Immunoconjugates

Abstract

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

Description

Technical Field

[0001] The present invention generally relates to immunoconjugates, and in particular to immunoconjugates comprising a mutant interleukin-2 polypeptide and an antibody that binds to PD-1. Furthermore, 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 the mutant immunoconjugates, and uses thereof. Background Art

[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 production, survival, and homeostasis. It has a length of 133 amino acids and is composed of four antiparallel amphipathic α-helices that form a quaternary structure essential for its function (Smith, Science 240, 1169-76 (1988); Bazan, Science 257, 410-413 (1992)). Sequences of IL-2 from different species are found in NCBI RefSeq Nos. 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 is composed of up to three individual subunits, the different associations of which can produce receptor forms with different affinities for IL-2. c, CD132) subunits produce a high-affinity trimeric IL-2 receptor. The dimeric IL-2 receptor composed of β subunits and γ subunits is called the medium-affinity IL-2R receptor. The α subunit forms a low-affinity monomeric IL-2 receptor. Although the medium-affinity dimeric IL-2 receptor binds to IL-2 with an affinity about 100 times lower than the high-affinity trimeric receptor, both dimeric and trimeric IL-2 receptor variants are able to transmit signals when IL-2 binds (Minami et al., Annu Rev Immunol 11, 245-268 (1993)). Therefore, α-subunit CD25 is not essential for IL-2 signal transduction. It confers high-affinity binding to its receptor, while β subunit CD122 and γ subunits are crucial for signal transduction (Krieg et al., Proc Natl Acad Sci 107, 11906-11 (2010)). Trimeric IL-2 receptor containing CD25 is expressed by (resting) CD4 + FoxP3 + Regulatory T (T reg They are also transiently induced on conventionally activated T cells, whereas in the resting state, these cells express only dimeric IL-2 receptors. reg Cells consistently express the highest levels of CD25 in vivo (Fontenot et al., Nature Immunol 6, 1142-51 (2005)).

[0004] IL-2 is mainly produced by activated T cells, especially CD4 + Helps T cell synthesis. It 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, contributes to the proliferation and differentiation of B cells and the immunoglobulin synthesis of B cells, and stimulates the production, proliferation and activation of natural killer (NK) cells (e.g., reviewed in 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] Its ability to expand lymphocyte populations in vivo and increase the effector functions of these cells confers IL-2 its antitumor effects, making IL-2 immunotherapy an attractive treatment option for certain metastatic cancers. Consequently, high-dose IL-2 therapy has been approved for patients with metastatic renal cell carcinoma and malignant melanoma.

[0006] However, IL-2 has a dual function in immune responses as it not only mediates the expansion and activity of effector cells but also plays a key role in maintaining peripheral immune tolerance.

[0007] The main mechanism of peripheral self-tolerance is the activation-induced cell death (AICD) of IL-2-induced T cells. AICD is a process in which fully activated T cells undergo programmed cell death by engaging death receptors (such as CD95 (also referred to as Fas)) or TNF receptors expressed on the cell surface. When antigen-activated T cells expressing high-affinity IL-2 receptors (after previous exposure to IL-2) during proliferation are stimulated again by antigen via T cell receptor (T cellreceptor, TCR) / CD3 complexes, the expression of Fas ligand (Fas ligand, FasL) and / or tumor necrosis factor (tumor necrosis factor, TNF) is induced, making cells sensitive to Fas-mediated apoptosis. This process is IL-2-dependent (Lenardo, Nature 353, 858-61 (1991)) and mediated by STAT5. Through the process of AICD in T lymphocytes, tolerance can be established not only to self-antigens, but also to persistent antigens that are clearly not part of the host constitution, such as tumor antigens.

[0008] In addition, IL-2 is also involved in maintaining peripheral CD4 + CD25 + Regulatory T (T reg ) cells (Fontenot et al., Nature Immunol 6, 1142-51 (2005); D'Cruz and Klein, Nature Immunol 6, 1152-59 (2005); Maloy and Powrie, Nature Immunol 6, 1171-72 (2005)), which are also called suppressor T cells. They inhibit the destruction of their (self) targets by effector T cells by making cell-to-cell contacts with the help and activation of suppressor T cells or by releasing immunosuppressive cytokines such as IL-10 or TGF-β. T regDepletion of cells was shown to enhance IL-2-induced anti-tumor immunity (Imai et al., Cancer Sci 98, 416-23 (2007)).

[0009] Therefore, IL-2 is not optimal for inhibiting tumor growth because, in the presence of IL-2, generated CTLs may recognize the tumor as self and undergo AICD, or the immune response may be suppressed by IL-2-dependent T reg Cell inhibition.

[0010] Another problem relevant to IL-2 immunotherapy is the side effect produced by recombinant human IL-2 treatment. Patients receiving high-dose IL-2 treatment often experience serious cardiovascular, lung, kidney, liver, gastrointestinal, neurological, skin, blood and systemic adverse events, and the adverse events need to be closely monitored and hospitalized. Most of these side effects can be explained by the development of so-called vascular (or capillary) leak syndrome (vascular leak syndrome, VLS), which is that the pathological increase of vascular permeability causes fluid extravasation (causing for example lung and skin edema and liver cell damage) and intravascular fluid depletion (causing blood pressure to drop and heart rate compensatory increase) in multiple organs. Except eliminating IL-2, VLS does not have other Therapeutic Methods. Low-dose IL-2 regimen has been tested in patients to avoid VLS, but the cost is suboptimal treatment results. VLS is thought to be caused by the release of proinflammatory cytokines such as tumor necrosis factor (TNF)-α from IL-2-activated NK cells, however, IL-2-induced pulmonary edema has recently been shown to be caused by direct binding of IL-2 to lung endothelial cells, which express low to moderate levels of functional αβγ IL-2 receptors (Krieg et al., Proc Nat Acad Sci USA 107, 11906-11 (2010)).

[0011] Several methods have been taken to overcome these problems associated with IL-2 immunotherapy. For example, it has been found that the combination of IL-2 and some anti-IL-2 monoclonal antibodies enhances the in vivo therapeutic effect of IL-2 (Kamimura et al., J Immunol 177, 306-14 (2006); Boyman et al., Science 311, 1924-27 (2006)). In an alternative approach, IL-2 has been mutated in various ways to reduce its toxicity and / or enhance its efficacy. Hu et al. (Blood 101, 4853-4861 (2003), U.S. Patent Publication No. 2003 / 0124678) have replaced the arginine residue at position 38 of IL-2 with tryptophan to eliminate the vascular permeability activity of IL-2. Shanafelt et al. (Nature Biotechnol 18, 1197-1202 (2000)) have mutated asparagine 88 to arginine so that the selectivity of T cells is enhanced to exceed the selectivity of NK cells. Heaton et al. (Cancer Res 53, 2597-602 (1993); U.S. Pat. No. 5,229,109) have introduced two mutations, Arg38Ala and Phe42Lys, to reduce proinflammatory cytokine secretion by NK cells. Gillies et al. (U.S. Pat. Publication No. 2007 / 0036752) have replaced three residues of IL-2 (Asp20Thr, Asn88Arg and Gln126Asp) that contribute to the affinity for the medium-affinity IL-2 receptor to reduce VLS. Gillies et al. (WO 2008 / 0034473) have also mutated the interface of IL-2 with CD25 by amino acid substitutions, Arg38Trp and Phe42Lys, to reduce interaction with CD25 and T cells. reg For the same purpose, Wittrup et al. (WO 2009 / 061853) have generated IL-2 mutants that have enhanced CD25 affinity but do not activate the receptor, thus acting as antagonists. The introduced mutations are intended to disrupt the interaction with the β and / or γ subunits of the receptor.

[0012] In WO 2012 / 107417, a novel IL-2 immunotherapy designed to overcome the above-mentioned problems associated with IL-2 immunotherapy (toxicity induced by VLS, tumor tolerance induced by AICD, and T cell resistance) is described. regA specific mutant IL-2 polypeptide that inhibits immunosuppression caused by cell activation (e.g., immunosuppression caused by IL-2 cell activation) is described. The phenylalanine residue at position 42 of IL-2 is substituted with alanine, the tyrosine residue at position 45 is substituted with alanine, and the leucine residue at position 72 is substituted with glycine, thereby substantially eliminating the binding of the mutant IL-2 polypeptide to the α subunit of the IL-2 receptor (CD25). Liu et al. describe an engineered interleukin antagonist (Liu et al., J Immunother. 2009; 32(9): 887–894).

[0013] In addition to the above methods, IL-2 immunotherapy can be improved by selectively targeting IL-2 to tumors, for example in the form of immunoconjugates comprising 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 may be able to evade this targeting by shedding, mutating, or downregulating the antibody's target antigen. Furthermore, in a tumor microenvironment that actively depletes lymphocytes, tumor-targeted IL-2 may not optimally engage effector cells such as cytotoxic T lymphocytes (CTLs).

[0015] Therefore, further improvements in IL-2 immunotherapy are still needed. One approach that could circumvent the problem of tumor targeting is to target IL-2 directly to effector cells, particularly CTLs.

[0016] Ghasemi et al. have described a fusion protein of IL-2 and NKG2D binding protein (Ghashemi et al., Nat Comm (2016) 7, 12878) for targeting IL-2 to cells carrying NKG2D, such as natural killer (NK) cells.

[0017] Programmed cell death protein 1 (PD-1 or CD279) is an inhibitory member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS and BTLA. PD-1 is a cell surface receptor and is 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). The structure of PD-1 is a monomeric type 1 transmembrane protein, which consists of an immunoglobulin variable-like extracellular domain and a cytoplasmic domain containing an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). Two ligands for PD-1, PD-L1 and PD-L2, have been identified and 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 not to other CD28 family members. PD-L1, a ligand for PD-1, 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 leads to a decrease in tumor-infiltrating lymphocytes, a decrease in T cell receptor-mediated proliferation, and immune evasion of cancer 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 cumulative 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 A1. Immunoconjugates that bind to PD-1 are described, for example, in WO 2018 / 184964 A1. Summary of the Invention

[0019] The present invention provides a novel approach for targeting a mutant form of IL-2, which has the advantageous property of directly targeting immune effector cells, such as cytotoxic T lymphocytes, rather than tumor cells. Targeting of immune effector cells is achieved by conjugating the mutant IL-2 molecule to an antibody that binds to PD-1.

[0020] The IL-2 mutants used in the present invention have been designed to overcome the problems associated with IL-2 immunotherapy, particularly toxicity induced by VLS, tumor tolerance induced by AICD, and tumor resistance induced by T cells. reg In addition to preventing tumors from escaping tumor targeting as described above, targeting the IL-2 mutant to immune effector cells can further increase the effectiveness of CTLs relative to immunosuppressive T cells. reg By using antibodies that bind to PD-1, the inhibition of T cell activity induced by the interaction of PD-1 with its ligand PD-L1 can be reversed, thereby further enhancing the immune response.

[0021] An IL-2 fusion protein comprising 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, the immunoconjugates of the present invention comprising antibodies that bind to PD-1 exhibited significantly superior in vivo anti-tumor efficacy compared to similar immunoconjugates targeting PD-L1 (see Example 4 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 amino acid substitutions F42A, Y45A, L72G and Q126T (numbered relative to the human IL-2 sequence SEQ ID NO: 90).

[0024] In another 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 amino acid substitutions F42A, Y45A, L72G, and Q126T (numbered relative to the human IL-2 sequence SEQ ID NO: 90); and wherein 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 present invention provides an immunoconjugate comprising an 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 (numbered relative to the human IL-2 sequence SEQ ID NO: 90); and wherein 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 according to the present invention, 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 amino acid sequence set forth in 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 comprising a first subunit and a second subunit. In some such embodiments, the Fc domain is an IgG class Fc domain, particularly an IgG1 subclass Fc domain, and / or a human Fc domain. In some embodiments, the antibody is an IgG class immunoglobulin, particularly an IgG1 subclass immunoglobulin.

[0027] In some embodiments, wherein the immunoconjugate comprises an Fc domain comprising a modification that promotes association of a first subunit and a second subunit of the Fc domain, in some embodiments, an amino acid residue in the CH3 domain of the first subunit of the Fc domain is substituted with an amino acid residue having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit, and the protrusion can be positioned in a cavity in the CH3 domain of the second subunit; and in the CH3 domain of the second subunit of the Fc domain, an amino acid residue is substituted with an amino acid residue having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit, and the protrusion in the CH3 domain of the first subunit can be positioned in the cavity. In some embodiments, 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) (numbered according to the Kabat EU index). In some such embodiments, in the first subunit of the Fc domain, in addition, the serine residue at position 354 is replaced by a cysteine ​​residue (S354C) or the glutamic acid residue at position 356 is replaced by a cysteine ​​residue (E356C), and in the second subunit of the Fc domain, in addition, the tyrosine residue at position 349 is replaced by a cysteine ​​residue (Y349C) (numbered according to the EU index of Kabat). In some embodiments, the mutant IL-2 polypeptide is fused at its amino-terminal amino acid to the carboxyl-terminal amino acid of one of the subunits of the Fc domain, particularly to the carboxyl-terminal amino acid of 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, wherein the immunoconjugate comprises an Fc domain, the Fc domain comprises one or more amino acid substitutions, the one or more amino acid substitutions reduce binding to Fc receptors, particularly Fc γ receptors, and / or reduce effector function, particularly antibody-dependent cell-mediated cytotoxicity (ADCC). In some such embodiments, the one or more amino acid substitutions are located at one or more positions of the group selected from 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, the immunoconjugates according to the invention comprise 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: 23 or 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. In some embodiments, the immunoconjugate consists essentially of a mutant IL-2 polypeptide and an IgG1 immunoglobulin molecule joined by a linker sequence.

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

[0031] Also provided is 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 the present invention under conditions suitable for expression of the immunoconjugate, and optionally (b) recovering the immunoconjugate. The present invention also provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to PD-1, the immunoconjugate being produced by the method.

[0032] The present invention also provides a pharmaceutical composition and a method for using the immunoconjugate of the present invention. The pharmaceutical composition comprises: the immunoconjugate of the present invention and a pharmaceutically acceptable carrier.

[0033] In particular, the present invention comprises an immunoconjugate according to the present invention for use as a medicament and for the treatment of a disease. In a particular embodiment, the disease is cancer.

[0034] The present invention also includes the use of an immunoconjugate according to the present invention in the preparation of a medicament for treating a disease. In a specific embodiment, the disease is cancer.

[0035] Also provided is a method of treating a disease in an individual, the method comprising administering to the individual a therapeutically effective amount of a composition comprising an immunoconjugate according to the present invention in a pharmaceutically acceptable form. In a specific embodiment, the disease is cancer.

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

[0037] definition

[0038] Unless otherwise defined below, the terms used herein are generally as used in the art.

[0039] Unless otherwise indicated, the term "interleukin-2" or "IL-2" as used herein refers to any natural IL-2 from any vertebrate source, including mammals such as primates (e.g., humans), and rodents (e.g., mice and rats). The term includes unprocessed IL-2 and any form of IL-2 produced by processing in cells. The term also encompasses naturally occurring IL-2 variants, such as splice variants or allelic variants. The amino acid sequence of an exemplary human IL-2 is shown in SEQ ID NO: 90. Unprocessed human IL-2 additionally comprises an N-terminal 20 amino acid signal peptide having an amino acid sequence shown in SEQ ID NO: 94, which is not present in mature IL-2 molecules.

[0040] As used herein, the term "IL-2 mutant" or "mutant IL-2 polypeptide" is intended to encompass any mutant form of the various forms of IL-2 molecules, including full-length IL-2, truncated forms of IL-2, and forms of IL-2 linked to another molecule, such as by fusion or chemical conjugation. When used with respect to IL-2, "full length" is intended to mean a mature, native-length IL-2 molecule. For example, full-length human IL-2 refers to a molecule having 133 amino acids (see, e.g., SEQ ID NO: 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. The mutation may involve 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.

[0041] The various forms of IL-2 are named herein with respect to the sequence shown in SEQ ID NO: 90. Various names may be used herein to indicate the same mutation. For example, a mutation at position 42 from phenylalanine to alanine may be designated as 42A, A42, A42A, A42B, A42C, A42D, A42E, A42F ... 42 , F42A or Phe42Ala.

[0042] As used herein, a "human IL-2 molecule" refers to 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 amino acid sequence set forth in SEQ ID NO: 90. Specifically, the sequence identity is at least about 95%, more specifically at least about 96%. In certain embodiments, the human IL-2 molecule is a full-length IL-2 molecule.

[0043] As used herein, the term "amino acid mutation" encompasses amino acid substitutions, deletions, insertions, and modifications. Any combination of substitutions, deletions, insertions, and modifications can be performed to obtain the final construct, provided that the final construct possesses the desired characteristics, such as reduced binding to CD25. Amino acid sequence deletions and insertions include amino-terminal and / or carboxyl-terminal deletions and insertions. 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. Non-conservative amino acid substitutions, i.e., replacement of one amino acid with another having different structural and / or chemical properties, are particularly preferred for the purpose of altering, for example, the binding characteristics of an IL-2 polypeptide. Preferred amino acid substitutions include replacement of a hydrophobic amino acid with a hydrophilic amino acid. Amino acid substitutions include replacement with non-naturally occurring amino acids or with naturally occurring amino acid derivatives of the twenty 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, and the like. It is contemplated that methods of altering amino acid side chain groups by methods other than genetic engineering, such as chemical modification, are also useful.

[0044] As used herein, a "wild-type" form of IL-2 is a form of IL-2 that is otherwise identical to a mutant IL-2 polypeptide, except that the wild-type form has a wild-type amino acid at each amino acid position of the mutant IL-2 polypeptide. For example, if the IL-2 mutant is 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 the IL-2 mutant is a fusion between IL-2 and another polypeptide encoded downstream of IL-2 (e.g., an antibody chain), the wild-type form of the IL-2 mutant is IL-2 having a wild-type amino acid sequence fused to the same downstream polypeptide. Furthermore, if the IL-2 mutant is a truncated form of IL-2 (a mutation or modified sequence within the non-truncated portion of IL-2), the wild-type form of the IL-2 mutant is a similarly truncated IL-2 having a wild-type sequence. For the purpose of comparing the IL-2 receptor binding affinity or biological activity of various forms of IL-2 mutants with the corresponding wild-type form of IL-2, the term wild-type encompasses forms of IL-2 that contain one or more amino acid mutations that do not affect IL-2 receptor binding compared to naturally occurring native IL-2, such as, for example, a substitution of cysteine ​​for alanine at a position corresponding to residue 125 of human IL-2. In some embodiments, wild-type IL-2 for purposes of the present invention comprises the amino acid substitution C125A. In certain embodiments according to the present invention, the wild-type IL-2 polypeptide compared to the mutant IL-2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:90. In other embodiments, the wild-type IL-2 polypeptide compared to the mutant IL-2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:95.

[0045] Unless otherwise indicated, the term "CD25" or "α subunit of the IL-2 receptor" as used herein refers to any native CD25 from any vertebrate source, including mammals such as primates (e.g., humans), and rodents (e.g., mice and rats). The term includes "full-length," unprocessed CD25, as well as any form of CD25 produced by processing in cells. The term also encompasses naturally occurring CD25 variants, such as splice variants or allelic variants. In certain embodiments, CD25 is human CD25. The amino acid sequence of human CD25 is found, for example, in UniProt Accession No. P01589 (version 185).

[0046] As used herein, the term "high affinity IL-2 receptor" refers to the heterotrimeric form of the IL-2 receptor, which is composed of the receptor gamma subunit (also known as the common cytokine receptor gamma subunit, gamma cor CD132, see UniProt accession number P14784 (version 192)), receptor β subunit (also known as CD122 or p70, see UniProt accession number P31785 (version 197)) and receptor α subunit (also known as CD25 or p55, see UniProt accession number P01589 (version 185)). In contrast, the term "intermediate affinity IL-2 receptor" refers to an IL-2 receptor that contains only γ and β subunits, but no α subunit (for review, see, e.g., Olejniczak and Kasprzak, Med Sci Monit 14, RA179-189 (2008)).

[0047] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antigen binding moiety and an antigen, or a receptor and its ligand). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (K D ) indicates that the dissociation constant is the dissociation rate constant and the association rate constant (k off and k on ). Thus, equivalent affinities can include different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by well-established methods known in the art, including those described herein. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0048] 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 instrumentation 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, for example, 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 another form of the receptor. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0049] "Regulatory T cells" or "T reg "Cells" are special types of CD4 T cells that can suppress the response of other T cells. + T cells. regThe cells are characterized by expressing the α subunit of the IL-2 receptor (CD25) and the transcription factor forkhead box P3 (FOXP3) (Sakaguchi, Annu Rev Immunol 22, 531-62 (2004)) and play a key role in the induction and maintenance of peripheral self-tolerance to antigens, including antigens expressed by tumors. reg Cells require IL-2 for their function and development as well as to induce their suppressive characteristics.

[0050] As used herein, the term "effector cell" refers to a population of lymphocytes that mediate the cytotoxic effects of IL-2. Effector cells include effector T cells, such as CD8 + Cytotoxic T cells, NK cells, lymphokine-activated killer (LAK) cells and macrophages / monocytes.

[0051] As used herein, the term "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 a signal sequence) / (SEQ ID NO: 97, protein with a signal sequence). See also UniProt accession number Q15116 (version 156). As used herein, "an antibody that binds to PD-1", "specifically binds to PD-1", "binds to PD-1" or "anti-PD-1 antibody" refers to an antibody that can bind to PD-1, particularly a PD-1 polypeptide expressed on the surface of a cell, with sufficient affinity so that the antibody can be used as a diagnostic and / or therapeutic agent targeting PD-1. In one embodiment, the extent of 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 using a biosensor system such as In certain embodiments, the antibody that binds to PD-1 has a binding affinity for 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 smaller, for example, from 10 -8 M to 10 -13 M, for example, from 10 -9 M to 10 -13In one embodiment, the extracellular domain (ECD) of human PD-1 (PD-1-ECD, see SEQ ID NO: 43) was used as an antigen, and the KD value of the binding affinity was determined by surface plasmon resonance assay.

[0052] "Specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific 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 an 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 immunoconjugates described herein specifically binds to PD-1.

[0053] As used herein, the term "polypeptide" refers to a molecule consisting of monomers (amino acids) linearly connected by amide bonds (also referred to as peptide bonds). The term "polypeptide" refers to any chain with two or more amino acids, rather than the specific length of the product. Therefore, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other terms used to refer to chains with two or more amino acids are included in the definition of "polypeptide," and the term "polypeptide" can be used in place of any of these terms, or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to post-expression modification products of the polypeptide, which include but are not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization with known protection / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. The polypeptide can be derived from a natural biological source or produced by recombinant technology, and is not necessarily translated from a specified nucleic acid sequence. It can be generated in any way, including by chemical synthesis. Polypeptides can have a definite three-dimensional structure, but they do not necessarily have such a 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 can adopt a large number of different conformations, are said to be unfolded.

[0054] An "isolated" polypeptide or variant or derivative thereof is intended to mean a polypeptide that is not in its native environment. No particular level of purification is 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 the 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.

[0055] "Percent (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the candidate sequence with the reference polypeptide sequence and introducing gaps (if necessary) to achieve the maximum percentage of sequence identity, and without considering any conservative substitutions as part of sequence identity. Alignments for determining percent amino acid sequence identity can be achieved in various ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full length of the compared sequences. However, for purposes herein, amino acid sequence identity values ​​are generated using the ggsearch program of the FASTA package, version 36.3.8c or higher, 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 a public server accessible at http: / / fasta.bioch.virginia.edu / fasta_www2 / index.cgi, using the ggsearch (global protein:protein) program and default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) to ensure global, rather than local, alignments. The percentage amino acid identity is given in the output alignment header.

[0056] The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), virally derived RNA, or plasmid DNA (pDNA). A polynucleotide may contain conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNA)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide.

[0057] "Isolated" nucleic acid molecules or polynucleotides refer to nucleic acid molecules, DNA or RNA, that have been removed from their natural environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered to be isolated for the purposes of the present invention. Additional examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially purified) polynucleotides in solution. Isolated polynucleotides include polynucleotide molecules that are contained in cells that normally contain polynucleotide molecules, but that are present extrachromosomally or at a chromosomal location different from their natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present invention, as well as positive and negative strand forms and double-stranded forms. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced by synthesis. In addition, polynucleotides or nucleic acids can be or can include regulatory elements, such as promoters, ribosome binding sites, or transcription terminators.

[0058] An "isolated polynucleotide (or nucleic acid) encoding [e.g., an immunoconjugate of the invention]" refers to one or more polynucleotide molecules encoding antibody heavy and light chains and / or IL-2 polypeptide (or fragments thereof), including such polynucleotide molecules in a single vector or separate vectors, as well as such nucleic acid molecules present at one or more locations in a host cell.

