2 polypeptide. In one embodiment, the mutant IL-2 polypeptide induces less activation-induced cell death (AICD) in T cells compared to the wild-type IL-2 polypeptide. In one embodiment, the mutant IL-2 polypeptide has a reduced in vivo toxicity profile compared to the wild-type IL-2 polypeptide. In one embodiment, the mutant IL-2 polypeptide has an increased serum half-life compared to the wild-type IL-2 polypeptide.

[0107] A particular mutant IL-2 polypeptide useful in the present invention contains five amino acid substitutions at positions corresponding to residues 3, 42, 45, 72, and 126 of human IL-2. Specific amino acid substitutions are T3A, F42A, Y45A, L72G, and Q126T. Another particular mutant IL-2 polypeptide useful in the present invention contains five amino acid substitutions at positions corresponding to residues 3, 42, 45, 72, and 88 of human IL-2. Specific amino acid substitutions are T3A, F42A, Y45A, L72G, and N88D. Another particular mutant IL-2 polypeptide useful in the present invention contains five amino acid substitutions at positions corresponding to residues 3, 42, 45, 72, and 88 of human IL-2. Specific amino acid substitutions are T3A, F42A, Y45A, L72G, and N88Q.

[0108] In addition to having mutations in the regions of IL-2 that form the IL-2 interface with CD25 or glycosylation sites, useful IL-2 mutants of the present invention may also have one or more mutations in the amino acid sequence outside of these regions. Such additional mutations in human IL-2 may confer additional benefits, such as increased expression or stability. For example, the cysteine ​​at position 125 may be replaced with a neutral amino acid such as serine, alanine, threonine, or valine, resulting in C125S IL-2, C125A IL-2, C125T IL-2, or C125V IL-2, respectively, as described in U.S. Patent No. 4,518,584. Deletion of the N-terminal alanine residue of IL-2, as described herein, can result in mutants such as des-A1 C125S or des-A1 C125A. Alternatively, or in combination, the IL-2 mutant may contain a mutation whereby the methionine normally present at position 104 in wild-type human IL-2 is replaced with a neutral amino acid such as alanine (see U.S. Pat. No. 5,206,344). The resulting mutants, e.g., des-A1 M104A IL-2, des-A1 M104A C125S IL-2, M104A IL-2, M104A C125A IL-2, des-A1 M104A C125A IL-2, or M104A C125S IL-2 (these and other mutants can be found in U.S. Pat. No. 5,116,943 and Weiger et al., Eur J Biochem 180, 295-300 (1989)), may be used in combination with the particular IL-2 mutants of the present invention.

[0109] Thus, in certain embodiments, the mutant IL-2 polypeptide comprises an additional amino acid mutation at a position corresponding to human IL-2 residue 125. In one embodiment, the additional amino acid mutation is the amino acid substitution C125A.

[0110] Those skilled in the art will be able to determine which additional mutations may confer additional advantages for purposes of the present invention. For example, it will be understood that amino acid mutations in the IL-2 sequence that reduce or eliminate the affinity of IL-2 for the intermediate affinity IL-2 receptor, such as D20T, N88R, or Q126D (see, e.g., US 2007 / 0036752), may not be suitable for inclusion in mutant IL-2 polypeptides according to the present invention.

[0111] In one embodiment, the mutant IL-2 polypeptide contains no more than 12, no more than 11, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, or no more than 5 amino acid mutations compared to the corresponding wild-type IL-2 sequence, e.g., the human IL-2 sequence of SEQ ID NO: 90. In a specific embodiment, the mutant IL-2 polypeptide contains no more than 5 amino acid mutations compared to the corresponding wild-type IL-2 sequence, e.g., the human IL-2 sequence of SEQ ID NO: 90.

[0112] In one embodiment, the mutant IL-2 polypeptide comprises the sequence of SEQ ID NO: 92. In one embodiment, the mutant IL-2 polypeptide consists of the sequence of SEQ ID NO:92.

[0113] In one embodiment, the mutant IL-2 polypeptide comprises the sequence of SEQ ID NO: 98. In one embodiment, the mutant IL-2 polypeptide consists of the sequence of SEQ ID NO:98.

[0114] In one embodiment, the mutant IL-2 polypeptide comprises the sequence of SEQ ID NO: 99. In one embodiment, the mutant IL-2 polypeptide consists of the sequence of SEQ ID NO:99.

[0115] Immunoconjugates The immunoconjugates described herein comprise an IL-molecule and an antibody. Such immunoconjugates significantly enhance the efficacy of IL-2 therapy by directly targeting IL-2, for example, to the tumor microenvironment. According to the present invention, the antibody contained in the immunoconjugate may be a whole antibody or immunoglobulin, or a portion or variant thereof that has biological function, such as antigen-specific binding affinity.

[0116] The general benefits of immunoconjugate therapy are readily apparent. For example, antibodies contained in the immunoconjugate recognize tumor-specific epitopes, targeting the immunoconjugate molecule to the tumor site. Thus, high concentrations of IL-2 can be delivered to the tumor microenvironment, thereby triggering the activation and proliferation of various immune effector cells described herein using immunoconjugates at doses significantly lower than those required with unconjugated IL-2. Furthermore, administering IL-2 in the form of an immunoconjugate allows for lower doses of the cytokine itself, limiting the potential for undesirable side effects of IL-2. Targeting IL-2 to specific body sites by immunoconjugates also reduces systemic exposure, thereby resulting in fewer side effects than those achieved with unconjugated IL-2. Additionally, the increased serum half-life of immunoconjugates compared to unconjugated IL-2 contributes to the efficacy of immunoconjugates. However, this feature of IL-2 immunoconjugates may again exacerbate potential side effects of the IL-2 molecule: the blood half-life of IL-2 immunoconjugates in the bloodstream is significantly longer than that of unconjugated IL-2, increasing the likelihood that IL-2 or other moieties of the fusion protein moiety will activate components commonly present in the vasculature. The same considerations apply to other fusion proteins containing IL-2 fused to another moiety, such as Fc or albumin, which increases the half-life of IL-2 in the blood. Therefore, immunoconjugates containing the mutant IL-2 polypeptides described herein and in WO 2012 / 107417, which have reduced toxicity compared to wild-type forms of IL-2, are particularly advantageous.

[0117] As described hereinabove, targeting IL-2 directly to immune effector cells rather than tumor cells may be advantageous for IL-2 immunotherapy.

[0118] Thus, the present invention provides mutant IL-2 polypeptides and antibodies that bind to PD-1, as described hereinabove. In one embodiment, the mutant IL-2 polypeptide and antibody form a fusion protein, i.e., the mutant IL-2 polypeptide shares a peptide bond with the antibody. In some embodiments, the antibody comprises an Fc domain composed of a first subunit and a second subunit. In a specific embodiment, the mutant IL-2 polypeptide is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the subunits of the Fc domain, optionally via a linker peptide. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an immunoglobulin molecule, particularly an immunoglobulin molecule of the IgG class, more particularly an immunoglobulin molecule of the IgG1 subclass. In one such embodiment, the mutant IL-2 polypeptide shares an amino-terminal peptide bond with one of the immunoglobulin heavy chains. In certain embodiments, the antibody is an antibody fragment. In some embodiments, the antibody comprises a Fab molecule or an scFv molecule. In one embodiment, the antibody is a Fab molecule. In another embodiment, the antibody is an scFv molecule. An immunoconjugate may also contain two or more antibodies. When two or more antibodies are contained in an immunoconjugate, for example, a first antibody and a second antibody, each antibody may be independently selected from various forms of antibodies and antibody fragments. For example, the first antibody may be a Fab molecule, and the second antibody may be an scFv molecule. In specific embodiments, the first antibody and the second antibody are scFv molecules, or each of the first antibody and the second antibody is a Fab molecule. In certain embodiments, each of the first antibody and the second antibody is a Fab molecule. In one embodiment, each of the first antibody and the second antibody binds to PD-1.

[0119] Immunoconjugate Format Exemplary immunoconjugate formats are described in PCT Publication No. WO 2011 / 020783, which is incorporated herein by reference in its entirety. These immunoconjugates comprise at least two antibodies. Thus, in one embodiment, an immunoconjugate according to the present invention comprises a mutant IL-2 polypeptide described herein and at least a first antibody and a second antibody. In certain embodiments, the first antibody and the second antibody are independently selected from the group consisting of Fv molecules (particularly, scFv molecules) and Fab molecules. In specific embodiments, the mutant IL-2 polypeptide shares an amino- or carboxy-terminal peptide bond with the first antibody, and the second antibody shares an amino- or carboxy-terminal peptide bond with either i) the mutant IL-2 polypeptide or ii) the first antibody. In certain embodiments, the immunoconjugate consists essentially of a mutant IL-2 polypeptide and the first and second antibodies, particularly Fab molecules, linked by one or more linker sequences. Such a format has the advantage of binding with high affinity to the target antigen (PD-1) but providing only monomeric binding to the IL-2 receptor, thereby avoiding targeting of the immunoconjugate to immune cells that bear IL-2 receptors at locations other than the target site. In certain embodiments, the mutant IL-2 polypeptide shares a carboxy-terminal peptide bond with a first antibody, particularly a first Fab molecule, and also shares an amino-terminal peptide bond with a second antibody, particularly a second Fab molecule. In another embodiment, the first antibody, particularly a first Fab molecule, shares a carboxy-terminal peptide bond with the mutant IL-2 polypeptide and also shares an amino-terminal peptide bond with a second antibody, particularly a second Fab molecule. In another embodiment, the first antibody, particularly a first Fab molecule, shares an amino-terminal peptide bond with the first mutant IL-2 polypeptide and also shares a carboxy-terminal peptide with a second antibody, particularly a second Fab molecule.In certain embodiments, the mutant IL-2 polypeptide shares a carboxy-terminal peptide bond with a first heavy chain variable region and an amino-terminal peptide bond with a second heavy chain variable region. In another embodiment, the mutant IL-2 polypeptide shares a carboxy-terminal peptide bond with a first light chain variable region and an amino-terminal peptide bond with a second light chain variable region. In another embodiment, the first heavy or light chain variable region is linked to the mutant IL-2 polypeptide by a carboxy-terminal peptide bond and to a second heavy or light chain variable region by an amino-terminal peptide bond. In another embodiment, the first heavy or light chain variable region is linked to the mutant IL-2 polypeptide by an amino-terminal peptide bond and to a second heavy or light chain variable region by a carboxy-terminal peptide bond. In one embodiment, the mutant IL-2 polypeptide shares a carboxy-terminal peptide bond with a first Fab heavy or light chain and an amino-terminal peptide bond with a second Fab heavy or light chain. In another embodiment, a first Fab heavy or light chain shares a carboxy-terminal peptide bond with a mutant IL-2 polypeptide and also shares an amino-terminal peptide bond with a second Fab heavy or light chain. In other embodiments, a first Fab heavy or light chain shares an amino-terminal peptide bond with a mutant IL-2 polypeptide and also shares a carboxy-terminal peptide bond with a second Fab heavy or light chain. In one embodiment, an immunoconjugate comprises a mutant IL-2 polypeptide that shares an amino-terminal peptide bond with one or more scFV molecules and also shares a carboxy-terminal peptide bond with one or more scFV molecules.

[0120] However, a particularly suitable format for the immunoconjugates according to the present invention comprises immunoglobulin molecules as antibodies. Exemplary immunoconjugate formats are described in PCT Application Publication No. WO2012 / 146628, which is incorporated herein by reference in its entirety.

[0121] Thus, in certain embodiments, an immunoconjugate comprises a mutant IL-2 polypeptide described herein and an immunoglobulin molecule, particularly an IgG molecule, more particularly an IgG1 molecule, that binds to PD-1. In one embodiment, the immunoconjugate comprises no more than one mutant IL-2 polypeptide. In one embodiment, the immunoglobulin molecule is human. In one embodiment, the immunoglobulin molecule comprises a human constant region, e.g., a human CH1, CH2, CH3, and / or CL domain. In one embodiment, the immunoglobulin comprises a human Fc domain, particularly a human IgG1 Fc domain. In one embodiment, the mutant IL-2 polypeptide shares an amino-terminal peptide bond or a carboxy-terminal peptide bond with the immunoglobulin molecule. In one embodiment, the immunoconjugate consists essentially of a mutant IL-2 polypeptide and an immunoglobulin molecule, particularly an IgG molecule, more particularly an IgG1 molecule, linked by one or more linker peptides. In a specific embodiment, the mutant IL-2 polypeptide is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the immunoglobulin heavy chains, optionally via a linker peptide.

[0122] The mutant IL-2 polypeptide may be fused to the antibody directly or via a linker peptide comprising one or more amino acids, typically about 2-20 amino acids. Linker peptides are known in the art and are described herein. Suitable non-immunogenic linker peptides include, for example, (G4S) n , (SG4) n , (G4S) n or G4 (SG4) n Linker peptides are included. "n" is generally an integer between 1 and 10, typically between 2 and 4. In one embodiment, the linker peptide has a length of at least 5 amino acids, in one embodiment between 5 and 100 amino acids, and in a further embodiment between 10 and 50 amino acids. In a particular embodiment, the linker peptide has a length of 15 amino acids. In one embodiment, the linker peptide has a length of (GxS) n or (GxS)n G m where G=glycine, S=serine, (x=3, n=3, 4, 5 or 6, m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5, m=0, 1, 2 or 3), in some embodiments x=4, n=2 or 3, and in further embodiments x=4, n=3. In a particular embodiment, the linker peptide is (GS) (SEQ ID NO: 93). In one embodiment, the linker peptide comprises the amino acid sequence of SEQ ID NO: 93 (or consists of the amino acid sequence of SEQ ID NO: 24).

[0123] In a specific embodiment, the immunoconjugate comprises a mutant IL-2 molecule and an immunoglobulin molecule, particularly an IgG1 subclass immunoglobulin molecule, that binds to PD-1, wherein the mutant IL-2 molecule is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the immunoglobulin heavy chains via a linker peptide of SEQ ID NO:93.

[0124] In a specific embodiment, the immunoconjugate comprises a mutant IL-2 molecule and an antibody that binds to PD-1, wherein the antibody comprises an Fc domain composed of a first subunit and a second subunit, particularly a human IgG1 Fc domain, and the mutant IL-2 molecule is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the subunits of the Fc domain via a linker peptide of SEQ ID NO:93.

[0125] PD-1 antibody The antibodies included in the immunoconjugates of the invention bind to PD-1, particularly human PD-1, and can direct mutant IL-2 polypeptides to PD-1-expressing target sites, e.g., associated with tumors, particularly to PD-1-expressing T cells.

[0126] Suitable PD-1 antibodies that may be used in the immunoconjugates of the invention are described in PCT Patent Application No. PCT / EP2016 / 073248, which is incorporated herein by reference in its entirety.

[0127] The immunoconjugates of the present invention may comprise two or more antibodies that can bind to the same or different antigens. However, in certain embodiments, each of these antibodies binds to PD-1. In one embodiment, the antibodies comprised in the immunoconjugates of the present invention are monospecific. In certain embodiments, the immunoconjugates comprise a single monospecific antibody, particularly a monospecific immunoglobulin molecule.

[0128] The antibody can be any type of antibody or fragment thereof that retains specific binding to PD-1, particularly human PD-1. Antibody fragments include, but are not limited to, Fv molecules, scFv molecules, Fab molecules, and F(ab')2 molecules. However, in certain embodiments, the antibody is a full-length antibody. In some embodiments, the antibody comprises an Fc domain composed of a first subunit and a second subunit. In some embodiments, the antibody is an immunoglobulin, particularly an IgG class, more particularly an IgG1 subclass immunoglobulin.

[0129] In some embodiments, the antibody is a monoclonal antibody.

[0130] In some embodiments, the antibody comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 74, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 75, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 76, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 77, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 78, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 79.

[0131] In some embodiments, the antibody comprises (a) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 74, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 75, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 76, and (b) a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 77, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 78, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 79. In some embodiments, the heavy and / or light chain variable regions are humanized variable regions. In some embodiments, the heavy and / or light chain variable regions comprise human framework regions (FR).

[0132] In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the antibody comprises a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 81. In certain embodiments, the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 80 and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 81.

[0133] In some embodiments, the antibody is a humanized antibody. In one embodiment, the antibody is an immunoglobulin molecule comprising a human constant region, particularly an immunoglobulin molecule of the IgG class comprising human CH1, CH2, CH3 and / or CL domains.

[0134] Fc domain In certain embodiments, the antibody contained in the immunoconjugate described herein comprises an Fc domain composed of a first subunit and a second subunit. The Fc domain of an antibody consists of a pair of polypeptide chains comprising the heavy chain domain of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which contains the IgG heavy chain constant domains CH2 and CH3. The two subunits of the Fc domain are capable of stable association with each other. In one embodiment, the immunoconjugate of the present invention comprises no more than one Fc molecule.

[0135] In one embodiment, the Fc domain of the antibody comprised in the immunoconjugate is an IgG Fc domain. In a particular embodiment, the Fc domain is an IgG1 Fc domain. In another embodiment, the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228, in particular the amino acid substitution S228P (Kabat EU index numbering). This amino acid substitution reduces in vivo Fab arm exchange of IgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In a further particular embodiment, the Fc domain is a human Fc domain. In an even more specific embodiment, the Fc domain is a human IgG1 Fc domain.

[0136] Fc domain modifications that promote heterodimerization The immunoconjugates described herein comprise a mutant IL-2 polypeptide, particularly a single (not more than one) mutant IL-2 polypeptide, fused to one or the other of the two subunits of the Fc domain; thus, the two subunits of the Fc domain are typically contained in two non-identical polypeptide chains. Recombinant coexpression of these polypeptides followed by dimerization allows for several possible combinations of the two polypeptides. To increase the yield and purity of immunoconjugates during recombinant production, it is advantageous to introduce modifications into the Fc domain of the antibody that promote the association of the desired polypeptides.

[0137] Thus, in a particular embodiment, the Fc domain of an antibody comprised in an immunoconjugate according to the invention comprises a modification that promotes the association of the first and second subunits of the Fc domain. The most extensive site of protein-protein interaction between the two subunits of a human IgG Fc domain is within the CH3 domain of the Fc domain. Thus, in one embodiment, the modification is in the CH3 domain of the Fc domain.

[0138] There are several approaches to modifications in the CH3 domain of an Fc domain to enhance heterodimerization, which are fully described in, for example, WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, in all such approaches, the CH3 domain of the first Fc domain subunit and the CH3 domain of the second Fc domain subunit are both engineered in a complementary manner such that each CH3 domain (or the heavy chain comprising it) is directed not to homodimerize with itself but to heterodimerize with another complementary engineered CH3 domain (such that the first CH3 domain and the second CH3 domain heterodimerize, and no homodimers are formed between the two first CH3 domains or the two second CH3 domains).

[0139] In a specific embodiment, the modification that promotes association of the first and second subunits of the Fc domain is a so-called "knob-in-hole" modification, which comprises a "knob" modification on one of the two subunits of the Fc domain and a "hole" modification on the other of the two subunits of the Fc domain.

[0140] Knob-into-hole technology has been described, for example, in U.S. Pat. No. 5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Generally, this method involves introducing a "protuberance" ("knob") into the interface of a first polypeptide and a corresponding "cavity" ("hole") into the interface of a second polypeptide, positioning the protuberance in the cavity to promote heterodimer formation and prevent homodimer formation. The protuberance is constructed by replacing small amino acid side chains at the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Complementary depressions of identical or similar size to the protrusions are created in the interface of a second polypeptide by replacing large amino acid side chains with smaller ones (eg, alanine or threonine).

[0141] Thus, in certain embodiments, in the CH3 domain of a first subunit of an Fc domain of an antibody comprised in an immunoconjugate, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit that can be repositioned within a cavity in the CH3 domain of a second subunit, and in the CH3 domain of a second subunit of an Fc domain, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit, within which the protrusion in the CH3 domain of the first subunit can be repositioned.

[0142] Preferably, said amino acid residues having larger side chain volumes are selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W).

[0143] Preferably, said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T) and valine (V).

[0144] The protrusions and depressions can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.

[0145] In a specific embodiment, in the CH3 domain of the first subunit of the Fc domain (the "knob" subunit), the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain (the "hole" subunit), the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, the second subunit of the Fc domain further replaces the threonine residue at position 366 with a serine residue (T366S) and the leucine residue at position 368 with an alanine residue (L368A) (numbering according to the Kabat EU index).

[0146] In yet a further embodiment, the first subunit of the Fc domain further comprises a replacement of the serine residue at position 354 with a cysteine ​​residue (S354C) or a replacement of the glutamic acid residue at position 356 with a cysteine ​​residue (E356C) (particularly, the replacement of the serine residue at position 354 with a cysteine ​​residue), and the second subunit of the Fc domain further comprises a replacement of the tyrosine residue at position 349 with a cysteine ​​residue (Y349C) (numbering according to the Kabat EU index). The introduction of these two cysteine ​​residues creates a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0147] In a specific embodiment, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A, and Y407V (numbering according to the Kabat EU index).

