mutant interleukin-2 (il-2) polypeptide, immunoconjugate, isolated polynucleotide, host cell of micr

专利摘要:
MUTANT INTERLEUKIN-2 (IL-2) POLYPEPIDS, IMMUNOCONJUGATES, POLYNUCLEOTIDE, EXPRESSION VECTOR, HOST CELL, METHOD OF PRODUCTION OF A MUTANT IL-2 POLYPEPTIDE AND USES OF I-2 MUTANT POLIPEPTIDE. These are mutant interleukin-2 polypeptides that exhibit reduced affinity for the IL-2 receptor a subunit, for use as immunotherapeutic agents. In addition, the invention relates to immunoconjugates that comprise said mutant IL-2 polypeptides, polynucleotide molecules that encode mutant or immunoconjugate IL-2 polypeptides, and host vectors and cells that comprise such polynucleotide molecules. The invention further relates to methods for producing the mutant or immunoconjugate IL-2 polypeptides, pharmaceutical compositions comprising the same and uses thereof.
公开号:BR112013018932B1
申请号:R112013018932-0
申请日:2012-02-07
公开日:2020-11-17
发明作者:Oliver Ast；Anne Freimoser-Grundschober；Christian Klein；Ekkehard Moessner；Pablo Umana；Peter Bruenker；Ralf Hosse；Sylvia Herter；Thomas U. Hofer；Valeria G. Nicolini
申请人:Roche Glycart Ag；
IPC主号:

专利说明:

FIELD OF THE INVENTION
[001] The present invention generally relates to mutant interleukin-2 polypeptides. More particularly, the inventions relate to mutant IL-2 polypeptides that exhibit enhanced properties for use as immunotherapeutic agents. In addition, the invention relates to immunoconjugates that comprise said mutant IL-2 polypeptides, polynucleotide molecules that encode mutant or immunoconjugate IL-2 polypeptides, and host vectors and cells that comprise such polynucleotide molecules. The invention further relates to methods for producing the mutant or immunoconjugate IL-2 polypeptides, pharmaceutical compositions comprising them, and uses thereof. BACKGROUND
[002] 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 generation, survival and homeostasis. This has a length of 133 amino acids and consists of four antiparallel amphipathic α-helices that form an indispensable quaternary structure of their function (Smith, Science 240, 1,169 to 76 (1988); Bazan, Science 257, 410 to 413 (1992)) . The IL-2 sequences of different species are found with the NCBI Ref Seq numbers NP000577 (human), NP032392 (mouse), NP446288 (rat) or NP517425 (chimpanzee).
[003] IL-2 mediates its action by binding to IL-2 receptors (IL-2R), which consist of up to three individual subunits, the different association of which may produce forms of receptor that differ in their affinity with IL- two. The association of α (CD25), β (CD122) and y (yc, CD132) subunits results in a trimeric receptor with high affinity for IL-2. The dimeric IL-2 receptor consisting of the β and y θ subunits called intermediate affinity IL-2R. The α subunit forms the monomeric IL-2 receptor with low affinity. Although the intermediate-affinity dimeric IL-2 receptor binds to IL-2 with approximately 100-fold lower affinity compared to the high-affinity trimeric receptor, both dimeric and trimeric IL-2 receptor variants are capable of transmitting signal by binding to IL-2 (Minami et al., Annu Rev Immunol 11,245 to 268 (1993)). Therefore, the α subunit, CD25, is not essential for IL-2 signaling. This gives high affinity binding to its receptor, while the β subunit, CD122, and the y subunit are crucial for signal transduction (Krieg et al., Proc Natl Acad Sei 107, 11,906 to 11 (2010)). The trimeric IL-2 receptors that include CD25 are expressed by regulatory T cells (Treg) (at rest) CD4 + forkhead box P3 (FoxP3) +. They are also transiently induced in conventional activated T cells, whereas at rest, these cells express only dimeric IL-2 receptors. Treg cells consistently express the highest level of CD25 in vivo (Fontenot et al., Nature Immunol 6, 1142 to 51 (2005)).
[004] IL-2 is synthesized mainly by activated T cells, in particular CD4 + helper T cells. This stimulates the proliferation and differentiation of T cells, induces the generation of cytotoxic T lymphocytes (CTLs) and the differentiation of peripheral blood lymphocytes into cytotoxic cells and lymphokine-activated killer cells (LAK), promotes cytokine and cytolytic molecule expression by T cells , facilitates the proliferation and differentiation of B cells and immunoglobulin synthesis by B cells, and stimulates the generation, proliferation and activation of natural killer cells (NK) (reviewed, for example, in Waldmann, Nat Rev Imunol 6, 595 a 601 (2009); Olejniczak and Kasprzak, Med Sci Monit 14, RA179 to 89 (2008); Malek, Annu Rev Imunol 26, 453 to 79 (2008)).