[0059] The term "expression cassette" refers to a polynucleotide produced by recombination or synthesis that has a series of specific nucleic acid elements that allow transcription of a specific nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassette comprises a polynucleotide sequence encoding an immunoconjugate of the present invention or a fragment thereof.

[0060] The term "vector" or "expression vector" refers to a DNA molecule used to introduce a specific gene operably associated therewith into a cell and to direct the expression of the specific gene in the cell. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of the host cell into which they have been 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 within the cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cell's transcription and / or translation machinery. In one embodiment, the expression vector of the present invention comprises an expression cassette comprising a polynucleotide sequence encoding the immunoconjugate of the present invention or a fragment thereof.

[0061] 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 primary transformed cells and progeny derived from the primary transformed cells, regardless of the number of passages. Progeny may not be completely identical to the nucleic acid content of the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected in the original transformed cells are included herein. Host cells are any type of cell system that can be used to produce the immunoconjugates of the present invention. Host cells include cultured cells, for example, mammalian cultured cells, such as HEK cells, CHO cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, as well as cells contained in transgenic animals, transgenic plants, or cultured plant or animal tissues, to name a few.

[0062] The term "antibody" exhibits antigen-binding activity and encompasses various antibody structures that exhibit antigen-binding activity, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments.

[0063] As used herein, the term "monoclonal antibody" refers to the antibody obtained from a substantially homogeneous antibody population, that is, except possible variant antibodies (for example, containing naturally occurring mutations or producing during the production of monoclonal antibody preparations, such variants are generally present in small amounts), the individual antibodies comprising the population have identity and / or combine identical epitopes. Contrary to the polyclonal antibody preparations typically comprising different antibodies for different determinants (epitopes), each monoclonal antibody in the monoclonal antibody preparation is directed to a single determinant on the antigen. Therefore, the modifier "monoclonal" represents that the feature of an antibody is obtained from a substantially homogeneous antibody population, and should not be construed as needing to produce an antibody by any ad hoc method. For example, the monoclonal antibody according to the present invention can be prepared by various techniques, including but not limited to hybridoma method, recombinant DNA method, phage display method, and the method utilizing a transgenic animal containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for the preparation of monoclonal antibodies are described herein.

[0064] "Isolated" antibodies are antibodies that have been separated from the components of their natural environment, i.e., antibodies that are not in their natural environment. No specific purification level is required. For example, isolated antibodies can be taken out from the natural or natural environment of the antibody. Recombinant antibodies expressed in host cells are considered to be separated for the purposes of the present invention, and natural or recombinant antibodies that have been separated, graded, or partially or substantially purified by any suitable technique are also considered to be separated for the purposes of the present invention. In this way, the immunoconjugates of the present invention are isolated. In certain embodiments, the antibodies are purified to a purity greater than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0065] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody that has a structure substantially similar to a native antibody structure.

[0066] An "antibody fragment" refers to a molecule other than an intact antibody that comprises 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.

[0067] The term "immunoglobulin molecule" refers to a protein with the structure of a naturally occurring antibody. For example, the IgG class immunoglobulin is a heterotetrameric glycoprotein of approximately 150,000 daltons, consisting of two light chains and two heavy chains bonded by disulfide bonds. From N-terminus to C-terminus, each heavy chain has a variable domain (VH) (also referred to as a variable heavy chain domain or a heavy chain variable region), followed by three constant domains (CH1, CH2, and CH3) (also referred to as a heavy chain constant region). Similarly, from N-terminus to C-terminus, each light chain has a variable domain (VL) (also referred to as a variable light chain domain or a light chain variable region), followed by a constant light chain (CL) domain (also referred to as a light chain constant region). The heavy chains of immunoglobulins can be assigned to one of five types: α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which are further divided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of immunoglobulins can be assigned to one of two types: kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. Immunoglobulins are essentially composed of two Fab molecules and an Fc domain connected by the immunoglobulin hinge region.

[0068] The term "antigen binding domain" refers to a portion of an antibody that includes a region that specifically binds to and is complementary to part or all of an antigen. The antigen binding domain can be provided by, for example, one or more antibody variable domains (also referred to as antibody variable regions). In particular, the antigen binding domain includes an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0069] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that participates in binding of an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th edition, WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity. As used herein, "Kabat numbering" in relation to variable region sequences refers to the numbering system proposed by Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0070] As used herein, the amino acid positions of all constant regions and constant domains of heavy and light chains are numbered according to the Kabat 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), and are referred to herein as "numbered according to Kabat" or "Kabat numbering." Specifically, the Kabat numbering system (see pages 647 to 660 of Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)) is used for the light chain constant domains CL of the kappa and lambda isotypes, and the Kabat EU index numbering system (see pages 661 to 723) is used for the heavy chain constant domains (CH1, hinge, CH2, and CH3), which is further clarified herein by being referred to in this context as "numbered according to the Kabat EU index."

[0071] As used herein, the term "hypervariable region" or "HVR" refers to the individual regions of an antibody variable domain that are hypervariable in sequence and determine antigen binding specificity, such as the "complementarity determining regions" ("CDRs").

[0072] Typically, an antibody comprises six CDRs; three in VH (CDR-H1, CDR-H2, CDR-H3) and three in VL (CDR-L1, CDR-L2, CDR-L3). CDRs are defined by a variety of methods / systems by those skilled in the art. These systems and / or definitions have been developed and improved 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 position of the structural loop region. The IMGT system is based on sequence variability and position 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 (e.g., abYsis: http: / / www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi) are available and known to those skilled in the art for analyzing antibody sequences and measuring CDRs.

[0073] Exemplary CDRs herein include (amino acid residue numbering according to the cited references, i.e., Chothia numbering as defined by Chothia and Contact, Kabat numbering as defined by Kabat, and IMGT numbering as defined by IMGT):

[0074] (a) the 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)) (“Chothia definition”);

[0075] (b) CDRs present 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”);

[0076] (c) antigenic contact points present at amino acid residues 30-36 (L1), 46-55 (L2), 89-96 (L3), 30-35 (H1), 47-58 (H2), and 93-101 (H3) (according to MacCallum et al., J. Mol. Biol. 262:732-745 (1996)) (“Contact Definition”); and

[0077] (d) CDRs present 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”).

[0078] Unless otherwise indicated, CDRs herein are determined according to the method described by Kabat et al. (supra). One skilled in the art will appreciate that CDR names may also be determined according to Chothia, supra, MacCallum, supra, Lefranc, supra, or any other scientifically accepted definition / system.

[0079] "Framework" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. The FR of a variable domain is typically composed of the following four FR domains: FR1, FR2, FR3, and FR4. Thus, HVR and FR sequences typically appear in the following order in VH (or VL): FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.

[0080] "Humanized" antibodies refer to antibodies comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In some aspects, a humanized antibody will comprise substantially all of at least one and generally two variable domains, wherein all or substantially all of the CDRs correspond to the CDRs of a non-human antibody, and all or substantially all of the FRs correspond to the FRs of a human antibody. A humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has undergone humanization.

[0081] A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human source utilizing a repertoire of human antibodies or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0082] The "class" of an antibody or immunoglobulin refers to the type of constant domain or region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these antibodies can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0083] The term "Fc domain" or "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the IgG heavy chain Fc regions may be slightly different, the human IgG heavy chain Fc region is generally defined as extending from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the antibody produced by the host cell can undergo post-translational cleavage of one or more (particularly one or two) amino acids from the heavy chain C-terminus. Therefore, the antibody produced by the host cell by expressing a specific nucleic acid molecule encoding a full-length heavy chain can include a full-length heavy chain, or the antibody can include a cleavage variant (also referred to herein as "cleaved variant heavy chain") of the full-length heavy chain. This may be a situation where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, according to the Kabat EU index numbering). Therefore, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (K447) of the Fc region may be present or absent. If not otherwise specified, the amino acid sequence of the heavy chain comprising the Fc domain (or the subunit of the Fc domain as defined herein) is expressed herein as not having a C-terminal glycine-lysine dipeptide. In one embodiment of the invention, the heavy chain comprising the subunit of the Fc domain as specified herein is included in the immunoconjugate according to the present invention, and the heavy chain comprises other C-terminal glycine-lysine dipeptide (G446 and K447, according to the EU index numbering of Kabat). In one embodiment of the invention, the heavy chain comprising the subunit of the Fc domain as specified herein is included in the immunoconjugate according to the present invention, and the heavy chain comprises other C-terminal glycine residues (G446, according to the EU index numbering of Kabat). The compositions of the present invention, such as pharmaceutical compositions as described herein, comprise immunoconjugate populations of the present invention. The immunoconjugate population can include molecules with full-length heavy chains and molecules with cleaved variant heavy chains. The immunoconjugate population can be composed of a mixture of molecules with full-length heavy chains and molecules with 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 an immunoconjugate comprising a heavy chain comprising a subunit of an Fc domain as specified herein and 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 composition comprising a population of immunoconjugates of the invention comprises an immunoconjugate comprising a heavy chain comprising a subunit of an Fc domain as specified herein and an additional C-terminal glycine residue (G446, numbered according to the EU index of Kabat). In one embodiment of the invention, such a composition comprises a population of immunoconjugates consisting 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 and an additional C-terminal glycine residue (G446, numbered according to the EU index of Kabat); and molecules comprising a heavy chain comprising a subunit of an Fc domain as specified herein and an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the EU index of Kabat). Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also known as the EU index), as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also above). As used herein, a "subunit" of an Fc domain refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide comprising the C-terminal constant region of an immunoglobulin heavy chain, which polypeptide is capable of stable self-association. For example, a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain.

[0084] "Modifications that promote the association of the first and second subunits of the Fc domain" are manipulations of the peptide backbone or post-translational modifications of the Fc domain subunits that reduce or prevent the association of a polypeptide comprising the Fc domain subunit with the same polypeptide to form a homodimer. As used herein, modifications that promote association particularly include separate modifications to each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) that are desired to associate, wherein the modifications complement each other to promote the association of the two Fc domain subunits. For example, modifications that promote association may alter the structure or charge of one or both of the Fc domain subunits so as to render their association sterically or electrostatically favorable, respectively. Thus, (hetero) dimerization occurs between a polypeptide comprising the first Fc domain subunit and a polypeptide comprising the second Fc domain subunit, which may be different in the sense that the additional components (e.g., antigen binding moieties) fused to each subunit are not identical. In some embodiments, modifications that promote association include amino acid mutations, particularly amino acid substitutions, in the Fc domain. In a specific embodiment, the association-promoting modifications comprise individual amino acid mutations, particularly amino acid substitutions, of each of the two subunits of the Fc domain.

[0085] When used in reference to antibodies, the term "effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptor), and B cell activation.

[0086] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that causes immune effector cells to lyse antibody-coated target cells. Target cells are cells that specifically bind to antibodies or derivatives thereof comprising an Fc region, and this specific binding is typically through the protein portion of the N-terminus of the Fc region. As used herein, the term "reduced ADCC" is defined as a reduction in the number of target cells lysed by the ADCC mechanism defined above within a given time at a given antibody concentration in the culture medium surrounding the target cells, and / or an increase in the antibody concentration necessary to achieve lysis of a given number of target cells within a given time by the ADCC mechanism in the culture medium surrounding the target cells. ADCC reduction is relative to ADCC mediated by the same antibody produced by the same type of host cell but not yet engineered using the same standard production, purification, formulation and storage methods (such methods are known to those skilled in the art). For example, the reduction in ADCC mediated by an antibody comprising an amino acid substitution that reduces ADCC in the Fc domain is relative to ADCC mediated by the same antibody without the amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, eg, PCT Publication No. WO 2006 / 082515 or PCT Publication No. WO 2012 / 130831).

[0087] "Activating Fc receptors" are Fc receptors that, upon engagement by the Fc domain of an antibody, initiate signaling events that stimulate cells bearing the receptor to perform effector functions. Human activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89).

[0088] As used herein, the terms "engineering, engineered, engineered" are considered to include any manipulation of the peptide backbone, or post-translational modification of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modifications to the amino acid sequence, glycosylation pattern, or side chain groups of individual amino acids, as well as combinations of these approaches.

[0089] "Decreased binding," e.g., reduced binding to an Fc receptor or CD25, refers to a decrease in affinity for the corresponding interaction, as measured, e.g., by SPR. For clarity, the term also encompasses a decrease in affinity to zero (or below the detection limit of the analytical method), i.e., complete elimination of the interaction. Conversely, "increased binding" refers to an increase in binding affinity for the corresponding interaction.

[0090] 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 through various interactions and in various configurations as described herein. In specific embodiments, the IL-2 molecule is fused to the antibody via a peptide linker. Specific immunoconjugates according to the present invention essentially consist of one (one) IL-2 molecule and one (one) antibody joined by one or more (one or more) linker sequences.

[0091] "Fusion" refers to the connection of the components (eg, an antibody and an IL-2 molecule) by peptide bonds, either directly or via one or more peptide linkers.

[0092] As used herein, the terms "first" and "second" with respect to Fc domain subunits, etc., are used for convenience in distinguishing when more than one type of moiety is present. Unless explicitly stated, the use of these terms is not intended to confer a particular order or orientation upon the immunoconjugates.

[0093] An "effective amount" of an agent is that amount required to produce a physiological change in the cells or tissues to which it is administered.

[0094] A "therapeutically effective amount" of an agent (e.g., a pharmaceutical composition) is an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or preventive result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents the adverse effects of a disease.

[0095] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.

[0096] The term "pharmaceutical composition" refers to a preparation that is in such form that the biological activity of the active ingredient contained therein is effective, and that contains no additional components that are unacceptably toxic to a subject to which the composition would be administered.

[0097] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical composition other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0098] As used herein, "treatment" (and grammatical variants thereof such as treat or treat) refers to an attempt to alter the natural course of the disease in the individual being treated, and may be performed for prevention or as a clinical intervention during clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, diminishing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the immunoconjugates of the invention are used to delay the development of the disease or slow the progression of the disease.

[0099] Detailed description of the embodiment

[0100] Mutant IL-2 polypeptide

[0101] The immunoconjugates according to the present invention comprise mutant IL-2 polypeptides having properties that are advantageous for immunotherapy. Specifically, the pharmacological properties of IL-2 that contribute to toxicity but are not essential for the efficacy of IL-2 are eliminated in the mutant IL-2 polypeptides. Such mutant IL-2 polypeptides are described in detail in WO 2012 / 107417, which is incorporated herein by reference in its entirety. As described above, different forms of IL-2 receptors are composed of different subunits and exhibit different IL-2 affinities. The medium-affinity IL-2 receptor, composed of the β and γ receptor subunits, is expressed on resting effector cells and is sufficient for IL-2 signaling. The high-affinity IL-2 receptor, which also comprises the α subunit of the receptor, is expressed primarily on regulatory T cells (Tregs). reg ) cells and activated effector cells, where the engagement of this receptor with IL-2 can promote T reg Cell-mediated immunosuppression or activation-induced cell death (AICD). Thus, without being bound by theory, reducing or eliminating the affinity of IL-2 for the α-subunit of the IL-2 receptor should reduce the down-regulation of effector cell function by IL-2-induced regulatory T cells and the development of tumor tolerance achieved through the AICD process. On the other hand, maintaining affinity for the intermediate-affinity IL-2 receptor should maintain the induction of IL-2 on the proliferation and activation of effector cells such as NK and T cells.

[0102] The mutant interleukin-2 (IL-2) polypeptide contained in the immunoconjugate according to the present invention comprises at least one amino acid mutation, each of which eliminates or reduces the affinity of the mutant IL-2 polypeptide for the α subunit of the IL-2 receptor and retains the affinity of the mutant IL-2 polypeptide for the intermediate affinity IL-2 receptor compared to the wild-type IL-2 polypeptide.

[0103] Mutants of human IL-2 (hIL-2) with reduced affinity for CD25 can be generated, for example, by amino acid substitution at amino acid position 35, 38, 42, 43, 45 or 72, or a combination thereof (numbering corresponding to the amino acid sequence of human IL-2 as set forth in 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. Specific IL-2 mutants useful in the immunoconjugates of the present 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 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, and L72K, more specifically an amino acid substitution selected from the group consisting of F42A, Y45A, and L72G. Compared to the wild-type form of the IL-2 mutants, these mutants exhibit substantially similar binding affinities to the intermediate-affinity IL-2 receptor and have greatly reduced affinities to the α subunit of the IL-2 receptor and the high-affinity IL-2 receptor.

[0104] Other characteristics of useful mutants may include the ability to induce proliferation of IL-2 receptor bearing T cells and / or NK cells, the ability to induce IL-2 signaling in IL-2 receptor bearing T cells and / or NK cells, the ability to cause NK cells to produce interferon (IFN)-γ as a secondary cytokine, reduced ability to induce peripheral blood mononuclear cells (PBMCs) to process (elaboration) of secondary cytokines (particularly IL-10 and TNF-α), reduced ability to activate regulatory T cells, reduced ability to induce T cell apoptosis, and reduced in vivo toxicity characteristics.

[0105] Specific mutant IL-2 polypeptides useful in the present invention comprise three amino acid mutations that eliminate or reduce the affinity of the mutant IL-2 polypeptide for the α subunit of the IL-2 receptor, while retaining the affinity of the mutant IL-2 polypeptide for the intermediate-affinity IL-2 receptor. In one embodiment, the three amino acid mutations are located 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 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, and L72K. In a specific embodiment, the three amino acid mutations are amino acid substitutions F42A, Y45A, and L72G (numbering corresponding to the amino acid sequence of human IL-2 as set forth in SEQ ID NO: 90).

[0106] Specific mutant IL-2 polypeptides useful in the present invention comprise four amino acid mutations that eliminate or reduce the affinity of the mutant IL-2 polypeptide for the α subunit of the IL-2 receptor, but retain the affinity of the mutant IL-2 polypeptide for the intermediate-affinity IL-2 receptor. In one embodiment, the three amino acid mutations are located 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 corresponding to the amino acid sequence of human IL-2 as set forth in SEQ ID NO: 90). In a specific embodiment, the three amino acid mutations are amino acid substitutions F42A, Y45A, L72G and N88D (numbered corresponding to the amino acid sequence of human IL-2 as shown in SEQ ID NO: 90). In a specific embodiment, the three amino acid mutations are amino acid substitutions F42A, Y45A, L72G and N88Q (numbered corresponding to the amino acid sequence of human IL-2 as shown in SEQ ID NO: 90).

[0107] In certain embodiments, the amino acid mutation reduces the affinity of the mutant IL-2 polypeptide for the α subunit of the IL-2 receptor by at least 5-fold, specifically at least 10-fold, more specifically at least 25-fold. In embodiments where there is 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, 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 eliminates the affinity of the mutant IL-2 polypeptide for the α subunit of the IL-2 receptor such that no binding is detected by surface plasmon resonance.

[0108] Substantially similar binding to the intermediate-affinity receptor, i.e., retention of the affinity of the mutant IL-2 polypeptide for the 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 affinity of the wild-type form of the IL-2 mutant. The IL-2 mutants of the present invention may exhibit greater than about 80%, or even greater than about 90%, of such affinity.

[0109] The combination of reducing the affinity of IL-2 for the α subunit of the IL-2 receptor and eliminating O-glycosylation of IL-2 results in an IL-2 protein with improved properties. For example, when the mutant IL-2 polypeptide is expressed in mammalian cells such as CHO or HEK cells, eliminating the O-glycosylation site results in a more homogeneous product.

[0110] Thus, in certain embodiments, the mutant IL-2 polypeptide comprises an additional amino acid mutation that eliminates the O-glycosylation site of IL-2 at a position corresponding to residue 3 of human IL-2. In one embodiment, the additional amino acid mutation that eliminates the O-glycosylation site of IL-2 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.

[0111] In certain embodiments, the mutant IL-2 polypeptide is substantially 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 amino acid sequence of SEQ ID NO:90 having at least one amino acid mutation, wherein the at least one amino acid mutation eliminates or reduces affinity of the mutant IL-2 polypeptide for the α subunit of the IL-2 receptor, while retaining affinity for the intermediate-affinity IL-2 receptor, compared to an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO:90 without the mutation. In another embodiment, the mutant IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO:95 having at least one amino acid mutation, wherein the at least one amino acid mutation eliminates or reduces affinity of the mutant IL-2 polypeptide for the α subunit of the IL-2 receptor, while retaining affinity for the intermediate-affinity IL-2 receptor, compared to an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO:95 without the mutation.

[0112] In a specific embodiment, the mutant IL-2 polypeptide can elicit one or more cellular responses selected from the group consisting of: proliferation of activated T lymphocytes, differentiation of activated T lymphocytes, cytotoxic T cell (CTL) activity, proliferation of activated B cells, differentiation of activated B cells, proliferation of natural killer (NK) cells, differentiation of NK cells, cytokine secretion by activated T cells or NK cells, and NK / lymphocyte activation killer (LAK) anti-tumor cytotoxicity.

[0113] In one embodiment, the mutant IL-2 polypeptide has a reduced ability to induce IL-2 signaling in regulatory T cells compared to 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 wild-type IL-2 polypeptide. In one embodiment, the mutant IL-2 polypeptide has a reduced in vivo toxicity profile compared to wild-type IL-2 polypeptide. In one embodiment, the mutant IL-2 polypeptide has an extended serum half-life compared to wild-type IL-2 polypeptide.

[0114] Specific mutant IL-2 polypeptides useful in the present invention comprise 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 specific mutant IL-2 polypeptide useful in the present invention comprises 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 specific mutant IL-2 polypeptide useful in the present invention comprises 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.

[0115] The IL-2 mutants used in the present invention may have one or more mutations in the amino acid sequence outside of these regions, in addition to mutations in the IL-2 region that forms the interface between IL-2 and CD25 or the glycosylation site. Such additional mutations in human IL-2 may provide additional advantages, such as enhanced expression or stability. For example, as described in U.S. Patent No. 4,518,584, the cysteine ​​at position 125 may be replaced with a neutral amino acid (such as serine, alanine, threonine, or valine), thereby producing C125S IL-2, C125A IL-2, C125T IL-2, or C125V IL-2, respectively. As described therein, the N-terminal alanine residue of IL-2 may also be deleted, thereby producing mutations such as des-A1 C125S or des-A1 C125A. Alternatively or in combination, IL-2 mutants may include mutations whereby the methionine normally present at position 104 of 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, such as 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 M104AC125S IL-2 (these and other mutants are described in U.S. Pat. No. 5,116,943 and Weiger et al., Eur J Biochem 180, 295-300 (1989)), can be used in combination with the specific IL-2 mutations of the present invention.

[0116] Thus, in certain embodiments, the mutant IL-2 polypeptide comprises an additional amino acid mutation at a position corresponding to residue 125 of human IL-2. In one embodiment, the additional amino acid mutation is the amino acid substitution C125A.

[0117] The skilled person will be able to determine which additional mutations may provide additional advantages for the 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 a mutant IL-2 polypeptide according to the present invention.

[0118] In one embodiment, the mutant IL-2 polypeptide comprises 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 amino acid sequence as shown in SEQ ID NO: 90). In a specific embodiment, the mutant IL-2 polypeptide comprises no more than 5 amino acid mutations compared to the corresponding wild-type IL-2 sequence (e.g., the human IL-2 amino acid sequence as shown in SEQ ID NO: 90).

[0119] In one embodiment, the mutant IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 92. In one embodiment, the mutant IL-2 polypeptide consists of the amino acid sequence of SEQ ID NO: 92.

[0120] In one embodiment, the mutant IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 98. In one embodiment, the mutant IL-2 polypeptide consists of the amino acid sequence of SEQ ID NO: 98.

[0121] In one embodiment, the mutant IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 99. In one embodiment, the mutant IL-2 polypeptide consists of the amino acid sequence of SEQ ID NO: 99.

[0122] Immunoconjugates

[0123] As described herein, immunoconjugates comprise IL molecules and antibodies. Such immunoconjugates significantly increase the efficacy of IL-2 therapy by directly targeting IL-2 (e.g., into the tumor microenvironment). According to the present invention, the antibody contained in the immunoconjugate can be a complete antibody or immunoglobulin, or a portion or variant thereof having a biological function (such as antigen-specific binding affinity).