[0148] In some embodiments, the second subunit of the Fc domain further comprises the amino acid substitutions H435R and Y436F (numbering according to the Kabat EU index).

[0149] In certain embodiments, the mutant IL-2 polypeptide is fused (optionally via a linker peptide) to the first subunit of the Fc domain (containing the "knob" modification). Without wishing to be bound by theory, fusion of the mutant IL-2 polypeptide to the knob-containing subunit of the Fc domain (further) minimizes the generation of immunoconjugates comprising two mutant IL-2 polypeptides (steric clash of the two knob-containing polypeptides).

[0150] Other techniques for CH3 modifications that enhance heterodimerization are contemplated as alternatives in the present invention and are described, for example, in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO 2013 / 157954, WO 2013 / 096291.

[0151] In one embodiment, the heterodimerization approach described in EP 1870459 is used instead. This approach is based on the introduction of oppositely charged amino acids at specific amino acid positions in the CH3 / CH3 domain interface between the two subunits of the Fc domain. One preferred embodiment of the antibody comprised in the immunoconjugate of the invention has the amino acid mutations R409D, K370E in one of the two CH3 domains (of the Fc domain), and D399K, E357K in the other CH3 domain of the Fc domain (numbering according to the Kabat EU index).

[0152] In another embodiment, the antibody comprised in the immunoconjugate of the invention comprises the amino acid mutation T366W in the CH3 domain of the first subunit of the Fc domain, the amino acid mutations T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and further amino acid mutations R409D;K370E in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations D399K;E357K in the CH3 domain of the second subunit of the Fc domain (numbering according to Kabat EU index).

[0153] In another embodiment, the antibody comprised in the immunoconjugate of the invention comprises the amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, or said antibody comprises the amino acid mutations Y349C, T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations S354C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and further comprises the amino acid mutations R409D, K370E in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations D399K, E357K in the CH3 domain of the second subunit of the Fc domain (all numbered according to Kabat EU index).

[0154] In one embodiment, the heterodimerization approach described in WO 2013 / 157953 is used instead. In one embodiment, the heterodimerization approach described in WO 2012 / 058768 is used instead. In one embodiment, the first CH3 domain comprises the amino acid mutation T366K and the second CH3 domain comprises the amino acid mutation L351D (numbering according to the Kabat EU index). In a further embodiment, the first CH3 domain comprises the additional amino acid mutation L351K. In a further embodiment, the second CH3 domain further comprises an amino acid mutation selected from Y349E, Y349D and L368E (preferably L368E) (numbering according to the Kabat EU index).

[0155] In one embodiment, the heterodimerization approach described in WO 2012 / 058768 is used instead. In one embodiment, the heterodimerization approach described in WO 2012 / 058768 is used instead. In one embodiment, the first CH3 domain comprises the amino acid mutations L351Y, Y407A, and the second CH3 domain comprises the amino acid mutations T366A, K409F. In a further embodiment, the second CH3 domain comprises an additional amino acid mutation at positions T411, D399, S400, F405, N390, or K392, such as: a) T411N, T411R, T411Q, T411K, T411D, T411E, or T411W; b) D399R, D399W, D399Y or D399K, c) S400E, S400D, S400R or S400K, d) F405I, F405M, F405T, F405S, F405V or F405W, e) N390R, N390K or N390D, f) K392V, K392M, K392R, K392L, K392F or K392E (numbering according to the Kabat EU index). In a further embodiment, the first CH3 domain comprises the amino acid mutations L351Y, Y407A and the second CH3 domain comprises the amino acid mutations T366V, K409F. In a further embodiment, the first CH3 domain comprises the amino acid mutation Y407A and the second CH3 domain comprises the amino acid mutations T366A, K409F. In a further embodiment, the second CH3 domain further comprises the amino acid mutations K392E, T411E, D399R and S400R (numbering according to the Kabat EU index).

[0156] In one embodiment, the heterodimerization approach described in WO 2011 / 143545 is used instead, e.g., with an amino acid modification at a position selected from the group consisting of 368 and 409 (numbering according to the Kabat EU index).

[0157] In one embodiment, the heterodimerization approach described in WO 2011 / 090762, which also uses the knob-in-hole technique described above, is used instead. In one embodiment, the first CH3 domain contains the amino acid mutation T366W and the second CH3 domain contains the amino acid mutation Y407A. In one embodiment, the first CH3 domain contains the amino acid mutation T366Y and the second CH3 domain contains the amino acid mutation Y407T (numbering according to the Kabat EU index).

[0158] In one embodiment, the antibody or Fc domain thereof included in the immunoconjugate is of the IgG2 subclass, and the heterodimerization approach described in WO 2010 / 129304 is used instead.

[0159] In an alternative embodiment, the modification that promotes association of the first and second subunits of the Fc domain comprises a modification that mediates electrostatic steering effects, e.g., as described in WO 2009 / 089004. Generally, this method involves replacing one or more amino acid residues at the interface of the two Fc domain subunits with charged amino acid residues such that homodimer formation is electrostatically unfavorable, but heterodimerization is electrostatically favorable. In one such embodiment, the first CH3 domain comprises an amino acid substitution at K392 or N392 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), preferably K392D or N392D), and the second CH3 domain comprises an amino acid substitution at D399, E356, D356, or E357 with a positively charged amino acid (e.g., lysine (K) or arginine (R), preferably D399K, E356K, D356K, or E357K, more preferably D399K and E356K). In a further embodiment, the first CH3 domain further comprises an amino acid substitution at K409 or R409 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), preferably K409D or R409D). In a further embodiment, the first CH3 domain also or alternatively comprises an amino acid substitution at K439 and / or K370 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D)) (all numbered according to the Kabat EU index).

[0160] In yet further embodiments, the heterodimerization approach described in WO 2007 / 147901 is used instead. In one embodiment, the first CH3 domain contains the amino acid mutations K253E, D282K, and K322D, and the second CH3 domain contains the amino acid mutations D239K, E240K, and K292D (numbering according to the Kabat EU index).

[0161] In yet another embodiment, the heterodimerization approach described in WO 2007 / 110205 may be used instead.

[0162] In one embodiment, the first subunit of the Fc domain comprises the amino acid substitutions K392D and K409D, and the second subunit of the Fc domain comprises the amino acid substitutions D356K and D399K (numbering according to the Kabat EU index).

[0163] Fc domain modifications that reduce Fc receptor binding and / or effector function The Fc domain confers desirable pharmacokinetic properties to immunoconjugates, including a long serum half-life and favorable tissue-to-blood distribution ratio, which contribute to favorable accumulation in target tissues. However, it may also result in undesirable targeting of immunoconjugates to cells expressing Fc receptors rather than to preferred antigen-bearing cells. Furthermore, simultaneous activation of the Fc receptor signaling pathway leads to cytokine release, and the combination of IL-2 polypeptides and long-half-life immunoconjugates, when administered systemically, can result in excessive cytokine receptor activation and severe side effects. Consistent with this, conventional IgG-IL-2 immunoconjugates have been associated with infusion reactions (see, e.g., King et al., J Clin Oncol 22, 4463-4473 (2004)).

[0164] Thus, in certain embodiments, the Fc domain of an antibody comprised in an immunoconjugate according to the present invention exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to the Fc domain of a native IgG1. In one such embodiment, the Fc domain (or an antibody comprising said Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% of the binding affinity to Fc receptors compared to a native IgG1 Fc domain (or an antibody comprising a native IgG1 Fc domain), and / or exhibits less than 50%, preferably less than 20%, more preferably less than 10%, and most preferably less than 5% of the effector function compared to a native IgG1 Fc domain (or an antibody comprising a native IgG1 Fc domain). In one embodiment, the Fc domain (or an antibody comprising said Fc domain) does not substantially bind to an Fc receptor and / or does not induce effector function. In a specific embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group consisting of CDC, ADCC, ADCP, and cytokine secretion. In a particular embodiment, the effector function is ADCC. In one embodiment, the Fc domain exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to a native IgG1 Fc domain. Substantially similar binding to FcRn is achieved when the Fc domain (or an antibody comprising said Fc domain) exhibits greater than about 70%, particularly greater than about 80%, and more particularly greater than about 90% of the binding affinity to FcRn compared to a native IgG1 Fc domain (or an antibody comprising a native IgG1 Fc domain).

[0165] In certain embodiments, the Fc domain is engineered to reduce its binding affinity to an Fc receptor and / or its effector function compared to an unengineered Fc domain. In certain embodiments, the Fc domain of an antibody included in the immunoconjugate contains one or more amino acid mutations that reduce the binding affinity of the Fc domain to an Fc receptor and / or its effector function. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutations reduce the binding affinity of the Fc domain to an Fc receptor. In one embodiment, the amino acid mutations reduce the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments where there are more than one amino acid mutations that reduce the binding affinity of the Fc domain to an Fc receptor, the combination of these amino acid mutations can reduce the binding affinity of the Fc domain to an Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold. In one embodiment, an antibody comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, and more particularly less than 5% of the binding affinity of an Fc receptor compared to an antibody comprising a non-engineered Fc domain. In a specific embodiment, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fc receptor. In some embodiments, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more particularly human FcγRIIIa, FcγRI, or FcγRIIa, most particularly human FcγRIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments, binding affinity to complement components, particularly C1q, is also reduced. In one embodiment, binding affinity to neonatal Fc receptor (FcRn) is not reduced.Substantially similar binding to FcRn, i.e., preservation of the binding affinity of the Fc domain to the receptor, is achieved when the Fc domain (or an antibody comprising said Fc domain) exhibits a binding affinity to FcRn that is greater than about 70% of the binding affinity of an unengineered form of the Fc domain (or an antibody comprising said unengineered form of Fc). The Fc domain, or an antibody comprised in an immunoconjugate of the invention comprising said Fc domain, may exhibit greater than about 80%, or even greater than about 90%, of such affinity. In certain embodiments, the Fc domain of the antibody comprised in the immunoconjugate is engineered to have reduced effector function compared to the unengineered Fc domain. Reduced effector function can include, but is not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immunoconjugate-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling to induce apoptosis, reduced crosslinking of target-bound antibodies, reduced dendritic cell maturation, or reduced T cell priming. In one embodiment, the reduced effector function is one or more selected from the group consisting of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a particular embodiment, the reduced effector function is reduced ADCC. In one embodiment, the reduced ADCC is less than 20% of the ADCC induced by a non-engineered Fc domain (or an antibody comprising a non-engineered Fc domain).

[0166] In one embodiment, the amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and / or the effector function is an amino acid substitution. In one embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329 (numbering according to Kabat EU index). In a more specific embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235 and P329 (numbering according to Kabat EU index). In some embodiments, the Fc domain comprises amino acid substitutions L234A and L235A (numbering according to Kabat EU index). In one such embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In one embodiment, the Fc domain comprises an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (numbering according to Kabat EU index). In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and an additional amino acid substitution at a position selected from E233, L234, L235, N297, and P331 (numbering according to the Kabat EU index). In a more specific embodiment, the additional amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a particular embodiment, the Fc domain comprises amino acid substitutions at positions P329, L234, and L235 (numbering according to the Kabat EU index). In a more specific embodiment, the Fc domain comprises the amino acid mutations L234A, L235A, and P329G ("P329G LALA," "PGLALA," or "LALAPG").In particular, in certain embodiments, each subunit of the Fc domain contains the amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbering), i.e., in each of the first and second subunits of the Fc domain, the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A), and the proline residue at position 329 is replaced with a glycine residue (P329G) (Kabat EU index numbering). In one such embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. The "P329G LALA" combination of amino acid substitutions almost completely abolishes Fcγ receptor (as well as complement) binding of the human IgG1 Fc domain, as described in WO 2012 / 130831, which is incorporated herein by reference in its entirety. WO 2012 / 130831 also describes methods for preparing such mutant Fc domains and determining their properties, such as Fc receptor binding or effector function.

[0167] IgG4 antibodies exhibit reduced binding affinity to Fc receptors and reduced effector functions compared to IgG1 antibodies. Thus, in some embodiments, the Fc domain of an antibody comprised in an immunoconjugate of the present invention is an IgG4 Fc domain, particularly a human IgG4 Fc domain. In one embodiment, the IgG4 Fc domain comprises an amino acid substitution at position S228, specifically the amino acid substitution S228P (numbering according to the Kabat EU index). To further reduce binding affinity to Fc receptors and / or their effector functions, in one embodiment, the IgG4 Fc domain comprises an amino acid substitution at position L235, specifically the amino acid substitution L235E (numbering according to the Kabat EU index). In another embodiment, the IgG4 Fc domain comprises an amino acid substitution at position P329, specifically the amino acid substitution P329G (numbering according to the Kabat EU index). In a specific embodiment, the IgG4 Fc domain comprises amino acid substitutions at positions S228, L235 and P329, specifically the amino acid substitutions S228P, L235E and P329G (numbering according to the Kabat EU index). Such IgG4 Fc domain mutants and their Fcγ receptor binding properties are described in WO 2012 / 130831, which is incorporated herein by reference in its entirety.

[0168] In specific embodiments, the Fc domain that exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to a native IgG1 Fc domain is a human IgG1 Fc domain comprising the amino acid substitutions L234A, L235A and optionally P329G, or a human IgG4 Fc domain comprising the amino acid substitutions S228P, L235E and optionally P329G (numbering according to the Kabat EU index).

[0169] In certain embodiments, the N-glycosylation of the Fc domain is ablated. In one such embodiment, the Fc domain comprises an amino acid mutation at position N297, specifically an asparagine to alanine (N297A) or an aspartic acid to aspartic acid (N297D) (numbering according to the Kabat EU index).

[0170] In addition to the Fc domains described herein and in WO 2012 / 130831, Fc domains with reduced Fc receptor binding and / or effector function also comprise substitutions of one or more of Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056) (numbering according to the Kabat EU index). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including so-called "DANA" Fc mutants with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0171] Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion, or modification using genetic or chemical methods well known in the art. Genetic methods include site-directed mutagenesis of the encoding DNA sequence, PCR, gene synthesis, etc. The exact nucleotide changes can be confirmed, for example, by sequencing.

[0172] Binding to Fc receptors can be readily measured, for example, by ELISA or surface plasmon resonance (SPR) using standard equipment such as a BIAcore instrument (Cytiva), which can be obtained by recombinant expression. Alternatively, the binding affinity of an Fc domain or an antibody containing an Fc domain to an Fc receptor can be assessed using a cell line known to express a particular Fc receptor, such as human NK cells expressing the FcγIIIa receptor.

[0173] The effector function of an Fc domain or an antibody containing an Fc domain can be measured by a method known in the art. Examples of in vitro assays for evaluating the ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362, Hellstrom et al., Proc Natl Acad Sci USA 83, 7059-7063 (1986), and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985), U.S. Patent No. 5,821,337, Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assay methods may be used (see, e.g., ACTI® Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA) and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, for example in an animal model (such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998)).

[0174] In some embodiments, binding of the Fc domain to complement components, particularly C1q, is reduced. Thus, in some embodiments in which the Fc domain is modified to reduce effector function, the reduced effector function includes reduced CDC. To determine whether an Fc domain, or an antibody comprising an Fc domain, can bind to C1q and therefore has CDC activity, a C1q binding assay may be performed. See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

[0175] Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006); WO 2013 / 120929).

[0176] Specific Aspects of the Invention In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to PD-1, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising amino acid substitutions F42A, Y45A, L72G, and Q126T (numbering relative to SEQ ID NO: 90 of the human IL-2 sequence); and the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 80, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 81. In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to PD-1, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising amino acid substitutions T3A, F42A, Y45A, L72G, C125A, and Q126T (numbering relative to SEQ ID NO: 90 of the human IL-2 sequence); and the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 80, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 81. In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to PD-1, wherein the mutant IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 92; and the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 80, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 81.In one embodiment of any of the above aspects of the invention, the antibody is an immunoglobulin of the IgG class comprising a human IgG1 Fc domain composed of a first subunit and a second subunit, wherein in the first subunit of the Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V), optionally, the threonine residue at position 366 is replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A) (Kabat EU index numbering), and further each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbering). In this embodiment, the mutant IL-2 polypeptide may be fused at its amino-terminal amino acid to the carboxy-terminal amino acid of the first subunit of the Fc domain via the linker peptide of SEQ ID NO: 93. In one aspect, the invention provides immunoconjugates comprising a polypeptide comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 21, a polypeptide comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 23 or SEQ ID NO: 22, and a polypeptide comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 35.

[0177] In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to PD-1, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising amino acid substitutions F42A, Y45A, L72G, and N88D (numbering relative to SEQ ID NO: 90 of the human IL-2 sequence); and the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 80, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 81.

[0178] In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to PD-1, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising the amino acid substitutions T3A, F42A, Y45A, L72G, N88D, and C125A (numbering relative to SEQ ID NO: 90 of the human IL-2 sequence); and the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 80, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 81. In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to PD-1, wherein the mutant IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 98; and the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 80, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 81. In one embodiment of any of the above aspects of the invention, the antibody is an immunoglobulin of the IgG class comprising a human IgG1 Fc domain composed of a first subunit and a second subunit, wherein in the first subunit of the Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V), optionally, the threonine residue at position 366 is replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A) (Kabat EU index numbering), and further each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbering). In this embodiment, the mutant IL-2 polypeptide may be fused at its amino-terminal amino acid to the carboxy-terminal amino acid of the first subunit of the Fc domain via the linker peptide of SEQ ID NO:93.In one aspect, the invention provides immunoconjugates comprising a polypeptide comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:21, a polypeptide comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:23 or SEQ ID NO:22, and a polypeptide comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:100.

[0179] In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to PD-1, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising amino acid substitutions F42A, Y45A, L72G, and N88Q (numbering relative to SEQ ID NO: 90 of the human IL-2 sequence); and the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 80, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 81. In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to PD-1, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising the amino acid substitutions T3A, F42A, Y45A, L72G, N88Q, and C125A (numbering relative to SEQ ID NO: 90 of the human IL-2 sequence); and the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 80, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 81. In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to PD-1, wherein the mutant IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 99; and the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 80, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 81.

[0180] In one embodiment of any of the above aspects of the invention, the antibody is an immunoglobulin of the IgG class comprising a human IgG1 Fc domain composed of a first subunit and a second subunit, wherein in the first subunit of the Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V), optionally, the threonine residue at position 366 is replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A) (Kabat EU index numbering), and further each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbering). In this embodiment, the mutant IL-2 polypeptide may be fused at its amino-terminal amino acid to the carboxy-terminal amino acid of the first subunit of the Fc domain via the linker peptide of SEQ ID NO: 93. In one aspect, the invention provides immunoconjugates comprising a polypeptide comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 21, a polypeptide comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 23 or SEQ ID NO: 22, and a polypeptide comprising an amino acid sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 31.

[0181] Bispecific antigen-binding molecules that bind to PD-1 and LAG3 Immunoconjugates of the invention include bispecific antigen-binding molecules, i.e., antigen-binding molecules that contain at least two antigen-binding moieties capable of specifically binding to two distinct antigenic determinants (e.g., PD-1 and LAG3).

[0182] The bispecific antigen-binding molecule comprised in the immunoconjugate of the invention binds to PD-1 and LAG3, particularly human PD-1 and human LAG3, and can direct mutant IL-2 polypeptides to target sites expressing PD-1 and / or LAG3, e.g., associated with tumors, particularly to T cells expressing PD-1 and / or LAG3.

[0183] According to specific embodiments of the present invention, the antigen-binding moieties comprised in the bispecific antigen-binding molecule are Fab molecules (i.e., antigen-binding domains composed of heavy and light chains, each comprising a variable domain and a constant domain). In one embodiment, the first and / or second antigen-binding moieties are Fab molecules. In one embodiment, the Fab molecules are human. In a specific embodiment, the Fab molecules are humanized. In yet another embodiment, the Fab molecules comprise human heavy and light chain constant domains.

[0184] Preferably, at least one of the antigen-binding portions is a crossover Fab molecule. Such modification reduces mismatches between heavy and light chains from different Fab molecules, thereby increasing the yield and purity of bispecific antigen-binding molecules during recombinant production. In certain crossover Fab molecules useful for bispecific antigen-binding molecules contained in the immunoconjugates of the present invention, the variable domains of the Fab light chain and the Fab heavy chain (VL and VH, respectively) are swapped. However, even with such domain swapping, the preparation of bispecific antigen-binding molecules may contain some by-products due to so-called Bence-Jones interactions between mismatched heavy and light chains (see Schaefer et al., PNAS, 108 (2011) 11187-11191). To further reduce mispairing of heavy and light chains from different Fab molecules and thereby increase the purity and yield of the desired bispecific antigen-binding molecule, oppositely charged amino acids may be introduced into specific amino acid positions in the CH1 and CL domains of either the PD-1-binding Fab molecule or the LAG3-binding Fab molecule, as further described herein. The charge modifications are made in either the conventional Fab molecule comprised in the bispecific antigen-binding molecule or the VH / VL crossover Fab molecule comprised in the bispecific antigen-binding molecule (but not both). In certain embodiments, the charge modifications are made in the conventional Fab molecule comprised in the bispecific antigen-binding molecule (which, in certain embodiments, binds to LAG3).