[005] Its ability to expand lymphocyte populations in vivo and increase the effector functions of these cells gives IL-2 antitumor effects, making IL-2 immunotherapy an attractive treatment option for certain metastatic cancers. Consequently, high-dose IL-2 treatment has been approved for use in patients with metastatic renal cell carcinoma and malignant melanoma.
[006] However, IL-2 has a dual role in the immune response in that it not only mediates the expansion and activity of effector cells, but is also crucially involved in maintaining peripheral immune tolerance.
[007] A major mechanism underlying peripheral self-tolerance is cell death induced by IL-2-induced activation (AICD) in T cells. AICD is a process by which fully activated T cells are subjected to programmed cell death through connection of death receptors expressed on cell surfaces such as CD95 (also known as Fas) or TNF receptor. When antigen-activated T cells that express a high-affinity IL-2 receptor (after previous exposure to IL-2) during proliferation are restimulated with antigen via the CD3 T-cell receptor (TCR), CD3 complex expression Fas (FasL) and / or tumor necrosis factor (TNF) is induced, making cells susceptible to Fas-mediated apoptosis. This process is dependent on IL-2 (Lenardo, Nature 353, 858 to 61 (1991)) and mediated by STAT5. Through the AICD process in T lymphocyte tolerance, it can not only be established for autoantigens, but also for persistent antigens that are clearly not part of the host's constitution, such as tumor antigens.
[008] In addition, IL-2 is also involved in the maintenance of peripheral regulatory T cells (Treg) CD4 + CD25 + (Fontenot et al., Nature Immunol 6, 1142 to 51 (2005); D'Cruz and Klein, Nature Immunol 6, 1,152 to 59 (2005); Maloy and Powrie, Nature Immunol 6, 1171 to 72 (2005), which are also known as suppressor T cells, which suppress the destruction by effector T cells of their (auto) target, through of cell-cell contact through inhibition of T cell activation and activation, or through the release of immunosuppressive cytokines such as IL-10 or TGF-β Depletion of Treg cells demonstrated the intensification of IL-2-induced antitumor immunity (Imai et al., Cancer Sci 98, 416 to 23 (2007)).
[009] Therefore, IL-2 is not ideal for inhibiting tumor growth, because of the presence of IL-2 or the generated CTLs can recognize the tumor as autonomous and be subjected to IACD or the immune response can be inhibited by Treg cells. dependent on IL-2.
[010] An additional issue regarding IL-2 immunotherapy is the side effects produced by treatment with recombinant human IL-2. Patients receiving high-dose IL-2 treatment often experience serious cardiovascular, pulmonary, renal, hepatic, gastrointestinal, neurological, cutaneous, hematological and systemic adverse events, which require intensive monitoring and patient management. Most of these side effects can be explained by the development of the so-called vascular (or capillary) leak syndrome (VLS), a pathological increase in vascular permeability that leads to fluid leakage in multiple organs (causing, for example, pulmonary and skin edema) and liver cell damage) and depletion of intravascular fluid (which causes a drop in blood pressure and a compensatory increase in heart rate). There is no treatment for VLS other than IL-2 withdrawal. Low-dose IL-2 regimens have been tested on patients to prevent VLS, however, in spending less than ideal therapeutic results. It is believed that VLS is caused by the release of pro-inflammatory cytokines, such as tumor necrosis factor (TNF) -α from IL-2 activated NK cells, however, it has recently been demonstrated that IL-2-induced pulmonary edema resulted from the direct binding of IL-2 to pulmonary endothelial cells, which expressed low levels of IL-2s functional αβy receptor intermediate (Krieg et al., Proc Nat Acad Sei USA 107, 11,906 to 11 (2010)).