[0124] The general benefits of immunoconjugate therapy are obvious. For example, the antibodies contained in the immunoconjugate recognize tumor-specific epitopes and cause the immunoconjugate molecules to target the tumor site. Therefore, high concentrations of IL-2 can be delivered to the tumor microenvironment, thereby using a much lower dose of immunoconjugate than that required for unconjugated IL-2 to cause the activation and proliferation of the various immune effector cells mentioned herein. In addition, since IL-2 is applied in the form of an immunoconjugate, a lower dose of the cytokine itself is allowed, the possibility of adverse side effects of IL-2 is limited, and targeting IL-2 to specific sites in the body by means of an immunoconjugate can also result in reduced systemic exposure and therefore less side effects than those obtained using unconjugated IL-2. In addition, the extended circulation half-life of the immunoconjugate compared to unconjugated IL-2 contributes to the efficacy of the immunoconjugate. However, this feature of IL-2 immunoconjugates may again exacerbate potential side effects of IL-2 molecules: because the circulating half-life of IL-2 immunoconjugates in the bloodstream is significantly prolonged relative to unconjugated IL-2, the likelihood that IL-2 or other parts of the fusion protein molecule will activate components normally present in the vasculature increases. The same issue applies to other fusion proteins containing IL-2 fused to another moiety (such as Fc or albumin), which can result in an extended half-life of IL-2 in the circulation. Therefore, it is particularly advantageous that immunoconjugates comprising mutant IL-2 polypeptides as described herein and in WO 2012 / 107417 have reduced toxicity compared to the wild-type form of IL-2.

[0125] As discussed above, targeting IL-2 directly to immune effector cells rather than tumor cells may be advantageous for IL-2 immunotherapy.

[0126] Therefore, the present invention provides mutant IL-2 polypeptides and antibodies that bind to PD-1 as previously described. In one embodiment, the mutant IL-2 polypeptide and the antibody form a fusion protein, i.e., the mutant IL-2 polypeptide and the antibody share a peptide bond. In some embodiments, the antibody comprises an Fc domain comprising 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 carboxyl-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 IgG-class immunoglobulin molecule, more particularly an IgG1-subclass immunoglobulin molecule. In one such embodiment, the mutant IL-2 polypeptide shares an amino-terminal peptide bond with one of the heavy chains of the immunoglobulin. In certain embodiments, the antibody is an antibody fragment. In some embodiments, the antibody is a Fab molecule or a scFv molecule. In one embodiment, the antibody is a Fab molecule. In another embodiment, the antibody is a scFv molecule. The immunoconjugate may also comprise more than one (one) antibody. When the immunoconjugate contains more than one antibody, such as a first antibody and a second antibody, each antibody can be independently selected from various forms of antibodies and antibody fragments. For example, the first antibody can be a Fab molecule, and the second antibody can be a scFv molecule. In a specific embodiment, each of the first antibody and the second antibody is an scFv molecule, or each of the first antibody and the second antibody is a Fab molecule. In a specific embodiment, 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.

[0127] Immunoconjugate format

[0128] 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 as described herein, and at least a first antibody and a second antibody. In a specific embodiment, the first antibody and the second antibody are independently selected from the group consisting of: an Fv molecule, particularly an scFv molecule; and a Fab molecule. In a specific embodiment, the mutant IL-2 polypeptide shares an amino-terminal peptide bond or a carboxyl-terminal peptide bond with the first antibody, and the second antibody shares an amino-terminal peptide bond or a carboxyl-terminal peptide bond with i) the mutant IL-2 polypeptide or ii) the first antibody. In a specific embodiment, the immunoconjugate consists essentially of the mutant IL-2 polypeptide and the first and second antibodies (particularly Fab molecules) joined by one or more linker sequences. This format has the following advantages: they bind to the target antigen (PD-1) with high affinity, but only provide monomeric binding to the IL-2 receptor, thereby avoiding targeting the immunoconjugate to immune cells bearing the IL-2 receptor at locations other than the target site. In a specific embodiment, the mutant IL-2 polypeptide shares a carboxyl-terminal peptide bond with the first antibody, in particular the first Fab molecule, and further shares an amino-terminal peptide bond with the second antibody, in particular the second Fab molecule. In another embodiment, the first antibody, in particular the first Fab molecule, shares a carboxyl-terminal peptide bond with the mutant IL-2 polypeptide, and further shares an amino-terminal peptide bond with the second antibody, in particular the second Fab molecule. In another embodiment, the first antibody, in particular the first Fab molecule, shares an amino-terminal peptide bond with the first mutant IL-2 polypeptide, and further shares a carboxyl-terminal peptide with the second antibody, in particular the second Fab molecule. In a specific embodiment, the mutant IL-2 polypeptide shares a carboxyl-terminal peptide bond with the first heavy chain variable region, and also shares an amino-terminal peptide bond with the second heavy chain variable region. In another embodiment, the mutant IL-2 polypeptide shares a carboxy-terminal peptide bond with the first light chain variable region and also shares an amino-terminal peptide bond with the second light chain variable region. In another embodiment, the first heavy or light chain variable region is joined to the mutant IL-2 polypeptide via a carboxy-terminal peptide bond and is also joined to the second heavy or light chain variable region via an amino-terminal peptide bond. In another embodiment, the first heavy or light chain variable region is joined to the mutant IL-2 polypeptide via an amino-terminal peptide bond and is also joined to the second heavy or light chain variable region via a carboxy-terminal peptide bond. In one embodiment, the mutant IL-2 polypeptide shares a carboxy-terminal peptide bond with the first Fab heavy or light chain and also shares an amino-terminal peptide bond with the second Fab heavy or light chain. In another embodiment, the first Fab heavy or light chain shares a carboxy-terminal peptide bond with the mutant IL-2 polypeptide and further shares an amino-terminal peptide bond with the second Fab heavy or light chain.In other embodiments, the first Fab heavy or light chain shares an amino-terminal peptide bond with the mutant IL-2 polypeptide and also shares a carboxy-terminal peptide bond with the second Fab heavy or light chain. In one embodiment, the 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.

[0129] However, a particularly suitable format of the immunoconjugate according to the invention comprises an immunoglobulin molecule as the antibody. Such immunoconjugate formats are described in WO 2012 / 146628, which is incorporated herein by reference in its entirety.

[0130] Thus, in a specific embodiment, an immunoconjugate comprises a mutant IL-2 polypeptide as 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, such as 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 or 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, joined by one or more linker sequences. 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.

[0131] The mutant IL-2 polypeptide can be fused to the antibody directly or via a linker peptide comprising one or more amino acids (usually about 2-20 amino acids). Linker peptides are known in the art and described herein. Suitable non-immunogenic linker peptides include, for example, (G4S) n 、(SG4) n 、(G4S) n or G4 (SG4) n Linker peptide. "n" is typically an integer from 1 to 10, typically from 2 to 4. In one embodiment, the linker peptide is at least 5 amino acids long, in one embodiment from 5 to 100 amino acids long, and in a further embodiment from 10 to 50 amino acids long. In a specific embodiment, the linker peptide is 15 amino acids long. In one embodiment, the linker peptide is (GxS) n or (GxS) n Gm , wherein G = glycine, S = serine, and (x = 3, n = 3, 4, 5 or 6, and m = 0, 1, 2 or 3) or (x = 4, n = 2, 3, 4 or 5 and m = 0, 1, 2 or 3), in one embodiment, x = 4 and n = 2 or 3, in another embodiment, x = 4 and n = 3. In a specific embodiment, the linker peptide is (G4S)3 (SEQ ID NO: 93). In one embodiment, the linker peptide has (or consists of) the amino acid sequence of SEQ ID NO: 93.

[0132] In a specific embodiment, the immunoconjugate comprises a mutant IL-2 molecule and an immunoglobulin molecule that binds to PD-1, particularly an IgG1 subclass immunoglobulin molecule, wherein the mutant IL-2 molecule is fused at its amino-terminal amino acid to the carboxyl-terminal amino acid of one of the immunoglobulin heavy chains via a linker peptide as shown in SEQ ID NO: 93.

[0133] 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 comprising 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 carboxyl-terminal amino acid of one of the subunits of the Fc domain via a linker peptide as shown in SEQ ID NO: 93.

[0134] PD-1 antibodies

[0135] The antibodies contained in the immunoconjugates of the present invention bind to PD-1, particularly human PD-1, and are capable of directing the mutant IL-2 polypeptide to target sites expressing PD-1, particularly T cells expressing PD-1, such as tumor-associated T cells.

[0136] Suitable PD-1 / antibodies that can be used in the immunoconjugates of the present invention are described in PCT Patent Application No. PCT / EP2016 / 073248, the entire contents of which are incorporated herein by reference.

[0137] The immunoconjugates of the present invention may comprise two or more (two or more) antibodies that can bind to the same or different antigens. However, in specific embodiments, each of these antibodies binds to PD-1. In one embodiment, the antibodies contained in the immunoconjugates of the present invention are monospecific. In a specific embodiment, the immunoconjugate comprises a single monospecific antibody, particularly a monospecific immunoglobulin molecule.

[0138] The antibody can be any type of antibody or fragment thereof that maintains 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 comprising a first subunit and a second subunit. In some embodiments, the antibody is an immunoglobulin, particularly an IgG-class immunoglobulin, more particularly an IgG1-subclass immunoglobulin.

[0139] In some embodiments, the antibody is a monoclonal antibody.

[0140] 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, an HVR-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.

[0141] In some embodiments, 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. In some embodiments, the heavy chain and / or light chain variable region is a humanized variable region. In some embodiments, the heavy chain and / or light chain variable region comprises a human framework region (FR).

[0142] 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 a specific embodiment, 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.

[0143] 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 IgG class immunoglobulin molecule comprising a human CH1, CH2, CH3 and / or CL domain.

[0144] Fc domain

[0145] In a specific embodiment, the antibody contained in the immunoconjugate according to the present invention comprises an Fc domain comprising a first subunit and a second subunit. The Fc domain of an antibody is composed 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 comprises a CH2 and CH3 IgG heavy chain constant domain. The two subunits of the Fc domain are capable of stably associating with each other. In one embodiment, the immunoconjugate of the present invention comprises no more than one Fc domain.

[0146] In one embodiment, the Fc domain of the antibody included in the immunoconjugate is an IgG Fc domain. In a specific 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, which comprises an amino acid substitution (Kabat EU index number) at position S228, particularly an amino acid substitution S228P. This amino acid substitution reduces the in vivo Fab arm exchange of IgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In another specific embodiment, the Fc domain is a human Fc domain. In an even more specific embodiment, the Fc domain is a human IgG1 Fc domain.

[0147] Fc domain modifications that promote heterodimerization

[0148] The immunoconjugates according to the present invention comprise a mutant IL-2 polypeptide, in particular a single (no more than one) mutant IL-2 polypeptide, fused to one or the other of the two subunits of the Fc domain, whereby the two subunits of the Fc domain are typically contained in two different polypeptide chains. Recombinant co-expression and subsequent dimerization of these polypeptides results in several possible combinations of the two polypeptides. In order to improve the yield and purity of immunoconjugates during recombinant production, it would be advantageous to introduce modifications into the Fc domain of the antibody that promote the association of the desired polypeptide.

[0149] Thus, in certain embodiments, the Fc domain of an antibody included in an immunoconjugate according to the present invention comprises a modification that promotes the association of the first and second subunits of the Fc domain. The most extensive protein-protein interaction site between the two subunits of the human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one embodiment, the modification is located in the CH3 domain of the Fc domain.

[0150] There are several methods for modifying the CH3 domain of the Fc domain to achieve heterodimerization, which are described in detail 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, and WO 2013096291. Typically, in all such methods, the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are engineered in a complementary manner such that each CH3 domain (or a heavy chain comprising the same) can no longer homodimerize with itself, but is forced to heterodimerize with the other complementarily engineered CH3 domain (such that the first and second CH3 domains heterodimerize and homodimers are not formed between the two first or two second CH3 domains).

[0151] In a specific embodiment, the modification that promotes the association of the first subunit and the second subunit of the Fc domain is a so-called "knob-to-hole" modification, which comprises a "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other of the two subunits of the Fc domain.

[0152] Knob-in-hole structure technology is described in, for example, US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and introducing a corresponding cavity ("hole") in the interface of a second polypeptide so that the protrusion can be positioned in the cavity to promote the formation of heterodimers and hinder the formation of homodimers. The protrusion is constructed by replacing the small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). A compensatory cavity having the same or similar size as the protrusion is created in the interface of the second polypeptide by replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine).

[0153] Thus, in a specific embodiment, in the CH3 domain of the first subunit of the Fc domain of the antibody comprised in the immunoconjugate, an amino acid residue is replaced by an amino acid residue having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit, and the protrusion is positionable in a cavity in the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, an amino acid residue is replaced by an amino acid residue having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit, and the protrusion in the CH3 domain of the first subunit is positionable in the cavity.

[0154] Preferably, the amino acid residue with a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W).

[0155] Preferably, the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T) and valine (V).

[0156] Protrusions and cavities can be produced by altering the nucleic acid encoding the polypeptide, for example by site-specific mutagenesis or by peptide synthesis.

[0157] In a specific embodiment, in the CH3 domain of the first subunit ("knob" subunit) of the Fc domain, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the CH3 domain of the second subunit ("hole" subunit) of the Fc domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain, additionally, 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 EU index of Kabat).

[0158] In yet another embodiment, in the first subunit of the Fc domain, additionally, the serine residue at position 354 is replaced by a cysteine ​​residue (S354C) or the glutamic acid residue at position 356 is replaced by a cysteine ​​residue (E356C) (particularly, the serine residue at position 354 is replaced by a cysteine ​​residue), and in the second subunit of the Fc domain, additionally, the tyrosine residue at position 349 is replaced by a cysteine ​​residue (Y349C) (numbering according to the EU index of Kabat). The introduction of these two cysteine ​​residues results in the formation of a disulfide bridge between the two subunits of the Fc domain, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0159] In a specific embodiment, the first subunit of the Fc domain comprises amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (numbering according to the Kabat EU index).

[0160] In some embodiments, the second subunit of the Fc domain further comprises amino acid substitutions H435R and Y436F (numbering according to the Kabat EU index).

[0161] In a specific embodiment, the mutant IL-2 polypeptide is fused to the first subunit of the Fc domain (comprising a "knob" modification) (optionally, via a linker peptide). Without wishing to be bound by theory, fusion of the mutant IL-2 polypeptide to the knob-containing subunit of the Fc domain will (further) minimize the production of immunoconjugates comprising two mutant IL-2 polypeptides (steric hindrance of the two knob-containing polypeptides).

[0162] Other CH3 modification techniques for implementing heterodimerization are envisioned as alternatives according to 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, WO2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO2013 / 157954, WO 2013 / 096291.

[0163] In one embodiment, the heterodimerization method described in EP 1870459 is used instead. This method is based on the introduction of charged amino acids with opposite charges at specific amino acid positions in the CH3 / CH3 domain interface between the two subunits of the Fc domain. A preferred embodiment of the antibody contained in the immunoconjugate of the present invention is the amino acid mutation R409D; K370E in one of the two CH3 domains (of the Fc domain), and the amino acid mutation D399K; E357K in the other of the CH3 domains of the Fc domain (numbered according to the Kabat EU index).

[0164] 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 and the amino acid mutations T366S, L368A, Y407V, and additionally the amino acid mutation R409D in the CH3 domain of the second subunit of the Fc domain; K370E in the CH3 domain of the first subunit of the Fc domain, and the amino acid mutation D399K; E357K in the CH3 domain of the second subunit of the Fc domain (numbering according to the Kabat EU index).

[0165] 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 the 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 additionally the amino acid mutation R409D; K370E in the CH3 domain of the first subunit of the Fc domain, and the amino acid mutation D399K; E357K in the CH3 domain of the second subunit of the Fc domain (all numbering according to the Kabat EU index).

[0166] In one embodiment, the heterodimerization method described in WO 2013 / 157953 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 another embodiment, the first CH3 domain comprises the additional amino acid mutation L351K. In another embodiment, the second CH3 domain further comprises an amino acid mutation selected from the group consisting of Y349E, Y349D, and L368E (preferably L368E) (numbering according to the Kabat EU index).

[0167] In one embodiment, the heterodimerization method 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 another embodiment, the second CH3 domain comprises a further amino acid mutation at position T411, D399, S400, F405, N390 or K392, e) N390R, N390K or N390D, e) K392V, K392M, K392R, K392L, K392F or K392E (according to Kabat EU index numbering). In another embodiment, the first CH3 domain comprises amino acid mutations L351Y and Y407A, and the second CH3 domain comprises amino acid mutations T366V and K409F. In another embodiment, the first CH3 domain comprises amino acid mutation Y407A, and the second CH3 domain comprises amino acid mutations T366A and K409F. In another embodiment, the second CH3 domain further comprises amino acid mutations K392E, T411E, D399R, and S400R (numbering according to EU index as in Kabat).

[0168] In one embodiment, the heterodimerization method described in WO 2011 / 143545 is used instead, eg, an amino acid modification is made at a position selected from the group consisting of 368 and 409 (numbered according to the Kabat EU index).

[0169] In one embodiment, the heterodimerization method described in WO 2011 / 090762 is used instead, which also uses the above-mentioned knob-in-hole technique. In one embodiment, the first CH3 domain comprises the amino acid mutation T366W, and the second CH3 domain comprises the amino acid mutation Y407A. In one embodiment, the first CH3 domain comprises the amino acid mutation T366Y, and the second CH3 domain comprises the amino acid mutation Y407T (numbering according to the Kabat EU index).

[0170] In one embodiment, the antibody or Fc domain thereof comprised in the immunoconjugate is of the IgG2 subclass and the heterodimerization approach described in WO 2010 / 129304 is used instead.

[0171] In an alternative embodiment, the modification that promotes the association of the first and second subunits of the Fc domain comprises a modification that mediates an electrostatic steering effect, for example as described in PCT Publication WO 2009 / 089004. Typically, this approach 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 becomes electrostatically unfavorable, but heterodimerization is electrostatically favorable. In one such embodiment, the first CH3 domain comprises an amino acid substitution with a negatively charged amino acid pair K392 or N392 (e.g., glutamic acid (E) or aspartic acid (D), preferably K392D or N392D), and the second CH3 domain comprises an amino acid substitution with a positively charged amino acid pair D399, E356, D356 or E357 (e.g., lysine (K) or arginine (R), preferably D399K, E356K, D356K or E357K, more preferably D399K and E356K). In another embodiment, the first CH3 domain further comprises an amino acid substitution of K409 or R409 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), preferably K409D or R409D). In another embodiment, the first CH3 domain further or alternatively comprises an amino acid substitution of 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).

[0172] In yet another embodiment, the heterodimerization method described in WO 2007 / 147901 is used instead.In one embodiment, the first CH3 domain comprises the amino acid mutations K253E, D282K and K322D, and the second CH3 domain comprises the amino acid mutations D239K, E240K and K292D (numbered according to the EU index of Kabat).

[0173] In yet another embodiment, the heterodimerization method described in WO 2007 / 110205 may be used instead.

[0174] In one embodiment, the first subunit of the Fc domain comprises amino acid substitutions K392D and K409D, and the second subunit of the Fc domain comprises amino acid substitutions D356K and D399K (numbering according to the Kabat EU index).

[0175] Fc domain modifications that reduce Fc receptor binding and / or effector function

[0176] The Fc domain confers favorable pharmacokinetic properties to the immunoconjugate, including a long serum half-life and a favorable tissue-blood distribution ratio that contribute to good accumulation in the target tissue. However, at the same time, it may cause the immunoconjugate to be undesirably targeted to cells expressing Fc receptors, rather than preferred antigen-bearing cells. In addition, co-activation of the Fc receptor signaling pathway can lead to cytokine release, which, combined with the long half-life of the IL-2 polypeptide and the immunoconjugate, leads to excessive activation of cytokine receptors and severe side effects after systemic administration. Consistent with this, conventional IgG-IL-2 immunoconjugates associated with infusion reactions have been described (see, e.g., King et al., J Clin Oncol 22, 4463-4473 (2004)).

[0177] Thus, in specific embodiments, the Fc domain of the antibody contained in the immunoconjugate according to the present invention exhibits reduced binding affinity to Fc receptors and / or reduced effector function compared to a native IgG1 Fc domain. 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 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 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 activated human Fcγ receptor, more particularly human FcγRIIIa, FcγRI or FcγRIIa, most particularly human FcγRIIIa. In one embodiment, the effector function is one or more effector functions selected from the group consisting of CDC, ADCC, ADCP and cytokine secretion. In a specific embodiment, the effector function is ADCC. In one embodiment, the Fc domain domain exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn) compared to the native IgG1 Fc domain domain. When the Fc domain (or an antibody comprising the Fc domain) exhibits greater than about 70% of the binding affinity of the native IgG1 Fc domain (or an antibody comprising the native IgG1 Fc domain) to FcRn, particularly greater than about 80%, more particularly greater than about 90%, substantially similar binding to FcRn is achieved.

[0178] In certain embodiments, the Fc domain is engineered to have a reduced binding affinity to Fc receptors and / or reduced effector functions compared to a non-engineered Fc domain. In a specific embodiment, the Fc domain of the antibody contained in the immunoconjugate comprises one or more amino acid mutations that reduce the binding affinity of the Fc domain to Fc receptors and / or effector functions. 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 mutation reduces the binding affinity of the Fc domain to Fc receptors. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to Fc receptors by at least 2 times, at least 5 times, or at least 10 times. In embodiments where there is more than one amino acid mutation that reduces the binding affinity of the Fc domain to Fc receptors, the combination of these amino acid mutations can reduce the binding affinity of the Fc domain to Fc receptors by at least 10 times, at least 20 times, or even at least 50 times. In one embodiment, the antibody comprising the engineered Fc domain exhibits less than 20% of the binding affinity for an Fc receptor, particularly less than 10%, more particularly less than 5%, 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 activated Fc receptor. In a specific embodiment, the Fc receptor is an activated 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 complementary components, particularly binding affinity to C1q, is also reduced. In one embodiment, binding affinity to the neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e., the binding affinity of the Fc domain for the receptor is maintained, is achieved when the Fc domain (or an antibody comprising the Fc domain) exhibits greater than about 70% of the binding affinity of a non-engineered form of the Fc domain (or an antibody comprising the non-engineered form of the Fc domain) for FcRn. The Fc domain or an antibody comprising the Fc domain included in the immunoconjugate of the present invention may exhibit greater than about 80% and even greater than about 90% of this affinity. In certain embodiments, the Fc domain of the antibody included in the immunoconjugate is engineered to have reduced effector function compared to a non-engineered Fc domain.The effector function of reduction may 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 immune complex mediated antigen presenting cells to antigen uptake, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling induced apoptosis, reduced target binding antibody crosslinking, reduced dendritic cell maturation, or reduced T cell sensitization. In one embodiment, the effector function of reduction is one or more selected from the group consisting of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a specific embodiment, the effector function of reduction is reduced ADCC. In one embodiment, the ADCC reduced is less than 20% of the ADCC induced by non-engineered Fc domains (or antibodies comprising non-engineered Fc domains).

[0179] In one embodiment, the amino acid mutation that reduces the binding affinity of Fc domains to Fc receptors and / or effector function is amino acid replacement.In one embodiment, Fc domains are included in the amino acid replacement (according to Kabat EU index numbering) at the position of the group selected from E233, L234, L235, N297, P331 and P329.In a more specific embodiment, Fc domains are included in the amino acid replacement (according to Kabat EU index numbering) at the position of the group selected from L234, L235 and P329. In certain embodiments, Fc domains include amino acid replacement L234A and L235A (according to Kabat EU index numbering).In such embodiment, Fc domains are IgG1 Fc domains, particularly human IgG1 Fc domains.In one embodiment, Fc domains include the amino acid replacement at position P329.In a more specific embodiment, amino acid replacement is P329A or P329G, particularly P329G (according to Kabat EU index numbering). In one embodiment, the Fc domains are included in the amino acid replacement at position P329, and the further amino acid replacement (according to the Kabat EU index numbering) at a position selected from E233, L234, L235, N297 and P331. In a more specific embodiment, the further amino acid replacement is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In a specific embodiment, the Fc domains are included in the amino acid replacement (according to the Kabat EU index numbering) at position P329, L234 and L235. In a more specific embodiment, the Fc domains include amino acid mutations L234A, L235A and P329G ("P329G LALA", "PGLALA" or "LALAPG"). Specifically, in a specific embodiment, each subunit of the Fc domain comprises 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) (according to the EU index numbering of Kabat). In one such embodiment, the Fc domain is an IgG1 Fc domain, in particular a human IgG1 Fc domain. The "P329G LALA" combination of amino acid substitutions almost completely eliminates Fcγ receptor (and complement) binding of the human IgG1 Fc domain, as described in PCT Publication No. WO 2012 / 130831, the entire contents of which are incorporated herein by reference.WO 2012 / 130831 also describes methods for preparing such mutant Fc domains and methods for determining their properties, such as Fc receptor binding or effector function.