[0185] First antigen-binding moiety The bispecific antigen-binding molecule comprised in the immunoconjugates of the present invention comprises at least one antigen-binding portion, particularly a Fab molecule, that binds to PD-1, particularly human PD-1 (the first antigen). In certain embodiments, the antigen-binding portion that binds to PD-1 is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains have been swapped / replaced with each other. In such embodiments, the antigen-binding portion that binds to LAG3 is a conventional Fab molecule. In alternative embodiments, the antigen-binding portion that binds to LAG3 is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains have been swapped / replaced with each other. In such embodiments, the antigen-binding portion that binds to PD-1 is a conventional Fab molecule.

[0186] In some embodiments, the 74th antigen-binding portion comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 75, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 76, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 77, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 78, and an amino acid sequence of SEQ ID NO: 79.

[0187] In some embodiments, the 74th antigen-binding portion comprises (a) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 75, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 76, and (b) a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 77, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 78, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 79.

[0188] In some embodiments, the first antigen-binding portion is a humanized antibody (derived from a humanized antibody). In one embodiment, the VH is a humanized VH and / or the VL is a humanized VL. In one embodiment, the first antigen-binding portion comprises the same CDRs as any of the above embodiments and further comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework. In some embodiments, the heavy and / or light chain variable regions comprise human framework regions (FR).

[0189] In some embodiments, the first antigen-binding portion comprises (a) a heavy chain variable region (VH) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 80. In some embodiments, the first antigen-binding portion comprises a light chain variable region (VL) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 81. In some embodiments, the second antigen-binding portion comprises (a) a heavy chain variable region (VH) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 80 and (b) a light chain variable region (VL) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 81.

[0190] In some embodiments, the first antigen-binding portion comprises a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 80, and a VL sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 81.

[0191] In certain embodiments, the second antigen-binding portion comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 80 and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 81.

[0192] In some embodiments, the first antigen-binding portion comprises the VH sequence of SEQ ID NO:80 and the VL sequence of SEQ ID NO:81.

[0193] In one embodiment, the first antigen-binding moiety comprises a human constant region. In one embodiment, the first antigen-binding moiety is a Fab molecule comprising a human constant region, particularly a human CH1 and / or CL domain. In one embodiment, no more than one antigen-binding moiety that binds to PD-1 is present in the bispecific antigen-binding molecule (i.e., the bispecific antigen-binding molecule provides monovalent binding to PD-1).

[0194] Second antigen-binding moiety The bispecific antigen-binding molecule comprised in the immunoconjugate of the present invention comprises at least one antigen-binding moiety, particularly a Fab molecule, that binds to LAG3, particularly human LAG3 (second antigen).

[0195] In certain embodiments, the antigen-binding portion that binds LAG3 is a conventional Fab molecule. In such embodiments, the antigen-binding portion that binds PD-1 is preferably a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or the constant domains CH1 and CL of the Fab heavy and light chains have been swapped / substituted for one another.

[0196] In alternative embodiments, the antigen-binding portion that binds PD-1 is a conventional Fab molecule. In such embodiments, the antigen-binding portion that binds LAG3 is a crossover Fab molecule as described herein, i.e., a Fab molecule in which the variable domains VH and VL or constant domains CH1 and CL of the Fab heavy and light chains have been swapped / substituted for one another.

[0197] In some embodiments, the second antigen-binding portion comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 82, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 83, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 84, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 85, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 86, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 87.

[0198] In some embodiments, the second antigen-binding portion comprises (a) a heavy chain variable region (VH) comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 82, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 83, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 84, and (b) a light chain variable region (VL) comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 85, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 86, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 87.

[0199] In some embodiments, the second antigen-binding portion is a humanized antibody (derived from a humanized antibody). In one embodiment, the VH is a humanized VH and / or the VL is a humanized VL. In one embodiment, the second antigen-binding portion comprises the same CDRs as any of the above embodiments and further comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework. In some embodiments, the heavy and / or light chain variable regions comprise human framework regions (FR).

[0200] In some embodiments, the second antigen-binding portion comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 88. In some embodiments, the second antigen-binding portion comprises a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:89.

[0201] In one embodiment, the second antigen-binding portion comprises a VH sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 88, and a VL sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 89.

[0202] In certain embodiments, the second antigen-binding portion comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 88 and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 89. In more specific embodiments, the second antigen-binding portion comprises the VH sequence of SEQ ID NO: 88 and the VL sequence of SEQ ID NO: 89.

[0203] In one embodiment, the second antigen-binding moiety comprises a human constant region. In one embodiment, the second antigen-binding moiety is a Fab molecule comprising a human constant region, particularly a human CH1 and / or CL domain. In particular, the light chain constant region may comprise the amino acid mutations described herein in a "charge-modified" state and / or, in the case of a crossover Fab molecule, may comprise deletions or substitutions of one or more (particularly two) N-terminal amino acids. In particular, the heavy chain constant region (particularly the CH1 domain) may comprise the amino acid mutations described herein in a "charge-modified" state.

[0204] In one embodiment, no more than one antigen-binding moiety that binds to LAG3 is present in the bispecific antigen-binding molecule (i.e., the bispecific antigen-binding molecule provides monovalent binding to LAG3).

[0205] Specific embodiments of the invention - PD 1-LAG3-IL2v_Q126T In a specific aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and a bispecific antigen-binding molecule that binds to PD-1 and LAG3, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising amino acid substitutions F42A, Y45A, L72G, and Q126T (numbering relative to SEQ ID NO: 90 of the human IL-2 sequence); and the bispecific antigen-binding molecule comprises: (i) a first antigen-binding moiety that binds to PD-1, comprising (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 80 and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 81; and (ii) a second antigen-binding moiety that binds to LAG3, comprising (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 88 and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 89.

[0206] In a specific aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and a bispecific antigen-binding molecule that binds to PD-1 and LAG3, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising amino acid substitutions T3A, F42A, Y45A, L72G, C125A, and Q126T (numbering relative to SEQ ID NO: 90 of the human IL-2 sequence); and the bispecific antigen-binding molecule comprises: (i) a first antigen-binding moiety that binds to PD-1, comprising (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 80 and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 81, and (ii) a second antigen-binding moiety that binds to LAG3, comprising (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 88 and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 89.

[0207] In a specific aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and a bispecific antigen-binding molecule that binds to PD-1 and LAG3, wherein the mutant IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO:92; and the bispecific antigen-binding molecule comprises: (i) a first antigen-binding moiety that binds to PD-1, the first antigen-binding moiety comprising (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:80 and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:81, and (ii) a second antigen-binding moiety that binds to LAG3, the second antigen-binding moiety comprising (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:88 and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:89.

[0208] In some embodiments according to any one of the above aspects, the first antigen-binding moiety comprises the variable domains VL and VH of the Fab light and heavy chains substituted for each other, and the second antigen-binding moiety is a (traditional) Fab molecule. In some such embodiments, in the constant domain CL of the second antigen-binding moiety, the amino acid at position 124 is substituted by a lysine (K) (Kabat numbering), and the amino acid at position 123 is substituted by a lysine (K) or an arginine (R), most particularly by an arginine (R) (Kabat numbering), and in the constant domain CHI of the second antigen-binding moiety, the amino acid at position 147 is substituted by a glutamic acid (E) (Kabat EU index numbering), and the amino acid at position 213 is substituted by a glutamic acid (E) (Kabat EU index numbering).

[0209] In some embodiments of any one of the above aspects, the bispecific antigen-binding molecule further comprises an Fc domain composed of a first subunit and a second subunit. In some such embodiments, the first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain (particularly to the first Fc domain subunit), and the second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the other of the subunits of the Fc domain (particularly to the second Fc domain subunit).

[0210] In a specific aspect, the present invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and a bispecific antigen binding molecule that binds to PD-1 and LAG3, wherein the mutant IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 92; The bispecific antigen-binding molecule comprises: (i) a first antigen-binding moiety that binds to PD-1, wherein the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other, and the first antigen-binding moiety is a Fab molecule comprising (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 80 and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 81; and (ii) a second antigen-binding moiety that binds to LAG3, wherein (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 88 and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 81. a (conventional) Fab molecule comprising a light chain variable region (VL) comprising the amino acid sequence of sequence number 89, wherein in the constant domain CL of the second antigen-binding portion the amino acid at position 124 is substituted by lysine (K) (Kabat numbering) and the amino acid at position 123 is substituted by lysine (K) or arginine (R) (Kabat numbering) (most particularly by arginine (R)), and in the constant domain CH1 of the second antigen-binding portion the amino acid at position 147 is substituted by glutamic acid (E) (Kabat EU index numbering) and the amino acid at position 213 is substituted by glutamic acid (E) (Kabat EU index numbering); and (iii) an Fc domain composed of a first subunit and a second subunit, Immunoconjugates are provided in which a first antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the Fc domain subunits (particularly the first Fc domain subunit), and a second antigen-binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of another of the Fc domain subunits (particularly the second Fc domain subunit).

[0211] In some embodiments of any of the above aspects of the invention, in the first subunit of the Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V), and optionally, the threonine residue at position 366 is replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A) (numbering according to the Kabat EU index). In some such embodiments, the first subunit of the Fc domain further comprises a replacement of the serine residue at position 354 with a cysteine ​​residue (S354C) or the glutamic acid residue at position 356 with a cysteine ​​residue (E356C) (particularly, the serine residue at position 354 is replaced with a cysteine ​​residue), and the second subunit of the Fc domain further comprises a replacement of the tyrosine residue at position 349 with a cysteine ​​residue (Y349C) (numbering according to the Kabat EU index).

[0212] In some embodiments of any of the above aspects of the invention, the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A), and the proline residue at position 329 is replaced with a glycine residue (P329G) in each of the first and second subunits of the Fc domain (numbering according to the Kabat EU index).

[0213] In some embodiments according to any of the above aspects of the invention, the Fc domain is a human IgG1 Fc domain.

[0214] In some embodiments according to any of the above aspects of the invention, the mutant IL-2 polypeptide is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of the first subunit of the Fc domain via a linker peptide of SEQ ID NO:93.

[0215] In certain specific embodiments, the immunoconjugate comprises a polypeptide comprising an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 68, a polypeptide comprising an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 69, a polypeptide comprising an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 70, and a polypeptide comprising an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 71. In even more specific embodiments, the bispecific antigen-binding molecule comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 68, a polypeptide comprising the amino acid sequence of SEQ ID NO: 69, a polypeptide comprising the amino acid sequence of SEQ ID NO: 70, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 71.

[0216] Polynucleotides The present invention further provides an isolated polynucleotide or fragment thereof encoding an immunoconjugate described herein, hi some embodiments, the fragment is an antigen-binding fragment.

[0217] Polynucleotides encoding the immunoconjugates of the present invention may be expressed as a single polynucleotide encoding the entire immunoconjugate, or as multiple (e.g., two or more) polynucleotides that are co-expressed. Polypeptides encoded by polynucleotides expressed together may associate, for example, via disulfide bonds or other means, to form a functional immunoconjugate. For example, the light chain portion of an antibody may be encoded by a separate polynucleotide derived from the portion of the immunoconjugate comprising the heavy chain of the antibody and a mutant IL-2 polypeptide. When co-expressed, the heavy chain polypeptide associates with the light chain polypeptide to form the immunoconjugate. In another example, the portion of the immunoconjugate comprising one of the two Fc domain subunits and the mutant IL-2 polypeptide may be encoded by a separate polynucleotide derived from the portion of the immunoconjugate comprising the other of the two Fc domain subunits. When co-expressed, the Fc domain subunits associate to form the Fc domain.

[0218] In some embodiments, the isolated polynucleotide encodes the entire immunoconjugate of the invention described herein, hi other embodiments, the isolated polynucleotide encodes a polypeptide included in an immunoconjugate of the invention described herein.

[0219] In one embodiment, an isolated polynucleotide of the invention encodes the heavy chain (e.g., immunoglobulin heavy chain) of an antibody comprised in an immunoconjugate and a mutant IL-2 polypeptide. In another embodiment, an isolated polynucleotide of the invention encodes the light chain of an antibody comprised in an immunoconjugate.

[0220] In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide of the present invention is RNA, for example, RNA in the form of messenger RNA (mRNA). The RNA of the present invention may be single-stranded or double-stranded.

[0221] Recombination Method Mutant IL-2 polypeptides useful in the present invention can be prepared by deletion, substitution, insertion, or modification using genetic or chemical methods well known in the art. Genetic methods include site-directed mutagenesis of the encoding DNA sequence, PCR, gene synthesis, etc. The exact nucleotide changes can be confirmed, for example, by sequencing. In this regard, the nucleotide sequence of native IL-2 is described in Taniguchi et al. (Nature 302, 305-10 (1983)), and nucleic acids encoding human IL-2 are available from public depositories such as the American Type Culture Collection (Rockville, MD). The sequence of native human IL-2 is set forth in SEQ ID NO: 19. Substitutions or insertions may include natural and unnatural amino acid residues. Amino acid modifications include well-known methods of chemical modification, such as the addition of glycosylation sites or the attachment of carbohydrates.

[0222] The immunoconjugates of the present invention may 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 the immunoconjugate (fragment), such as those 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 readily isolated and sequenced using conventional procedures. In one embodiment, vectors, preferably expression vectors, are provided that contain one or more 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 immunoconjugate (fragment), along with appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination. See, for example, the techniques described in Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, NY (1989); and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, NY (1989). An expression vector may be part of a plasmid, a virus, or a nucleic acid fragment. An expression vector contains an expression cassette into which a polynucleotide (i.e., coding region) encoding an immunoconjugate (fragment) is cloned in operative association with a promoter and / or other transcription or translation control elements. As used herein, a "coding region" is a portion of a nucleic acid consisting of codons that are translated into amino acids. A "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, but is considered part of the coding region (if present). However, 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 can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate vectors. Furthermore, any vector can contain a single coding region or two or more coding regions; for example, a vector of the present invention can encode one or more polypeptides that are separated into final proteins post- or co-translationally via proteolytic cleavage. Additionally, a vector, polynucleotide, or nucleic acid of the present invention can encode a heterologous coding region, which may or may not be fused to a polynucleotide encoding an immunoconjugate of the present invention, or a variant or derivative thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as secretory signal peptides or heterologous functional domains. Operable association occurs when the coding region for a gene product, such as a polypeptide, is associated with one or more regulatory sequences in such a way that expression of the gene product is under the influence or control of the one or more regulatory sequences. Two DNA fragments (such as a polypeptide coding region and its associated promoter) are "operably linked" if induction of promoter function results in transcription of mRNA encoding the desired gene product, and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression control sequences to direct expression of the gene product or the ability of the DNA template to be transcribed. Thus, a promoter region is said to be operably linked to a polypeptide-encoding nucleic acid if the promoter is capable of effecting transcription of that nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells. In addition to the promoter, other transcriptional regulatory elements, such as enhancers, operators, repressors, and transcription termination signals, may be operably linked to the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcriptional control regions are disclosed herein. A variety of transcriptional regulatory regions are known to those skilled in the art.These include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (e.g., the immediate early promoter linked to intron A), Simian Virus 40 (e.g., the early promoter), and retroviruses (e.g., Rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes, such as actin, heat shock proteins, bovine growth hormone, and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Further suitable transcriptional control regions include tissue-specific promoters and enhancers, and inducible promoters (e.g., tetracycline-inducible promoters). Similarly, various translational control elements are known to those skilled in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly internal ribosome entry sites, or IRES, also known as CITE sequences). The expression cassette may also contain other features, such as an origin of replication and / or chromosomal integration elements, such as the long terminal repeats (LTRs) of retroviruses or the inverted terminal repeats (ITRs) of adeno-associated viruses (AAV).

[0223] Polynucleotide 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 a polypeptide encoded by a polynucleotide of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells possess a signal peptide or secretory leader sequence that is cleaved from the mature protein upon initiation of transport of the growing protein chain across the rough endoplasmic reticulum. Those skilled in the art will recognize that polypeptides secreted by vertebrate cells typically possess a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to generate the secreted or "mature" form of the polypeptide. For example, human IL-2 is translated with a 20-amino acid signal sequence at the N-terminus of the polypeptide, which is then cleaved to generate the mature 133-amino acid human IL-2. In certain embodiments, a native signal peptide, such as the IL-2 signal peptide or an immunoglobulin heavy or light chain signal peptide, is used, or a functional derivative of such a sequence that retains the ability to direct the secretion of an operably associated polypeptide is used. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof may be used. For example, the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

[0224] DNA encoding short protein sequences that can be used to facilitate subsequent purification (e.g., a histidine tag) or serve to label the immunoconjugate may be included at or within the ends of the immunoconjugate (fragment) encoding polynucleotide.

[0225] In further embodiments, host cells are provided that comprise one or more polynucleotides of the invention. In certain embodiments, host cells are provided that comprise one or more vectors of the invention. The polynucleotides and vectors may incorporate any of the features described herein in connection with the polynucleotides and vectors, respectively, alone or in combination. In one such embodiment, the host cell comprises (e.g., is transformed or transfected with) one or more vectors that comprise one or more polynucleotides encoding an immunoconjugate of the invention. As used herein, the term "host cell" refers to any type of cell line that can be engineered to produce an immunoconjugate of the invention or a fragment thereof. Suitable host cells for replicating and supporting the expression of immunoconjugates are well known in the art. Such cells may be transfected or transduced, where appropriate, with a particular expression vector, and large quantities of the vector-containing cells may be grown to inoculate a large-scale fermenter, yielding sufficient quantities of immunoconjugate for clinical use. Suitable host cells include prokaryotic microorganisms (e.g., Escherichia coli) or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, etc. For example, polypeptides may be produced in bacteria, particularly if glycosylation is not required. After expression, the polypeptide may be isolated from the bacterial cell paste in a soluble fraction and further purified. In addition to prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeast, are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized" to produce polypeptides with partially or completely human glycosylation patterns. See Gerngross, Nat Biotech 22, 1409-1414 (2004) and Li et al., Nat Biotech 24, 210-215 (2006). Suitable host cells for the expression of (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells.Numerous baculovirus strains have been identified and may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe the PLANTIBODIES™ technology for producing antibodies in transgenic plants). 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 the SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (e.g., 293 or 293T cells described in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (e.g., Mather et al., Annals of NY Acad Sci 383, 44-68 (1982)), MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include dhfr. -Examples include Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)), and myeloma cell lines such as YO, NS0, P3X63, and Sp2 / 0. For a review of specific mammalian host cells suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). Host cells include cultured cells, such as cultured mammalian cells, yeast cells, insect cells, bacterial cells, and plant cells, to name just a few, but also cells contained in transgenic animals, transgenic plants, or cultured plants or animal tissues. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a lymphocytic cell (e.g., a Y0, NS0, Sp20 cell).

[0226] Standard techniques for expressing foreign genes in these systems are known in the art. Cells expressing mutant IL-2 polypeptides fused to either the heavy or light chain of an antibody may also be engineered to express the other antibody chain, such that the fusion product of the expressed mutant IL-2 is an antibody containing both a heavy and a light chain.

[0227] In one embodiment, a method of producing an immunoconjugate according to the invention is provided, the method comprising culturing a host cell comprising one or more polynucleotides encoding the immunoconjugate as provided herein under conditions suitable for expression of the immunoconjugate, and optionally recovering the immunoconjugate from the host cell (or host cell medium).

[0228] In the immunoconjugates of the present invention, the mutant IL-2 polypeptide may be genetically fused to the antibody or chemically conjugated to the antibody. Genetic fusion of the IL-2 polypeptide to the antibody can be designed so that the IL-2 sequence is fused directly to the polypeptide or indirectly fused via a linker sequence. The composition and length of the linker can be determined and tested for effectiveness according to methods well known in the art. Specific linker peptides are described herein. Additional sequences, such as endopeptidase recognition sequences, can be included as needed to incorporate cleavage sites for separating the individual components of the fusion. Additionally, IL-2 fusion proteins can be chemically synthesized using polypeptide synthesis methods (e.g., Merrifield solid-phase synthesis), as are well known in the art. The mutant IL-2 polypeptide can also be chemically conjugated to other molecules (e.g., antibodies) using well-known chemical conjugation methods. Bifunctional cross-linking reagents (e.g., homofunctional and heterofunctional cross-linking reagents well known in the art) can be used for this purpose. The type of cross-linking reagent used will depend on the nature of the molecule being coupled to IL-2 and can be readily identified by one of skill in the art. Alternatively, or in addition, the mutant IL-2 and / or the molecule intended to be conjugated may be chemically derivatized so that the two can be conjugated in separate reactions, as is also well known in the art.