[011] Several approaches have been taken to overcome these problems associated with IL-2 immunotherapy. For example, it has been found that the combination of IL-2 with certain anti-IL-2 monoclonal antibodies enhances the effects of treating IL-2 in vivo (Kamimura et al., J Immunol 177, 306 to 14 (2006); Boyman et al., Science 311, 1,924 to 27 (2006)). In an alternative approach, IL-2 has been mutated in several ways to reduce its toxicity and / or increase its effectiveness. Hu et al. (Blood 101,4853 to 4861 (2003), Patent Publication No. US2003 / 0124678) replaced the arginine residue at position 38 of IL-2 with tryptophan to eliminate IL-2 vasopermeability activity. Shanafelt et al. (Nature Biotechnol 18, 1,197 to 1,202 (2000)) mutated asparagine 88 to arginine to enhance selectivity for T cells over NK cells. Heaton et al. (Cancer Res 53, 2597-602 (1993); U.S. Patent No. 5,229,109) introduced two mutations, Arg38Ala and Phe42Lys, to reduce the secretion of pro-inflammatory cytokines from NK cells. Gillies et al. (Patent Publication No. US2007 / 0036752) replaced three IL-2 residues (Asp20Thr, Asn88Arg and Gln126Asp) that contribute to the affinity for the intermediate-affinity IL-2 receptor to reduce VLS. Gillies et al. (WO 2008/0034473) also mutated the IL-2 interface with CD25 by substituting the amino acid Arg38Trp and Phe42Lys to reduce interaction with CD25 and activation of Treg cells to enhance efficacy. With the same objective, Wittrup et al. (WO 2009/061853) produced IL-2 mutants that enhanced the affinity for CD25, but did not activate the receptor, thus acting as antagonists. The introduced mutations were aimed at breaking the interaction with the β- and / or Y subunit of the receptor.
[012] However, none of the known IL-2 mutants have been shown to overcome all the problems mentioned above associated with IL-2 immunotherapy, namely, toxicity caused by VLS induction, tumor tolerance caused by AICD induction and immunosuppression caused by activation of Treg cells. Thus, there remains a need in the art for further intensification of the therapeutic utility of IL-2 proteins. BRIEF DESCRIPTION OF THE INVENTION
[013] The present invention is based, in part, on the recognition that the interaction of IL-2 with the α-subunit of the trimeric IL-2 receptor with high affinity is responsible for the problems associated with IL-2 immunotherapy.
[014] Consequently, in a first aspect, the invention provides a mutant interleukin-2 (IL-2) polypeptide comprising a first amino acid mutation that abolishes or reduces the affinity of the mutant IL-2 polypeptide for the IL receptor -2 of high affinity and preserves the affinity of the mutant IL-2 polypeptide for the intermediate affinity IL-2 receptor, each compared to a wild-type IL-2 polypeptide. In one embodiment, said first amino acid mutation is in a position corresponding to residue 72 of human IL-2. In one embodiment, said first amino acid mutation is an amino acid substitution, selected from the group of L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. In a more specific embodiment, said first amino acid mutation is the substitution of amino acid L72G. In certain embodiments, the mutant IL-2 polypeptide comprises a second amino acid mutation that abolishes or reduces affinity of the mutant IL-2 polypeptide to the high affinity IL-2 receptor and preserves the affinity of the mutant IL-2 polypeptide for the intermediate affinity IL-2 receptor, each compared to a wild-type IL-2 polypeptide. In one embodiment, said second amino acid mutation is at a position selected from positions corresponding to residues 35, 38, 42, 43 and 45 of human IL-2. In a specific embodiment, said second amino acid mutation is in a position corresponding to residue 42 of human IL-2. In a more specific embodiment, said second amino acid mutation is an amino acid substitution selected from the group of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, and F42K. Still in a more specific embodiment, said second amino acid mutation is the substitution of amino acid F42A. In certain embodiments, the mutant interleukin-2 polypeptide comprises a third amino acid mutation that abolishes or reduces the affinity of the mutant IL-2 polypeptide for the high-affinity IL-2 receptor and preserves the affinity of the IL-2 polypeptide mutant for the intermediate affinity IL-2 receptor, each compared to a wild-type IL-2 polypeptide. In a particular embodiment, the mutant interleukin-2 polypeptide comprises three amino acid mutations that abolish or reduce the affinity of the mutant IL-2 polypeptide for the high-affinity IL-2 receptor and preserve the affinity of the IL-2 polypeptide mutant for the intermediate affinity IL-2 receptor, each compared to a wild-type IL-2 polypeptide, wherein said three amino acid mutations are in positions corresponding to residues 42, 45 and 72 of human IL-2 . In one embodiment, said three amino acid mutations are amino acid substitutions selected from the group of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. In a specific embodiment, said three amino acid mutations are the amino acid substitutions F42A, Y45A and L72G. In certain embodiments, the mutant interleukin-2 polypeptide further comprises an amino acid mutation that eliminates the IL-2 glycosylation site at a position corresponding to human IL-2 residue 3. In one embodiment, said amino acid mutation which eliminates the IL-2 glycosylation site at a position corresponding to human IL-2 residue 3 is an amino acid substitution selected from the group of T3A, T3G, T3Q, T3E, T3N , T3D, T3R, T3K, and T3P. In a specific embodiment, the amino acid mutation that eliminates the IL-2 glycosylation site at a position corresponding to human IL-2 residue 3 is T3A. In certain embodiments, the mutant IL-2 polypeptide is essentially a full-length IL-2 molecule, particularly a full-length human IL-2 molecule.