[0180] Compared with IgG1 antibody, IgG4 antibody shows the binding affinity to Fc acceptor and the effector function of reduction.Therefore, in some embodiments, the Fc domain of the antibody included in the immunoconjugate of the present invention is IgG4 Fc domain, particularly human IgG4 Fc domain.In one embodiment, IgG4 Fc domain is included in the aminoacid replacement at position S228, particularly aminoacid replacement S228P (according to Kabat EU index numbering).In order to further reduce its binding affinity to Fc acceptor and / or its effector function, in one embodiment, IgG4 Fc domain is included in the aminoacid replacement at position L235, particularly aminoacid replacement L235E (according to Kabat EU index numbering).In another embodiment, IgG4 Fc domain comprises the aminoacid replacement at P329 position, particularly aminoacid replacement P329G (according to Kabat's EU index numbering). In a specific embodiment, the IgG4 Fc domain comprises amino acid substitutions at positions S228, L235, and P329, particularly amino acid substitutions S228P, L235E, and P329G (numbered according to the EU index of Kabat). Such IgG4 Fc domain mutants and their Fcγ receptor binding properties are described in PCT Publication No. WO 2012 / 130831, the entire contents of which are incorporated herein by reference.

[0181] In a specific embodiment, the Fc domain that exhibits reduced binding affinity to an Fc receptor and / or reduced effector function compared to a native IgGl Fc domain is a human IgGl Fc domain comprising amino acid substitutions L234A, L235A, and optionally P329G, or a human IgG4 Fc domain comprising amino acid substitutions S228P, L235E, and optionally P329G (numbering according to the EU index as in Kabat).

[0182] In certain embodiments, N-glycosylation of the Fc domain has been eliminated. In one such embodiment, the Fc domain comprises an amino acid mutation at position N297, particularly an amino acid substitution replacing asparagine with alanine (N297A) or aspartic acid (N297D) (numbering according to the EU index of Kabat).

[0183] In addition to the Fc domains described above and in PCT Publication No. WO 2012 / 130831, Fc domains with reduced Fc receptor binding and / or decreased effector function also include those with substitutions of one or more of Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056) (numbered according to the EU index of Kabat). Such Fc mutants include Fc mutants with substitutions at two or more of amino acids 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant, in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0184] 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 can include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, etc. The correct nucleotide changes can be verified, for example, by sequencing.

[0185] Binding to Fc receptors can be readily determined using standard instruments (such as a BIAcore instrument (Cytiva)), for example, by ELISA or by surface plasmon resonance (SPR), and Fc receptors can be obtained, for example, by recombinant expression. Alternatively, cell lines known to express specific Fc receptors (such as human NK cells expressing FcγIIIa receptors) can be used to assess the binding affinity of an Fc domain or an antibody comprising an Fc domain to an Fc receptor.

[0186] Fc domains, or effector functions of antibodies comprising Fc domains, can be measured by methods known in the art. Examples of in vitro assays for assessing ADCC activity of molecules 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 assays can be used (see, for example, ACTI for flow cytometry). TM Non-radioactive cytotoxicity assay (CellTechnology, Inc. Mountain View, CA); and CytoTox Non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can 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).

[0187] In some embodiments, the binding of the Fc domain to complement components, particularly C1q, is reduced. Thus, in some embodiments, the Fc domain is engineered to have reduced effector function, including reduced CDC. A C1q binding assay can be performed to determine whether the Fc domain or an antibody comprising the Fc domain is capable of binding to C1q and therefore has CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can 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)).

[0188] FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, eg, Petkova, SB et al., Int'l. Immunol. 18(12): 1759-1769 (2006); WO 2013 / 120929).

[0189] Particular aspects of the invention

[0190] In one 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 amino acid substitutions F42A, Y45A, L72G, and Q126T (numbered relative to the human IL-2 sequence SEQ ID NO: 90); and wherein 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 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 amino acid substitutions T3A, F42A, Y45A, L72G, C125A, and Q126T (numbered relative to the human IL-2 sequence SEQ ID NO: 90); and wherein 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 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 comprises the amino acid sequence of SEQ ID NO: 92; and wherein 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 according to any of the aforementioned aspects of the present invention, the antibody is an IgG class immunoglobulin comprising a human IgG1 Fc domain consisting 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), 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), and wherein further, each subunit of the Fc domain comprises amino acid substitutions L234A, L235A, and P329G (numbering according to the Kabat EU index). In this embodiment, the mutant IL-2 polypeptide can be fused at its amino-terminal amino acid to the carboxyl-terminal amino acid of the first subunit of the Fc domain via a linker peptide as shown in SEQ ID NO: 93.In one aspect, the invention provides an immunoconjugate 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.

[0191] In one 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 amino acid substitutions F42A, Y45A, L72G, and N88D (numbered relative to the human IL-2 sequence SEQ ID NO: 90); and wherein 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.

[0192] In one 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 amino acid substitutions T3A, F42A, Y45A, L72G, N88D, and C125A (numbered relative to the human IL-2 sequence SEQ ID NO: 90); and wherein 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 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 comprises the amino acid sequence of SEQ ID NO: 98; and wherein 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 according to any of the aforementioned aspects of the present invention, the antibody is an IgG class immunoglobulin comprising a human IgG1 Fc domain consisting 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), 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), and wherein further, each subunit of the Fc domain comprises amino acid substitutions L234A, L235A, and P329G (numbering according to the Kabat EU index). In this embodiment, the mutant IL-2 polypeptide can be fused at its amino-terminal amino acid to the carboxyl-terminal amino acid of the first subunit of the Fc domain via a linker peptide as shown in SEQ ID NO: 93. In one aspect, the invention provides an immunoconjugate 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:100.

[0193] In one 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 amino acid substitutions F42A, Y45A, L72G, and N88Q (numbered relative to the human IL-2 sequence SEQ ID NO: 90); and wherein 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 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 amino acid substitutions T3A, F42A, Y45A, L72G, N88Q, and C125A (numbered relative to the human IL-2 sequence SEQ ID NO: 90); and wherein 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 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 comprises the amino acid sequence of SEQ ID NO: 99; and wherein 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.

[0194] In one embodiment according to any of the aforementioned aspects of the present invention, the antibody is an IgG class immunoglobulin comprising a human IgG1 Fc domain consisting 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), 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), and wherein further, each subunit of the Fc domain comprises amino acid substitutions L234A, L235A, and P329G (numbering according to the Kabat EU index). In this embodiment, the mutant IL-2 polypeptide can be fused at its amino-terminal amino acid to the carboxyl-terminal amino acid of the first subunit of the Fc domain via a linker peptide as shown in SEQ ID NO: 93. In one aspect, the invention provides an immunoconjugate 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:31.

[0195] Bispecific antigen-binding molecules that bind to PD-1 and LAG3

[0196] The immunoconjugates of the present invention comprise bispecific antigen binding molecules, ie, antigen binding molecules comprising at least two antigen binding moieties capable of specific binding to two different antigenic determinants, such as PD-1 and LAG3.

[0197] The bispecific antigen-binding molecules contained in the immunoconjugates of the present invention bind to PD-1 and LAG3, in particular human PD-1 and human LAG3, and are capable of directing the mutant IL-2 polypeptide to target sites expressing PD-1 and / or LAG3, in particular to T cells expressing PD-1 and / or LAG3, such as T cells associated with tumors.

[0198] According to a specific embodiment of the present invention, the antigen binding moiety included in the bispecific antigen binding molecule is a Fab molecule (i.e., an antigen binding domain consisting of a heavy chain and a light chain, each antigen binding domain comprising a variable domain and a constant domain). In one embodiment, the first and / or second antigen binding moiety is a Fab molecule. In one embodiment, the Fab molecule is human. In a specific embodiment, the Fab molecule is humanized. In another embodiment, the Fab molecule comprises human heavy chain and light chain constant domains.

[0199] Preferably, at least one antigen binding portion in the antigen binding portion is a cross Fab molecule. This modification reduces the mispairing of the heavy chain and light chain from different Fab molecules, thereby improving the yield and purity of bispecific antigen binding molecules in recombinant production. In the specific cross Fab molecules of the bispecific antigen binding molecules that can be used for inclusion in the immunoconjugates of the present invention, the variable domains (VL and VH, respectively) of the Fab light chain and the Fab heavy chain are exchanged. However, even if this domain exchange is performed, due to the so-called Bence Jones type interaction between the heavy chain and the light chain of the mispairing, the preparation of bispecific antigen binding molecules may include certain by-products (see Schaefer et al., PNAS, 108 (2011) 11187-11191). In order to further reduce the mispairing of the heavy chain and light chain from different Fab molecules and thus improve the purity and yield of the desired bispecific antigen binding molecules, charged amino acids with opposite charges can be introduced at the specific amino acid positions of the CH1 and CL domains of any one of the Fab molecules in conjunction with PD-1 or in conjunction with LAG3, as further described herein. The charge modification is performed in either a conventional Fab molecule contained in the bispecific antigen-binding molecule or in a VH / VL crossover Fab molecule contained in the bispecific antigen-binding molecule (but not in both). In a specific embodiment, the charge modification is performed in a conventional Fab molecule contained in the bispecific antigen-binding molecule (which binds LAG3 in a specific embodiment).

[0200] First antigen binding moiety

[0201] The bispecific antigen-binding molecules contained in the immunoconjugates of the present invention comprise at least one antigen-binding portion, in particular a Fab molecule, that binds to PD-1 (in particular, human PD-1 (first antigen)). In a specific embodiment, the antigen-binding portion that binds to PD-1 is a cross-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 are exchanged / replaced with each other. In such embodiments, the antigen-binding portion that binds to LAG3 is a conventional Fab molecule. In an alternative embodiment, the antigen-binding portion that binds to LAG3 is a cross-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 are exchanged / replaced with each other. In such embodiments, the antigen-binding portion that binds to PD-1 is a conventional Fab molecule.

[0202] In some embodiments, the first antigen binding portion comprises: a CDR-H1 comprising the amino acid sequence as shown in SEQ ID NO:74, a CDR-H2 comprising the amino acid sequence as shown in SEQ ID NO:75, a CDR-H3 comprising the amino acid sequence as shown in SEQ ID NO:76, a CDR-L1 comprising the amino acid sequence as shown in SEQ ID NO:77, a CDR-L2 comprising the amino acid sequence as shown in SEQ ID NO:78, and a CDR-L3 comprising the amino acid sequence as shown in SEQ ID NO:79.

[0203] In some embodiments, the first antigen binding portion comprises: (a) a heavy chain variable region (VH) comprising: a CDR-H1 comprising the amino acid sequence as shown in SEQ ID NO:74, a CDR-H2 comprising the amino acid sequence as shown in SEQ ID NO:75, and a CDR-H3 comprising the amino acid sequence as shown in SEQ ID NO:76; and (b) a light chain variable region (VL) comprising: a CDR-L1 comprising the amino acid sequence as shown in SEQ ID NO:77, a CDR-L2 comprising the amino acid sequence as shown in SEQ ID NO:78, and a CDR-L3 comprising the amino acid sequence as shown in SEQ ID NO:79.

[0204] In some embodiments, the first antigen binding portion is (or is 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 CDRs of any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In some embodiments, the heavy chain and / or light chain variable region comprises a human framework region (FR).

[0205] In some embodiments, the first antigen binding portion 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. In some embodiments, the first 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: 81. In some embodiments, the first antigen binding portion 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.

[0206] 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.

[0207] In some embodiments, the first 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.

[0208] 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.

[0209] 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, there is no more than one antigen binding moiety that binds to PD-1 in the bispecific antigen binding molecule (i.e., the bispecific antigen binding molecule provides monovalent binding to PD-1).

[0210] Second antigen binding moiety

[0211] The bispecific antigen-binding molecule comprised in the immunoconjugate of the present invention comprises at least one antigen-binding portion, particularly a Fab molecule, that binds to LAG3, particularly human LAG3 (second antigen).

[0212] In certain embodiments, the antigen-binding moiety that binds to LAG3 is a conventional Fab molecule. In certain embodiments, the antigen-binding moiety 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 are exchanged / replaced with each other.

[0213] In alternative embodiments, the antigen binding moiety that binds to PD-1 is a conventional Fab molecule. In such embodiments, the antigen binding moiety 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 are exchanged / replaced with each other.

[0214] In some embodiments, the second antigen-binding portion comprises: a CDR-H1 comprising the amino acid sequence of SEQ ID NO:82, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:83, a CDR-H3 comprising the amino acid sequence of SEQ ID NO:84, a CDR-L1 comprising the amino acid sequence of SEQ ID NO:85, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:86, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:87.

[0215] In some embodiments, the second antigen binding portion comprises: (a) a heavy chain variable region (VH) comprising: a CDR-H1 comprising the amino acid sequence as shown in SEQ ID NO:82, a CDR-H2 comprising the amino acid sequence as shown in SEQ ID NO:83, and a CDR-H3 comprising the amino acid sequence as shown in SEQ ID NO:84; and (b) a light chain variable region (VL) comprising: a CDR-L1 comprising the amino acid sequence as shown in SEQ ID NO:85, a CDR-L2 comprising the amino acid sequence as shown in SEQ ID NO:86, and a CDR-L3 comprising the amino acid sequence as shown in SEQ ID NO:87.

[0216] In some embodiments, the second antigen binding portion is (or is 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 CDRs of any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In some embodiments, the heavy chain and / or light chain variable region comprises a human framework region (FR).

[0217] In some embodiments, the second antigen binding portion comprises 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: 88. In some embodiments, the second 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: 89.

[0218] In some embodiments, 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.

[0219] In a specific embodiment, the second antigen-binding portion comprises: (a) a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 88; and (b) a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 89. In another specific embodiment, the second antigen-binding portion comprises the VH amino acid sequence set forth in SEQ ID NO: 88 and the VL amino acid sequence set forth in SEQ ID NO: 89.

[0220] In one embodiment, the second antigen binding portion comprises a human constant region. In one embodiment, the second antigen binding portion is a Fab molecule comprising a human constant region, particularly a human CH1 and / or CL domain. Specifically, the light chain constant region may comprise amino acid mutations under "charge modification" as described herein and / or if in a cross-Fab molecule, the deletion or substitution of one or more (particularly two) N-terminal amino acids may be included. Specifically, the heavy chain constant region (particularly the CH1 domain) may comprise amino acid mutations under "charge modification" as described herein.

[0221] In one embodiment, no more than one antigen-binding moiety that binds LAG3 is present in the bispecific antigen-binding molecule (ie, the bispecific antigen-binding molecule provides monovalent binding to LAG3).

[0222] Particular aspects of the invention - PD1-LAG3-IL2v_Q126T

[0223] 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 is a human IL-2 molecule comprising amino acid substitutions F42A, Y45A, L72G, and Q126T (numbered relative to the human IL-2 sequence SEQ ID NO: 90); and wherein the bispecific antigen-binding molecule comprises: (i) a first antigen-binding portion that binds to PD-1, the first antigen-binding portion 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 portion that binds to LAG3, the second antigen-binding portion 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.

[0224] 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 is a human IL-2 molecule comprising amino acid substitutions T3A, F42A, Y45A, L72G, C125A, and Q126T (numbered relative to the human IL-2 sequence SEQ ID NO: 90); and wherein the bispecific antigen-binding molecule comprises: (i) a first antigen-binding portion that binds to PD-1, the first antigen-binding portion 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 portion that binds to LAG3, the second antigen-binding portion 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.

[0225] 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; and wherein the bispecific antigen-binding molecule comprises: (i) a first antigen-binding portion that binds to PD-1, the first antigen-binding portion 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 portion that binds to LAG3, the second antigen-binding portion 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.

[0226] In some embodiments according to any of the above aspects, the first antigen binding moiety is a Fab molecule in which the variable domains VL and VH of the Fab light chain and the Fab heavy chain replace each other, and the second antigen binding moiety is a (conventional) 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 lysine (K) (according to Kabat numbering) and the amino acid at position 123 is substituted by lysine (K) or arginine (R) (according to Kabat numbering) (most particularly substituted by arginine (R)), and in the constant domain CH1 of the second antigen binding moiety, the amino acid at position 147 is substituted by glutamic acid (E) (according to the EU index of Kabat) and the amino acid at position 213 is substituted by glutamic acid (E) (according to the EU index of Kabat).

[0227] In some embodiments according to any of the above aspects, the bispecific antigen binding molecule further comprises an Fc domain consisting 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 N-terminus of the first subunit of the Fc domain), and the second antigen binding moiety is fused at the C-terminus of the Fab heavy chain to the N-terminus of the other subunit of the Fc domain (particularly to the N-terminus of the second subunit of the Fc domain).

[0228] 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; and

[0229] The bispecific antigen-binding molecule comprises (i) a first antigen-binding moiety that binds to PD-1, wherein the first antigen-binding moiety is a Fab molecule (in which the variable domains VL and VH of the Fab light chain and Fab heavy chain are replaced with each other), which 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; and (ii) a second antigen-binding moiety that binds to LAG3, wherein the second antigen-binding moiety is a (conventional) Fab molecule, which 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. NO:89, wherein in the constant domain CL of the second antigen-binding portion, the amino acid at position 124 is substituted by lysine (K) and the amino acid at position 123 is substituted by lysine (K) or arginine (R), most particularly by arginine (R) (according to Kabat numbering), and in the constant domain CH1 of the second antigen-binding portion, the amino acid at position 147 is substituted by glutamic acid (E) (according to the Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (according to the Kabat EU index); and (iii) an Fc domain composed of a first subunit and a second subunit,

[0230] wherein the first antigen binding portion 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 N-terminus of the first subunit of the Fc domain), and the second antigen binding portion is fused at the C-terminus of the Fab heavy chain to the N-terminus of the other subunit of the Fc domain (particularly to the N-terminus of the second subunit of the Fc domain).

[0231] In some embodiments according to 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 EU index of Kabat). In some such embodiments, in the first subunit of the Fc domain, additionally the serine residue at position 354 is replaced by a cysteine ​​residue (S354C) or the glutamic acid residue at position 356 is replaced by a cysteine ​​residue (E356C) (in particular the serine residue at position 354 is replaced by a cysteine ​​residue), and in the second subunit of the Fc domain, additionally the tyrosine residue at position 349 is replaced by a cysteine ​​residue (Y349C) (numbering according to the EU index of Kabat).

[0232] In some embodiments according to any of the above aspects of the invention, 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) (numbering according to the EU index of Kabat).

[0233] In some embodiments according to any of the above aspects of the invention, the Fc domain is a human IgG1 Fc domain.

[0234] 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 carboxyl-terminal amino acid of the first subunit of the Fc domain via a linker peptide of SEQ ID NO: 93.

[0235] In a specific embodiment, the immunoconjugate comprises a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 68, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 69, a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 70, and a polypeptide comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 71. In another specific embodiment, 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.

[0236] polynucleotides

[0237] The present invention also provides an isolated polynucleotide encoding an immunoconjugate as described herein or a fragment thereof. In some embodiments, the fragment is an antigen-binding fragment.

[0238] The polynucleotide encoding the immunoconjugate of the present invention can be expressed as a single polynucleotide encoding a complete immunoconjugate, or as a plurality of (e.g., two or more) polynucleotides of co-expression. The polypeptides encoded by the co-expressed polynucleotides can associate to form a functional immunoconjugate via, for example, a disulfide bond or other means. For example, the light chain portion of an antibody can be encoded by a separate polynucleotide from an immunoconjugate portion comprising the heavy chain portion of the antibody and a mutant IL-2 polypeptide. When co-expressed, the heavy chain polypeptide will associate with the light chain polypeptide to form an immunoconjugate. In another example, the immunoconjugate portion comprising one subunit of two Fc domain subunits and a mutant IL-2 polypeptide can be encoded by a separate polynucleotide from an immunoconjugate portion comprising the other subunit of the two Fc domain subunits. When co-expressed, the Fc domain subunits will associate to form an Fc domain.

[0239] In some embodiments, the isolated polynucleotide encodes a complete immunoconjugate according to the invention as described herein. In other embodiments, the isolated polynucleotide encodes a polypeptide comprised in an immunoconjugate according to the invention as described herein.

[0240] In one embodiment, the isolated polynucleotides of the present invention encode the heavy chain (e.g., immunoglobulin heavy chain) and mutant IL-2 polypeptide of the antibody contained in the immunoconjugate. In another embodiment, the isolated polynucleotides of the present invention encode the light chain of the antibody contained in the immunoconjugate.

[0241] In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide of the present invention is RNA, for example in the form of messenger RNA (mRNA). The RNA of the present invention can be single-stranded or double-stranded.

[0242] Recombination methods

[0243] 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 may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. Correct nucleotide changes can be verified, for example, by sequencing. In this regard, the nucleotide sequence of native IL-2 has been described by 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 shown in SEQ ID NO: 19. Substitutions or insertions may involve both natural and non-natural amino acid residues. Amino acid modifications include well-known chemical modification methods, such as the addition of glycosylation sites or carbohydrate attachment.

[0244] The immunoconjugates of the present invention can be obtained, for example, by solid-state peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant production. For recombinant production, one or more polynucleotides encoding the immunoconjugates (fragments), for example, as described above, are separated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such polynucleotides can be easily separated and sequenced using conventional methods. In one embodiment, a vector is provided, preferably an expression vector, comprising one or more polynucleotides in 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 immunoconjugates (fragments) and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, synthetic technology, and in vivo recombination / genetic recombination. See, for example, Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY (1989). The expression vector can be part of a plasmid, a virus, or can be a nucleic acid fragment. The expression vector includes an expression cassette into which the polynucleotide encoding the immunoconjugate (fragment) (i.e., coding region) is cloned in operable 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 that is composed of codons that are translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into amino acids, it (if present) can be considered to be a part of the coding region, while any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, 5' and 3' untranslated regions, etc., are not part of the coding region. Two or more coding regions may be present in a single polynucleotide construct (e.g., on a single vector), or in separate polynucleotide constructs (e.g., on separate (different) vectors). In addition, any vector may contain a single coding region, or may comprise two or more coding regions, such as a vector of the present invention that may encode one or more polypeptides that are separated into final proteins by proteolytic cleavage after translation or during translation. In addition, the vectors, polynucleotides or nucleic acids of the present invention may encode heterologous coding regions, which may be fused or unfused 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 is when the coding region of a gene product (e.g., a polypeptide) is associated with one or more regulatory sequences in a manner such that the expression of the gene product is under the influence or control of the regulatory sequences. If the induction of promoter function results in the transcription of mRNA encoding the desired gene product, and if the nature of the bond between the two DNA fragments does not interfere with the ability of the expression regulatory sequence to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed, then the two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated". Thus, if the promoter is able to affect the transcription of the nucleic acid, the promoter region will be operably associated with the nucleic acid encoding the polypeptide. The promoter can be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells. In addition to the promoter, other transcription control elements, such as enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein. A variety of transcription control regions are known to those skilled in the art. These transcription control regions include, but are not limited to, those that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (e.g., immediate early promoter in conjunction with intron-A), simian virus 40 (e.g., early promoter), and retroviruses (such as, for example, Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes (such as actin, heat shock protein, bovine growth hormone, and rabbit beta-globin), as well as other sequences capable of controlling gene expression in eukaryotic cells. Other suitable transcription control regions include tissue-specific promoters and enhancers and inducible promoters (e.g., tetracycline-inducible promoters). Similarly, various translation control elements are known to those of ordinary skill in the art. These translation control elements include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly internal ribosome entry sites, or IRES, also known as CITE sequences). The expression cassette may also include other features, such as an origin of replication, and / or chromosomal integration elements, such as retroviral long terminal repeats (LTRs), or adeno-associated virus (AAV) inverted terminal repeats (ITRs).

[0245] The polynucleotides and nucleic acid coding regions of the present invention may be associated with additional coding regions encoding secretory or signal peptides that direct the secretion of the polypeptides encoded by the polynucleotides of the present invention. 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 once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those skilled in the art will recognize that polypeptides secreted by vertebrate cells typically have a signal peptide fused to the N-terminus of the polypeptide that is cleaved from the translated polypeptide to produce 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 subsequently cleaved to produce the mature 133-amino acid human IL-2. In certain embodiments, a native signal peptide (e.g., an 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 a polypeptide to which it is operably associated. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof may be used. For example, the wild-type leader sequence can be replaced by the leader sequence of human tissue plasminogen activator (TPA) or mouse beta-glucuronidase.

[0246] DNA encoding a short protein sequence that can be used to facilitate subsequent purification (eg, a histidine tag) or to aid in labeling the immunoconjugate can be included internally or at the end of the immunoconjugate (fragment) encoding polynucleotide.