[0229] The immunoconjugates of the present invention comprise antibodies. Methods for producing 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 can be constructed using solid-phase peptide synthesis, recombinantly produced (e.g., as described in U.S. Pat. No. 4,186,567), or obtained, for example, by screening combinatorial libraries containing variable heavy chains and variable light chains (see, e.g., U.S. Pat. No. 5,969,108 to McCafferty). Immunoconjugates, antibodies, and methods for producing them are also described, for example, in PCT Application Publication Nos. WO2011 / 020783, WO2012 / 107417, and WO2012 / 146628, the entire contents of which are incorporated herein by reference.

[0230] Antibodies of any animal species can be used in the immunoconjugates of the present invention. Non-limiting antibodies useful in the present invention can be of murine, primate, or human origin. If the immunoconjugate is intended for use in humans, chimeric forms of antibodies in which the antibody constant region is of human origin can be used. Humanized or fully human forms of antibodies can also be prepared according to methods well known in the art (see, for example, U.S. Patent No. 5,565,332 to Winter). Humanization can be achieved by various methods, including, but not limited to, (a) grafting CDRs of a non-human (e.g., donor antibody) into the framework and constant regions of a human (e.g., recipient antibody) with or without retaining critical framework residues (e.g., those important for maintaining good antigen-binding affinity or antibody function); (b) grafting only non-human specificity-determining regions (SDRs or a-CDRs; residues important for antibody-antigen interactions) into human framework and constant regions; or (c) grafting entire non-human variable domains but "cloaking" them with human-like sections by replacing surface residues.Humanized antibodies and methods for their production are reviewed, e.g., by Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity-determining region (SDR) 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 "guided selection" of FR shuffling).Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from human antibody consensus sequences of particular subgroups of heavy or light chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., Baca et al. al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0231] Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr Opin Pharmacol. 5, 368-74 (2001) and Lonberg, Curr Opin Immunol 20, 450-459 (2008). Human antibodies can also be prepared by administering immunogens to transgenic animals that have been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci that replace the endogenous immunoglobulin loci, or are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HuMab® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology; and U.S. Patent Application Publication No. 2007 / 0061900, which describes VelociMouse® technology. The human variable regions from intact antibodies produced by such animals may be further modified, for example, by combining them with different human constant regions.

[0232] Human antibodies can also be produced by hybridoma-based methods. Human myeloma cell lines and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)). Human antibodies produced via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include, for example, U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines), and Ni, Xiandai Mianyixue, 26(4):265-268(2006) (which describes human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Histology and Histopathology, 20(3):927-937(2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91(2005).

[0233] Human antibodies may also be produced by isolation from human antibody libraries, as described herein.

[0234] Antibodies useful in the present invention may be isolated by screening combinatorial libraries for antibodies with one or more desired activities. Methods for screening combinatorial libraries are reviewed, for example, in Lerner et al., Nature Reviews 16:498-508 (2016). For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with desired binding properties. Such methods are reviewed, for example, in Frenzel et al., mAbs 8:1177-1194 (2016); Bazan et al., Human Vaccines and Immunotherapeutics 8:1817-1828 (2012) and Zhao et al., Critical Reviews in Biotechnology 36:276-289 (2016), as well as Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and Marks and Bradbury in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003).

[0235] In one particular phage display method, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR), randomly recombined into phage libraries, and then screened for antigen-binding phage, as described by Winter et al., Annual Review of Immunology 12:433-455 (1994). Phages typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources yield high-affinity antibodies against immunogens without the need to construct hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) without immunization to provide a single source of antibodies against a wide range of non-self and self antigens, as described by Griffiths et al., EMBO Journal 12:725-734 (1993). Finally, naive libraries can also be synthetically generated by cloning unrearranged V gene segments from stem cells, using PCR primers containing random sequences to encode highly variable CDR3 regions, and achieving reordering in vitro, as described by Hoogenboom and Winter, Journal of Molecular Biology 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent Nos. 5,750,373, 7,985,840, 7,785,903, and 8,679,490, as well as U.S. Patent Application Publication Nos. 2005 / 0079574, 2007 / 0117126, 2007 / 0237764, and 2007 / 0292936. Further examples of methods known in the art for screening combinatorial libraries for antibodies with the desired activity(ies) include ribosome and mRNA display, and methods for antibody display and selection in bacteria, mammalian cells, insect cells, or yeast cells.Methods for yeast surface display are, for example, outlined in Scholler et al. Methods in Molecular Biology 503:135-56 (2012) and Cherf et al. Methods in Molecular Biology 1319:155-175 (2015) and Zhao et al. Methods in Molecular Biology 889:73-84 (2012). Methods for ribosome display are, for example, described in He et al. in Nucleic Acids Research 25:5132-5134 (1997) and Hanes et al. in PNAS 94:4937-4942 (1997).

[0236] Further chemical modification of the immunoconjugates of the invention may be desirable. For example, immunogenicity and half-life issues may be improved by conjugation to substantially linear polymers such as polyethylene glycol (PEG) or polypropylene glycol (PPG) (see, e.g., WO 87 / 00056).

[0237] Immunoconjugates prepared as described herein may be purified by techniques known in the art, such as, for example, high-performance liquid chromatography, ion-exclusion chromatography, gel electrophoresis, affinity chromatography, and size-exclusion chromatography. The actual conditions used to purify a particular protein will depend, in part, on factors such as net charge, hydrophobicity, and hydrophilicity, and will be apparent to those of skill in the art. For affinity chromatography purification of antibodies, a ligand receptor or antigen may be used to which the immunoconjugate binds. For example, an antibody that specifically binds to a mutant IL-2 polypeptide may be used. For affinity chromatography purification of the immunoconjugates of the present invention, a matrix containing protein A or G may be used. For example, sequential protein A or G affinity chromatography and size-exclusion chromatography may be used to isolate the immunoconjugate, essentially as described in the Examples. The purity of the immunoconjugate can be determined by any of a variety of well-known analytical methods, such as gel electrophoresis and high-performance liquid chromatography.

[0238] Compositions, Formulations, and Routes of Administration In a further aspect, the present invention provides a pharmaceutical composition comprising an immunoconjugate described herein for use in any of the following methods of treatment. In one embodiment, the pharmaceutical composition comprises any of the immunoconjugates provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises any of the immunoconjugates provided herein and at least one additional therapeutic agent (e.g., those described below).

[0239] Also provided is a method of producing an immunoconjugate of the invention in a form suitable for in vivo administration, the method comprising: (a) obtaining an immunoconjugate according to the invention; and (b) combining the immunoconjugate with at least one pharmaceutically acceptable carrier, thereby formulating a formulation of the immunoconjugate for in vivo administration.

[0240] Pharmaceutical compositions of the present invention comprise a therapeutically effective amount of an immunoconjugate dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that are generally non-toxic to recipients at the dosages and concentrations employed, i.e., do not cause adverse, allergic, or other untoward reactions when administered to animals, such as humans. The preparation of pharmaceutical compositions containing immunoconjugates and, optionally, additional active ingredients is known in the art in view of the present disclosure, as exemplified in Remington's Pharmaceutical Sciences, 18th Ed., Mack Printing Company, 1990, incorporated herein by reference. Furthermore, it will be understood that for administration to animals (e.g., humans), the formulation must meet sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biological Standards or other national equivalents. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonicity agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and the like, as materials known to those skilled in the art, and combinations thereof (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed., Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0241] The immunoconjugates of the invention (and any additional therapeutic agents) may be administered by any suitable means, including parenteral, intrapulmonary, intranasal, or, if desired, intralesional administration for localized treatment. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration may be by any suitable route, for example, injection, such as intravenous or subcutaneous injection, depending on whether administration is brief or chronic.

[0242] Parenteral compositions include those designed for administration by injection (e.g., subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal, or intraperitoneal injection). For injection, the immunoconjugates of the present invention may be formulated in an aqueous solution, preferably in a physiologically compatible buffer, such as Hank's solution, Ringer's solution, or physiological saline buffer. The solution may contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the immunoconjugates may be in powder form for constitution before use with a suitable vehicle, e.g., sterile pyrogen-free water. Sterile injectable solutions are prepared by incorporating the immunoconjugates of the present invention in the required amount in an appropriate solvent, with various other ingredients, as required, as listed below. Sterilization can be readily accomplished, for example, by filtration through sterile filtration membranes. Typically, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and / or other ingredients. In the case of sterile powders for preparing sterile injectable solutions, suspensions, or emulsions, the preferred preparation method is vacuum drying or freeze-drying, which yields a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered liquid medium. The liquid medium should be appropriately buffered, if necessary, and the liquid diluent is first rendered isotonic with sufficient saline or glucose prior to injection. The composition must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms, such as bacteria and fungi. It will be understood that endotoxin contamination should be kept to a minimum, for example, below 0.5 ng / mg protein.Suitable pharmaceutically acceptable carriers include, but are not limited to, buffers, e.g., phosphate, citrate and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methylparaben or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, tacrolimus, hydroxybenzoates ... Proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or 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, and the like. Optionally, suspensions may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl acetate or triglycerides, or liposomes.

[0243] The active ingredient can also be incorporated into colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions, for example, by microcapsules prepared by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained-release preparations may also be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, for example, films or microcapsules. In certain embodiments, sustained absorption of injectable compositions can be achieved by using agents that delay absorption in the compositions, such as aluminum monostearate, gelatin, or combinations thereof.

[0244] In addition to the compositions already described, the immunoconjugates can also be formulated as depot preparations. Such long-acting preparations can be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Thus, for example, the immunoconjugates can be formulated with suitable polymers or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as sparingly soluble salts.

[0245] Pharmaceutical compositions containing the immunoconjugates of the present invention may be prepared by conventional mixing, dissolving, emulsifying, encapsulating, encapsulating, 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 the processing of proteins into pharmaceutically usable preparations. The appropriate formulation depends on the selected route of administration.

[0246] The immunoconjugate may be formulated in the composition in a free acid or free base, neutral or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or free base. These include acid addition salts, such as those formed with the free amino groups of the proteinaceous composition, or those formed with organic 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, potassium, ammonium, calcium, and ferric hydroxide; or organic bases such as isopropylamine, trimethylamine, histidine, and procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than the corresponding free base forms.

[0247] Therapeutic methods and compositions Any of the immunoconjugates provided herein may be used in methods of treatment. The immunoconjugates of the invention may be used as immunotherapeutic agents, for example, in the treatment of cancer.

[0248] For use in therapeutic methods, the immunoconjugates of the invention will be formulated, dosed, and administered in a manner consistent with good medical practice, with factors to consider in this context including the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of administration of the agent, the method of administration, the administration schedule, and other factors known to medical practitioners.

[0249] The immunoconjugates of the present invention may be particularly useful in treating disease states in which stimulation of the host's immune system is beneficial, particularly conditions in which an enhanced cellular immune response is desirable. These may include disease states in which the host immune response is insufficient or deficient. Disease states in which the immunoconjugates of the present invention may be administered include, for example, tumors or infections in which the cellular immune response is a critical mechanism for specific immunity. The immunoconjugates of the present invention may be administered as is or in any suitable pharmaceutical composition.

[0250] In one aspect, an immunoconjugate of the present invention is provided for use as a pharmaceutical. In a further aspect, an immunoconjugate of the present invention is provided for use in the treatment of a disease. In certain embodiments, an immunoconjugate of the present invention is provided for use in a method of treatment. In one embodiment, the present invention provides an immunoconjugate described herein for use in the treatment of a disease to an individual in need thereof. In certain embodiments, the present invention provides an immunoconjugate for use in a method of treating an individual having a disease, comprising administering to the individual a therapeutically effective amount of the immunoconjugate. In certain embodiments, the disease being treated is a proliferative disorder. In certain embodiments, the disease is cancer. In certain embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease being treated is cancer. In further embodiments, the present invention provides an immunoconjugate for use in stimulating the immune system. In certain embodiments, the present invention provides an immunoconjugate for use in a method for stimulating the immune system in an individual, comprising administering to the individual an effective amount of the immunoconjugate, thereby stimulating the immune system. An "individual" according to any of the above embodiments is a mammal, preferably a human. "Stimulation of the immune system" according to any of the above embodiments may include any one or more of a general increase in immune function, an increase in T cell function, an increase in B cell function, restoration of lymphocyte function, an increase in IL-2 receptor expression, an increase in T cell responsiveness, an increase in natural killer cell activity or lymphokine-activated killer (LAK) cell activity, etc.

[0251] In a further aspect, the present invention provides the use of an immunoconjugate of the invention in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of a disease in an individual in need thereof. In one embodiment, the medicament is for use in a method of treating a disease, comprising administering a therapeutically effective amount of the medicament to an individual having the disease. In certain embodiments, the disease being treated is a proliferative disorder. In certain embodiments, the disease is cancer. In one embodiment, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease being treated is cancer. In a further embodiment, the medicament is for stimulating the immune system. In a further embodiment, the medicament is for use in a method for stimulating the immune system in an individual, comprising administering to the individual an effective amount of the immunoconjugate, thereby stimulating the immune system. The "individual" according to any of the above embodiments may be a mammal, preferably a human. "Stimulating the immune system" in any of the above embodiments may include any one or more of a general increase in immune function, an increase in T cell function, an increase in B cell function, restoration of lymphocyte function, an increase in IL-2 receptor expression, an increase in T cell responsiveness, an increase in natural killer cell activity or lymphokine-activated killer (LAK) cell activity, and the like.

[0252] In a further aspect, the present invention provides a method for treating a disease in an individual. In one embodiment, the method comprises administering to an individual having such a disease a therapeutically effective amount of an immunoconjugate of the present invention. In one embodiment, a composition is administered to the individual, comprising the immunoconjugate of the present invention in a pharmaceutically acceptable form. In certain embodiments, the disease being treated is a proliferative disorder. In certain embodiments, the disease is cancer. In certain embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease being treated is cancer. In a further aspect, the present invention provides a method for stimulating the immune system in an individual, comprising administering to the individual an effective amount of the immunoconjugate, thereby stimulating the immune system. The "individual" according to any of the above embodiments may be a mammal, preferably a human. "Stimulating the immune system" in any of the above embodiments may include any one or more of a general increase in immune function, an increase in T cell function, an increase in B cell function, restoration of lymphocyte function, an increase in IL-2 receptor expression, an increase in T cell responsiveness, an increase in natural killer cell activity or lymphokine-activated killer (LAK) cell activity, and the like.

[0253] In certain embodiments, the disease to be treated is a proliferative disorder, particularly cancer. Non-limiting examples of cancer include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, stomach cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other cell proliferative disorders that can be treated using the immunoconjugates of the present invention include, but are not limited to, neoplasms located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary gland, testes, ovaries, thymus, thyroid gland), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, chest region, and genitourinary system. Precancerous conditions or lesions and cancer metastasis are also included. In certain embodiments, the cancer is selected from the group consisting of kidney cancer, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer, prostate cancer, and bladder cancer. Those skilled in the art will readily recognize that in many cases, an immunoconjugate may not provide a cure but may only provide a partial benefit. In some embodiments, a physiological change that has some benefit is also considered therapeutically beneficial. Thus, in some embodiments, the amount of immunoconjugate that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount." The subject, patient, or individual in need of treatment is typically a mammal, and more particularly, a human.

[0254] In some embodiments, an effective amount of an immunoconjugate of the invention is administered to a cell, hi other embodiments, a therapeutically effective amount of an immunoconjugate of the invention is administered to an individual to treat a disease.

[0255] The appropriate dosage of the immunoconjugates of the present invention (whether used alone or in combination with one or more other additional therapeutic agents) for the prevention or treatment of disease will vary depending on the type of disease being treated, the route of administration, the patient's weight, the type of molecule (e.g., whether it contains an Fc domain or not), the severity and course of the disease, whether the immunoconjugate is administered for prophylactic or therapeutic purposes, previous or current therapeutic interventions, the patient's medical history and response to the immunoconjugate, and the judgment of the attending physician. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient in the composition and the appropriate dose for the individual subject. Various administration schedules are contemplated herein, including, but not limited to, single or multiple administrations over various time periods, bolus administration, and pulse infusion.

[0256] The immunoconjugate is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, an initial candidate dosage for administration to the patient of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of the immunoconjugate may be used, whether by one or more separate administrations or by continuous infusion, for example. A typical daily dosage may range from about 1 μg / kg to 100 mg / kg, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on symptoms, treatment is usually continued until a desired suppression of disease symptoms occurs. One exemplary dosage of the immunoconjugate ranges from about 0.005 mg / kg to about 10 mg / kg. In other non-limiting examples, the dosage may be about 1 microgram / kg body weight, about 5 micrograms / kg body weight, about 10 micrograms / kg body weight, about 50 micrograms / kg body weight, about 100 micrograms / kg body weight, about 200 micrograms / kg body weight, about 350 micrograms / kg body weight, about 500 micrograms / kg body weight, about 1 milligram / kg body weight, about 5 milligrams / kg body weight, about 10 milligrams / kg body weight, about 50 milligrams / kg body weight, about 100 milligrams / kg body weight, about 200 milligrams / kg body weight, about 350 milligrams / kg body weight, about 500 milligrams / kg body weight, about 1000 mg / kg body weight or more per administration, including any range derivable therein. Non-limiting examples of ranges derived from the numbers recited herein include ranges such as about 5 mg / kg body weight to about 100 mg / kg body weight, about 5 micrograms / kg body weight to about 500 milligrams / kg body weight, based on the above numbers. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 5.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the patient. Such dosages may be administered intermittently, for example, weekly or every three weeks (e.g., the patient receives about two to about 20, or, for example, about six, doses of the immunoconjugate). An initial higher loading dose may be administered, followed by one or more lower doses. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0257] The immunoconjugates of the present invention are generally used in an amount effective to achieve their intended purpose. For use in treating or preventing a disease state, the immunoconjugates of the present invention, or pharmaceutical compositions thereof, are administered or applied in a therapeutically effective amount. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0258] For systemic administration, a therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays. The IC 50 A dose may be formulated in animal models to achieve a circulating concentration range including 100 mg / kg / day. Such information can be used to more accurately determine useful doses in humans.

[0259] Initial dosages can also be estimated from in vivo data, e.g., from animal models, using techniques well known in the art. Those skilled in the art will readily be able to optimize administration to humans based on the animal data.

[0260] Dosage and dosing intervals may be adjusted individually to provide plasma concentrations of the immunoconjugate sufficient to maintain therapeutic efficacy. Typical patient dosages for administration by injection range from about 0.1 to 50 mg / kg / day, typically about 0.5 to 1 mg / kg / day. Therapeutically effective plasma concentrations may be achieved by administering multiple doses each day. Plasma levels may be measured, for example, by HPLC.

[0261] In cases of local administration or selective uptake, the effective local concentration of the immunoconjugate may be unrelated to plasma concentration. One skilled in the art will be able to optimize a therapeutically effective local dosage without undue experimentation.

[0262] Therapeutically effective dosages of the immunoconjugates described herein generally provide therapeutic benefit without causing substantial toxicity. The toxicity and therapeutic efficacy of immunoconjugates can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Cell culture assays and animal studies can be used to determine the LD 50 (the dose that is lethal to 50% of the population) and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index, which is the LD 50 / ED 50 The therapeutic index may be expressed as a ratio of ED to ED . Immunoconjugates that exhibit large therapeutic indices are preferred. In one embodiment, the immunoconjugates described in the present invention exhibit a high therapeutic index. Data obtained from cell culture assays and animal studies can be used in formulating a dosage range appropriate for human use. Dosages are preferably administered at or above the ED with little or no toxicity. 50 The blood concentration range includes the range of 0.01 to 0.01 mg / kg of the active ingredient. The dosage may vary within this range depending on various factors, such as the dosage form used, the route of administration utilized, the condition of the subject, etc. The actual formulation, route of administration, and dosage to be administered may be selected by the individual physician in view of the patient's condition. (See, for example, Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1, which is incorporated herein by reference in its entirety.)

[0263] The attending physician of a patient treated with an immunoconjugate of the invention will know how and when to discontinue, interrupt, or adjust administration due to toxicity, organ failure, etc. Conversely, the attending physician will also know to adjust treatment to higher levels if the clinical response is inadequate (without causing toxicity). The magnitude of an administered dose in the management of a target disorder will vary depending on the severity of the condition being treated, the route of administration, etc. The severity of the condition may, for example, be assessed, in part, by standard prognostic evaluation methods. Furthermore, the dose, and perhaps the frequency of administration, will also vary according to the age, weight, and response of the individual patient.

[0264] The maximum therapeutic dose of an immunoconjugate comprising a mutant IL-2 polypeptide described herein may be increased from that used for an immunoconjugate comprising wild-type IL-2.

[0265] Other drugs and treatments The immunoconjugates of the present invention may be administered in combination with one or more other therapeutic agents. For example, the immunoconjugates of the present invention may be administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any agent administered to treat a condition or disease in an individual in need of such treatment. Such additional therapeutic agents may include active ingredients appropriate for the particular indication being treated, preferably active ingredients with complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is an immunosuppressant, a cytostatic, a cell adhesion inhibitor, a cytotoxic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of cells to apoptosis-inducing factors. In certain embodiments, the additional therapeutic agent is an anticancer agent, such as a microtubule-disrupting agent, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, hormone therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an antiangiogenic agent.