[015] The invention further provides a mutant interleukin-2 polypeptide linked to a non-IL-2 moiety. In certain embodiments, said non-IL-2 portion is a bleaching portion. In certain embodiments, said non-IL-2 portion is a chemical antigen-binding portion. In one embodiment, said antigen-binding chemical moiety is an antibody. In another embodiment, said antigen-binding chemical moiety is an antibody fragment. In a more specific embodiment, said antigen-binding chemical portion is selected from a Fab molecule and an scFv molecule. In a particular embodiment, said antigen-binding chemical portion is a Fab molecule. In another embodiment, said antigen-binding chemical portion is an scFv molecule. In particular embodiments, the mutant IL-2 polypeptide is linked to a first and a second non-IL-2 moiety. In such an embodiment, the mutant interleukin-2 polypeptide shares a carboxy terminal peptide bond with said first non-IL-2 portion and an amino terminal peptide link with said second non-IL-2 portion. In one embodiment, said antigen-binding chemical moiety is an immunoglobulin molecule. In a more specific embodiment, said antigen-binding chemical moiety is a class of IgG, particularly a subclass of IgGi, an immunoglobulin molecule. In certain embodiments, said antigen-binding chemical portion is directed to an antigen presented on a tumor cell or in a tumor cell environment, particularly an antigen selected from the group of Fibroblast Activating Protein (FAP), the A1 domain of Tenascin-C (TNC A1), the A2 domain of Tenascin-C (TNC A2), the Extra B Domain of Fibronectin (EDB), Carcinoembryonic Antigen (CEA) and the Chondroitin Sulfate Proteoglycan associated with Melanoma (MCSP).
[016] Also provided by the invention is an immunoconjugate comprising a mutant IL-2 polypeptide as described in the present invention, and an antigen-binding chemical moiety. In an embodiment of the immunoconjugate according to the invention, the mutant IL-2 polypeptide shares a carbon or amino terminal peptide bond with said antigen-binding chemical moiety. In particular embodiments, the immunoconjugate comprises the first and second chemical portions of antigen binding. In such an embodiment, the mutant IL-2 polypeptide comprised in the immunoconjugate according to the invention shares a carbon or amino terminal peptide bond with a first antigen-binding chemical portion and a second antigen-binding chemical portion shares a bond of a carboxy or amino terminal peptide with i) the mutant IL-2 polypeptide or ii) said first antigen-binding chemical moiety. In one embodiment, the antigen-binding chemical portion comprised in the immunoconjugate according to the invention is an antibody, in another embodiment, said antigen-binding chemical portion is an antibody fragment. In a specific embodiment, said antigen-binding chemical portion is selected from a Fab molecule and an scFv molecule. In a particular embodiment, said antigen-binding chemical moiety is a Fab molecule. In another particular embodiment, said antigen-binding moiety is an immunoglobulin molecule. In a more specific embodiment, said antigen-binding chemical moiety is a class of IgG, particularly a subclass of IgGi, an immunoglobulin molecule. In certain embodiments, said chemical antigen-binding portion is directed to an antigen presented on a tumor cell or in a tumor cell environment, particularly an antigen selected from the group of Fibroblast Activating Protein (FAP), the A1 domain of Tenascin-C (TNC A1), the A2 domain of Tenascin-C (TNC A2), the Extra B Domain of Fibronectin (EDB), Carcinoembryonic Antigen (CEA) and the Chondroitin Sulfate Proteoglycan associated with Melanoma (MCSP).
[017] The invention further provides isolated polynucleotides that encode a mutant IL-2 polypeptide or an immunoconjugate as described in the present invention, expression vectors that comprise said polynucleotides, and host cells that comprise the polynucleotides or expression vectors.
[018] Also provided is a method of producing a mutant IL-2 polypeptide or an immunoconjugate as described in the present invention, a pharmaceutical composition comprising a mutant IL-2 polypeptide or an immunoconjugate as described in the present invention and a carrier pharmaceutically acceptable, and methods of using a mutant IL-2 polypeptide or an immunoconjugate as described in the present invention.