[0247] In another embodiment, a host cell comprising one or more polynucleotides of the present invention is provided. In certain embodiments, a host cell comprising one or more vectors of the present invention is provided. The polynucleotides and vectors may be incorporated, alone or in combination, into any of the features described herein for the polynucleotides and vectors, respectively. In one such embodiment, the host cell comprises one or more vectors (e.g., transformed or transfected with one or more vectors) comprising one or more polynucleotides encoding the immunoconjugates of the present invention. As used herein, the term "host cell" refers to any type of cell system that can be engineered to produce the immunoconjugates of the present invention or fragments thereof. Host cells suitable for replication and support expression of immunoconjugates are well known in the art. Such cells can be appropriately transfected or transduced with specific expression vectors, and large quantities of cells containing the vectors can be grown for inoculating large-scale fermenters to obtain sufficient amounts of immunoconjugates for clinical applications. Suitable host cells include prokaryotic microorganisms, such as Escherichia coli, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, etc. For example, polypeptides can be produced in bacteria, particularly when glycosylation is not required. After expression, the polypeptide can be separated from the bacterial cell paste in a soluble fraction and can be further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding polypeptides, including fungi and yeast strains whose glycosylation pathways have been "humanized", thereby resulting in the production of polypeptides with partial or complete human glycosylation patterns. See Gerngross, Nat Biotech 22, 1409-1414 (2004) and Li et al., Nat Biotech 24, 210-215 (2006). Suitable host cells for expressing (glycosylated) polypeptides also come from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains that can be used with insect cells have been identified, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES for producing antibodies in transgenic plants). TMVertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells, as described, for example, in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells, as described, for example, in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (as described, for example, in Mather et al., Annals NY Acad Sci 383, 44-68 (1982)), MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr - CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines, such as YO, NSO, P3X63 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). Host cells include cultured cells, such as mammalian cultured cells, yeast cells, insect cells, bacterial cells and plant cells, to name a few, and also include cells comprised 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 Chinese hamster ovary (CHO) cell, human embryonic kidney (HEK) cell or lymphocyte (e.g., Y0, NSO, Sp20 cell).

[0248] Standard techniques for expressing exogenous genes in these systems are known in the art. Cells expressing a mutant IL-2 polypeptide fused to either the heavy or light chain of an antibody can be engineered to also express the other of the antibody chains so that the expressed mutant IL-2 fusion product is an antibody having both heavy or light chains.

[0249] In one embodiment, a method of producing an immunoconjugate according to the invention is provided, wherein the method comprises culturing a host cell comprising one or more polynucleotides encoding an immunoconjugate as provided herein under conditions suitable for expression of the immunoconjugate, and optionally recovering the immunoconjugate from the host cell (or host cell culture medium).

[0250] In the immunoconjugates of the present invention, the mutant IL-2 polypeptide can be genetically fused to the antibody or chemically conjugated to the antibody. The 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 to the polypeptide via a linker sequence. The composition and length of the linker can be determined according to methods well known in the art, and the efficacy of the linker can be tested. Specific linker peptides are described herein. If desired, additional sequences (e.g., endopeptidase recognition sequences) can also be included to incorporate cleavage sites to separate the individual components of the fusion. Alternatively, the IL-2 fusion protein can be chemically synthesized using polypeptide synthesis methods well known in the art (e.g., Merrifield solid-phase synthesis). The mutant IL-2 polypeptide can be chemically conjugated to other molecules (e.g., antibodies) using well-known chemical conjugation methods. Bifunctional cross-linkers (such as homofunctional and heterofunctional cross-linkers well known in the art) can be used for this purpose. The type of cross-linker used depends on the nature of the molecule to which the IL-2 is conjugated and can be readily identified by those skilled in the art. Alternatively or additionally, the mutant IL-2 and / or the molecule to which it is intended to be conjugated may be chemically derivatized so that both the mutant IL-2 and / or the molecule to which it is intended to be conjugated may be conjugated in separate reactions, as is also well known in the art.

[0251] The immunoconjugates of the present invention include antibodies. Methods for producing antibodies are well known in the art (see, for example, Harlow and Lane, "Antibodies, a laboratory manual", Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be recombinantly produced (e.g., as described in U.S. Patent No. 4,186,567), or can be obtained, for example, by screening a combinatorial library comprising variable heavy chains and variable light chains (see, for example, U.S. Patent No. 5,969,108 of McCafferty). Immunoconjugates, antibodies, and methods for preparing same are also described in detail in, for example, PCT Publications WO 2011 / 020783, WO 2012 / 107417, and WO 2012 / 146628, each of which is incorporated herein by reference in its entirety.

[0252] Antibodies of any animal species can be used in the immunoconjugates of the present invention. Non-limiting antibodies that can be used in the present invention can be of mouse, primate or human origin. If the immunoconjugate is intended for human use, a chimeric antibody can be used, wherein the constant region of the antibody is from a human. Humanized or fully humanized 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) transplanting non-human (e.g., donor antibody) CDRs onto human (e.g., acceptor antibody) frameworks and constant regions, with or without retaining key framework residues (e.g., key framework residues important for maintaining good antigen binding affinity or antibody function), (b) transplanting only non-human specific determining regions (SDRs or a-CDRs; residues that are crucial for antibody-antigen interactions) onto human frameworks and constant regions, or (c) transplanting entire non-human variable domains, but "hiding" them with human-like segments by replacing surface residues. Humanized antibodies and methods for their preparation are reviewed in, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in, e.g., Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "surface remodeling"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the "guided selection" method for FR shuffling).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 the consensus sequence of human antibodies having a particular subset of light or heavy 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)). 993)); 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 FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

[0253] Various techniques known in the art can be used to produce human antibodies. 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 be prepared in the following manner: an immunogen is administered to a transgenic animal that has been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigenic stimulation. Such animals typically contain all or part of a human immunoglobulin locus that replaces an endogenous immunoglobulin locus, or exists outside the chromosome of the animal or is randomly integrated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin locus is typically 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, description of XENOMOUSE. TM US Patent Nos. 6,075,181 and 6,150,584; Description US Patent No. 5,770,429; describes KM US Patent No. 7,041,870, and describes Human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0254] Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma 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). Additional methods include, for example, those described in U.S. Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, 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).

[0255] Human antibodies can also be generated by isolation from human antibody libraries, as described herein.

[0256] The antibodies useful in the present invention can be isolated by screening the combinatorial library for antibodies with one or more desired activities. Methods for screening combinatorial libraries are summarized in, for example, 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 to obtain antibodies with desired binding characteristics. 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), and in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001), and Marks and Bradbury, Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003).

[0257] In certain phage display methods, the entire collection of VH and VL genes is cloned individually by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phage, as described in Winter et al., Annual Review of Immunology 12:433-455 (1994). Phage typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, all natural repertoires (e.g., all natural repertoires from humans) can be cloned to provide a single source of antibodies to a wide range of non-self and self antigens without any immunization, as described by Griffiths et al. in EMBO Journal 12:725-734 (1993). Finally, natural libraries are also synthesized by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and perform in vitro rearrangement as described by Hoogenboom and Winter in 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 and U.S. Patent Publication Nos. 2005 / 0079574, 2007 / 0117126, 2007 / 0237764 and 2007 / 0292936. Other examples of methods known in the art for screening combinatorial libraries of antibodies with one or more desired activities include ribosome and mRNA display, and methods for displaying antibodies and selecting antibodies to bacteria, mammalian cells, insect cells or yeast cells. Methods for yeast surface display are summarized in, for example, 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 described in, for example, He et al., Nucleic Acids Research 25:5132-5134 (1997) and Hanes et al., PNAS 94:4937-4942 (1997).

[0258] It may be desirable to further chemically modify the immunoconjugates of the invention. For example, by conjugation to substantially linear polymers such as polyethylene glycol (PEG) or polypropylene glycol (PPG), the problems of immunogenicity and short half-life may be ameliorated (see, for example, WO 87 / 00056).

[0259] The immunoconjugates prepared as described herein can be purified by techniques known in the art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and will be apparent to those skilled in the art. For affinity chromatography purification, an antibody, ligand, receptor, or antigen that binds to the immunoconjugate can be used. For example, an antibody that specifically binds to a mutant IL-2 polypeptide can be used. For affinity chromatography purification of the immunoconjugates of the invention, a matrix having protein A or protein G can be used. For example, sequential protein A or G affinity chromatography and size exclusion chromatography can be used to separate the immunoconjugates, essentially as described in the Examples. The purity of the immunoconjugate can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high pressure liquid chromatography, and the like.

[0260] Compositions, formulations and routes of administration

[0261] In another aspect, the present invention provides pharmaceutical compositions comprising the immunoconjugates described herein, for example, for use in any of the following treatment methods. 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, for example, as described below.

[0262] Also provided is a method of producing an immunoconjugate of the present invention in a form suitable for in vivo administration, the method comprising (a) obtaining an immunoconjugate according to the present invention, and (b) formulating the immunoconjugate with at least one pharmaceutically acceptable carrier, thereby formulating the immunoconjugate for in vivo administration.

[0263] The pharmaceutical composition of the present invention comprises the immunoconjugate of therapeutically effective amount dissolved or dispersed in a pharmaceutical carrier. The phrase "pharmaceutically or pharmacologically acceptable" refers to that the molecular entity and composition are generally non-toxic to the recipient at the dosage and concentration adopted, i.e., when applied to an animal (such as, for example, a human), no adverse, allergic or other adverse reactions will be produced. According to the present disclosure, the preparation of pharmaceutical compositions containing immunoconjugates and optional other active ingredients will be known to those skilled in the art, as illustrated by Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, which is incorporated herein by reference. In addition, for animal (e.g., human) administration, it should be understood that the preparation should meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Office of Biological Standards or other national / regional authorities. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, the like, and combinations thereof, as would be known to one of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Printing Company, 1990, pp. 1289-1329, which is incorporated herein by reference). Except in the event that any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is contemplated.

[0264] The immunoconjugates of the present invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and if desired for local treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be carried out by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-lived or long-term.

[0265] 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 can be formulated in an aqueous solution, preferably in a physiologically compatible buffer (such as Hanks solution, Ringer solution or physiological saline buffer). The solution may contain a formulation agent (formulatory agent), such as a suspending agent, a stabilizer and / or a dispersant. Alternatively, the immunoconjugate can be in powder form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use. Sterile injectable solutions are prepared by incorporating the immunoconjugates of the present invention into a suitable solvent in the desired amount together with the various other ingredients listed below as needed. For example, sterility can be easily achieved by filtration through a sterile filtration membrane. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile solvent containing a basic dispersion medium and / or other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsions, the preferred preparation method is vacuum drying or lyophilization techniques, which produce a powder of the active ingredient plus any additional required ingredients from a previously sterile-filtered liquid medium. If necessary, the liquid medium should be suitably buffered, and sufficient saline or glucose should first be used to make the liquid diluent isotonic before injection. The composition must be stable under the conditions of manufacture and storage and preserved to resist the contaminating effects of microorganisms such as bacteria and fungi. It should be understood that endotoxin contamination should be kept to a minimum, for example, at a safety level below 0.5 ng / mg protein. Suitable pharmaceutical carriers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as Serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, dextran, and the like. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to allow the preparation of high concentration solutions.Additionally, suspensions of the active compound may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes.

[0266] The active ingredient can be embedded in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared, for example, by coacervation techniques or by interfacial polymerization; embedded in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules); or embedded in coarse emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th edition, Mack Printing Company, 1990). Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing polypeptides, which are in the form of shaped articles such as films or microcapsules. In particular embodiments, prolonged absorption of the injectable composition can be achieved by using an agent that delays absorption (such as, for example, aluminum monostearate, gelatin, or a combination thereof) in the composition.

[0267] In addition to the compositions described previously, immunoconjugates can also be formulated as depot preparations. Such long-acting preparations can be administered by implantation (e.g., subcutaneous or intramuscular implantation) or by intramuscular injection. Thus, for example, immunoconjugates can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or with ion exchange resins, or formulated as slightly soluble derivatives, such as slightly soluble salts.

[0268] Pharmaceutical compositions comprising the immunoconjugates of the present invention can be prepared by conventional mixing, dissolving, emulsifying, encapsulating, embedding, or lyophilizing methods. Pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants that facilitate processing of the protein into pharmaceutically acceptable formulations. The appropriate formulation depends on the chosen route of administration.

[0269] The immunoconjugates can be formulated as compositions in free acid or base, neutral or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or free base. These pharmaceutically acceptable salts include acid addition salts, such as acid addition salts formed with the free amino groups of the protein composition, or acid addition salts formed with inorganic acids such as hydrochloric acid or phosphoric acid or organic acids such as acetic acid, oxalic acid, tartaric acid or mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide; or organic bases such as isopropylamine, trimethylamine, histidine or procaine. Pharmaceutically acceptable salts tend to be more soluble in aqueous and other protic solvents than the corresponding free base forms.

[0270] Methods of treatment and compositions

[0271] Any of the immunoconjugates provided herein can be used in therapeutic methods.The immunoconjugates of the invention can be used as immunotherapeutic agents, for example, for the treatment of cancer.

[0272] For use in therapeutic methods, the immunoconjugates of the present invention will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this context include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to practitioners.

[0273] The immunoconjugates of the present invention are particularly useful for treating disease states in which stimulation of the host immune system is beneficial, particularly diseases in which an enhanced cellular immune response is desired. These disease states may include those in which the host immune response is insufficient or absent. Disease states in which the immunoconjugates of the present invention may be administered include, for example, tumors or infections in which a cellular immune response is a key mechanism of specific immunity. The immunoconjugates of the present invention may be administered per se or in any suitable pharmaceutical composition.

[0274] In one aspect, the immunoconjugates of the present invention are provided for use as a medicament. In other aspects, the immunoconjugates of the present invention are provided for use in treating a disease. In certain embodiments, the immunoconjugates of the present invention are provided for use in a method of treatment. In one embodiment, the present invention provides an immunoconjugate as described herein for use in treating a disease in an individual in need thereof. In certain embodiments, the present invention provides an immunoconjugate for use in a method of treating an individual suffering from a disease, the method comprising administering a therapeutically effective amount of the immunoconjugate to the individual. In certain embodiments, the disease to be treated is a proliferative disorder. In a specific embodiment, the disease is cancer. In certain embodiments, the method further comprises administering a therapeutically effective amount of at least one additional therapeutic agent to the individual, for example, if the disease to be treated is cancer, an anticancer agent is used. In other 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 of stimulating the immune system of an individual, the method comprising administering an effective amount of the immunoconjugate to the individual to stimulate the immune system. The "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 the following: general enhancement of immune function, enhancement of T cell function, enhancement of B cell function, restoration of lymphocyte function, increased expression of IL-2 receptors, enhancement of T cell reactivity, enhancement of natural killer cell activity or lymphokine-activated killer (LAK) cell activity, and the like.

[0275] In another aspect, the present invention provides use of an immunoconjugate of the present invention in the manufacture or preparation of a medicament. In one embodiment, the medicament is used to treat a disease in an individual in need thereof. In one embodiment, the medicament is used in a method of treating a disease, the method comprising administering a therapeutically effective amount of the medicament to an individual having the disease. In certain embodiments, the disease to be treated is a proliferative disorder. In a specific embodiment, the disease is cancer. In one embodiment, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, such as an anticancer agent if the disease to be treated is cancer. In another embodiment, the medicament is used to stimulate the immune system. In another embodiment, the medicament is used in a method of stimulating the immune system of an individual, the method comprising administering to the individual an effective amount of the medicament to stimulate the immune system. According to any of the above embodiments, the "individual" can be a mammal, preferably a human. According to any of the above embodiments, "stimulation of the immune system" can include any one or more of the following: general enhancement of immune function, enhancement of T cell function, enhancement of B cell function, restoration of lymphocyte function, increased expression of IL-2 receptor, enhanced T cell reactivity, enhancement of natural killer cell activity or lymphokine-activated killer (LAK) cell activity, etc.

[0276] In another aspect, the present invention provides a method for treating a disease in an individual. In one embodiment, the method comprises administering a therapeutically effective amount of an immunoconjugate of the present invention to an individual suffering from the disease. In one embodiment, a composition comprising a pharmaceutically acceptable form of the immunoconjugate of the present invention is administered to the individual. In certain embodiments, the disease to be treated is a proliferative disorder. In a specific embodiment, the disease is cancer. In certain embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, such as an anticancer agent if the disease to be treated is cancer. In another aspect, the present invention provides a method for stimulating the immune system of an individual, comprising administering to the individual an effective amount of an immunoconjugate to stimulate the immune system. The "individual" according to any of the above embodiments can be a mammal, preferably a human. "Stimulation of the immune system" according to any of the above embodiments can include any one or more of the following: general enhancement of immune function, enhancement of T cell function, enhancement of B cell function, restoration of lymphocyte function, increase in IL-2 receptor expression, enhancement of T cell reactivity, enhancement of natural killer cell activity or lymphokine-activated killer (LAK) cell activity, and the like.

[0277] In certain embodiments, the disease to be treated is a proliferative disorder, particularly cancer. Non-limiting examples of cancer include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and renal cancer. Other cell proliferation disorders that can be treated using the immunoconjugates of the present invention include, but are not limited to, tumors located in the following parts: abdomen, bones, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary gland, testicles, ovaries, thymus, thyroid gland), eyes, head and neck, nervous system (central and peripheral nervous system), lymphatic system, pelvis, skin, soft tissue, spleen, chest, and urogenital system. Precancerous conditions or lesions and cancer metastasis are also included. In certain embodiments, the cancer is selected from the group consisting of: renal cancer, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer, prostate cancer, and bladder cancer. It will be readily appreciated by those skilled in the art that, in many cases, the immunoconjugate may not provide a cure, but may only provide a partial benefit. In some embodiments, physiological changes with some benefit are also considered therapeutically beneficial. Thus, in some embodiments, the amount of the immunoconjugate that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount." The subject, patient, or individual in need of treatment is typically a mammal, more particularly a human.

[0278] In some embodiments, an effective amount of an immunoconjugate of the invention is administered to a cell. In other embodiments, a therapeutically effective amount of an immunoconjugate of the invention is administered to an individual to treat a disease.

[0279] For the prevention or treatment of disease, the appropriate dosage of the immunoconjugates of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the patient's weight, the type of molecule (e.g., containing or not containing an Fc domain), the severity and course of the disease, whether the immunoconjugate is administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's clinical history and response to the immunoconjugate, and the judgment of the attending physician. In any case, the practitioner responsible for administration will determine the concentration and appropriate dosage of the active ingredient in the composition for the individual subject. Various dosing schedules are contemplated herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulse infusions.

[0280] The immunoconjugate is appropriately administered to the patient once or in a series of treatments. Depending on the type and severity of the disease, an immunoconjugate of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg-10 mg / kg) can be the initial candidate dose for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. Depending on the above factors, a typical daily dose can range from about 1 μg / kg to 100 mg / kg or more. For repeated administration over several days or longer, depending on the disease, treatment will typically continue until desired disease symptom suppression occurs. An exemplary dosage of the immunoconjugate should be in the range of about 0.005 mg / kg to about 10 mg / kg. In other non-limiting examples, dosages can also include about 1 μg / kg / body weight, about 5 μg / kg / body weight, about 10 μg / kg / body weight, about 50 μg / kg / body weight, about 100 μg / kg / body weight, about 200 μg / kg / body weight, about 350 μg / kg / body weight, about 500 μg / kg / body weight, about 1 mg / kg / body weight, about 5 mg / kg / body weight, about 10 mg / kg / body weight, about 50 mg / kg / body weight, about 100 mg / kg / body weight, about 200 mg / kg / body weight, about 350 mg / kg / body weight, about 500 mg / kg / body weight, to about 1000 mg / kg / body weight or more per administration, and any range derived therefrom. In the non-limiting examples of the scope that can be deduced from the numerals listed herein, the scope of about 5mg / kg / body weight to about 100mg / kg / body weight, about 5 micrograms / kg / body weight to about 500 milligrams / kg / body weight etc. can be applied based on the above numerical value. Therefore, one or more dosages of about 0.5mg / kg, 2.0mg / kg, 5.0mg / kg or 10mg / kg (or any combination thereof) can be applied to the patient. Such dosage can be applied intermittently, for example, weekly or every three weeks (for example, so that the patient receives about twice to about 20 times, or for example, an immunoconjugate of about six doses). An initial higher loading dose can be applied, followed by one or more lower dosages. However, other dosage regimens may be available. The progress of this therapy can be easily monitored by conventional techniques and determination.

[0281] The immunoconjugates of the present invention will generally be used in an amount effective to achieve the intended purpose. For use in treating or preventing a condition, 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.

[0282] For systemic administration, the therapeutically effective dose can be estimated initially based on in vitro assays (such as cell culture assays). A dose can then be formulated in animal models to achieve an IC including that determined in cell culture.50 Such information can be used to more accurately determine useful doses in humans.

[0283] Initial doses can also be estimated based on in vivo data (e.g., animal models) using techniques well known in the art. One of ordinary skill in the art can easily optimize administration to humans based on animal data.

[0284] The amount and interval of the dose can be adjusted individually to provide plasma levels of the immunoconjugate sufficient to maintain the therapeutic effect. Common patient dosages administered by injection range from about 0.1 to 50 mg / kg / day, typically from about 0.5 to 1 mg / kg / day. Therapeutically effective plasma levels can be achieved by administering multiple doses per day. Levels in plasma can be measured, for example, by HPLC.

[0285] In cases of local administration or selective uptake, the effective local concentration of the immunoconjugate may not be related to plasma concentration. One skilled in the art will be able to optimize a therapeutically effective local dose without undue experimentation.

[0286] The therapeutically effective dose of the immunoconjugates described herein will generally provide therapeutic benefit without causing substantial toxicity. The toxicity and therapeutic efficacy of the immunoconjugates can be determined by standard pharmaceutical methods in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine the LD 50 (dose that is lethal to 50% of the population) and ED 50 (the dose that is therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD 50 / ED 50 Immunoconjugates that exhibit a large therapeutic index are preferred. In one embodiment, the immunoconjugates according to the present invention exhibit a high therapeutic index. Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages suitable for use in humans. Dosages are preferably within the range of EDs that include little or no toxicity. 50 The dosage may vary within this range depending on a variety of factors, such as the dosage form employed, the route of administration utilized, the condition of the subject, and the like. The exact formulation, route of administration, and dosage can be selected by the individual physician based on the patient's condition. (See, for example, Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Chapter 1, page 1, the entire contents of which are incorporated herein by reference).

[0287] The attending physician of the patient being treated with the immunoconjugate of the present invention should know how and when to terminate, interrupt or adjust administration due to toxicity, organ dysfunction, etc. Conversely, if the clinical response is insufficient (excluding toxicity), the attending physician will also know to adjust the treatment to a higher level. The size of the dose administered in the management of the target disorder will vary with the severity of the condition to be treated, the route of administration, etc. For example, the severity of the condition can be assessed in part by standard prognostic assessment methods. In addition, the dosage and possible dosage frequency will also vary according to the age, weight and response of the individual patient.

[0288] The maximum therapeutic dose of an immunoconjugate comprising a mutant IL-2 polypeptide as described herein can be increased relative to the maximum therapeutic dose used for an immunoconjugate comprising wild-type IL-2.

[0289] Other medications and treatments

[0290] The immunoconjugates according to the present invention can be administered in combination with one or more other agents for treatment. For example, the immunoconjugates of the present invention can be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" includes any agent administered to treat the symptoms or diseases of an individual in need of such treatment. Such additional therapeutic agents may include any active ingredients suitable for the specific indications being treated, preferably active ingredients with complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is an immunomodulator, a cell growth inhibitor, a cell adhesion inhibitor, a cytotoxic agent, a cell apoptosis activator, or an agent that increases the sensitivity of cells to apoptosis-inducing agents. In a specific embodiment, the additional therapeutic agent is an anticancer agent, such as a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormone therapy, a kinase inhibitor, a receptor antagonist, a tumor cell apoptosis activator, or an anti-angiogenic agent.

[0291] Such other agents are suitably present in combination in amounts effective for their intended purpose. The effective amount of such other agents depends on the amount of immunoconjugate used, the type of disorder or treatment, and other factors discussed above. The immunoconjugates are generally used in the same dosages and routes of administration as described herein, or at about 1% to 99% of the dosages described herein, or in any dosage and by any route determined empirically / clinically to be appropriate.

[0292] Such combination therapies include combined administration (wherein two or more therapeutic agents are included in the same or different compositions) and separate administration. In the case of separate administration, the administration of the immunoconjugate of the present invention can be performed before, simultaneously with, and / or after the administration of the additional therapeutic agent and / or adjuvant. The immunoconjugate of the present invention can also be used in combination with radiotherapy.

[0293] Products

[0294] In another aspect of the present invention, an article of manufacture is provided that contains a substance that can be used to treat, prevent, and / or diagnose the above-mentioned conditions. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous (IV) solution bags, and the like. The container can be formed from a variety of materials, such as glass or plastic. The container holds a composition that, by itself or in combination with another composition, is effective for treating, preventing, and / or diagnosing the condition, and the container can have a sterile access port (e.g., the container can be an IV solution bag or vial with 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. In addition, the article of manufacture may include (a) a first container containing a composition, wherein the composition comprises an immunoconjugate of the present invention; and (b) a second container containing a composition, wherein the composition comprises an additional cytotoxic agent or other therapeutic agent. The article of manufacture in this embodiment of the present invention may further include a package insert indicating that the compositions can be used to treat a specific condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising 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 desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes. BRIEF DESCRIPTION OF THE DRAWINGS

[0295] Figure 1A to Figure 1B . A mouse alternative to the IL2v construct targeting mouse PD1 (using the V domain from rat to target mouse PD1). Figure 1A Shown is P1AG9991, a bivalent mouse IgG1 DA PG targeting mouse PD1, with human IL2v fused to the C-terminus of the Fc DD-chain. Figure 1B Shown is P1AG8304, a bivalent mouse IgG1 DAPG targeting mouse PD1, in which human IL2v Q126T is fused to the C-terminus of the Fc DD-chain.