[0266] Such other agents are preferably present in combination in amounts effective for the intended purpose. The effective amount of such other agents will depend on the amount of immunoconjugate used, the type of disorder or treatment, and other factors discussed above. Immunoconjugates are generally used in the same dosages and via the routes of administration described herein, or at about 1-99% of the dosages described herein, or at any dosage and via any route determined empirically / clinically appropriate.

[0267] Such combination therapy as described above encompasses combined administration (two or more therapeutic agents in the same or separate compositions) and separate administration, where administration of the immunoconjugates of the invention may occur before, simultaneously with, and / or after administration of the additional therapeutic agent(s) and / or adjuvant. The immunoconjugates of the invention may also be used in combination with radiation therapy.

[0268] manufactured goods Another aspect of the present invention provides an article of manufacture containing materials useful for the treatment, prevention, and / or diagnosis of the aforementioned disorders. The article of manufacture comprises a container and a label or package insert affixed to or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV infusion bags, etc. The container may be formed from a variety of materials, such as glass or plastic. The container holds the composition by itself or in combination with another composition effective for treating, preventing, and / or diagnosing a condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an immunoconjugate of the present invention. The label or package insert indicates that the composition is used to treat a selected condition. The article of manufacture may further comprise (a) a first container containing a composition comprising an antibody of the present invention; and (b) a second container containing a composition comprising an additional cytotoxic or other therapeutic agent. The article of manufacture in this embodiment of the invention may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively or additionally, the article of manufacture may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes. [Brief explanation of the drawings]

[0269] [Figure 1A-1B] Mouse surrogates of mouse PD1-targeting IL2v constructs (targeting mouse PD1 using rat-derived V domains). Figure 1A shows P1AG9991, a bivalent mouse PD1-targeting mouse IgG1 DA PG with human IL2v fused to the C-terminus of the Fc DD-chain. Figure 1B shows P1AG8304, a bivalent mouse PD1-targeting mouse IgG1 DA PG with human IL2v Q126T fused to the C-terminus of the Fc DD-chain. [Figures 2A-2C] Human PD1 / LAG3-targeted IL2v constructs. Figure 2A shows P1AF4801, a bispecific human PD1 / LAG3-targeted IgG1 PG LALA crossMab with human IL2v fused to the C-terminus of the Fc knob chain. Figure 2B shows P1AF7951, a bispecific human PD1 / LAG3-targeted IgG1 PG LALA crossMab with human IL2v Q126T fused to the C-terminus of the Fc knob chain. Figure 2C shows P1AA6888, a monospecific human PD1 IgG1 PG LALA used as a control. [Figure 3] Proliferation of NK92 cells upon treatment with PD1-IL2v and several PD1-IL2v variants for 3 days was determined by measuring ATP levels with CellTiter Glo. [Figures 4A-4C] The proliferation of CD8 T cells (Fig. 4A), NK cells (Fig. 4B), and CD4 T cells (Fig. 4C) in PBMCs upon 5-day treatment with PD1-IL2v and several PD1-IL2v variants was determined by flow cytometry. [Figures 5A-5C] Activation of CD8 T cells (Figure 5A), NK cells (Figure 5B), and CD4 T cells (Figure 5C) in PBMCs upon treatment with PD1-IL2v and several PD1-IL2v variants for 5 days was determined by measuring CD25 upregulation by flow cytometry. Proliferation of NK92 cells upon treatment with PD1-IL2v and several PD1-IL2v variants for 3 days in Figure 4 was determined by measuring ATP levels with CellTiter Glo. [Figures 6A-6D] The proliferation of CD8 T cells and NK cells in PBMCs was determined by flow cytometry after 4 days of treatment with FAP-IL2v and several FAP-IL2v variants. Figure 6A shows FAP-IL2v(G4S)5, FAP-IL2v_D20T_Selectikine, FAP-IL2v_E215V, FAP-IL2v_E95A, FAP-IL2v_E95A, and FAP-IL2v. Figure 6B shows FAP-IL2v_L12A, FAP-IL2v_L12A_L19A, FAP-IL2v_T133K, and FAP-IL2v. Figure 6C shows FAP-IL2v_L12A_L80A, FAP-IL2v_L19V, FAP-IL2v_N88T, FAP-IL2v_N119K, and FAP-IL2v. Figure 6D shows FAP-IL2v_Q22A, FAP-IL2v_Q126T, FAP-IL2v_S87A, FAP-IL2v_S130A and FAP-IL2v. [Figures 7A-7B] Activation of CD8 T cells (Fig. 7A) and NK cells (Fig. 7B) in PBMCs upon 4-day treatment with PD1-IL2v and several PD1-IL2v variants was determined by measuring CD25 upregulation by flow cytometry. [Figure 8A-8B] Activation of CD8 T cells (Fig. 8A) and NK cells (Fig. 8B) in PBMCs upon 4-day treatment with PD1-IL2v and several PD1-IL2v variants was determined by measuring CD25 upregulation by flow cytometry. [Figures 9A-9C] The proliferation of NK cells (Fig. 9A), CD8 T cells (Fig. 9B), and CD4 T cells (Fig. 9C) in PBMCs upon treatment with FAP-IL2v and selected FAP-IL2v variants for 5 days was determined by flow cytometry. [Figures 10A-10C] Activation of NK cells (Fig. 10A), CD8 T cells (Fig. 10B), and CD4 T cells (Fig. 10C) in PBMCs upon 5-day treatment with FAP-IL2v and selected FAP-IL2v variants was determined by measuring CD25 upregulation by flow cytometry. [Figures 11A-11B] STAT5 phosphorylation was determined by flow cytometry in CD4 T cells (Fig. 11A), regulatory T cells (Fig. 11B), CD8 T cells (Fig. 11C), and NK cells (Fig. 11D) upon treatment of PBMCs with FAP-IL2v and selected FAP-IL2v variants. [Figures 12A-12C] The proliferation of CD8 T cells (Fig. 12A), NK cells (Fig. 12B), and CD4 T cells (Fig. 12C) in PBMCs upon 5-day treatment with FAP-IL2v, FAP-IL2v Q126T, and PD1-IL2v Q126T was determined by flow cytometry. [Figures 13A-13C] Activation of CD8 T cells (FIG. 13A), NK cells (FIG. 13B), and CD4 T cells (FIG. 13C) in PBMCs upon 5-day treatment with FAP-IL2v, FAP-IL2v Q126T, and PD1-IL2v Q126T was determined by measuring CD25 upregulation by flow cytometry. [Figures 14A-14G]IL-2 signaling (STAT5-P) in PD1-blocked and PD-1-expressing CD4+ cells cultured together. IL-2 signaling (STAT5-P) is shown as the frequency of STAT5-P in human PD1+ (solid line) and PD-1 pre-blocked (dotted line) CD4 T cells upon 12 min exposure to PD1-IL2v mutants. Mean ± SEM of four donors is shown. Figure 14A shows PD1-IL2v, PD1-IL2v and PD1-pre-blockade, PD1-IL2v_Q126T, PD1-IL2v_Q126T and PD1-pre-blockade, PD1-IL2v_K8S, PD1-IL2v_K8S and PD1-pre-blockade. Figure 14B shows PD1-IL2v, PD1-IL2v and PD1-pre-block, PD1-IL2v_Q126T, PD1-IL2v_Q126T and PD1-pre-block, PD1-IL2v_L12A, PD1-IL2v_L12A and PD1-pre-block. Figure 14C shows PD1-IL2v, PD1-IL2v and PD1-pre-block, PD1-IL2v_Q126T, PD1-IL2v_Q126T and PD1-pre-block, PD1-IL2v_N88D, PD1-IL2v_N88D and PD1-pre-block. Figure 14D shows PD1-IL2v, PD1-IL2v and PD1-pre-block, PD1-IL2v_Q126T, PD1-IL2v_Q126T and PD1-pre-block, PD1-IL2v_L19V, PD1-IL2v_L19V and PD1-pre-block. Figure 14E shows PD1-IL2v, PD1-IL2v and PD1-pre-block, PD1-IL2v_Q126T, PD1-IL2v_Q126T and PD1-pre-block, PD1-IL2v_H79S, PD1-IL2v_H79S and PD1-pre-block. Figure 14F shows PD1-IL2v, PD1-IL2v and PD1-pre-block, PD1-IL2v_Q126T, PD1-IL2v_Q126T and PD1-pre-block, PD1-IL2v_D109A, PD1-IL2v_D109A and PD1-pre-block. Figure 14G shows PD1-IL2v, PD1-IL2v and PD1-pre-block, PD1-IL2v_Q126T, PD1-IL2v_Q126T and PD1-pre-block, PD1-IL2v_L80A, PD1-IL2v_L80A and PD1-pre-block. [Figures 15A-15B] IL-2 signaling (STAT5-P) PD-1-expressing CD4+ T cells (FIG. 15A) show the frequency and mean fluorescence intensity (MFI) of selected PD1-, FAP-, and NKG2D-IL2 variants on PD-1+ CD4 T cells (FIG. 15B). Potency measurements in PD1+ CD4 T cells reflect PD1-mediated delivery of IL-2v relative to PD1-independent delivery of the FAP-IL-2v construct. Mean ± SEM of four donors is shown. [Figures 16A-16B] Frequency of CMV-specific CD4 T cells upon restimulation with CMV protein pp65 and in the presence of the indicated treatments in combination with pp65 (Figure 16A). Fold increase in frequency of CMV-specific CD4 T cells by normalizing each response to pp65 alone indicates specific compound effects in expanding antigen-specific T cell responses (Figure 16B). Mean ± SEM of five donors is shown. [Figure 17] Percentage of Treg-mediated suppression of granzyme B production by T conjugates in the presence or absence of the indicated immunoconjugates over 5 days of coculture. Median of 6 donors. p is calculated using one-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 18] IL-2 signaling (STAT5-P) PD-1-expressing CD4+ T cells. Frequency of potency of selected PD1-, LAG-3, FAP-IL2 variants on PD-1+, LAG-3+ and PD-1-, LAG-3- CD4 T cells. Potency measurements in PD1+, LAG-3+ CD4 T cells reflect PD1- (LAG-3)-mediated delivery of IL-2v versus PD-1 / LAG-3-independent delivery of FAP-IL-2v constructs. Mean ± SEM of three donors is shown. [Figure 19]Percentage of Treg-mediated suppression of granzyme B production by T cells in 5 days of coculture in the presence or absence of the indicated immunoconjugates. Median of 10 donors. p is calculated using one-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 20] Percentage of internalized molecules at 0.6 nM by activated CD4 T cells after 3 h incubation at 37° C. Median of 4 donors. p is calculated using one-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 21] We present the results of efficacy experiments using the PD1-IL2vQ126T variant and a mouse surrogate of the PD-IL2v MAb as single agents. The Panc02-Fluc pancreatic cancer cell line was subcutaneously injected into Black 6-huIL2RBG transgenic mice to test tumor growth inhibition (TGI) in a subcutaneous model. Tumor size was measured using a vernier caliper. Treatment began when tumors reached 100 mm3. The amount of antibody injected per mouse was 2 mg / kg for the muPD1-IL2vQ126T variant and 0.5 mg / kg for the muPD1-IL2v qw. Treatment continued for 3 weeks. The PD1-IL2vQ126T variant mediated significantly greater efficacy in terms of tumor growth inhibition compared to the vehicle and PD1-IL2v groups. The PD1-IL2vQ126T molecule was well tolerated, and no clinical signs or weight loss were observed. [Table 0] TIFF2026504860000002.tif254170TIFF2026504860000003.tif253170TIFF2026504860000004.tif254170TIFF2026504860000005.tif254170TIFF2026504860000006.tif254170TIFF2026504860000007.tif253170TIFF2026504860000008.tif254170TIFF2026504860000009.tif254170TIFF2026504860000010.tif254170TIFF2026504860000011.tif254170TIFF2026504860000012.tif254170TIFF2026504860000013.tif254170TIFF2026504860000014.tif254170TIFF2026504860000015.tif254170TIFF2026504860000016.tif254170TIFF2026504860000017.tif254170TIFF2026504860000018.tif254170TIFF2026504860000019.tif254170TIFF2026504860000020.tif254170TIFF2026504860000021.tif254170TIFF2026504860000022.tif254170TIFF2026504860000023.tif254170TIFF2026504860000024.tif254170TIFF2026504860000025.tif254170TIFF2026504860000026.tif254170TIFF2026504860000027.tif254170TIFF2026504860000028.tif254170TIFF2026504860000029.tif254170TIFF2026504860000030.tif254170TIFF2026504860000031.tif254170TIFF2026504860000032.tif254170TIFF2026504860000033.tif254170TIFF2026504860000034.tif254170TIFF2026504860000035.tif255170TIFF2026504860000036.tif255170TIFF2026504860000037.tif166170. [Example]

[0270] The following are examples of methods and compositions of the present invention. Given the general description provided above, it will be understood that various other embodiments may be practiced.

[0271] Example 1 Example 1A. Molecules The molecules tested in the examples below consist of the amino acid sequences set forth in Table A. [Table A] TIFF2026504860000039.tif255169TIFF2026504860000040.tif22170

[0272] Example 1B. Generation and Analysis of Human PD1 and FAP IgG-IL2v Variants The antibody-IL2v variant fusion constructs described herein were produced in HEK cells. In some cases (surface plasmon resonance measurements), the supernatant was used directly without prior purification (Table 1). For all other assays, the protein was first purified by Protein A affinity chromatography and size exclusion chromatography. Analysis of the final product consisted of monomer content determination (by analytical size exclusion chromatography) and percentage of the main peak (determined by non-reducing capillary SDS electrophoresis: CE-SDS).

[0273] Production of IgG-like protein in HEK293 EBNA cells Antibody-IL2v variant fusion constructs were generated by transient transfection of HEK293 EBNA cells. Cells were centrifuged, and the medium was replaced with prewarmed CD CHO medium (Thermo Fisher Scientific, catalog no. 10743029). The expression vector was mixed in CD CHO medium, PEI (Polyethylenimine, Polysciences, Inc., catalog no. 23966-1) was added, the solution was vortexed, and incubated at room temperature for 10 minutes. Cells (2 μl / ml) were then mixed with the vector / PEI solution, transferred to a flask, and incubated at 37°C in a shaking incubator with a 5% CO2 atmosphere for 3 hours. After incubation, Excell medium (80% of the total volume) containing supplements was added (W. Zhou and A. Kantardjieff, Mammalian Cell Cultures for Biologics Manufacturing, DOI:10.1007 / 978-3-642-54050-9; 2014). One day after transfection, supplements (12% of the total volume) were added. After 4–7 days, cell supernatants were harvested by centrifugation and subsequent filtration (0.2 μm filter). Proteins were purified from the harvested supernatants using standard methods described below.

[0274] Titer determination (PA-HPLC) Quantification of the Fc-containing construct in the supernatant was performed by Protein A-HPLC on an Agilent HPLC system equipped with a UV detector. The supernatant was injected into a POROS 20 A (Applied Biosystems), washed with 10 mM Tris, 50 mM Glycine, 100 mM NaCl, pH 8.0, and eluted with the same buffer at pH 2.0. The elution peak area at 280 nm was integrated and converted to concentration using a calibration curve with standards analyzed in the same run.

[0275] Purification of IgG-like proteins Proteins were purified from filtered cell culture supernatants according to standard protocols. Briefly, Fc-containing proteins were purified from cell culture supernatants by Protein A-affinity chromatography (equilibration buffer: 20 mM sodium citrate, 20 mM sodium phosphate, pH 7.5; elution buffer: 20 mM sodium citrate, pH 3.0). Elution was achieved at pH 3.0, followed immediately by neutralization of the sample pH. Proteins were concentrated by centrifugation (Millipore Amicon® ULTRA-15 (facility number: UFC903096)), and aggregated proteins were separated from monomeric proteins by size-exclusion chromatography in 20 mM histidine, 140 mM sodium chloride, pH 6.0.

[0276] Analysis of IgG-like proteins The concentration of purified protein was determined by measuring absorbance at 280 nm using the mass extinction coefficient calculated based on the amino acid sequence according to Pace et al., Protein Science, 1995, 4, 2411-1423. Protein purity and molecular weight were analyzed by CE-SDS in the presence and absence of reducing agents using a LabChip GXII or LabChip GX Touch (Perkin Elmer). Aggregate content was determined by HPLC chromatography at 25°C using an analytical size-exclusion column (TSKgel G3000 SW XL or UP-SW3000) equilibrated in running buffer (200 mM KH2PO4, 250 mM KCl pH 6.2, 0.02% NaN3). [Table 1] [Table 2] [Table 3]

[0277] result IgG-IL2v variant constructs produced in HEK cells were tested from the supernatant without prior purification but either after quantification by Protein A titration (Table 1) or after purification. Quality analysis of the purified material revealed that the product peaks were 57.8% (if any) or 87%-100% by analytical size exclusion chromatography analysis (Table 2) and 88-99% by non-reducing capillary electrophoresis (Table 3).

[0278] conclusion All IgG-IL2v variants were produced with good quality, except for IL2v Q22A, IL2v Q126N and IL2v Q126E.

[0279] Example 1C. Affinity of purified FAP-IL2v variants to recombinant human IL2Rβ-gamma-Fc heterodimer setting Equipment:Biacore T200 Chip: CM5 (number 697) Fc1-4: Anti-human Fc specific (Roche (in-house)) Capture: 10 nM FAP-IL2v variant for 40 seconds Analyte: Human IL2R beta-gamma Fc (P1AD7029-002) Running buffer: HBS-EP Temperature: 25℃ Dilution ratio: 5-fold dilution in 3.2-2000 nM HBS-EP Flow: 30 μl / min Combine: 240 seconds Dissociation: 120 or 600 seconds Regeneration: 10 mM glycine pH 2.1, 30 seconds

[0280] Surface plasmon resonance (SPR) experiments were performed on a Biacore T200 using HBS-EP+ as the running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005% surfactant P20 (BR-1006-69, Cytiva)). Anti-human Fc-specific antibodies (in-house manufactured by Roche) were directly immobilized on a CM5 chip (Cytiva) by amine coupling. FAP-IL2v variants were captured at 10 nM for 40 s. A 5-fold diluted serum sample of human IL2R beta-gamma Fc was passed over the ligand at 30 μl / min for 240 s to record the association phase. The dissociation phase was monitored for 120 or 600 s and triggered by switching from the sample solution to HBS-EP+. The chip surface was regenerated after each cycle using a single 30-s injection of 10 mM glycine pH 2.1. Bulk refractive index differences were corrected by subtracting the response obtained with reference flow cell 1. Affinity constants were derived from the kinetic rate constants by fitting to 1:1 Langmuir binding using Biaeval software (Cytiva).

[0281] sample The following samples were analyzed for binding to human IL2R beta-gamma-Fc (Table 4). [Table 4]

[0282] result Affinity determination of 14 FAP-IL2v variants to recombinant human IL2R beta-gamma-Fc heterodimer Affinity measurements for human IL2R beta-gamma showed very slow off-rates reaching the limits of the instrument, resulting in unrealistic KDs. However, the decrease in affinity can still be assessed (Table 5). [Table 5]

[0283] conclusion FAP-IL2v variants were purified and their affinity for IL2R beta-gamma-Fc was measured. The D20T, Q126T, and N88T variants showed reduced affinity for human IL2R beta-gamma-Fc. The double mutation L12A / L19A showed slightly reduced affinity for human IL2R beta-gamma-Fc.

[0284] Example 1D. Characterization of PD1-IL2v variants Binding assessment of PD1-IL2v variants from supernatants to recombinant human IL2R beta-gamma-Fc heterodimer setting Equipment:Biacore T200 Chip: CM5 (number 593) Fc1-4: Anti-human Fab specific (Cytiva 28-9583-25) Capture: The supernatant was captured until it reached approximately 200 RU. Analyte: Human IL2R beta-gamma Fc (P1AD7029-002) Running buffer: HBS-EP Temperature: 25℃ Dilution: Single injection at 300 nM Flow: 30 μl / min Join: 120 seconds Dissociation: 120 seconds Regeneration: 10 mM glycine pH 2.1, 2 x 30 sec

[0285] Surface plasmon resonance (SPR) experiments were performed on a Biacore T200 using HBS-EP+ as the running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005% surfactant P20 (BR-1006-69, Cytiva)). Anti-human Fc-specific antibodies (Cytiva 28-9583-25) were directly immobilized on a CM5 chip (Cytiva) by amine coupling. PD1-IL2v variants were captured from the supernatant until they reached approximately 200 RU. A single injection of 300 nM recombinant human IL2R beta-gamma Fc was passed over the ligand at 30 μl / min for 120 s to record the association phase. The dissociation phase was monitored for 120 s and triggered by switching from the sample solution to HBS-EP+. The chip surface was regenerated after each cycle using two 30-second injections of 10 mM glycine, pH 2.1. Bulk refractive index differences were corrected by subtracting the response obtained with reference flow cell 1. The ratio of response units after binding compared to response units after capture was calculated. Additionally, binding curves were visually inspected to identify variants with faster dissociation rates.