[0296] Figures 2A to 2C .IL2v construct targeting human PD1- / LAG3. Figure 2A Shown is P1AF4801, a bispecific IgG1 PG LALA crossMab targeting human PD1- / LAG3, in which human IL2v is fused to the C-terminus of the Fc knob chain. Figure 2BShown is P1AF7951, a bispecific IgG1 PG LALA crossMab targeting human PD1- / LAG3, in which human IL2v Q126T is fused to the C-terminus of the Fc knob chain. Figure 2C Shown is P1AA6888, a monospecific human PD1 IgG1 PGLALA used as a control.

[0297] Figure 3 The proliferation of NK92 cells after 3 days of treatment with PD1-IL2v and several PD1-IL2v variants was determined by measuring ATP levels using CellTiter Glo.

[0298] Figure 4 A to Figure 4 C. Flow cytometry was used to determine the expression of CD8 T cells in PBMCs after 5 days of treatment with PD1-IL2v and several PD1-IL2v variants ( Figure 4 A) NK cells ( Figure 4 B) and CD4 T cells ( Figure 4 C) proliferation.

[0299] Figure 5 A to Figure 5 C. CD8 T cells within PBMCs after 5 days of treatment with PD1-IL2v and several PD1-IL2v variants as determined by measuring CD25 upregulation via flow cytometry. Figure 5 A) NK cells ( Figure 5 B) and CD4 T cells ( Figure 5 C) activation.

[0300] Figure 4 The proliferation of NK92 cells after 3 days of treatment with FAP-IL2v and several FAP-IL2v variants was determined by measuring ATP levels using CellTiter Glo.

[0301] Figure 6 A to Figure 6 D. Proliferation of CD8 T cells and NK cells in PBMCs after 4 days of treatment with FAP-IL2v and several FAP-IL2v variants was determined by flow cytometry. Figure 6 A shows FAP-IL2v(G4S)5, FAP-IL2v_D20T_Selectikine, FAP-IL2v_E215V, FAP-IL2v_E95A, FAP-IL2v_E95A, and FAP-IL2v. Figure 6 B 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 6 D shows FAP-IL2v_Q22A, FAP-IL2v_Q126T, FAP-IL2v_S87A, FAP-IL2v_S130A, and FAP-IL2v.

[0302] Figure 7 A to Figure 7 B. CD8 T cells within PBMCs after 4 days of treatment with PD1-IL2v and several PD1-IL2v variants were determined by measuring CD25 upregulation via flow cytometry. Figure 7 A) and NK cells ( Figure 7 B) activation.

[0303] Figure 8 A to Figure 8 B. CD8 T cells within PBMCs after 4 days of treatment with PD1-IL2v and several PD1-IL2v variants were determined by measuring CD25 upregulation via flow cytometry. Figure 8 A) and NK cells ( Figure 8 B) activation.

[0304] Figure 9 A to Figure 9 C. Flow cytometry was used to determine the expression of NK cells in PBMCs after 5 days of treatment with FAP-IL2v and selected FAP-IL2v variants. Figure 9 A), CD8 T cells ( Figure 9 B) and CD4 T cells ( Figure 9 C) proliferation.

[0305] Figure 10 A to Figure 10 C. NK cell upregulation within PBMCs after 5 days of treatment with FAP-IL2v and selected FAP-IL2v variants was determined by measuring CD25 upregulation via flow cytometry. Figure 10 A), CD8 T cells ( Figure 10 B) and CD4 T cells ( Figure 10 C) activation.

[0306] Figure 11 A to Figure 11 D. CD4 T cells ( Figure 11 A) Regulatory T cells ( Figure 11 B) CD8 T cells ( Figure 11 C) and NK cells ( Figure 11 D) STAT5 phosphorylation.

[0307] Figure 12 A to Figure 12 C. Flow cytometry was used to determine the CD8 T cells in PBMC after 5 days of treatment with FAP-IL2v, FAP-IL2vQ126T and PD1-IL2vQ126T ( Figure 12 A) NK cells ( Figure 12 B) and CD4 T cells ( Figure 12 C) proliferation.

[0308] Figure 13 A to Figure 13 C. CD8 T cells within PBMCs after 5 days of treatment with FAP-IL2v, FAP-IL2vQ126T, and PD1-IL2v Q126T were determined by measuring CD25 upregulation via flow cytometry. Figure 13 A) NK cells ( Figure 13 B) and CD4 T cells ( Figure 13 C) activation.

[0309] Figure 14 A to Figure 14 G. IL-2 signaling (STAT5-P) in PD1-blocked and PD-1-expressing CD4+ T cells cultured together. IL-2 signaling (STAT5-P) is depicted as STAT5-P frequency in human PD1+ (solid line) and PD-1 pre-blocked (dashed line) CD4 T cells 12 minutes after exposure to the PD1-IL2v mutant. Mean ± SEM of 4 donors is shown. Figure 14 A shows PD1-IL2v, PD1-IL2v and PD1-preblocking, PD1-IL2v_Q126T, PD1-IL2v_Q126T and PD1-preblocking, PD1-IL2v_K8S, PD1-IL2v_K8S and PD1-preblocking. Figure 14 B shows PD1-IL2v, PD1-IL2v and PD1-preblocking, PD1-IL2v_Q126T, PD1-IL2v_Q126T and PD1-preblocking, PD1-IL2v_L12A, PD1-IL2v_L12A and PD1-preblocking. Figure 14 C shows PD1-IL2v, PD1-IL2v and PD1-preblocking, PD1-IL2v_Q126T, PD1-IL2v_Q126T and PD1-preblocking, PD1-IL2v_N88D, PD1-IL2v_N88D and PD1-preblocking. Figure 14D shows PD1-IL2v, PD1-IL2v and PD1-preblocking, PD1-IL2v_Q126T, PD1-IL2v_Q126T and PD1-preblocking, PD1-IL2v_L19V, PD1-IL2v_L19V and PD1-preblocking. Figure 14 E shows PD1-IL2v, PD1-IL2v and PD1-preblocking, PD1-IL2v_Q126T, PD1-IL2v_Q126T and PD1-preblocking, PD1-IL2v_H79S, PD1-IL2v_H79S and PD1-preblocking. Figure 14 F shows PD1-IL2v, PD1-IL2v and PD1-preblocking, PD1-IL2v_Q126T, PD1-IL2v_Q126T and PD1-preblocking, PD1-IL2v_D109A, PD1-IL2v_D109A and PD1-preblocking. Figure 14 G shows PD1-IL2v, PD1-IL2v and PD1-preblocking, PD1-IL2v_Q126T, PD1-IL2v_Q126T and PD1-preblocking, PD1-IL2v_L80A, PD1-IL2v_L80A and PD1-preblocking.

[0310] Figure 15 A to Figure 15 B.IL-2 signaling (STAT5-P) in CD4+ T cells expressing PD-1 ( Figure 15 A) Frequency and mean fluorescence intensity (MFI) showing the potency of selected PD1-, FAP-, and NKG2D-IL2 variants on PD-1+ CD4 T cells ( Figure 15 B). Efficacy measurements in PD1+CD4 T cells reflect PD1-mediated delivery of IL-2v versus PD1-independent delivery of the FAP-IL-2v construct. Mean ± SEM of 4 donors are shown.

[0311] Figure 16 A to Figure 16 B. Frequency of CMV-specific CD4 T cells upon restimulation with the CMV protein pp65 and in the presence of the indicated treatments in combination with pp65 ( Figure 16 A). The fold increase in CMV-specific CD4 T cell frequency normalized to the corresponding response to pp65 alone shows the effect of specific compounds in amplifying antigen-specific T cell responses ( Figure 16 B) Shown are the mean ± SEM of 5 donors.

[0312] Figure 17Percent Treg-mediated inhibition of granzyme B production by Tconv during 5-day co-culture in the presence or absence of the indicated immunoconjugates. Median of 6 donors. P values ​​were calculated using one-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0313] Figure 18 IL-2 signaling (STAT5-β) on PD-1-expressing CD4+ T cells. Efficacy frequency of selected PD1-, LAG-3, and FAP-IL2 variants on PD-1+, LAG-3+, and PD-1-, LAG-3- CD4 T cells. Efficacy measurements in PD1+, LAG-3+ CD4 T cells reflect PD1-(LAG-3)-mediated IL-2v delivery versus PD-1 / LAG-3-independent FAP-IL-2v construct delivery. Mean ± SEM of 3 donors is shown.

[0314] Figure 19 Percent Treg-mediated inhibition of granzyme B production by Tconv during 5-day coculture in the presence or absence of the indicated immunoconjugates. Median of 10 donors. P values ​​were calculated using one-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0315] Figure 20 Percentage of activated CD4 T cells internalizing the molecule at 0.6 nM after 3 hours of incubation at 37°C. Median of 4 donors. P values ​​were calculated using one-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0316] Figure 21 .The results of efficacy experiments of the PD1-IL2vQ126T variant and the murine surrogate of PD-IL2v Mab as single agents are presented. The Panc02-Fluc pancreatic cancer cell line was subcutaneously injected into black 6-huIL2RBG transgenic mice to study tumor growth inhibition (TGI) in a subcutaneous model. Tumor size was measured using a caliper. Treatment was started when the tumor reached 100 mm3. For the muPD1-IL2vQ126T variant, the amount of antibody injected per mouse was 2 mg / kg, and for muPD1-IL2vqw, the amount of antibody injected was 0.5 mg / kg. Treatment lasted for 3 weeks. Compared with the vehicle and PD1-IL2v groups, the PD1-IL2vQ126T variant mediated significantly superior efficacy in tumor growth inhibition. The PD1-IL2vQ126T molecule was well tolerated, and no clinical signs or weight loss were observed.

[0317] Amino acid sequence

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372] Examples

[0373] The following are examples of methods and compositions of the present invention.It should be understood that various other embodiments may be practiced, given the general description provided above.

[0374] Example 1

[0375] Example 1A. Molecules

[0376] The molecules tested in the following examples consisted of the amino acid sequences according to Table A.

[0377] Table A. Molecules generated and tested in the Examples.

[0378]

[0379]

[0380] Example 1B. Production and Analysis of Human PD1 and FAP IgG-IL2v Variants

[0381] Antibody IL2v variant fusion constructs as described herein are produced in HEK cells. In some cases (surface plasmon resonance measurement), supernatant is directly used without prior purification (Table 1). In all other assays, protein is first purified by Protein A affinity chromatography and size exclusion chromatography. Final product analysis consists of the following: monomer content determination (by analyzing size exclusion chromatography) and main peak percentage (by following non-reducing capillary SDS electrophoresis determination: CE-SDS).

[0382] Production of IgG-like proteins in HEK293 EBNA cells

[0383] Antibody IL2v variant fusion constructs were generated by transient transfection of HEK293 EBNA cells. The cells were centrifuged and then replaced with preheated CD CHO culture medium (Thermo Fisher, catalog number 10743029). The expression vector was mixed in CD CHO culture medium, PEI (polyethyleneimine, Polysciences, Inc, catalog number 23966-1) was added, the solution was vortexed and incubated at room temperature for 10 minutes. The cells (2 Mio / ml) were then mixed with the vector / PEI solution, transferred to a flask, and placed in a shaking incubator and incubated at 37°C for 3 hours in an atmosphere of 5% CO2. After incubation, Excell culture medium (W. Zhou and A. Kantardjieff, Mammalian Cell Cultures for Biologics Manufacturing, DOI: 10.1007 / 978-3-642-54050-9; 2014) containing supplements (80% of the total volume) was added. One day after transfection, supplements (feed, 12% of the total volume) were added. After 7 days, the cell supernatant was harvested by centrifugation and subsequent filtration (0.2 μm filter), and proteins were purified from the harvested supernatant using standard methods as shown below.

[0384] Titer determination (PA-HPLC)

[0385] Fc-containing constructs in the supernatant were quantified by Protein A HPLC on an Agilent HPLC system with UV detection. The supernatant was injected onto a POROS20A (Applied Biosystems), washed with 10 mM Tris, 50 mM glycine, 100 mM NaCl (pH 8.0), and eluted in 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.

[0386] Purification of IgG-like proteins

[0387] Proteins were purified from filtered cell culture supernatants according to standard protocols. Briefly, Fc-containing proteins were purified from filtered cell culture supernatants using 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 by immediate pH neutralization of the sample. The elution was performed by centrifugation (Millipore Proteins were concentrated using ULTRA-15 (Cat. No. UFC903096), and aggregated proteins were separated from monomeric proteins by size exclusion chromatography in 20 mM histidine, 140 mM sodium chloride (pH 6.0).

[0388] Analysis of IgG-like proteins

[0389] The concentration of the purified protein was determined by measuring the absorbance at 280 nm using the mass extinction coefficient calculated based on the amino acid sequence according to the method described in Pace et al. (Protein Science, 1995, 4, 2411-1423). The purity and molecular weight of the protein were analyzed by CE-SDS using a LabChip GXII or LabChip GX Touch (Perkin Elmer) (Perkin Elmer) in the presence and absence of a reducing agent. Aggregation content was determined at 25 ° C by HPLC chromatography using an analytical size exclusion column (TSKgel G3000 SW XL or UP-SW3000, Tosoh Bioscience) equilibrated in running buffer (200 mM KH PO , 250 mM KCl pH 6.2, 0.02% NaN ).

[0390] Table 1. Titer determination of harvested HEK supernatants by Protein A-HPLC.

[0391]

[0392]

[0393] Table 2. Monomeric product peak, high molecular weight (HMW) and low molecular weight (LMW) by-products determined by analytical size exclusion chromatography.

[0394]

[0395]

[0396] Table 3. Major product peaks determined by non-reducing CE-SDS.

[0397]

[0398] result

[0399] IgG-IL2v variant constructs produced in HEK cells were tested without prior purification from the supernatant, but after quantification by Protein A titer determination (Table 1) or after purification. Mass analysis of the purified material showed product peaks at 57.8 (in one case) or between 87% and 100% by analytical size exclusion chromatography (Table 2) and between 88 and 99 by non-reducing capillary electrophoresis (Table 3).

[0400] in conclusion

[0401] With the exception of IL2v Q22A, IL2v Q126N and IL2v Q126E, all IgG-IL2v variants were produced in good quality.

[0402] Example 1C. Affinity setting of purified FAP-IL2v variants for recombinant human IL2Rβ-γ-Fc heterodimer

[0403]

[0404] Surface plasmon resonance (SPR) experiments were performed on a Biacore T200 using HBS-EP+ as a 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 (Roche in-house) were directly immobilized on a CM5 chip (Cytiva) by amine coupling. FAP-IL2v variants were captured at 10 nM for 40 s. A five-fold dilution series of human IL2Rβ-γ-Fc was passed over the ligand at a rate of 30 μl / min for 240 sec 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+. After each cycle, the chip surface was regenerated using a 30-second injection of 10 mM glycine pH 2.1. Bulk refractive index differences were corrected by subtracting the response obtained on reference flow cell 1. Affinity constants were derived from kinetic rate constants by fitting to 1 :1 Langmuir binding using Biaeval software (Cytiva).

[0405] sample

[0406] The following samples were analyzed for binding to human IL2Rβ-γ-Fc (Table 4).

[0407] Table 4. Description of samples analyzed for binding to human IL2Rβ-γ-Fc.

[0408]

[0409] result

[0410] Affinity determination of fourteen FAP-IL2v variants for recombinant human IL2Rβ-γ-Fc heterodimer

[0411] Affinity measurements for human IL2Rβ-γ showed very slow off-rates, reaching the limits of the instrument and giving unrealistic KD. However, reduced affinity could still be assessed (Table 5).

[0412] Table 5. Kinetic constants (1:1 Langmuir binding).

[0413]

[0414]

[0415] in conclusion

[0416] FAP-IL2v variants were purified and their affinity for IL2Rβ-γ-Fc was measured. The D20T, Q126T, and N88T variants exhibited reduced affinity for human IL2Rβ-γ-Fc. The double mutant L12A / L19A exhibited slightly reduced affinity for human IL2Rβ-γ-Fc.

[0417] Example 1D. Characterization of PD1-IL2v variants

[0418] Binding assessment setup of PD1-IL2v variants from supernatants to recombinant human IL2Rβ-γ-Fc heterodimers

[0419]

[0420] Surface plasmon resonance (SPR) experiments were performed on a Biacore T200 using HBS-EP+ as a running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005% surfactant P20 (BR-1006-69, Cytiva)). Anti-human Fab-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 to achieve approximately 200 RU. A single injection of 300 nM recombinant human IL2Rβ-γ-Fc was passed over the ligand at a rate of 30 μl / min for 120 sec to record the association phase. The dissociation phase was monitored for 120 s and triggered by switching from the sample solution to HBS-EP+. After each cycle, the chip surface was regenerated using two 30 sec injections of 10 mM glycine pH 2.1. Bulk refractive index differences were corrected by subtracting the response obtained on reference flow cell 1. The ratio of post-binding response units to post-capture response units was calculated. In addition, binding curves were visually inspected to identify variants with faster off-rates.

[0421] Affinity of purified PD1-IL2v variants for recombinant human IL2Rβ-γ-Fc heterodimer

[0422]

[0423] Surface plasmon resonance (SPR) experiments were performed on a Biacore T200 using HBS-EP+ as 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 (Roche in-house) were directly immobilized on a CM5 chip (Cytiva) by amine coupling. The PD1-IL2v variant was captured at 10 nM for 40 s. A five-fold dilution series of human IL2Rβ-γ-Fc was passed over the ligand at a rate of 30 μl / min for 240 sec 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+. After each cycle, the chip surface was regenerated using a 30 s injection of 10 mM glycine pH 2.1. Bulk refractive index differences were corrected by subtracting the response obtained on reference flow cell 1. Affinity constants were derived from kinetic rate constants by fitting to 1 :1 Langmuir binding using Biaeval software (Cytiva).

[0424] sample

[0425] The following samples were analyzed for binding to human IL2Rβ-γ-Fc (Table 6).

[0426] Table 6. Description of samples analyzed for binding to human IL2Rβ-γ-Fc.

[0427] Captured molecules TAPIR ID form PD1 376 IgG-huIL2v K8S P1AE0488 Supernatant / Purified PD1 376 IgG-huIL2v L12V P1AE0489 Supernatant / Purified PD1 376 IgG-huIL2v L12A P1AE0490 Supernatant / Purified PD1 376 IgG-huIL2v L19V P1AE0491 Supernatant / Purified PD1 376 IgG-huIL2v T51P P1AE0492 in the supernatant) PD1 376 IgG-huIL2v H79S P1AE0493 Supernatant / Purified PD1 376 IgG-huIL2v L80A P1AE0494 Supernatant / Purified PD1 376 IgG-huIL2v N88A P1AE0495 Supernatant / Purified PD1 376 IgG-huIL2v N88Q P1AE0496 Supernatant / Purified PD1 376 IgG-huIL2v D109A P1AE0497 Supernatant / Purified PD1 376 IgG-huIL2v Q126N P1AE0498 in the supernatant) PD1 376 IgG-huIL2v Q126E P1AE0499 in the supernatant) FAP IgG-huIL2v P1AD1317-002 Purified PD1 379 IgG-huIL2v P 1AD4370-002 Purified hu IL2Rβ-γ-Fc P1AD7029-002 Antigen used as analyte, purified

[0428] result

[0429] Binding evaluation of PD1-IL2v variants from supernatants to recombinant human IL2Rβ-γ-Fc heterodimers

[0430] The resonance units after capture and after binding were recorded, the ratio was calculated and compared with IL2v without additional mutations (Table 7).

[0431] Table 7. Resonance units after capture and binding and their ratios were used to identify variants with reduced binding to human IL2Rβ-γ-Fc.

[0432]

[0433]

[0434] Determination of the affinity of PD1-IL2v variants for recombinant human IL2Rβ-γ-Fc heterodimers

[0435] Affinity measurements for human IL2Rβ-γ showed very slow off-rates, reaching the limits of the instrument and giving unrealistic KD. However, reduced affinity could still be assessed (Table 8).

[0436] Table 8. Kinetic constants (1:1 Langmuir binding).

[0437]

[0438] in conclusion

[0439] Twelve IL2v variants from supernatants were tested to identify candidates with reduced binding to IL2Rβ-γ-Fc. Six variants behaved like the parental IL2v (K8S, L12A, L19V, H79S, L80A, D109A), two variants were not expressed correctly (Q126N, Q126E), one variant had slower association and faster dissociation than IL2v (N88Q), two variants had slower association than IL2v (N88A, L12V), and one variant lost binding (T51P).

[0440] Nine variants were purified and their affinity for IL2Rβ-γ-Fc was measured. The N88A and N88Q variants were confirmed to have reduced affinity for human IL2Rβ-γ-Fc. L12V and L19V may have slightly reduced affinity for human IL2Rβ-γ-Fc, but otherwise performed very closely to the parental IL2v.

[0441] Example 1E. Design of a Mouse Surrogate for the IL2v Immunoconjugate Targeting Mouse PD1

[0442] To facilitate in vivo efficacy studies in mouse cancer models, mouse surrogates of IL2v immunoconjugates targeting mouse PD1 were generated. To reduce immunogenicity, all constant antibody domains in these constructs correspond to mouse sequences. In contrast, the V domain sequences of anti-mouse PD1 antibodies are derived from rats. Due to the cross-reactivity of human IL2v with the mouse IL2 receptor, human IL2v has been used in both constructs.

[0443] These mouse surrogate constructs bind bivalently to mouse PD1 via the N-terminal Fab arms on the Fc DD- and Fc KK+ chains, which additionally carry the C-terminal IL2v( Figure 1A P1AG9991) or its Q126T mutant protein ( Figure 1B Heterodimerization was achieved by applying complementary charges in the murine IgG1 CH3 domains (Fc DD- and Fc KK+ chains, respectively), and binding to activating Fcγ receptors and complement component C1q was abolished by introducing DA PG mutations in the murine IgG1 CH2 Fc domain of the antibody. These immunoconjugates are schematically depicted in Figure 1A and Figure 1B middle.

[0444] Example 1F. Production and purification of a mouse surrogate of an IL2v immunoconjugate targeting mouse PD1

[0445] A mouse surrogate of the IL2v immunoconjugate targeting PD1 was produced and purified at WuXi Biologics. They were transiently expressed in HEK293 (P1AG9991, expression system "Transient2.0") or CHO (P1AG8304, expression system "Transient2.5") and purified in a 2-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).

[0446] For P1AG9991, protein purity was determined by SEC-HPLC (monomer peak 99.3%), non-reduced CE-SDS (main peak 97.8%), reduced CE-SDS (sum of peaks for all 3 different chains 99.9%), and protein identity was confirmed by LC-MS. It was determined that the endotoxin level was 0.34 EU / mg, and the final concentration was 1.1 mg / mL. For P1AG8304, protein purity was determined by SEC-HPLC (monomer peak 99.4%), non-reduced CE-SDS (main peak 98.8%), reduced CE-SDS (sum of peaks for all 3 different chains 97.2%), and protein identity was confirmed by LC-MS. It was determined that the endotoxin level was 0.2 EU / mg, and the final concentration was 2.1 mg / mL.

[0447] Example 1G. Design of a bispecific immunoconjugate targeting human PD1- / LAG3-IL2v

[0448] For checkpoint inhibition targeting not only PD1 but also LAG3, bispecific immunoconjugates targeting human PD1 / LAG3IL2v were generated. These immunoconjugates monovalently bind to both human PD1 and human LAG3 via the N-terminal Fab arm. To avoid light chain mispairing, the V-domains of the human PD1 binder were crossed, while charge complementarity was introduced into the CH1 and Ck domains of the human LAG3 Fab. Heterodimerization of the two heavy chains was achieved using knobs-into-holes technology, and binding to activating Fcγ receptors and complement component C1q was abolished by introducing the PG LALA mutation into the CH2 Fc domain of the antibody. The HC knob chain additionally carried the C-terminal IL2v (P1AF4801) or its Q126T mutant (P1AF7951). As a control, a monospecific human PD1 IgG1 PG LALA lacking the IL2v cytokine fusion was also generated. These immunoconjugates and PD1 IgG (P1AA6888) are schematically depicted in Figure 2A 、 Figure 2B and Figure 2C middle.

[0449] Example 1H. Production and purification of IL2v bispecific immunoconjugates targeting human PD1- / LAG3

[0450] Production and purification of P1AF4801 were 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%), 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. Production and purification of P1AF7951 were 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%), 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. P1AA6888 was produced and purified at Roche, Penzberg, Germany. HEKExpi293F 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.

[0451] Example 2. Selection of IL2v Q126T

[0452] Example 2A. Proliferation of NK92 cells with PD1-IL2v variants

[0453] We evaluated the proliferation of the NK cell line NK92 after 3 days of treatment with a panel 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 at the IL2 receptor compared to IL2v in a non-targeted setting. Variant N88Q completely lost its activity in inducing NK92 proliferation, and variant N88A had minimal activity at the IL2 receptor, so we did not consider further evaluation of these two variants. Variants L12V, H79S, and D190A retained similar activity to the parent IL2v and were therefore not further characterized. Variants K8S, L12A, L19V, and L80A had at least two-fold reduced activity at the IL2 receptor and were selected for more detailed characterization.

[0454] NK92 proliferation

[0455] NK92 cells were harvested, counted and assessed for viability. The cells were washed three times with PBS to remove residual IL2 and resuspended in RPMI1640, 10% FCS, 1% glutamine without IL2. The washed NK92 cells were incubated in a cell culture incubator for 2 hours (IL2 starvation). After starvation, the cells were resuspended in fresh culture medium without IL2 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 culture medium without IL2), Proleukin (1.5 μg / ml final concentration) or culture medium (control wells) to a final volume of 100 μl per well. The plate was incubated in an incubator for 3 days.

[0456] After 3 days, 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 solution was added to each well. After incubation for 10 min, the remaining aggregates were resuspended by pipetting, and 150 μl of the mixture was transferred to a white flat-bottomed plate. Luminescence was measured using a Tecan Spark 10M multimode reader.

[0457] Example 2B. PBMC proliferation and activation with PD1-IL2v variants

[0458] We then tested the activity of four selected new PD1-IL2v variants (K8S, L12A, L19V, and L80A) on PBMCs and compared them with the parental PD1-IL2v and FAP-IL2v molecules. After five days of treatment with the PD1-IL2v variants PD1-IL2v or FAP-IL2v, the proliferation of CD8 T cells, CD4 T cells, and NK cells was measured by flow cytometry ( Figure 4 A to Figure 4C) and upregulation of CD25 as a marker of CD8 T cell, NK cell, and CD4 T cell activation ( Figure 5 A to Figure 5 C) The results generated using NK92 cells were confirmed in this experiment; all tested novel variants showed reduced activity in inducing proliferation and activation of CD8 T cells, CD4 T cells, and NK cells compared to the two parental IL2v molecules. However, the reduction in activity observed for these variants was considered insufficiently robust. Therefore, characterization of these variants was not expanded, and additional variants were designed.

[0459] PBMC proliferation and activation

[0460] 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, CSFE stock solution (2 mM in DMSO) was diluted 1:20 in PBS. PBMCs were resuspended in preheated PBS to 1 Mio / ml, 1 μl of CFSE solution was added to 1 ml of cell suspension, and the cells were immediately mixed. For optimal labeling, cells were incubated at 37°C for 15 min. 10 ml of preheated culture medium (RPMI1640, 10% FCS, 1% glutamine) was then added to stop the labeling reaction. The cells were centrifuged at 400 g for 10 min and resuspended in fresh culture medium to 1 Mio / ml and incubated at 37°C for another 30 min. Finally, the cells were washed once with culture medium and resuspended in fresh culture medium and used directly or stored in an incubator overnight. The labeled PBMCs were seeded in 96-well round-bottom plates (100'000 cells per well) and treated with the specified molecules for 5 days. After incubation for five days, the cells were washed once with FACS buffer and stained at 4 ° C for 30 min with a mixture of 20 μl anti-human CD3 APC-Cy7 (300318, BioLegend), anti-human CD8 APC (344722, BioLegend), and anti-human CD56 BV421 (318328, BioLegend) in FACS buffer. The PBMCs were then washed twice with FACS buffer, then fixed with 1% PFA in FACS buffer and fluorescence was measured with BD Fortessa. Proliferation was determined by measuring the CFSE dilution of CD8 T cells (CD3+CD8+), CD4 T cells (CD3+CD8-), and NK cells (CD3-CD56+), and activation was determined by upregulating Cd25 on CD8 T cells, CD4 T cells, and NK cells.

[0461] Example 2C. NK92 cell proliferation with newly designed FAP-IL2v variants

[0462] The combination of the new IL2v variant and the previously tested variant was designed to achieve a higher reduction in IL2 receptor activity than the previously tested IL2v variant. We tested the variants in the FAP-IL2v format and compared their activity to the parent FAP-IL2v molecule. In a first step, the variants were tested for proliferation induction using NK92 cells ( Figure 6 A to Figure 6 D). Proliferation was measured after three days of treatment with IL2v variants. IL2v variants D20T_Selectikine, L12A_L19A, L12A_L80A, N88T and Q126T showed the strongest reduction in activity compared to the parent IL2v. We selected these variants plus two variants, Q22A and S130A, and tested their ability to induce CD8 T cells and NK cell proliferation and activation in PBMCs. Proliferation was measured after 4 days of treatment ( Figure 7 A to Figure 7 B) and upregulation of CD25 as an immune cell activation marker ( Figure 8 A to Figure 8 B). Similarly, the results obtained with NK92 cells were confirmed in PBMCs. Variants D20T_Selectikine and N88T showed the strongest reduction in activity, which was considered too strong, and these variants were not evaluated further. The other tested variants will be evaluated in additional experiments.

[0463] NK92 proliferation

[0464] NK92 cells were harvested, counted and assessed for viability. The cells were washed three times with PBS to remove residual IL2 and resuspended in RPMI1640, 10% FCS, 1% glutamine without IL2. The washed NK92 cells were incubated in a cell culture incubator for 2 hours (IL2 starvation). After starvation, the cells were resuspended in fresh culture medium without IL2 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 culture medium without IL2), Proleukin (1.5 μg / ml final concentration) or culture medium (control wells) to a final volume of 100 μl per well. The plate was incubated in an incubator for 3 days.

[0465] After 3 days, 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 solution was added to each well. After incubation for 10 min, the remaining aggregates were resuspended by pipetting, and 150 μl of the mixture was transferred to a white flat-bottomed plate. Luminescence was measured using a Tecan Spark 10M multimode reader.

[0466] PBMC proliferation and activation

[0467] Freshly isolated PBMCs from healthy donors were labeled with cell proliferation dye eFluor670 (65-0840-85, BioLegend). Briefly, PBMCs were washed twice with PBS and resuspended in PBS to a final concentration of 10 Mio cells / ml. At the same time, a 10 μM solution of cell proliferation dye eFluor 670 was prepared by diluting the stock solution (5 mM) in preheated PBS. The PBMCs were mixed and pre-diluted cell proliferation dye was added at a ratio of 1:1 to obtain a final concentration of 5 μM. The PBMCs were incubated at 37 ° C for 10 min. Four times the volume of cold culture medium was then added to stop the labeling reaction, the cells were washed three times with culture medium, and resuspended in fresh culture medium at a rate of 1 million cells / ml. The labeled PBMCs were seeded in 96-well round-bottom plates (100,000 cells per well) and treated with the specified molecules for 4 days. After incubation, the cells were washed twice with PBS, stained with 50 μl / well reconstructed fluorescent reactive live / dead dye (L34976, Invitrogen), and incubated at room temperature for 20 min. 150 μl FACS buffer was then added to each well, and the plate was centrifuged at 400 g for 4 min. The supernatant was removed and the cells were stained with a mixture of 50 μl CD3 PE-Cy5 (555341, BD Bioscience), CD8 BV711 (301044, BioLegend), CD25 PE-Dazzle 594 (356126, BioLegend), CD56 BV421 (318328, BioLegend) in FACS buffer at 4 ° C for 30 min. PBMCs were then washed twice with FACS buffer, then fixed in FACS buffer with 1% PFA, and fluorescence was measured using 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 was measured by upregulation of CD25 on the corresponding cells.

[0468] Example 2D. PBMC proliferation and activation with selected FAP-IL2v variants

[0469] We then tested the newly selected FAP-IL2v variants on PBMCs. 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, were measured and compared with the activity of the parental FAP-IL2v.

[0470] PBMCs were treated with selected FAP-IL2v variants and parental FAP-IL2v for five days and immune cell proliferation was analyzed ( Figure 9 A to Figure 9 C) and activation ( Figure 10 A to Figure 10 C). As shown in the previous tests, variants Q22A and S130A behaved similarly to the parental IL2v, variants L12A_L19A and L12A_L80A showed approximately 10-fold reduced activity, and variant Q126T showed more than 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 in CD4 T cells, regulatory T cells, CD8 T cells, and NK cells as a direct marker of IL2 receptor activation ( Figure 11 A to Figure 11 D). As previously mentioned, Q22A and S130A had similar activities to the parental IL2v. Variants L12A_L19A, L12A_L80A, and Q126T showed reduced activity, with Q126T showing slightly lower activity than the other two variants. Of all the IL2v variants tested, the IL2v variant Q126T was selected as the most promising candidate because its reduction in activity compared to the parental FAP-IL2v was within our target range.

[0471] PBMC proliferation and activation

[0472] Freshly isolated PBMCs from healthy donors were labeled with cell proliferation dye eFluor670 (65-0840-85, BioLegend). Briefly, PBMCs were washed twice with PBS and resuspended in PBS to a final concentration of 10 Mio cells / ml. At the same time, a 10 μM solution of cell proliferation dye eFluor 670 was prepared by diluting the stock solution (5 mM) in preheated PBS. The PBMCs were mixed and pre-diluted cell proliferation dye was added at a ratio of 1:1 to obtain a final concentration of 5 μM. The PBMCs were incubated at 37 ° C for 10 min. Four times the volume of cold culture medium was then added to stop the labeling reaction, the cells were washed three times with culture medium, and resuspended in fresh culture medium at a rate of 1 million cells / ml. The labeled PBMCs were seeded in 96-well round-bottom plates (100,000 cells per well) and treated with the specified molecules for 5 days. After incubation, the cells were washed twice with PBS, stained with 50 μl / well reconstructed fluorescent reactive live / dead dye (L34976, Invitrogen), and incubated at room temperature for 20 min. 150 μl FACS buffer was then added to each well, and the plate was centrifuged at 400 g for 4 min. The supernatant was removed and the cells were stained with a mixture of 50 μl CD3 PE-Cy5 (555341, BD Bioscience), CD4 BV605 (317438, BioLegend), CD8 BV711 (301044, BioLegend), CD25 PE-Dazzle594 (356126, BioLegend), CD56 BV421 (318328, BioLegend) in FACS buffer at 4 ° C for 30 min. PBMCs were then washed twice with FACS buffer, fixed with 1% PFA in 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 was measured by upregulation of CD25 on the corresponding cells.

[0473] Example 2E. Activity of PD1-IL2v Q126T and FAP-IL2v Q126T

[0474] In the next step, PD1-IL2v Q126T and FAP-IL2v Q126T were compared with FAP-IL2v to confirm the results generated so far. PBMCs were treated with the three molecules for five days and the proliferation of CD8 T cells, NK cells and CD4 T cells was determined ( Figure 12 A to Figure 12 C) and activation ( Figure 13 A to Figure 13 C). PD1-IL2v Q126T and FAP-IL2v Q126T have comparable activity against CD4 T cells, CD8 T cells, and NK cells, but activity is significantly reduced compared to the parental FAP-IL2v.

[0475] PBMC proliferation and activation

[0476] Freshly isolated PBMCs from healthy donors were labeled with cell proliferation dye eFluor670 (65-0840-85, BioLegend). Briefly, PBMCs were washed twice with PBS and resuspended in PBS to a final concentration of 10 Mio cells / ml. At the same time, a 10 μM solution of cell proliferation dye eFluor 670 was prepared by diluting the stock solution (5 mM) in preheated PBS. The PBMCs were mixed and pre-diluted cell proliferation dye was added at a ratio of 1:1 to obtain a final concentration of 5 μM. The PBMCs were incubated at 37 ° C for 10 min. Four times the volume of cold culture medium was then added to stop the labeling reaction, the cells were washed three times with culture medium, and resuspended in fresh culture medium at a rate of 1 million cells / ml. The labeled PBMCs were seeded in 96-well round-bottom plates (100,000 cells per well) and treated with the specified molecules for 5 days. After incubation, the cells were washed twice with PBS, stained with 50 μl / well reconstituted fluorescent reactive live / dead dye (L34957, Invitrogen), and incubated at room temperature for 15-30 min. 150 μl FACS buffer was then added to each well, and the plate was centrifuged at 400 g for 4 min. The supernatant was removed and the cells were stained with a mixture of 30 μl 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 at 4 ° C for 30 min. 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 was measured by upregulation of CD25 on the corresponding cells.

[0477] STAT5 phosphorylation

[0478] Freshly isolated PBMCs from healthy donors were seeded in warm culture medium (RPMI1640, 10% FCS, 2mM glutamine) and placed in 96-well round-bottom plates (200,000 cells / well). The plate was centrifuged at 300g for 10min and the supernatant was removed. The cells were resuspended in 100 μl of culture medium containing IL2v molecules and stimulated at 37°C for 20min. In order to maintain the phosphorylation state, the cells were fixed at 37°C for 10min with an equal amount of preheated Cytofix buffer (554655, BD Bioscience) immediately after stimulation. The plate was then centrifuged at 350g for 5min and the supernatant was removed. In order to allow intracellular staining, the cells were permeabilized at 4°C for 30min in 100 μl Phosflow Perm buffer III (558050, BD Bioscience). The cells were then washed twice with 150 μl of cold FACS buffer and separated into two 96-well round-bottom plates and each stained in a refrigerator for 60 min with 20 μl of antibody mixture I or II. Antibody mixture I was used to stain the pSTAT5 in CD4 T cells and regulatory T cells, and antibody mixture II was used to stain the pSTAT5 in CD8 T cells and NK cells. The cells were then washed twice with FACS buffer and resuspended in 200 μl FACS buffer containing 2% PFA per well. CD8 T cells (CD3+CD8+), NK cells (CD3-CD56+), CD4 T cells (CD4+) and Treg (CD4+CD25+FoxP3+) were analyzed using BD flow cytometer gating.

[0479] Table 9. FACS Antibody Cocktail I (CD4 T Cells and Regulatory T Cells)

[0480]

[0481] Table 10. FACS Antibody Cocktail II (CD8 T Cells and NK Cells)

[0482]

[0483] Example 3. PD1-IL2v Q126T and PD1-LAG3-IL2v Q126T

[0484] Example 3A. IL-2R Signaling Assay

[0485] In one aspect, assays are provided to determine the potency and cis / trans signaling of PD-1-IL-2v immunoconjugates (e.g., comprising at least one binding domain that binds to PD-1 coupled to an IL-2 polypeptide having additional mutations).

[0486] For this purpose, CD4 T cells were sorted from healthy donor PBMCs using CD4 beads (Miltenyi, #130-045-101) and activated for 3 days in the presence of 1 μg / ml plate-bound anti-CD3 (overnight pre-coating, clone OKT3, #317315, BioLegend) and 1 μg / ml soluble anti-CD28 (clone CD28.2, #302923, BioLegend) antibodies to induce PD-1 expression. Three days later, cells were harvested and washed several times to remove endogenous cytokines, and half of the cells were labeled with Cell Trace Violet (CTV) (5 μM, 5 minutes at room temperature (RT); C34557, Thermo Scientific) and the other half were unlabeled.

[0487] Then, unlabeled cells were incubated with saturating concentrations of competitive anti-PD-1 antibody (internal molecule, 10 μg / ml) at RT for 30 min, followed by several washing steps to remove excess unbound anti-PD-1 antibody. 6 cells / ml) and PD-1 + CTV-labeled cells (25 μl, 6 × 10 6 cells / ml) were co-cultured in a V-bottom plate at a 1:1 ratio and then treated with increasing concentrations of therapeutic immunoconjugates (50 μl, 1:10 dilution step) at 37°C for 12 minutes. To maintain the phosphorylation state, an equal amount of Phosphoflow fixation buffer I (100 μl, 557870, BD Bioscience) was added after incubation with various constructs for 12 minutes. The cells were then incubated at 37°C for another 30 minutes and then permeabilized overnight with Phosphoflow PermBuffer III (558050, BD Bioscience) at -80°C. The next day, the phosphorylated form of STAT-5 was stained using an anti-STAT-5P antibody (47 / Stat5 (pY694) clone, 562076, BD Bioscience) at 4°C for 30 minutes.

[0488] Cells were collected on a fluorescence activated cell sorting (FACS) BD-LSRFortessa (BD Bioscience) instrument. The frequency of STAT-5P was determined using FlowJo (v10) and graphed using GraphPad Prism (v8).

[0489] PD-1 +Dose-response curves on T cells provide information on the potency of the evaluated molecules in signaling through IL-2R. In addition, dose-response curves on T cells pretreated with a competing anti-PD-1 antibody to prevent PD-1-mediated delivery demonstrate the potency of molecules that provide IL-2R signaling independent of PD-1 expression.

[0490] CMV-specific restimulation assay

[0491] In the context of chronic viral infection, an in vitro assay has been developed to evaluate the effect of PD-1 targeting of mutant IL-2v delivery to dysfunctional antigen-specific T cells. To avoid limiting the number of suitable donors for the assay, given that approximately 80% of the population is CMV seropositive, a CMV immunogenic viral protein (pp65) was used as a wake-up antigen for T cells. Therefore, healthy human donor peripheral blood mononuclear cells (PBMCs) were stimulated with CMV-pp65 (Miltenyi, #130-093-435) in the presence of different constructs at a concentration of 0.6 nM. Forty-three hours later, the cells were immunized with IL-2v by adding a protein transport inhibitor (GolgiPlug TM #555029, BD Bioscience; and GolgiStop TM #554724, BD Bioscience) to block protein transport from the Golgi apparatus, and then incubate the cells for an additional 5 hours at 37°C. The cells were then washed and surface-stained with anti-human CD3, CD4, CD8, CD62L, and CD45RO antibodies, followed by fixation / 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.

[0492] Inhibition assay

[0493] In one aspect, an assay is provided to evaluate whether a PD-1-IL-2v immunoconjugate can reverse the expression of conventional T cells (T conv ) regulatory T cells (T) reg ) inhibition. In some cases, T conv and T reg Separated and labeled.

[0494] In certain aspects, CD4 Treg cells are isolated using a two-step Treg cell isolation kit (Miltenyi, #130-094-775). + CD25 + CD127 dim T reg In parallel, CD4+ CD25 - T conv : Negative fractions were collected after CD25 positive selection (Miltenyi, #130-092-983) and then CD4 + Enrichment (Miltenyi, #130-045-101). conv The cells were labeled with carboxyfluorescein succinimidyl ester (CFSE, eBioscience, #65-0850-84) and T reg They were labeled with Cell Trace Violet (CTV, ThermoFisher scientific, C34557) to be able to distinguish them and follow the proliferation of the two populations. - CD25 - In the presence of PBMC, T conv and T reg The cells were cultured together for 5 days to provide allotype-specific stimulation.

[0495] In certain aspects, on day 5, the cells were stained with a protein transport inhibitor (GolgiPlug TM #555029, BD Bioscience; and GolgiStop TM #554724, BD Bioscience) for 5 hours to enhance the accumulation of cytokines in the Golgi complex. conv In the presence or absence of T reg The ability to secrete granzyme B (GrzB) in the presence of β-catenin was calculated using the following formula: reg inhibition:

[0496]

[0497] Among them, % cytokines ( Tconv+Treg±免疫缀合物) For the existence of T reg ± In the case of therapeutic immunoconjugates, T conv Secreted cytokine levels, % cytokine (Tconv) For the absence of T reg In the case of T conv The levels of secreted cytokines were calculated using one-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0498] Internalization assay

[0499] In one aspect, assays are provided to assess the internalization of different immunoconjugates. For this purpose, PBMCs were isolated by density gradient centrifugation using Ficoll-Paque (Sigma-Aldrich). Following manufacturer's instructions, PBMCs were isolated from 10 μg of cultured cells by using a CD4 positive selection kit (Miltenyi, #130-045-101). 8 CD4 T cells were then sorted by beads starting with 100 PBMCs. CD4 T cells were then sorted at 2-4 x 10 6 Cells / well were seeded in 12-well plates pre-coated with 1 μg / ml anti-CD3 (pre-coated overnight, clone OKT3, #317315, BioLegend) and cultured at 37°C for 3 days.

[0500] CD4 T cells activated for three days were incubated in duplicate at 4 ° C for 30 minutes in the presence of immunoconjugates in FACS tubes. The cells were then washed and divided into two groups, one of which was incubated for another 3 hours at 37 ° C, and the other group was immediately stained with PE-labeled anti-PGLALA secondary antibodies and anti-CD4 antibodies (eBioscience), and then fixed with BD Cell Fix. After incubation for 3 hours, the second group of cells was also stained with PE-labeled anti-PGLALA secondary antibodies and anti-CD4 before fixation. Cells were then collected at LSRFortessa (BD Biosciences), and data analysis was performed using FlowJo (V10), and graphing was performed using GraphPad Prism (v8). The expression levels of detectable antibodies on the cell surface at 4 ° C were compared with those at 37 ° C to calculate the percentage of internalized molecules at 37 ° C. P (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) was calculated using one-way analysis of variance.

[0501] Example 3B. Activated PD-1 after treatment with increasing doses of PD-1-IL-2v immunoconjugate + and PD-1 - IL-2R signaling on CD4 T cells (STAT5-P)

[0502] By treating activated PD-1 expressing cells (PD-1 + ) and PD-1 negative (PD-1 -The aim was to determine the dependency of the PD-1-IL-2v immunoconjugate on PD-1 expression on T cells in order to deliver IL-2R signaling.

[0503] To this end, CD4 T cells were sorted from healthy donor PBMCs using CD4 beads (#130-045-101, Miltenyi) and activated for 3 days in the presence of 1 μg / ml plate-bound anti-CD3 (pre-coated overnight, clone OKT3, #317315, BioLegend) and 1 μg / ml soluble anti-CD28 (clone CD28.2, #302923, BioLegend) antibodies to induce PD-1 expression. Three days later, cells were harvested and washed several times to remove endogenous cytokines, and half of the cells were labeled with Cell Trace Violet (CTV) (5 μM, 5 minutes at room temperature (RT); C34557, Thermo Scientific) and the other half were unlabeled.

[0504] Then, unlabeled cells were incubated with saturating concentrations of competitive anti-PD-1 antibody (internal molecule, 10 μg / ml) at RT for 30 min, followed by several washing steps to remove excess unbound anti-PD-1 antibody. 6 cells / ml) and PD-1 + CTV-labeled cells (25 μl, 6 × 10 6 cells / ml) were co-cultured in a V-bottom plate at a 1:1 ratio and then treated with increasing concentrations of therapeutic immunoconjugates (50 μl, 1:10 dilution step) at 37°C for 12 minutes. To maintain the phosphorylation state, an equal amount of Phosphoflow fixation buffer I (100 μl, 557870, BD Bioscience) was added after incubation with various constructs for 12 minutes to allow IL-2R signaling after binding to PD-1. The cells were then incubated at 37°C for another 30 minutes for fixation and then permeabilized overnight with Phosphoflow PermBuffer III (558050, BD Bioscience) at -80°C. The next day, the phosphorylated form of STAT-5 was stained for 30 minutes at 4°C using an anti-STAT-5P antibody (47 / Stat5 (pY694) clone, 562076, BD Bioscience).

[0505] Cells were collected on a BD-LSRFortessa (BD Bioscience) flow cytometer (FACS). The frequency of STAT-5P was determined using FlowJo (V10) and plotted using GraphPad Prism (V8).

[0506] Figure 14 A to Figure 14 The data in G show that selected PD1-IL2 variants are + and PD-1 - Differences in the efficacy of signaling through IL-2R on CD4 T cells. + Potency measurements in CD4 T cells reflect PD1-mediated delivery of IL-2v and PD1 - PD1-independent delivery of IL-2v in CD4 T cells.

[0507] In Table 11, the EC50 of cells pre-blocked with PD-1 was divided by the EC50 of cells pre-blocked with PD1 + The EC50 of T cells was used to calculate the STAT-5PEC50 fold increase between PD1-mediated delivery of IL-2v and PD-1-independent delivery of each PD1-IL2v immunoconjugate molecule. This provides evidence for the strength of PD1-dependent delivery of IL2v for each IL2v mutant. In addition, the EC50 fold increase between various PD1-IL2v immunoconjugates and PD1-IL2v was calculated by dividing the EC50 of the new mutant by the EC50 of PD1-IL2v. This indicates that the PD1-IL2v immunoconjugate loses its potency in signaling through IL-2R due to its reduced affinity.