[0286] Affinity of purified PD1-IL2v variants to recombinant human IL2R beta-gamma-Fc heterodimer Equipment:Biacore T200 Chip: CM5 (number 643) Fc1-4: Anti-human Fc specific (Roche (in-house)) Capture: 10 nM FAP-IL2v variant for 40 seconds Analyte: Human IL2R beta-gamma Fc (P1AD7029-002) Running buffer: HBS-EP Temperature: 25℃ Dilution ratio: 5-fold dilution in 3.2-2000 nM HBS-EP Flow: 30 μl / min Combine: 240 seconds Dissociation: 120 or 600 seconds Regeneration: 10 mM glycine pH 2.1, 30 seconds

[0287] Surface plasmon resonance (SPR) experiments were performed on a Biacore T200 using HBS-EP+ as the running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005% surfactant P20 (BR-1006-69, Cytiva)). Anti-human Fc-specific antibodies (in-house manufactured by Roche) were directly immobilized on a CM5 chip (Cytiva) by amine coupling. PD1-IL2v variants were captured at 10 nM for 40 s. A 5-fold diluted serum sample of human IL2R beta-gamma Fc was passed over the ligand at 30 μl / min for 240 s to record the association phase. The dissociation phase was monitored for 120 or 600 s and triggered by switching from the sample solution to HBS-EP+. The chip surface was regenerated after each cycle using a single 30-s injection of 10 mM glycine pH 2.1. Bulk refractive index differences were corrected by subtracting the response obtained with reference flow cell 1. Affinity constants were derived from the kinetic rate constants by fitting to 1:1 Langmuir binding using Biaeval software (Cytiva).

[0288] sample The following samples were analyzed for binding to human IL2R beta-gamma-Fc (Table 6). [Table 6]

[0289] result Binding assessment of PD1-IL2v variants from supernatants to recombinant human IL2R beta-gamma-Fc heterodimer The resonance units after capture and after binding were recorded and the ratio was calculated and compared to IL2v without further mutations (Table 7). [Table 7]

[0290] Affinity determination of PD1-IL2v variants to recombinant human IL2R beta-gamma-Fc heterodimer Affinity measurements for human IL2R beta-gamma showed very slow off-rates that reached the limits of the instrument, giving unrealistic KDs. However, the decrease in affinity can still be assessed (Table 8). [Table 8]

[0291] conclusion Twelve variants of IL2v were tested from supernatant to identify candidates with reduced binding to IL2R beta-gamma-Fc: six variants behaved as the parent IL2v (K8S, L12A, L19V, H79S, L80A, D109A), two variants failed to express properly (Q126N, Q126E), one variant had slower binding and faster dissociation than IL2v (N88Q), two variants had slower binding than IL2v (N88A, L12V), and one variant lost binding (T51P).

[0292] Nine variants were purified and their affinity for IL2R beta-gamma-Fc was measured. The N88A and N88Q variants were confirmed to have reduced affinity for human IL2R beta-gamma-Fc. L12V and L19V behaved very similarly to the parent IL2v, although their affinity for human IL2R beta-gamma-Fc may be slightly reduced.

[0293] Example 1E. Design of a mouse surrogate for a mouse PD1-targeted IL2v immunoconjugate To facilitate in vivo efficacy studies in mouse models of cancer, we generated mouse surrogates of PD1-targeted IL2v immunoconjugates targeting mouse PD1. To reduce immunogenicity, all constant antibody domains in these constructs correspond to mouse sequences. In contrast, the V domain sequences of the anti-mouse PD1 antibody are derived from rats. Due to the cross-reactivity of human IL2v with the mouse IL2 receptor, human IL2v was used in both constructs.

[0294] These murine surrogate constructs bivalently bind to mouse PD1 via the N-terminal Fab arms on the Fc DD- and Fc KK+ chains, with the Fc DD- chain additionally bearing either an IL2v (P1AG9991 shown in Figure 1A) or Q126T mutein (P1AG8304 shown in Figure 1B) at its C-terminus. Heterodimerization was achieved by adding complementary charges to the mouse IgG1 CH3 domain (Fc DD- and Fc KK+ chains, respectively), and binding to activating Fcγ receptors and complement component C1q was abrogated by introducing a DA PG mutation into the mouse IgG1 CH2 Fc domain of the antibody. These immunoconjugates are shown schematically in Figures 1A and B.

[0295] Example 1F. Generation and Purification of a Mouse Surrogate of a Mouse PD1-Targeted IL2v Immunoconjugate A mouse surrogate of the PD1-targeted IL2v immunoconjugate was generated and purified at WuXi Biologics. They were transiently expressed in either HEK293 (P1AG9991, expression system "Transient2.0") or CHO (P1AG8304, expression system "Transient2.5") cells and purified by a two-column DSP process: 1. MabSelectSuRe LX affinity chromatography (equilibration and first wash: 50 mM Tris-HCl, 150 mM NaCl, pH 7.4; second wash: 50 mM Tris-HCl, 150 mM NaCl, pH 7.4, 0.1% Triton 100 / 114; elution: 100 mM Arg, 140 mM NaCl, pH 3.4; neutralization: 1 M Arg, pH 9.1); and 2. Superdex200 size exclusion chromatography (equilibration and formulation buffer: 20 mM histidine-HCl, 140 mM NaCl, pH 6.0).

[0296] For P1AG9991, protein purity was determined by SEC-HPLC (99.3% monomer peak), non-reduced CE-SDS (97.8% main peak), and reduced CE-SDS (99.9% total peaks for all three distinct chains), and protein identity was confirmed by LC-MS. The endotoxin level was determined to be 0.34 EU / mg, and the final concentration was 1.1 mg / mL. For P1AG8304, protein purity was determined by SEC-HPLC (99.4% monomer peak), non-reduced CE-SDS (98.8% main peak), and reduced CE-SDS (97.2% total peaks for all three distinct chains), and protein identity was confirmed by LC-MS. The endotoxin level was determined to be 0.2 EU / mg, and the final concentration was 2.1 mg / mL.

[0297] Example 1G. Design of a bispecific human PD1- / LAG3-targeted IL2v immunoconjugate We generated bispecific human PD1- / LAG3-targeted IL2v immunoconjugates for checkpoint inhibition targeting not only PD1 but also LAG3. These immunoconjugates monovalently bind human PD1 and human LAG3 via the N-terminal Fab arm. To avoid mispairing of light chains, the V domains of the human PD1 binder were cross-linked, while charge complementarity was introduced into the CH1 and CK domains of the human LAG3 Fab. Heterodimerization of the two heavy chains was achieved by applying knob-into-hole technology, and the introduction of the PG-LALA mutation in the CH2 Fc domain of the antibody abolished binding to activating Fcγ receptors and the complement component C1q. The HC knob chain additionally bears a C-terminal IL2v (P1AF4801) or its Q126T mutein (P1AF7951). For comparison, we also generated a monospecific human PD1 IgG1 PG-LALA antibody lacking the IL2v cytokine fusion. These immunoconjugates and PD1 IgG (P1AA6888) are shown schematically in Figures 2A, 2B, and 2C.

[0298] Example 1H. Generation and Purification of a Bispecific Human PD1- / LAG3-Targeted IL2v Immunoconjugate The production and purification of P1AF4801 was outsourced to Proteros Biostructures GmbH, Martinsried, Germany. HEK293F cells were transiently transfected, and the antibody was purified by affinity chromatography (MabSelect Sure) and preparative size-exclusion chromatography. Protein purity was determined by SEC-HPLC (monomer peak >94.2%) and non-reducing CE-SDS (main peak >88%), and protein identity was confirmed by LC-MS. Endotoxin levels were determined to be <0.5 EU / mg, and the final concentration was 2.96 mg / mL. The production and purification of P1AF7951 was performed at Roche, Zurich, Switzerland. HEK293 cells were transiently transfected, and the antibody was purified by affinity chromatography (MabSelect Sure), cation exchange chromatography (PorosXS), and preparative size-exclusion chromatography. Protein purity was determined by SEC-HPLC (monomer peak >99.1%) and non-reducing CE-SDS (main peak >98.7%), and protein identity was confirmed by LC-MS. Endotoxin levels were determined to be ≤0.07 EU / mg, and the final concentration was 3.51 mg / mL. Production and purification of P1AA6888 was performed at Roche, Penzberg, Germany. HEK Expi293F cells were transiently transfected, and the antibody was purified by affinity chromatography (MabSelect Sure) and preparative size-exclusion chromatography (Superdex 200). Protein purity was determined by SEC-HPLC (monomer peak 100%) and non-reducing CE-SDS (main peak 98.1%). Endotoxin levels were determined to be <0.16 EU / mL, and the final concentration was 5.5 mg / mL.

[0299] Example 2. Selection of IL2v Q126T Example 2A. Expansion of NK92 cells with PD1-IL2v variants We evaluated the proliferation of the NK cell line NK92 upon 3-day treatment with a set of nine newly designed IL2v variants containing single amino acid exchanges fused to the PD1 antibody and compared their activity with that of the parental PD1-IL2v molecule (Figure 3). The goal was to identify IL2v variants with reduced but still detectable activity against the IL2 receptor compared to IL2v in a non-targeted setting. Because variant N88Q completely lost its activity in inducing NK92 proliferation and variant N88A had minimal activity against the IL2 receptor, we discarded both variants for further evaluation. Variants L12V, H79S, and D190A retained activity similar to that of the parental IL2v and were therefore not further characterized. Variants K8S, L12A, L19V, and L80A showed at least a 2-fold reduction in activity against the IL2 receptor and were selected for more detailed characterization.

[0300] NK92 proliferation NK92 cells were harvested, counted, and assessed for viability. Cells were washed three times with PBS to remove residual IL2 and resuspended in IL2-free medium (RPMI 1640, 10% FCS, 1% glutamine). The washed NK92 cells were incubated in a cell incubator for 2 hours (IL2 starvation). After starvation, the cells were resuspended in fresh IL2-free medium to 200,000 cells / ml. 50 μl of the cell suspension was then transferred to each well of a 96-well cell-culture-treated flat-bottom plate and supplemented with 50 μl of diluted antibody (in IL2-free medium), Proleukin (final concentration 1.5 μg / ml), or medium (control wells) to reach a final volume of 100 μl per well. The plates were incubated in the incubator for 3 days.

[0301] After 3 days, the CellTiter-Glo (Promega) reagent and cell culture plates were equilibrated to room temperature. CellTiter-Glo solution was prepared as described in the manufacturer's instructions, and 100 μl of the solution was added to each well. After a 10-minute incubation, the remaining aggregates were resuspended with a pipette, and 150 μl of the mixture was transferred to a white flat-bottom plate. Luminescence was measured using a Tecan Spark 10M multimode reader.

[0302] Example 2B. PBMC proliferation and activation by PD1-IL2v variants We then tested four selected new PD1-IL2v variants (K8S, L12A, L19V, and L80A) for their activity against PBMCs and compared them with the parental PD1-IL2v and FAP-IL2v molecules. After 5 days of treatment with PD1-IL2v variants, PD1-IL2v, or FAP-IL2v, we measured the proliferation of CD8 T cells, CD4 T cells, and NK cells (Figure 4A-C) and the upregulation of CD25 as a marker of CD8 T cell, NK cell, and CD4 T cell activation (Figure 5A-C) by flow cytometry. The results obtained with NK92 cells could be confirmed in this experiment; all new variants tested showed reduced activity compared to the two parental IL2v molecules in inducing the proliferation and activation of CD8 T cells, CD4 T cells, and NK cells. However, the reduced activity observed with these variants was not considered sufficiently strong. Therefore, characterization of these variants was not extended, and additional variants were designed.

[0303] PBMC proliferation and activation Freshly isolated PBMCs from healthy donors were labeled with CFSE (5(6)-carboxyfluorescein diacetate N-succinimidyl ester, 21888, Sigma-Aldrich). Briefly, PBMCs were washed once with PBS. In parallel, CFSE stock solution (2 mM in DMSO) was diluted 1:20 in PBS. PBMCs were resuspended at 1 μl / ml in prewarmed PBS, and 1 μl of CFSE solution was added to 1 ml of cell suspension, followed by immediate mixing. For optimal labeling, cells were incubated at 37°C for 15 min. The labeling reaction was then stopped by adding 10 ml of prewarmed medium (RPMI 1640, 10% FCS, 1% glutamine). Cells were spun down at 400 g for 10 min, resuspended in 1 μl / ml fresh medium, and incubated at 37°C for an additional 30 min. Finally, cells were washed once with medium, resuspended in fresh medium, and either used directly or stored overnight in an incubator. Labeled PBMCs were seeded into 96-well round-bottom plates (100,000 cells / well) and treated with the indicated molecules for 5 days. After incubation, cells were washed once with FACS buffer and stained with 20 μl of a mixture of anti-human CD3 APC-Cy7 (300318, BioLegend), anti-human CD8 APC (344722, BioLegend), and anti-human CD56 BV421 (318328, BioLegend) in FACS buffer for 30 minutes at 4°C. PBMCs were then washed twice with FACS buffer, fixed with 1% PFA in FACS buffer, and fluorescence was measured using a BD Fortessa. Proliferation was determined by measuring CFSE dilution of CD8 T cells (CD3+CD8+), CD4 T cells (CD3+CD8-), and NK cells (CD3-CD56+), and activation was determined by Cd25 upregulation on CD8 T cells, CD4 T cells, and NK cells.

[0304] Example 2C. NK92 Cell Expansion with Newly Designed FAP-IL2v Variants Combinations of new IL2v variants with previously tested variants were designed to achieve a greater reduction in activity against the IL2 receptor compared to previously tested IL2v variants. We tested the variants in the FAP-IL2v format and compared their activity with the parent FAP-IL2v molecule. In a first step, we tested the proliferation induction of the variants using NK92 cells (Figures 6A-D). Proliferation was measured after 3 days of treatment with the IL2v variants. The IL2v variants D20T_Selectikine, L12A_L19A, L12A_L80A, N88T, and Q126T had the strongest reduction in activity compared to the parent IL2v. We selected these variants plus two variants, Q22A and S130A, and tested them for their ability to induce proliferation and activation of CD8 T cells and NK cells in PBMCs. Proliferation (Figures 7A-B) and CD25 upregulation as a marker of immune cell activation (Figures 8A-B) were measured 4 days after treatment. Again, the results obtained with NK92 cells could be confirmed with PBMCs. Variants D20T_Selectikine and N88T had the strongest reduction in activity, which was considered too strong, and these variants were not evaluated further. Other test variants were evaluated in additional experiments.

[0305] NK92 proliferation NK92 cells were harvested, counted, and assessed for viability. Cells were washed three times with PBS to remove residual IL2 and resuspended in IL2-free medium (RPMI 1640, 10% FCS, 1% glutamine). The washed NK92 cells were incubated in a cell incubator for 2 hours (IL2 starvation). After starvation, the cells were resuspended in fresh IL2-free medium to 200,000 cells / ml. 50 μl of the cell suspension was then transferred to each well of a 96-well cell-culture-treated flat-bottom plate and supplemented with 50 μl of diluted antibody (in IL2-free medium), Proleukin (final concentration 1.5 μg / ml), or medium (control wells) to reach a final volume of 100 μl per well. The plates were incubated in the incubator for 3 days.

[0306] After 3 days, the CellTiter-Glo (Promega) reagent and cell culture plates were equilibrated to room temperature. CellTiter-Glo solution was prepared as described in the manufacturer's instructions, and 100 μl of the solution was added to each well. After a 10-minute incubation, the remaining aggregates were resuspended with a pipette, and 150 μl of the mixture was transferred to a white flat-bottom plate. Luminescence was measured using a Tecan Spark 10M multimode reader.

[0307] PBMC proliferation and activation Freshly isolated PBMCs from healthy donors were labeled with the cell proliferation dye eFluor 670 (65-0840-85, BioLegend). Briefly, PBMCs were washed twice with PBS and resuspended in PBS to a final concentration of 10 μM cells / ml. In parallel, a 10 μM solution of Cell Proliferation Dye eFluor 670 was prepared by diluting the stock solution (5 mM) in pre-warmed PBS. PBMCs were mixed, and pre-diluted Cell Proliferation Dye was added at a 1:1 ratio to obtain a final concentration of 5 μM. PBMCs were incubated at 37°C for 10 min. The labeling reaction was then stopped by adding four volumes of cold medium, and the cells were washed three times with medium and resuspended in fresh medium at 1 million cells / ml. The labeled PBMCs were seeded into 96-well round-bottom plates (100,000 cells / well) and treated with the indicated molecules for 4 days. After incubation, cells were washed twice with PBS and stained with 50 μl / well of reconstituted fluorescently reactive Live / Dead dye (L34976, Invitrogen) and incubated at room temperature for 20 minutes. Then, 150 μl of FACS buffer was added per well, and the plate was centrifuged at 400×g for 4 minutes. The supernatant was removed, and cells were stained with 50 μl of a mixture of CD3 PE-Cy5 (555341, BD Bioscience), CD8 BV711 (301044, BioLegend), CD25 PE-Dazzle 594 (356126, BioLegend), and CD56 BV421 (318328, BioLegend) in FACS buffer for 30 minutes at 4°C. PBMCs were then washed twice with FACS buffer, fixed with 1% PFA in FACS buffer, and fluorescence was measured on a BD flow cytometer. Proliferation was determined by measuring proliferation dye dilution of CD8 T cells (CD3+CD8+) and NK cells (CD3-CD56+) and T cell activation by upregulation of CD25 on the respective cells.

[0308] Example 2D. Proliferation and activation of PBMCs by selected FAP-IL2v variants We then tested the newly selected FAP-IL2v variants on PBMCs and measured proliferation and activation of CD8 T cells, CD4 T cells, and NK cells, as well as STAT5 phosphorylation in CD8 T cells, CD4 T cells, regulatory T cells, and NK cells, and compared the activity with that of the parent FAP-IL2v.

[0309] PBMCs were treated with selected FAP-IL2v variants and the parental FAP-IL2v for 5 days, and immune cell proliferation (Figure 9A-C) and activation (Figure 10A-C) were analyzed. As seen in previous studies, variants Q22A and S130A behaved similarly to the parental IL2v, variants L12A_L19A and L12A_L80A had approximately 10-fold reduced activity, and variant Q126T had over 20-fold reduced activity in inducing proliferation and immune cell activation compared to the parental FAP-IL2v. In a second experiment, PBMCs were treated with the same set of FAP-IL2v variants, and STAT5 phosphorylation was measured as a direct marker of IL2 receptor activation in CD4 T cells, regulatory T cells, CD8 T cells, and NK cells (Figure 11A-D). As previously mentioned, Q22A and S130A have similar activity to the parental IL2v. The variants L12A_L19A, L12A_L80A, and Q126T had reduced activity, with Q126T being slightly less active than the other two. The IL2v variant Q126T was selected as the most promising candidate among all the IL2v variants tested because its reduced activity compared to the parent FAP-IL2v was within the range we were aiming for.

[0310] PBMC proliferation and activation Freshly isolated PBMCs from healthy donors were labeled with the cell proliferation dye eFluor 670 (65-0840-85, BioLegend). Briefly, PBMCs were washed twice with PBS and resuspended in PBS to a final concentration of 10 million cells / ml. In parallel, a 10 μM solution of Cell Proliferation Dye eFluor 670 was prepared by diluting the stock solution (5 mM) in pre-warmed PBS. PBMCs were mixed, and pre-diluted Cell Proliferation Dye was added at a 1:1 ratio to obtain a final concentration of 5 μM. PBMCs were incubated at 37°C for 10 min. The labeling reaction was then stopped by adding four volumes of cold medium, and the cells were washed three times with medium and resuspended in fresh medium at 1 million cells / ml. The labeled PBMCs were seeded into 96-well round-bottom plates (100,000 cells / well) and treated with the indicated molecules for 5 days. After incubation, cells were washed twice with PBS and stained with 50 μl / well of reconstituted fluorescently reactive Live / Dead dye (L34976, Invitrogen) and incubated at room temperature for 20 minutes. Then, 150 μl of FACS buffer was added per well, and the plate was centrifuged at 400×g for 4 minutes. The supernatant was removed, and cells were stained with 50 μl of a mixture of CD3 PE-Cy5 (555341, BD Bioscience), CD4 BV605 (317438, BioLegend), CD8 BV711 (301044, BioLegend), CD25 PE-Dazzle 594 (356126, BioLegend), and CD56 BV421 (318328, BioLegend) in FACS buffer for 30 minutes at 4°C. PBMCs were then washed twice with FACS buffer, fixed with 1% PFA in FACS buffer, and fluorescence was measured on a BD flow cytometer. Proliferation was determined by measuring proliferation dye dilution of CD8 T cells (CD3+CD8+), CD4 T cells (CD3+CD4+), and NK cells (CD3-CD56+), as well as T cell activation by upregulation of CD25 on each cell.

[0311] Example 2E. Activity of PD1-IL2v Q126T and FAP-IL2v Q126T In a next step, we compared PD1-IL2v Q126T and FAP-IL2v Q126T with FAP-IL2v to confirm the results obtained so far. PBMCs were treated with the three molecules for 5 days, and the proliferation (Figures 12A-C) and activation (Figures 13A-C) of CD8 T cells, NK cells, and CD4 T cells were determined. PD1-IL2v Q126T and FAP-IL2v Q126T had comparable activity on CD4 T cells, CD8 T cells, and NK cells, but their activity was significantly reduced compared to the parent FAP-IL2v.

[0312] PBMC proliferation and activation Freshly isolated PBMCs from healthy donors were labeled with the cell proliferation dye eFluor 670 (65-0840-85, BioLegend). Briefly, PBMCs were washed twice with PBS and resuspended in PBS to a final concentration of 10 mio cells / ml. In parallel, a 10 μM solution of Cell Proliferation Dye eFluor 670 was prepared by diluting the stock solution (5 mM) in pre-warmed PBS. PBMCs were mixed, and pre-diluted Cell Proliferation Dye was added at a 1:1 ratio to obtain a final concentration of 5 μM. PBMCs were incubated at 37°C for 10 min. The labeling reaction was then stopped by adding four volumes of cold medium, and the cells were washed three times with medium and resuspended in fresh medium at 1 million cells / ml. The labeled PBMCs were seeded into 96-well round-bottom plates (100,000 cells / well) and treated with the indicated molecules for 5 days. After incubation, cells were washed twice with PBS and stained with 50 μl / well of reconstituted fluorescently reactive Live / Dead dye (L34957, Invitrogen) and incubated at room temperature for 15–30 min. Then, 150 μl of FACS buffer was added per well, and the plate was centrifuged at 400×g for 4 min. The supernatant was removed, and cells were stained with 30 μl of a mixture of CD3 BUV359 (563546, BD Bioscience), CD4 PE (300508, BioLegend), CD8 FITC (344704, BioLegend), CD25 PE-Cy7 (302612, BioLegend), and CD56 BV421 (318328, BioLegend) in FACS buffer for 30 min at 4°C. PBMCs were then washed twice with FACS buffer and fluorescence was measured using a BD flow cytometer. Proliferation was determined by measuring proliferation dye dilution of CD8 T cells (CD3+CD8+), CD4 T cells (CD3+CD4+), and NK cells (CD3-CD56+) and T cell activation by upregulation of CD25 on each cell.

[0313] STAT5 phosphorylation Freshly isolated PBMCs from healthy donors were seeded in warm medium (RPMI 1640, 10% FCS, 2 mM glutamine) in 96-well round-bottom plates (200,000 cells / well). The plates were centrifuged at 300 g for 10 min, and the supernatant was removed. The cells were resuspended in 100 μl of medium containing IL2v molecules and stimulated at 37°C for 20 min. To preserve the phosphorylation state, the cells were immediately fixed after stimulation with an equal volume of prewarmed Cytofix buffer (554655, BD Bioscience) at 37°C for 10 min. The plates were then centrifuged at 350 g for 5 min, and the supernatant was removed. To enable intracellular staining, the cells were permeabilized in 100 μl of Phosflow Perm buffer III (558050, BD Bioscience) at 4°C for 30 min. The cells were then washed twice with 150 μl of cold FACS buffer and divided into two 96-well round-bottom plates. Each plate was stained with 20 μl of antibody mix I or II for 60 minutes in a refrigerator. Antibody mix I was used to stain pSTAT5 in CD4 T cells and regulatory T cells, and antibody mix II was used to stain pSTAT5 in CD8 T cells and NK cells. The cells were then washed twice with FACS buffer and resuspended in 200 μl of FACS buffer containing 2% PFA per well. Analysis was performed using a BD flow cytometer, gating on CD8 T cells (CD3+CD8+), NK cells (CD3-CD56+), CD4 T cells (CD4+), and Tregs (CD4+CD25+FoxP3+). [Table 9] [Table 10]

[0314] Example 3. PD1-IL2v Q126T and PD1-LAG3-IL2v Q126T Example 3A. Assay IL-2R signaling assay In one aspect, an assay is provided for determining the efficacy of a PD-1-IL-2v immunoconjugate (e.g., comprising at least one binding domain that binds PD-1 conjugated to an IL-2 polypeptide having additional mutations) to modulate cis / trans-signaling.

[0315] To this end, CD4 T cells from PBMCs of healthy donors were sorted using CD4 beads (Miltenyi, no. 130-045-101) and activated for 3 days in the presence of 1 μg / ml plate-bound anti-CD3 (overnight pre-coated, clone OKT3, no. 317315, BioLegend) and 1 μg / ml soluble anti-CD28 (clone CD28.2, no. 302923, BioLegend) antibodies to induce PD-1 expression. After 3 days, cells were harvested and washed several times to remove endogenous cytokines. Half of the cells were labeled with Cell Trace Violet (CTV) (5 μM, 5 min at room temperature (RT); C34557, Thermo Scientific), while the other half remained unlabeled.

[0316] Unlabeled cells were then incubated with a saturating concentration of a competing anti-PD-1 antibody (in-house molecule, 10 μg / ml) for 30 min at room temperature, followed by several washing steps to remove excess unbound anti-PD-1 antibody. PD-1 pre-blocked cells (25 μl, 6 × 10 6 cells / ml) in a V-bottom plate containing PPD-1 + CTV-labeled cells (25 μl, 6 x 10 6After 1:1 co-culture with 1:1 STAT5 (cells / ml), cells were treated with increasing concentrations of the therapeutic immunoconjugate (50 μl, 1:10 dilution steps) for 12 min at 37°C. To preserve the phosphorylation status, an equal volume of Phosphoflow Fix Buffer I (100 μl, 557870, BD Bioscience) was added after the 12 min incubation with the various constructs. Cells were then incubated for an additional 30 min at 37°C before being permeabilized overnight at -80°C with Phosphoflow PermBuffer III (558050, BD Bioscience). The following day, phosphorylated STAT-5 was stained for 30 min at 4°C using an anti-STAT-5P antibody (47 / Stat5(pY694) clone, 562076, BD Bioscience).

[0317] Cells were acquired using a fluorescence-activated cell sorting (FACS) BD-LSRFortessa (BD Bioscience) instrument. STAT-5P frequencies were determined using FlowJo (v10) and plotted using GraphPad Prism (v8).

[0318] PD-1 + Dose-response curves on T cells provide information about the efficacy of the evaluated molecules in signaling through IL-2R. Furthermore, dose-response curves on T cells pretreated with a competing anti-PD-1 antibody to prevent PD-1-mediated delivery demonstrate the efficacy of molecules in providing IL-2R signaling independent of PD-1 expression.

[0319] CMV-specific restimulation assay An in vitro assay has been developed to evaluate the effect of PD-1 targeting to deliver a mutant version of IL-2v to dysfunctional antigen-specific T cells in the setting of chronic viral infection. To circumvent the limitations on the number of donors suitable for this assay, we use the CMV immunogenic viral protein (pp65) as a retrieval antigen for T cells, where nearly 80% of the population is CMV serologically positive. Therefore, healthy human donor peripheral blood mononuclear cells (PBMCs) are stimulated with CMV-pp65 (Miltenyi, no. 130-093-435) in the presence of different constructs at a concentration of 0.6 nM. After 43 h, protein transport inhibitors (GolgiPlug) were added. (商標) No. 555029, BD Bioscience; and GolgiStop (商標) Protein transport from the Golgi is blocked by adding BD Bioscience (No. 554724) and subsequently incubating the cells at 37°C for an additional 5 hours. Cells were then washed and surface stained with anti-human CD3, CD4, CD8, CD62L, and CD45RO antibodies before fixation and permeabilization with FoxP3 Transcription Factor Staining Buffer Set (eBioscience). Finally, intracellular staining for IL-2, IFN-γ, and Ki67 (all from eBioscience) was performed to measure cytokine secretion and cell proliferation.

[0320] Suppression assay In one embodiment, the PD-1-IL-2v immunoconjugate inhibits conventional T cells (T conv ) effector function of regulatory T cells (T reg ) assays are provided to assess whether the suppression can be reversed. con v and T reg is isolated and labeled.

[0321] In certain embodiments, CD4 + CD25 + CD127 dim T regare isolated using a two-step Regulatory T cell Isolation Kit (Miltenyi, no. 130-094-775). In parallel, the negative fraction of CD25 positive selection (Miltenyi, no. 130-092-983) is collected and then CD4 + By enrichment (Miltenyi, no. 130-045-101), CD4 + CD25 - T conv Isolate T conv was labeled with carboxyfluorescein succinimidyl ester (CFSE, eBioscience, no. 65-0850-84) and T reg can be labeled with Cell Trace Violet (CTV, ThermoFisher scientific, C34557) to distinguish them and track the growth of both populations. conv and T reg CD4 from an unrelated donor - CD25 - They are cultured together for 5 days in the presence of PBMCs with or without treatment to provide specific stimulation.

[0322] In certain embodiments, cytokine accumulation within the Golgi complex is enhanced by applying Protein Transport Inhibitors (GolgiPlug™ No. 555029, BD Bioscience; and GolgiStop™ No. 554724, BD Bioscience) for 5 hours prior to FACS staining. reg T grown in the presence and absence of conv This assay measures the ability of T cells to secrete granzyme B (GrzB). reg Inhibition is calculated using the following formula:

number

[0323] Internalization assay In one embodiment, an assay is provided to assess the internalization of different immunoconjugates. To this end, PBMCs are isolated by density gradient centrifugation using Ficoll-Paque (Sigma-Aldrich). CD4 T cells are isolated from 10 8 Starting with PBMCs, bead-sort using a CD4 positive selection kit (Miltenyi, no. 130-045-101) according to the manufacturer's instructions. CD4 T cells are then cultured at 2-4 x 10 in RPMI 10% FBS in the presence of 1 µg / ml soluble anti-CD28 (clone CD28.2, no. 302923, BioLegend). 6 Cells were seeded at 1000 cells / well into 12-well plates pre-coated with 1 μg / ml anti-CD3 (overnight pre-coating, clone OKT3, no. 317315, BioLegend) and cultured at 37°C for 3 days.

[0324] CD4 T cells activated for 3 days were incubated in duplicate in FACS tubes in the presence of the immunoconjugate at 4°C for 30 minutes. The cells were then washed and divided into two groups; one was incubated for an additional 3 hours at 37°C, while the other was immediately stained with PE-labeled anti-PGLALA secondary antibody and anti-CD4 antibody (eBioscience) and then fixed with BD Cell Fix. After 3 hours of incubation, the second group of cells was also stained with PE-labeled anti-PGLALA secondary antibody and anti-CD4 before fixation. Cells were then acquired using an LSRFortessa (BD Biosciences), and data analysis was performed in FlowJo (V10) and plotted in GraphPad Prism (v8). The expression level of detectable antibody on the cell surface at 4°C was compared with that at 37°C to calculate the percentage of internalized molecules at 37°C. p was calculated using one-way ANOVA ( * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001). Example 3B. Activated PD-1 upon treatment with increasing doses of PD-1-IL-2v immunoconjugate + and PD-1 - IL-2R signaling (STAT5-P) on CD4 T cells

[0325] The efficacy and cis / trans-signaling of the PD-1-IL-2v immunoconjugate were assessed by measuring activated PD-1 expression (PD-1 + ) and PD-1 negative (PD-1 - IL-2R signaling was measured by treating (anti-PD-1 pretreated) CD4 T cells with increasing concentrations of the immunoconjugate. The objective was to determine the dependence of the PD-1-IL-2v immunoconjugate on PD-1 expression on T cells to deliver IL-2R signaling.

[0326] To this end, CD4 T cells from PBMCs of healthy donors were sorted using CD4 beads (product number 130-045-101, Miltenyi) and activated for 3 days in the presence of 1 μg / ml plate-bound anti-CD3 (overnight pre-coated, clone OKT3, product number 317315, BioLegend) and 1 μg / ml soluble anti-CD28 (clone CD28.2, product number 302923, BioLegend) antibodies to induce PD-1 expression. After 3 days, cells were harvested and washed several times to remove endogenous cytokines. Half of the cells were labeled with Cell Trace Violet (CTV) (5 μM, 5 min at room temperature (RT); product number C34557, Thermo Scientific), while the other half were left unlabeled.

[0327] The unlabeled cells were then incubated with a saturating concentration of a competing anti-PD-1 antibody (in-house molecule, 10 μg / ml) for 30 min at room temperature, followed by several washing steps to remove excess unbound anti-PD-1 antibody. The PD-1 pre-blocked unlabeled cells (25 μl, 6 × 10 6 cells / ml) in a V-bottom plate containing PPD-1 + CTV-labeled cells (25 μl, 6 x 10 6 After co-culture with PD-1 (cells / ml) at a 1:1 ratio, cells were treated with increasing concentrations of therapeutic immunoconjugates (50 μl, 1:10 dilution steps) for 12 min at 37°C. To preserve the phosphorylation status, an equal volume of Phosphoflow Fix Buffer I (100 μl, 557870, BD Bioscience) was added after the 12 min incubation with the various constructs to allow IL-2R signaling upon binding to PD-1. Cells were then fixed by incubation for an additional 30 min at 37°C and then permeabilized overnight at -80°C with Phosphoflow PermBuffer III (558050, BD Bioscience). The following day, phosphorylated forms of STAT-5 were stained for 30 min at 4°C using an anti-STAT-5P antibody (47 / Stat5(pY694) clone, 562076, BD Bioscience).

[0328] Cells were acquired using a flow cytometer (FACS) BD-LSR Fortessa (BD Bioscience) instrument. STAT-5P frequencies were determined using FlowJo (v10) and plotted using GraphPad Prism (v8).

[0329] The data in Figures 14A-G show that PD-1 + and PD-1 - Figure 1 shows the differential potency of selected PD1-IL2 variants in signaling through IL-2R on CD4 T cells. + Efficacy measurements in CD4 T cells are based on PD1 - Reflects PD1-mediated delivery of IL-2v versus PD1-independent delivery of IL-2v in CD4 T cells.

[0330] Table 11 shows the fold increase in STAT-5P EC50 between PD1-mediated and PD-1-independent delivery of IL-2v for each PD1-IL2v immunoconjugate molecule compared to the EC50 for PD-1 pre-blocked cells. + The EC50 values ​​were calculated by dividing the EC50 of the PD1-IL2v immunoconjugates by the EC50 of the T cells. This provides evidence for the strength of PD1-dependent delivery of IL2v for each IL2v mutant. Furthermore, the EC50 fold increase between various PD1-IL2v immunoconjugates and PD1-IL2v was calculated by dividing the EC50 of the new mutants by the EC50 of PD1-IL2v. This indicated a loss of efficacy of the PD1-IL2v immunoconjugates in signaling through the IL-2R due to their reduced affinity. [Table 11]

[0331] In this particular assay, several additional mutant immunoconjugates were shown to inhibit PD-1 + It has similar potency to PD1-IL2v in signaling through the IL-2R on T cells, but -On T cells, they show reduced activity, such as Q126T and L12A, which have 56.5-fold and 44.5-fold increased cis-activity, respectively. Others, such as N88D and N88Q, also show reduced activity on PD-1. + Although they have reduced activity in signaling through the IL-2R on T cells, others maintain the same characteristics of PD1-IL2v (Figures 14A-G). Table 11 shows the PD-1-IL2v clones obtained from four donors. + and PD-1 - The EC50 and area under the curve (AUC) of dose-response STAT-5 phosphorylation for each mutant on CD4 T cells are shown.

[0332] Example 3B. Activated PD-1 upon treatment with increasing doses of PD-1-IL-2v, FAP-IL2v, and NKG2D-IL2v immunoconjugates + IL-2R signaling (STAT5-P) on CD4 T cells In this experiment, we assessed the differential potency of signaling through IL-2R upon binding to PD-1 on PD-1-expressing CD4 T cells between PD1-IL2vQ126T, FAP-IL2vQ126T, and NKG2D-IL2vQ126T in a dose-dependent manner relative to PD1-IL2v and FAP-IL2v, using STAT5 phosphorylation as a readout.

[0333] To this end, CD4 T cells were sorted from PBMCs of healthy donors using CD4 beads (product number 130-045-101, Miltenyi) and activated for 3 days in the presence of 1 μg / ml plate-bound anti-CD3 (overnight pre-coated, clone OKT3, product number 317315, BioLegend) and 1 μg / ml soluble anti-CD28 (clone CD28.2, product number 302923, BioLegend) antibodies to induce PD-1 expression. After 3 days, cells were harvested and washed several times to remove endogenous IL-2. Cells (50 μl, 4*106 cells / ml) were seeded into V-bottom plates and then treated with increasing concentrations of therapeutic antibodies (50 μl, 1:10 dilution steps with a highest concentration of 66 nM) for 12 min at 37°C. To preserve the phosphorylation state, an equal volume of Phosphoflow Fix Buffer I (100 μl, BD product number 557870) was added immediately after the 12-minute incubation with the various constructs. Cells were then incubated for an additional 30 minutes at 37°C before being permeabilized overnight at 80°C with Phosphoflow PermBuffer III (BD product number 558050). The following day, phosphorylated STAT-5 was stained for 30 minutes at 4°C using anti-STAT-5P antibody (47 / Stat5(pY694) clone, BD product number 562076).

[0334] Cells were acquired using a FACS BD-LSR Fortessa (BD Bioscience). STAT-5P frequencies were determined using FlowJo (V10) and plotted using GraphPad Prism (V8).

[0335] The data in Figures 15A-B show that PD-1 + Figure 1 shows the differential potency of selected PD1-, FAP- and NKG2D-IL2 variants in CD4 T cells. + Efficacy measurements in CD4 T cells reflect PD1-mediated delivery of IL-2v versus PD1-independent delivery of FAP-IL-2v and FAP-IL2vQ126T.

[0336] Table 12 shows the PD-1 responses obtained from four donors. +The frequency and dose-response STAT-5 phosphorylation EC50 as mean fluorescence intensity (MFI) for IL-2v mutants on CD4 T cells are shown.

[0337] This experiment demonstrates PD-1-dependent and -independent delivery of IL-2 mutants to the IL-2R. The FAP-targeted antibody behaves like untargeted IL2v in this experiment due to the lack of FAP expression by activated CD4 T cells. Untargeted IL2vQ126T is 6.5-fold less potent than untargeted IL2v, whereas PD1-IL2vQ126T is only 1.5-fold less potent than PD1-IL2v. These findings support the PD1-IL2vQ126T-mediated PD1-mediated delivery of IL-2 mutants to the IL-2R. + While sharing similar IL-2R signaling on T cells, we predict fewer IL-2-mediated off-target effects. NKG2D-IL2vQ126T, like FAP-IL2vQ126T, has reduced potency against activated CD4 T cells (Figures 15A-B). [Table 12]

[0338] Example 3C. Expansion of CMV-specific CD4 T cell effector function upon treatment with PD1-IL2v immunoconjugates To evaluate the ability of PD-1-targeted and non-targeted IL-2v and IL-2vQ126T to expand antigen-specific CD4 T cell responses in the context of chronic viral infection, we used the CMV immunogenic viral protein (pp65) as a recall antigen. Therefore, healthy human donor peripheral blood mononuclear cells (PBMCs) were stimulated with CMV-pp65 (product number 130-093-435, Miltenyi) in the presence of different constructs at a concentration of 0.6 nM. After 43 h, protein transport from the Golgi was blocked by adding protein transport inhibitors (GolgiPlug™ product number 555029, BD Bioscience; and GolgiStop™ product number 554724, BD Bioscience) followed by incubating the cells at 37 °C for an additional 5 h. Cells were then washed and surface stained with anti-human CD3, CD4, CD8, CD62L, and CD45RO antibodies, followed by fixation and permeabilization with FoxP3 Transcription Factor Staining Buffer Set (eBioscience). Finally, intracellular staining for IL-2, IFN-γ, and Ki67 (all from eBioscience) was performed to measure cytokine production and cell proliferation.

[0339] Cells were acquired on a FACS BD-LSR Fortessa (BD Bioscience). The frequency of IFN-γ+ cells was determined with FlowJo (V10) and plotted with GraphPad Prism (v8). p is calculated using one-way ANOVA ( * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001).

[0340] This experiment shows that PD1-IL2v and PD1-IL2vQ126T increase the frequency of CMV-specific CD4 T cells compared to pp65 alone or pp65 in combination with FAP-IL2v and FAP-IL2vQ126T (Figure 16A). It also shows that PD1-IL2vQ126T increases the frequency of CMV-specific CD4 T cells secreting IFN-γ approximately five-fold 48 hours after restimulation, significantly better than pp65 stimulation alone (Figure 16B).