[0508] Table 11. PD-1 obtained from 4 donors + and PD-1 - EC50 and area under the curve (AUC) of dose-response STAT-5 phosphorylation for each mutant on CD4 T cells.

[0509]

[0510] In this particular assay, some further mutated immunoconjugates showed + It has similar potency to PD1-IL2v in signaling through IL-2R on T cells, but - The cis-activities of Q126T and L12A were 56.5-fold and 44.5-fold higher on T cells, respectively. +The activity of signaling through IL-2R on T cells was also reduced, while others maintained the same characteristics of PD1-IL2v ( Figure 14 A to Figure 14 G) Table 11 shows the PD-1 + and PD-1 - EC50 and area under the curve (AUC) of dose-response STAT-5 phosphorylation for each mutant on CD4 T cells.

[0511] Example 3B. Activated PD-1 after treatment with increasing doses of PD-1-IL-2v, FAP-IL2v, and NKG2D-IL2v immunoconjugates + IL-2R signaling on CD4 T cells (STAT5-P)

[0512] In this experiment, the potency of PD1-IL2vQ126T, FAP-IL2vQ126T, and NKG2D-IL2vQ126T relative to PD1-IL2v and FAP-IL2v in signaling through IL-2R upon engagement with PD-1 on PD-1-expressing CD4 T cells was assessed in a dose-dependent manner using STAT5 phosphorylation as a readout.

[0513] For this purpose, CD4 T cells were sorted from healthy donor PBMCs using CD4 beads (#130-045-101, Miltenyi) and activated for 3 days in the presence of 1 μg / ml plate-bound anti-CD3 (overnight pre-coated, clone OKT3, #317315, BioLegend) and 1 μg / ml soluble anti-CD28 (clone CD28.2, #302923, BioLegend) antibodies to induce PD-1 expression. Three days later, 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 treatment antibodies (50 μl, 1:10 dilution step, highest concentration was 66 nM) at 37 ° C for 12 min. To maintain the phosphorylation state, an equal amount of Phosphoflow Fixation Buffer I (100 μl, 557870, BD) was added immediately after incubation with various constructs for 12 minutes. The cells were then incubated at 37°C for another 30 minutes and then permeabilized overnight with Phosphoflow PermBuffer III (558050, BD) at 80°C. The next day, the phosphorylated form of STAT-5 was stained using an anti-STAT-5P antibody (47 / Stat5 (pY694) clone, 562076, BD) at 4°C for 30 minutes.

[0514] These cells were acquired using a FACS BD-LSR Fortessa (BD Bioscience). The frequency of STAT-5p was determined using FlowJo (V10) and plotted using GraphPad Prism (V8).

[0515] Figure 15 A to Figure 15 The data in panel B show that PD-1 + Differential potency of selected PD1-, FAP-, and NKG2D-IL2 variants in CD4 T cells. + Potency measurements in CD4 T cells reflected PD1-mediated delivery of IL-2v versus PD1-independent delivery of FAP-IL-2v and FAP-IL2vQ126T.

[0516] Table 12 shows the dose-response EC50 of STAT-5 phosphorylation as a function of PD-1 obtained from 4 donors. + Frequency and mean fluorescence intensity (MFI) of IL-2v mutants on CD4 T cells.

[0517] This experiment shows PD-1-dependent and -independent delivery of IL-2 mutants to IL-2R. In this experiment, the FAP-targeting antibody behaved similarly to the non-targeted IL2v, as activated CD4 T cells lack FAP expression. The non-targeted IL2vQ126T was 6.5-fold less potent than the non-targeted IL2v, while the PD1-IL2vQ126T was only 1.5-fold less potent than the PD1-IL2v. These findings predict fewer IL-2-mediated off-target effects while also reducing the PD1-mediated off-target effects. + NKG2D-IL2vQ126T has similar IL-2R signaling on T cells through PD1-IL2vQ126T. NKG2D-IL2vQ126T has reduced efficacy as FAP-IL2vQ126T on activated CD4 T cells ( Figure 15 A to Figure 15 B).

[0518] Table 12. (AB) Dose-Response EC50 of STAT-5 Phosphorylation as a Function of PD-1 + Frequency and mean fluorescence intensity (MFI) of IL-2v mutants on CD4 T cells. Mean ± SEM of 4 donors.

[0519]

[0520] Example 3C. Expansion of CMV-specific CD4 T cell effector function following treatment with PD1-IL2v immunoconjugate

[0521] To evaluate the ability of IL-2v and IL-2vQ126T targeting and non-targeting PD-1 to amplify antigen-specific CD4 T cell responses in the context of chronic viral infection, CMV immunogenic viral protein (pp65) was used as a wake-up antigen. Therefore, healthy human donor peripheral blood mononuclear cells (PBMCs) were stimulated with CMV-pp65 (#130-093-435, Miltenyi) in the presence of different constructs at a concentration of 0.6 nM. Forty-three hours later, the cells were amplified by the addition of a protein transport inhibitor (GolgiPlug TM #555029, BD Bioscience; and GolgiStop TM #554724, BD Bioscience) to block protein transport from the Golgi apparatus, and then incubate the cells for another 5 hours at 37°C. The cells were then washed and surface-stained with anti-human CD3, CD4, CD8, CD62L, and CD45RO antibodies, followed by fixation / 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.

[0522] These cells were obtained using a FACS BD-LSR Fortessa (BD Bioscience). The frequency of IFN-γ+ was determined using FlowJo (V10) and plotted using GraphPad Prism (V8). P values ​​were calculated using one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0523] This experiment showed that PD1-IL2v and PD1-IL2vQ126T increased the frequency of CMV-specific CD4 T cells compared to pp65 alone or in combination with FAP-IL2v and FAP-IL2vQ126T ( Figure 16 A). It also showed that PD1-IL2vQ126T increased the frequency of CMV-specific CD4 T cells secreting IFN-γ by approximately 5-fold after 48 hours of restimulation and was significantly superior to pp65 stimulation alone ( Figure 16 B).

[0524] Table 13 depicts the frequency of CMV-specific CD4 T cells after reactivation with the CMV protein pp65 and the fold increase in the frequency of CMV-specific CD4 T cells elicited by the indicated treatments in combination with pp65.

[0525] Table 13. Frequency of CMV-specific CD4 T cells after reactivation with the CMV protein pp65 and fold increase in CMV-specific CD4 T cell frequency elicited by the indicated treatments in combination with pp65. Mean ± SEM of 5 donors.

[0526]

[0527] Example 3D. Treatment with PD1-IL2v immunoconjugates from T cells reg Restoring T during suppression conv Effector function

[0528] To assess the ability to recover conventional T cells (Tconv) from Treg suppression, a suppressive function assay was established in which Tconv and Treg were combined with CD4 T cells from unrelated donors. - CD25 - Cells were co-cultured for 5 days for allospecific stimulation in the presence or absence of immunoconjugates.

[0529] In certain aspects, CD4 Treg cells are isolated using a two-step Treg cell isolation kit (Miltenyi, #130-094-775). + CD25 + CD127 dim T reg In parallel, CD4 + CD25 - T conv : Negative fractions were collected after CD25 positive selection (Miltenyi, #130-092-983) and then CD4 + Enrichment (Miltenyi, #130-045-101). conv The cells were labeled with carboxyfluorescein succinimidyl ester (CFSE, eBioscience, #65-0850-84) and T reg They were labeled with Cell Trace Violet (CTV, ThermoFisher scientific, C34557) to be able to distinguish them and follow the proliferation of the two populations. - CD25 - In the presence of PBMC, T conv and T reg The cells were cultured together for 5 days to provide allotype-specific stimulation.

[0530] In certain aspects, on day 5, the cells were stained with a protein transport inhibitor (GolgiPlug TM#555029, BD Bioscience; and GolgiStop TM #554724, BD Bioscience) for 5 hours to enhance cytokine accumulation in the Golgi complex. These cells were acquired using a BD-LSR Fortessa FACS scanner (BD Bioscience). Data were analyzed using FlowJo (v10) and graphed using GraphPad Prism (v8).

[0531] Measuring proliferation of T conv In the presence or absence of T reg The ability to secrete granzyme B (GrzB) in the presence of β-catenin was calculated using the following formula: reg inhibition:

[0532]

[0533] Among them, % cytokines ( Tconv+Treg±免疫缀合物) For the existence of T reg ± In the case of therapeutic immunoconjugates, T conv Secreted cytokine levels, % cytokine (Tconv) For the absence of T reg In the case of T conv The levels of secreted cytokines were calculated using one-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001)

[0534] Figure 17 Median and individual values ​​from 10 donors from independent experiments are shown, and Table 14 shows the median values.

[0535] Figure 17 The data in

[15] showed that Tregs inhibited 91% of granzyme B secretion by Tconv when untreated. 0.6 nM of PD1-IL2v and PD1-IL2vQ126T reduced the inhibition rate to 25.6% and 13.6%, respectively, and thus were able to restore 74% and 86% of granzyme B secretion by Tconv from Treg suppression ( Figure 4 and Table 14). 0.6 nM of the non-targeted versions FAP-IL2v and FAP-IL2vQ126T reduced the inhibition rate to 87.6% and 92.7%, respectively, and thus could only restore 12.3% and 7.2% of granzyme B secretion ( Figure 17Additional combination of non-targeted FAP-IL2v and FAP-IL2vQ126T with 66 nM parental blocking anti-PD-1 antibody modestly reduced Treg suppression to 69.2% and 84.5%, respectively, restoring 30.7% and 15.5% of granzyme B secretion by T conv ( Figure 17 and Table 14).

[0536] Table 14. Percentage of Treg-mediated suppression of granzyme B production by Tconv and percentage of restored granzyme B production by Tconv. Median of 6 donors.

[0537]

[0538] Example 3E. Activated PD-1 after treatment with increasing doses of PD-1-(LAG-3)-IL-2v immunoconjugate + and PD-1 - IL-2R signaling on CD4 T cells (STAT5-P)

[0539] By treating activated PD-1 expressing cells (PD-1 + ) and PD-1 negative (PD-1 - The efficacy and cis / trans signaling of PD-1-IL-2v, PD1-LAG3-IL2v, and FAP-IL2v immunoconjugates as a function of IL-2R signaling were measured using CD4 T cells pretreated with (anti-PD-1). The goal was to determine the dependence of the PD-1-(LAG3)-IL-2v immunoconjugate on PD-1 and LAG-3 expression by T cells in order to deliver IL-2R signaling.

[0540] To this end, CD4 T cells were sorted from healthy donor PBMCs using CD4 beads (#130-045-101, Miltenyi) and activated for 3 days in the presence of 1 μg / ml plate-bound anti-CD3 (pre-coated overnight, clone OKT3, #317315, BioLegend) and 1 μg / ml soluble anti-CD28 (clone CD28.2, #302923, BioLegend) antibodies to induce PD-1 expression. Three days later, cells were harvested and washed several times to remove endogenous cytokines, and half of the cells were labeled with Cell Trace Violet (CTV) (5 μM, 5 minutes at room temperature (RT); C34557, Thermo Scientific) and the other half were unlabeled.

[0541] Then, unlabeled cells were incubated with saturating concentrations of competitive anti-PD-1 and anti-LAG-3 antibodies (internal molecules, 10 μg / ml) at RT for 30 min, followed by several washing steps to remove excess unbound anti-PD-1 antibodies. 6 cells / ml) and PD-1 + CTV-labeled cells (25 μl, 6 × 10 6 cells / ml) were co-cultured in a V-bottom plate at a 1:1 ratio and then treated with increasing concentrations of therapeutic immunoconjugates (50 μl, 1:10 dilution step) at 37°C for 12 minutes. To maintain the phosphorylation state, an equal amount of Phosphoflow fixation buffer I (100 μl, 557870, BD Bioscience) was added after incubation with various constructs for 12 minutes to allow IL-2R signaling after binding to PD-1. The cells were then incubated at 37°C for another 30 minutes for fixation and then permeabilized overnight with Phosphoflow PermBuffer III (558050, BD Bioscience) at -80°C. The next day, the phosphorylated form of STAT-5 was stained for 30 minutes at 4°C using an anti-STAT-5P antibody (47 / Stat5 (pY694) clone, 562076, BD Bioscience).

[0542] Cells were collected on a BD-Symphony A5 flow cytometer (FACS) instrument (BD Bioscience). The frequency of STAT-5P was determined using FlowJo (V10) and plotted using GraphPad Prism (V8).

[0543] Figure 18 The data in the present study show that selected PD1-, LAG3-, and FAP-IL2v variants are + , LAG-3 + and PD-1 - , LAG-3 - Differences in the efficacy of signaling through IL-2R on CD4 T cells. + , LAG-3 + Potency measurements in CD4 T cells reflect PD1-(LAG-3)-mediated delivery of IL-2v and PD1 - , LAG-3 - PD1-(LAG-3)-independent delivery of IL-2v in CD4 T cells.

[0544] In Table 15, the EC50 of cells pre-blocked with PD-1 (LAG-3) was divided by the EC50 of cells pre-blocked with PD-1 (LAG-3). + , LAG-3 + The EC50 of T cells was used to calculate the STAT-5PEC50 increase fold between the PD-1-(LAG-3)-mediated delivery of IL-2v and the PD-1, LAG-3-independent delivery of each PD1-IL2v immunoconjugate molecule. This provides evidence for the strength of the PD-1-(LAG-3)-dependent delivery of IL2v for each IL2v mutant. In addition, the EC50 increase fold between various PD1-(LAG-3)-IL2v immunoconjugates and PD1-IL2v was calculated by dividing the EC50 of the new mutant by the EC50 of PD1-IL2v. This indicates that the PD1-IL2v immunoconjugate loses its potency in signal transduction through IL-2R due to its reduced affinity.

[0545] Due to the avidity gain of co-targeting of PD-1 and LAG-3, PD1-LAG-3-IL2v and PD1-LAG-3-IL2vQ126T were significantly associated with the PD-1 + , LAG-3 + The efficacy of PD-1-IL2v on T cells was higher than that of PD-1-IL2v, 126.6-fold and 6.5-fold, respectively. - , LAG3 - The efficacy on T cells was also increased ( Figure 18 and Table 15). However, PD1-LAG3-IL2v and PD1-LAG3-IL2vQ126T were not significantly associated with PD-1 + , LAG-3 + The cis-active windows on T cells are PD-1 - , LAG3 - The cis-active windows of PD1-IL2v and PD1-IL2vQ126T on T cells were 1105-fold and 485-fold higher, respectively. Figure 18 and Table 15).

[0546] Table 15. Dose-Response EC50 of STAT-5 Phosphorylation as a Function of PD-1 + LAG-3 + and PD-1 - LAG-3 - Frequency of IL-2v mutants on CD4 T cells. Mean ± SEM of 3 donors.

[0547] Example 3F. Treatment with PD-1-(LAG-3)-IL-2v immunoconjugates from T cells reg Restoring T during suppressionconv Effector function

[0548] To evaluate the ability of PD1-LAG3-IL2v and PD1-LAG3-IL2vQ126T to restore Tconv from Treg suppression, a suppressive function assay was established in which Tconv and Treg were combined with CD4 Tregs from unrelated donors. - CD25 - They were co-cultured for 5 days to elicit allospecific stimulation in the presence or absence of immunoconjugates as previously described in Example 3D.

[0549] Figure 19 The data in

[15] showed that Tregs inhibited 88% of granzyme B secretion by Tconv when untreated. 0.6 nM of PD1-IL2v and PD1-IL2vQ126T reduced the inhibition rate to -92.2% and -106.3%, respectively, and thus were able to not only recover from Treg suppression but also further increase granzyme B secretion by Tconv to 192% and 206% ( Figure 19 and Table 16). 0.6 nM of the non-targeted version of FAP-IL2v reduced the inhibition rate to 33% and was thus able to restore 66.38% of granzyme B secretion ( Figure 6 and Table 16). 0.6 nM of PD1-LAG3-IL2v and PD1-LAG3-IL2vQ126T reduced the inhibition rate to -293.25 and -171-4, respectively, and increased the secretion of granzyme B to 393% and 271.42% ( Figure 19 and Table 16). Figure 6 Median and individual values ​​from 10 donors from independent experiments are shown, and the median values ​​are shown in Table 16. P (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) were calculated using one-way ANOVA.

[0550] Table 16. Percentage of Treg-mediated suppression of granzyme B production by Tconv and percentage of restored granzyme B production by Tconv. Median of 10 donors.

[0551]

[0552] Example 3G. Internalization of PD-1-(LAG-3)-IL-2v Immunoconjugate by Activated CD4 T Cells

[0553] IL-2, once bound to IL-2R, induces internalization of the IL-2 / IL2R complex, which may represent a pooling of immunoconjugates that affects exposure. For this purpose, PBMCs were isolated by density gradient centrifugation using Ficoll-Paque (Sigma-Aldrich). PBMCs were isolated from 10 μg of cultured cells using a CD4 positive selection kit (Miltenyi, #130-045-101) according to the manufacturer's instructions. 8 CD4 T cells were then sorted by microbeads starting with 100 PBMCs. CD4 T cells were then sorted at 2-4 x 10 6 Cells / well were seeded in 12-well plates pre-coated with 1 μg / ml anti-CD3 (pre-coated overnight, clone OKT3, #317315, BioLegend) and cultured at 37°C for 3 days.

[0554] Three-day activated CD4 T cells were incubated in duplicate at 4 ° C in the presence of parental anti-PD-1, PD1-IL2v, PD1-LAG3-IL2v, FAP-IL2v, PD1-IL2vQ126T, PD1-LAG3-IL2vQ126T and FAP-IL2vQ126T in FACS tubes. The cells were then washed and divided into two groups, one of which was incubated for another 3 hours at 37 ° C, and the other was immediately stained with PE-labeled anti-PGLALA secondary antibody and anti-CD4 antibody (eBioscience), followed by fixation with BD Cell Fix. After incubation for 3 hours, the second group of cells was also stained with PE-labeled anti-PGLALA secondary antibody and anti-CD4 antibody before fixation. The cells were then collected at LSRFortessa (BD Biosciences), and data analysis was performed using FlowJo (V10), and graphing was performed using GraphPad Prism (v8). The expression level of detectable antibodies on the cell surface at 4°C was compared with the expression level 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 values ​​were calculated using one-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0555] The parental anti-PD1 was used as a negative control for internalization because it remains in the extracellular part of the cell membrane. 89.8% of FAP-IL2v and 76.5% of FAP-IL2vQ126T were internalized after 3 hours of incubation, followed by PD1-IL2v with 71% internalization. Interestingly, due to the higher affinity for PD-1 and further reduced affinity for IL-2R, only 47.4% of PD1-IL2vQ126T was internalized after 3 hours. By targeting IL-2v to PD-1 and LAG-3 with PD1-LAG3-IL2v, internalization was further reduced to 25%, and by further reducing the affinity for IL-2R with IL-2vQ126T, internalization remained unchanged (28%) ( Figure 20 and Table 17).

[0556] Table 17. Percentage of activated CD4 T cells internalizing molecules at 0.6 nM after 3 h incubation at 37°C. Median of 4 donors

[0557] 0.6 nM compound Internalization percentage Parental anti-PD-1 13.58815 PD1-IL2v 71.10301 PD1-LAG3-IL2v 25.65488 FAP-IL2v 89.8058 PD1-IL2v Q126T 48.44752 PD1-LAG3-IL2v Q126T 28.12816 FAP-IL2v Q126T 76.56525

[0558] Example 4

[0559] In vivo efficacy of a murine surrogate of the PD1-IL2vQ126T immunoconjugate in a syngeneic model of mouse tumor cell lines. - Panc02-Fluc subcutaneous syngeneic model

[0560] The murine surrogate PD1-IL2vQ126T immunoconjugate was tested in the mouse pancreatic cancer cell line Panc02-Fluc injected subcutaneously into Black 6-huIL2RBG transgenic mice.

[0561] Panc02-H7 cells (mouse pancreatic cancer) were originally obtained from the MD Anderson Cancer Center (Texas, USA) and stored in the Roche-Glycart internal cell bank after expansion. The Panc02-H7-Fluc cell line was produced internally by calcium transfection and subcloning technology. Panc02-H7-Fluc was cultured in RPMI medium containing 10% FCS (Sigma), 500ug / ml hygromycin and 1% Glutamax. Under 5% CO2, cells were cultured at 37°C in a water-saturated atmosphere. The 14th passage was used for transplantation. The cell viability was 94.7%. Using a 1ml tuberculin syringe (BD Biosciences), 2x10 per animal were used. 5 The cells were injected subcutaneously into the flank of mice in 100 μl of RPMI cell culture medium (Gibco).

[0562] Female 6-huIL2RBG transgenic mice (Breed 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-hour light / 12-hour dark daily cycle according to regulatory guidelines (GV-Solas; Felasa; TierschG). Following arrival, the animals were maintained for one week to acclimate to their new environment and were observed. Continuous health status monitoring was performed regularly.

[0563] On day 0 of the study, mice were injected subcutaneously with 2x10 5 Panc02-Fluc cells were randomly divided into groups and weighed. 3 Fifteen days after the start of the experiment, mice were injected intravenously with the muPD1-IL2vQ126T variant, muPD-IL2v, or vehicle once weekly for three weeks. All mice were injected intravenously with 200 μl of the appropriate solution. Mice in the vehicle group were injected with histidine buffer, and the treatment groups were injected intravenously with 2 mg / kg of muPD1-IL2vQ126T or 0.5 mg / kg of muPD1-IL2v once weekly for three weeks. To obtain the appropriate amount of immunoconjugate per 200 μl, the stock solution was diluted with histidine buffer as needed.

[0564] Figure 21 The results showed that the muPD1-IL2vQ126T variant mediated better efficacy in tumor growth inhibition compared with the vehicle and muPD1-IL2v groups. Mice injected with the muPD1-IL2vQ126T variant tolerated the treatment well.

[0565] Table 18.

[0566]

[0567] ***

[0568] Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, these descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims

1. 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 (numbered relative to the human IL-2 sequence SEQ ID NO: 90).

2. 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 (numbered relative to the human IL-2 sequence SEQ ID NO: 90); and wherein 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.

3. 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 (numbered relative to the human IL-2 sequence SEQ ID NO: 90); and 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 an amino acid substitution T3A and / or an 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, wherein the immunoconjugate comprises no more than one mutant IL-2 polypeptide.

7. The immunoconjugate according to any one of claims 1 to 6, wherein the antibody comprises an Fc domain composed of a first subunit and a second subunit. The immunoconjugate according to claim 7 , wherein the Fc domain is an IgG class Fc domain, particularly an IgG1 subclass Fc domain.

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

10. The immunoconjugate according to any one of claims 1 to 9, wherein the antibody is an immunoglobulin of the IgG class, in particular an immunoglobulin of the IgG1 subclass.

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. The immunoconjugate according to any one of claims 7 to 11, wherein in the CH3 domain of the first subunit of the Fc domain, amino acid residues are replaced by amino acid residues having a larger side chain volume, thereby generating a protrusion in the CH3 domain of the first subunit, and the protrusion is positionable in a cavity in the CH3 domain of the second subunit; and in the CH3 domain of the second subunit of the Fc domain, amino acid residues are replaced by amino acid residues having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second subunit, and the protrusion in the CH3 domain of the first subunit is positionable in the cavity.

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. The immunoconjugate of claim 13, wherein in the first subunit of the Fc domain, additionally, the serine residue at position 354 is replaced by a cysteine ​​residue (S354C) or the glutamic acid residue at position 356 is replaced by a cysteine ​​residue (E356C); and in the second subunit of the Fc domain, additionally, the tyrosine residue at position 349 is replaced by a cysteine ​​residue (Y349C) (numbering according to the Kabat EU index).

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 carboxyl-terminal amino acid of one of the subunits of the Fc domain, in particular to the carboxyl-terminal amino acid of 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. 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, particularly Fcγ receptors, and / or reduce effector function, particularly antibody-dependent cell-mediated cytotoxicity (ADCC).

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

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

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. The immunoconjugate of any one of claims 1 to 20, consisting essentially of a mutant IL-2 polypeptide and an IgG1 immunoglobulin molecule joined by a linker sequence.

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

23. One or more vectors, in particular expression vectors, comprising one or more polynucleotides according to claim 22.

24. A host cell comprising one or more polynucleotides according to claim 22 or one or more vectors according to claim 23.

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

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

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

29. The immunoconjugate of any one of claims 1 to 21 or 26 for use in treating a disease.

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

31. Use of the immunoconjugate according to any one of claims 1 to 21 or 26 in the preparation of a medicament for treating a disease.

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

33. A method of treating a disease in a subject, the method comprising administering to the subject 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. The method of claim 33, wherein the disease is cancer.

35. A method of stimulating the immune system of an individual, the method comprising administering to the 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. The invention as hereinbefore described.

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