[0341] Table 13 shows the frequency of CMV-specific CD4 T cells upon restimulation with the CMV protein pp65 and the fold increase in frequency of CMV-specific CD4 T cells induced by the indicated treatments in combination with pp65. [Table 13]

[0342] Example 3D. T upon treatment with PD1-IL2v immunoconjugate reg T from suppression conv Rescue of effector function To assess the ability of conventional T cells (Tconv) to rescue Treg suppression, we established a suppressive function assay in which Tconv and Treg were cultured for 5 days with unrelated donor-derived CD4-CD25- cells for specific stimulation in the presence or absence of immunoconjugates.

[0343] In certain embodiments, CD4 + CD25 + CD127 dim T reg were isolated using a two-step Regulatory T cell Isolation Kit (Miltenyi, no. 130-094-775). In parallel, the negative fraction of CD25 positive selection (Miltenyi, no. 130-092-983) was collected and then isolated using a CD4 + By enrichment (Miltenyi, no. 130-045-101), CD4 + CD25 - Tconv was isolated. conv was labeled with carboxyfluorescein succinimidyl ester (CFSE, eBioscience, no. 65-0850-84) and T reg were labeled with Cell Trace Violet (CTV, ThermoFisher scientific, C34557) to distinguish them and to track the growth of both populations. conv and T reg CD4 from an unrelated donor - CD25 - They were cultured together for 5 days in the presence of PBMCs with or without treatment to provide specific stimulation.

[0344] In certain embodiments, cytokine accumulation within the Golgi complex is monitored by injecting Protein Transport Inhibitors (GolgiPlug) prior to FACS staining. (商標) No. 555029, BD Bioscience; and GolgiStop (商標) The cells were enhanced by applying 500 mg of erythrocyte stimulating factor (BST) (number 554724, BD Bioscience) for 5 hours. Cells were acquired on a FACS BD-LSR Fortessa (BD Bioscience). Data analysis was performed using FlowJo (V10) and plotted using GraphPad Prism (v8). T reg T grown in the presence and absence of conv The ability of T cells to secrete granzyme B (GrzB) was measured. reg Inhibition is calculated using the following formula:

number

[0345] Figure 17 shows the median and individual values ​​for 10 donors from an independent experiment, and Table 14 shows the median values.

[0346] The data in Figure 17 show that Tregs suppress 91% of granzyme B secretion by Tconv when left untreated. 0.6 nM PD1-IL2v and PD1-IL2vQ126T reduce the suppression to 25.6% and 13.6%, respectively, thus rescuing 74% and 86% of granzyme B secretion by Tconv from Treg suppression (Figure 4 and Table 14). 0.6 nM of the non-targeting versions, FAP-IL2v and FAP-IL2vQ126T, reduce the suppression to 87.6% and 92.7%, respectively, thus rescuing only 12.3% and 7.2% of granzyme B secretion (Figure 17 and Table 14). Further combination of non-targeting FAP-IL2v and FAP-IL2vQ126T with 66 nM of the parent blocking anti-PD-1 antibody slightly reduced Treg suppression to 69.2% and 84.5%, respectively, resulting in a 30.7% and 15.5% rescue of granzyme B secretion by T cells (Figure 17 and Table 14). [Table 14]

[0347] Example 3E. Activated PD-1 upon treatment with escalating doses of PD-1-(LAG-3)-IL-2v immunoconjugate + and PD-1 - IL-2R signaling (STAT5-P) on CD4 T cells The efficacy and cis / trans-signaling of PD-1-IL-2v, PD1-LAG3-IL2v, and FAP-IL2v immunoconjugates were assessed by immunohistochemistry using immunohistochemistry with activated PD-1 expression (PD-1 + ) and PD-1 negative (PD-1 - ) (anti-PD-1 pre-treated) CD4 T cells were treated with increasing concentrations of the immunoconjugate to measure IL-2R signaling. We aimed to determine the dependency of the PD-1-(LAG3)-IL-2v immunoconjugate on PD-1 and LAG-3 expression in T cells to deliver IL-2R signaling.

[0348] To this end, CD4 T cells from PBMCs of healthy donors were sorted using CD4 beads (product number 130-045-101, Miltenyi) and activated for 3 days in the presence of 1 μg / ml plate-bound anti-CD3 (overnight pre-coated, clone OKT3, product number 317315, BioLegend) and 1 μg / ml soluble anti-CD28 (clone CD28.2, product number 302923, BioLegend) antibodies to induce PD-1 expression. After 3 days, cells were harvested and washed several times to remove endogenous cytokines. Half of the cells were labeled with Cell Trace Violet (CTV) (5 μM, 5 min at room temperature (RT); product number C34557, Thermo Scientific), while the other half were left unlabeled.

[0349] The unlabeled cells were then incubated with saturating concentrations of competing anti-PD-1 and anti-LAG3 antibodies (in-house molecules, 10 μg / ml) for 30 min at room temperature, followed by several washing steps to remove excess unbound anti-PD-1 antibodies. The PD-1 pre-blocked unlabeled cells (25 μl, 6 × 10 6 cells / ml) in a V-bottom plate containing PPD-1 + CTV-labeled cells (25 μl, 6 x 10 6After co-culture with PD-1 (cells / ml) at a 1:1 ratio, cells were treated with increasing concentrations of therapeutic immunoconjugates (50 μl, 1:10 dilution steps) for 12 min at 37°C. To preserve the phosphorylation status, an equal volume of Phosphoflow Fix Buffer I (100 μl, 557870, BD Bioscience) was added after the 12 min incubation with the various constructs to allow IL-2R signaling upon binding to PD-1. Cells were then fixed by incubation for an additional 30 min at 37°C and then permeabilized overnight at -80°C with Phosphoflow PermBuffer III (558050, BD Bioscience). The following day, phosphorylated forms of STAT-5 were stained for 30 min at 4°C using an anti-STAT-5P antibody (47 / Stat5(pY694) clone, 562076, BD Bioscience).

[0350] Cells were acquired using a flow cytometer (FACS) BD-SymphonyA5 (BD Bioscience) instrument. STAT-5P frequencies were determined using FlowJo (v10) and plotted using GraphPad Prism (v8).

[0351] The data in Figure 18 show that PD-1 + ,LAG-3 + and PD-1 - ,LAG-3 - Figure 1 shows the differential efficacy of PD1-, LAG3-, and FAP-IL2v variants in signaling through the IL-2R on CD4 T cells. + ,LAG-3 + Efficacy measurements in CD4 T cells are based on PD1 - ,LAG-3 - Reflects PD1-(LAG-3)-mediated delivery of IL-2v versus PD1-(LAG-3)-independent delivery of IL-2v in CD4 T cells.

[0352] Table 15 shows the STAT-5P EC50 fold increase between PD-1-(LAG-3)-mediated and PD-1, LAG-3-independent delivery of IL-2v for each PD1-IL2v immunoconjugate molecule, as compared to the EC50 for PD1 (LAG-3) preblocked cells. + ,LAG-3 + The EC50 values ​​were calculated by dividing the EC50 of the PD1-IL2v immunoconjugates by the EC50 of the T cells. This provides evidence for the strength of PD-1-(LAG-3)-dependent delivery of IL2v for each IL2v mutant. Furthermore, the EC50-fold increase between various PD1-(LAG-3)-IL2v immunoconjugates and PD1-IL2v was calculated by dividing the EC50 of the new mutants by the EC50 of PD1-IL2v. This indicated a loss of efficacy of the PD1-IL2v immunoconjugates in signaling through the IL-2R due to their reduced affinity.

[0353] PD1-LAG-3-IL2v and PD1-LAG-3-IL2vQ126T were more potent than PD1-IL2v due to the avidity gain of PD-1 and LAG-3 co-targeting. + , LAG-3 + PD1-LAG3-IL2v and PD1-LAG3-IL2vQ126T have 126.6-fold and 6.5-fold higher potency against PD-1-, LAG 3- T cells, respectively. However, their potency against PD-1-, LAG 3- T cells is also increased (Figure 18 and Table 15). Despite this, the cis-activity window of PD1-LAG3-IL2v and PD1-LAG3-IL2vQ126T is 158- and 305-fold higher on PD-1- than on PD-1-LAG 3- T cells, respectively. + , LAG-3 + 1105 and 485-fold higher on T cells, respectively (Figure 18 and Table 15). [Table 15]

[0354] Example 3F. T upon treatment with PD-1-(LAG-3)-IL-2v immunoconjugate reg T from suppression convRescue of effector function To assess the ability of PD1-LAG3-IL2v and PD1-LAG3-IL2vQ126T to rescue Tconv from Treg suppression, Tconv and Treg were co-cultured with CD4-CD25- cells from unrelated donors for 5 days to establish a suppressive function assay that elicits specific stimulation in the presence or absence of immunoconjugates as previously described in Example 3D.

[0355] The data in Figure 19 show that Tregs suppress 88% of granzyme B secretion by Tconv when left untreated. 0.6 nM PD1-IL2v and PD1-IL2vQ126T not only reduced the suppression to -92.2% and -106.3%, respectively, thus rescuing Treg suppression, but also further enhanced granzyme B secretion by Tconv to 192% and 206%, respectively (Figure 19 and Table 16). 0.6 nM non-targeted FAP-IL2v reduced the suppression to 33%, thus rescuing 66.38% of granzyme B secretion (Figure 6 and Table 16). 0.6 nM PD1-LAG3-IL2v and PD1-LAG3-IL2vQ126T reduced the inhibition to -293.25 and -171-4, respectively, resulting in an increase in granzyme B secretion to 393% and 271.42% (Figure 19 and Table 16). Figure 6 shows the median and individual values ​​for 10 donors from an independent experiment, and Table 16 shows the median values. p is calculated using one-way ANOVA ( * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001). [Table 16]

[0356] Example 3G. Internalization of PD-1-(LAG-3)-IL-2v Immunoconjugates by Activated CD4 T Cells IL-2 bound to IL-2R induces the internalization of the IL-2 / IL2R complex, which may represent a sink for immunoconjugates affecting exposure. To this end, PBMCs were isolated by density gradient centrifugation using Ficoll-Paque (Sigma-Aldrich). CD4 T cells were isolated from 10 8 Starting with PBMCs, CD4 T cells were bead-sorted using a CD4 positive selection kit (Miltenyi, no. 130-045-101) according to the manufacturer's instructions. CD4 T cells were then cultured at 2–4 × 10 in RPMI 10% FBS in the presence of 1 μg / ml soluble anti-CD28 (clone CD28.2, no. 302923, BioLegend). 6 The cells were seeded at 1000 cells / well onto a 12-well plate pre-coated with 1 μg / ml anti-CD3 (overnight pre-coating, clone OKT3, no. 317315, BioLegend) and cultured at 37°C for 3 days.

[0357] CD4 T cells activated for 3 days were incubated in duplicate in FACS tubes in the presence of parental anti-PD-1, PD1-IL2v, PD1-LAG3-IL2v, FAP-IL2v, PD1-IL2vQ126T, PD1-LAG3-IL2vQ126T, or FAP-IL2vQ126T for 30 minutes at 4°C. The cells were then washed and divided into two groups. One group was incubated for an additional 3 hours at 37°C, while the other group was immediately stained with PE-labeled anti-PGLALA secondary antibody and anti-CD4 antibody (eBioscience) and then fixed with BD Cell Fix. After the 3-hour incubation, the second group of cells was also stained with PE-labeled anti-PGLALA secondary antibody and anti-CD4 antibody before fixation. Cells were then acquired using an LSRFortessa (BD Biosciences), and data analysis was performed using FlowJo (v10) and plotted using GraphPad Prism (v8). The expression level of detectable antibody on the cell surface at 4°C was compared with that at 37°C, and the frequency of positive cells at 37°C was subtracted from the frequency of positive cells at 4°C to calculate the percentage of internalized molecules at 37°C. p is calculated using one-way ANOVA ( * p<0.05,** p<0.01, *** p<0.001, **** p<0.0001).

[0358] The parental anti-PD1 antibody was used as a negative control for internalization because it remains in the extracellular portion of the cell membrane. 89.8% of FAP-IL2v and 76.5% of FAP-IL2vQ126T are internalized after 3 hours of incubation, followed by 71% of PD1-IL2v. Interestingly, due to its higher affinity for PD-1 and reduced affinity for IL-2R, only 47.4% of PD1-IL2vQ126T is internalized after 3 hours. Internalization was further reduced to 25% by targeting IL-2v to PD-1 and LAG-3 with PD1-LAG3-IL2v and remained unchanged (28%) by further reducing affinity for IL-2R with IL-2vQ126T (Figure 20 and Table 17). [Table 17]

[0359] Example 4 In vivo efficacy of murine surrogate PD1-IL2vQ126T immunoconjugate in syngeneic models of mouse tumor cell lines. - Panc02-Fluc subcutaneous syngeneic model The murine surrogate PD1-IL2vQ126T immunoconjugate was tested in the murine pancreatic cancer cell line Panc02-Fluc injected subcutaneously into Black 6-huIL2RBG transgenic mice.

[0360] Panc02-H7 cells (mouse pancreatic carcinoma) were initially obtained from MD Anderson Cancer Center (Texas, USA) and deposited in the Roche-Glycart internal cell bank after expansion. The Panc02-H7-Fluc cell line was generated in-house by calcium transfection and subcloning techniques. Panc02-H7-Fluc were cultured in RPMI medium containing 10% FCS (Sigma), 500 μg / ml hygromycin, and 1% Glutamax. Cells were cultured at 37°C in a water-saturated atmosphere with 5% CO2. Passage 14 was used for transplantation. Cell viability was 94.7%. 2 × 10 cells per animal were cultured. 5 Cells were injected subcutaneously into the flank of mice in 100 μl of RPMI cell culture medium (Gibco) using a 1 ml tuberculin syringe (BD Biosciences, Germany).

[0361] Female Black6-huIL2RBG transgenic mice (housed at Charles Rivers, Lyon, France), 7–8 weeks old at the start of the experiment, were maintained under specific pathogen-free conditions with a 12-h light / 12-h dark diurnal cycle according to the adopted guidelines (GV-Solas; Felasa; TierschG). After arrival, the mice were kept for one week for acclimatization and observation. Continuous health monitoring was performed at regular intervals.

[0362] Mice were randomly selected, weighed, and then injected into 2 x 10 5Panc02-Fluc cells were subcutaneously injected. 15 days after tumor cell injection (tumor volume >100 mm3), mice were intravenously injected with muPD1-IL2vQ126T variant, muPD-IL2v, or vehicle once a week for 3 weeks. All mice were intravenously injected with 200 μl of the appropriate solution. Mice in the vehicle group were injected with histidine buffer, while treatment groups received muPD1-IL2vQ126T variant at 2 mg / kg iv qw or muPD1-IL2v at 0.5 mg / kg iv qw for 3 weeks. Stock solutions were diluted with histidine buffer as needed to obtain the appropriate amount of immunoconjugate per 200 μl.

[0363] Figure 21 shows that the muPD1-IL2vQ126T variant mediated superior efficacy in tumor growth inhibition compared to the vehicle and muPD1-IL2v groups. Mice injected with the muPD1-IL2vQ126T variant tolerated the treatment well. [Table 18]

[0364] The foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, but the illustrations 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 entireties.

Claims

1. 1. An immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to PD-1, wherein the mutant IL-2 polypeptide is a human IL-2 molecule containing the amino acid substitutions F42A, Y45A, L72G, and Q126T (numbering relative to SEQ ID NO: 90 of the human IL-2 sequence).

2. 1. An immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to PD-1, wherein said mutant IL-2 polypeptide is a human IL-2 molecule containing amino acid substitutions F42A, Y45A, L72G, and Q126T (numbering relative to SEQ ID NO:90 of the human IL-2 sequence); and said antibody (a) a heavy chain variable region (VH) comprising: a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 74; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 75; and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 76; and (b) a light chain variable region (VL) comprising: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 77; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 78; and CDR-L3 comprising the amino acid sequence of SEQ ID NO:

79.

10. An immunoconjugate comprising:

3. an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to PD-1, wherein said mutant IL-2 polypeptide is a human IL-2 molecule comprising amino acid substitutions F42A, Y45A, L72G, and Q126T (numbering relative to SEQ ID NO: 90 of the human IL-2 sequence); an immunoconjugate, wherein the antibody comprises: (a) a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 80; and (b) a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

81.

4. The immunoconjugate of any one of claims 1 to 3, wherein the mutant IL-2 polypeptide further comprises the amino acid substitution T3A and / or the amino acid substitution C125A.

5. The immunoconjugate of any one of claims 1 to 4, wherein the mutant IL-2 polypeptide comprises the sequence of SEQ ID NO:

92.

6. The immunoconjugate of any one of claims 1 to 5, comprising no more than one mutant IL-2 polypeptide.

7. The immunoconjugate of any one of claims 1 to 6, wherein the antibody comprises an Fc domain composed of a first subunit and a second subunit.

8. The Fc domain is of the IgG class, particularly IgG １ The immunoconjugate of claim 7 which is an Fc domain of a subclass.

9. The immunoconjugate of claim 6 or 7, wherein the Fc domain is a human Fc domain.

10. The antibody is of the IgG class, particularly IgG １ The immunoconjugate of any one of claims 1 to 9, which is a subclass of immunoglobulin.

11. The immunoconjugate of any one of claims 7 to 10, wherein the Fc domain comprises a modification that promotes association of the first and second subunits of the Fc domain.

12. 12. The immunoconjugate of any one of claims 7 to 11, wherein an amino acid residue in the CH3 domain of the first subunit of the Fc domain has been replaced with an amino acid residue having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit that can be positioned within a cavity in the CH3 domain of the second subunit, and wherein an amino acid residue in the CH3 domain of the second subunit of the Fc domain has been replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit into which the protrusion in the CH3 domain of the first subunit can be positioned.

13. 13. The immunoconjugate of any one of claims 7 to 12, wherein in the first subunit of the Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V), and optionally the threonine residue at position 366 is replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A) (numbering according to the Kabat EU index).

14. 14. The immunoconjugate of claim 13, wherein the first subunit of the Fc domain further comprises a replacement of the serine residue at position 354 with a cysteine ​​residue (S354C) or a replacement of the glutamic acid residue at position 356 with a cysteine ​​residue (E356C), and the second subunit of the Fc domain further comprises a replacement of the tyrosine residue at position 349 with a cysteine ​​residue (Y349C) (Kabat EU index numbering).

15. 15. The immunoconjugate of any one of claims 7 to 14, wherein the mutant IL-2 polypeptide is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the subunits of the Fc domain, in particular the first subunit of the Fc domain, optionally via a linker peptide.

16. The immunoconjugate of claim 15, wherein the linker peptide has the amino acid sequence of SEQ ID NO:

93.

17. 16. The immunoconjugate of any one of claims 7 to 15, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors, in particular Fcγ receptors, and / or effector function, in particular antibody-dependent cell-mediated cytotoxicity (ADCC).

18. 18. The immunoconjugate of claim 17, wherein the one or more amino acid substitutions are at one or more positions selected from the group of L234, L235, and P329 (Kabat EU index numbering).

19. 19. The immunoconjugate of any one of claims 7 to 18, wherein each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering).

20. 20. The immunoconjugate of any one of claims 1 to 19, comprising a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:21, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:22, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO:

35.

21. Mutant IL-2 polypeptide and IgG linked by a linker sequence １ The immunoconjugate of any one of claims 1 to 20, consisting essentially of immunoglobulin molecules.

22. One or more isolated polynucleotides encoding the immunoconjugate of any one of claims 1 to 21.

23. 23. One or more vectors, particularly expression vectors, comprising the polynucleotide of claim 22.

24. 24. A host cell comprising the polynucleotide of claim 22 or the vector of claim 23.

25. 25. A method for producing an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to PD-1, the method comprising: (a) culturing the host cell of claim 24 under conditions suitable for expression of the immunoconjugate; and, optionally, (b) recovering the immunoconjugate.

26. 26. An immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to PD-1, produced by the method of claim 25.

27. A pharmaceutical composition comprising the immunoconjugate of any one of claims 1 to 21 or 26 and a pharmaceutically acceptable carrier.

28. An immunoconjugate according to any one of claims 1 to 21 or 26 for use as a medicament.

29. 27. The immunoconjugate of any one of claims 1 to 21 or 26 for use in the treatment of disease.

30. 30. The immunoconjugate for use in treating a disease according to claim 29, wherein the disease is cancer.

31. 27. Use of the immunoconjugate of any one of claims 1 to 21 or 26 in the manufacture of a medicament for the treatment of a disease.

32. 32. The use according to claim 31 , wherein the disease is cancer.

33. 27. A method of treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of a composition comprising the immunoconjugate of any one of claims 1 to 21 or 26 in a pharmaceutically acceptable form.

34. 34. The method of claim 33, wherein the disease is cancer.

35. 27. A method of stimulating the immune system of an individual, comprising administering to said individual an effective amount of a composition comprising the immunoconjugate of any one of claims 1 to 21 or 26 in a pharmaceutically acceptable form.

36. 10. The invention as hereinbefore described.

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