[019] In particular, the invention encompasses a mutant IL-2 polypeptide or an immunoconjugate as described in the present invention for use in treating a disease in an individual in need thereof. In a particular embodiment, said disease is cancer. In a particular embodiment, the individual is a human.
[020] Also covered by the invention is the use of the mutant or immunoconjugate IL-2 polypeptide as described in the present invention for the manufacture of a medicament for treating a disease in an individual in need thereof.
[021] Additionally, a method of treating disease in an individual is comprised of administering to said individual a therapeutically effective amount of a composition comprising a mutant IL-2 polypeptide or an immunoconjugate as described in the present invention. Said disease is preferably cancer.
[022] Also provided is a method of stimulating an individual's immune system, which comprises administering to said individual an effective amount of a composition comprising the mutant or immunoconjugate IL-2 polypeptide described in the present invention in a pharmaceutically acceptable form. DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS
[023] The terms are used in the present invention as generally used in the art, unless otherwise defined below.
[024] The term "interleukin-2" or "IL-2" as used in the present invention, refers to any IL-2 native to any vertebrate source, including mammals such as primates (eg, humans) and rodents (eg mice and rats), unless otherwise indicated. The term covers unprocessed IL-2 as well as any form of IL-2 that results from processing in the cell. The term also encompasses naturally occurring variants of IL-2, for example, splice variants or allelic variants. The amino acid sequence of an exemplary human IL-2 is shown in SEQ ID NO: 1. Human unprocessed IL-2 additionally comprises an N-terminal 20 amino acid signal peptide that has the sequence of SEQ ID NO: 272, which is absent in the mature IL-2 molecule.
[025] The term "mutant IL-2" or "mutant IL-2 polypeptide" as used in the present invention is intended to encompass any mutant forms of various forms of the IL-2 molecule including full-length IL-2, truncated forms of IL-2 and forms in which IL-2 is linked to another molecule such as by chemical fusion or conjugation. "Full length" when used in reference to IL-2 is intended to mean the mature IL-2 molecule of natural length. For example, human full-length IL-2 refers to a molecule that has 133 amino acids (see, for example, SEQ ID NO: 1). The various forms of IL-2 mutants are characterized by the fact that they have at least one amino acid mutation that affects the interaction of IL-2 with CD25. This mutation may involve the replacement, 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, an IL-2 mutant may be referred to in the present invention as an IL-2 mutant peptide sequence, an IL-2 mutant polypeptide, IL-2 mutant protein, or mutant analog of IL-2.
[026] The designation of various forms of IL-2 is made in the present invention in relation to the sequence shown in SEQ ID NO: 1. Various designations can be used in the present invention to indicate the same mutation. For example, a phenylalanine mutation at position 42 to alanine can be indicated as 42A, A42, A42, F42A or Phe42Ala.
[027] The term "amino acid mutation" as used in the present invention is intended to encompass amino acid substitutions, deletions, insertions and modifications. Any combination of substitution, deletion, insertion and modification can be done to arrive at the final construct, as long as the final construct has the desired characteristics, for example, reduced binding to CD25. Deletions and insertions of amino acid sequence include deletions and insertions of the carboxy terminal and / or amino acid amino. An example of a terminal deletion is the deletion of the alanine residue at position 1 of human full-length IL-2. The preferred amino acid mutations are amino acid substitutions. For the purpose of altering, for example, the binding characteristics of an IL-2 polypeptide, non-conservative amino acid substitutions, that is, replacement of one amino acid with another amino acid that has different chemical and / or structural properties, are particularly preferred shares. Amino acid substitutions include replacing a hydrophobic amino acid with a hydrophilic amino acid. Amino acid substitutions include substitution with non-naturally occurring amino acid derivatives or with naturally occurring amino acid from the twenty standard amino acids (eg, 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using chemical or genetic methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis and the like. It is contemplated that methods of altering the side chain group of an amino acid by methods other than genetic engineering, such as chemical modification, may also be useful.
[028] As used in the present invention, a "wild-type" form of IL-2 is a form of IL-2 that is otherwise the same as the 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., unfused or conjugated IL-2 for any other molecule), the wild-type form of that mutant is native full-length IL-2 . If the IL-2 mutant is a fusion between IL-2 and another polypeptide encoded downstream of IL-2 (for example, an antibody chain), the wild-type form of that IL-2 mutant is IL-2 with a sequence of wild-type amino acids fused to the same polypeptide downstream. In addition, if the IL-2 mutant is a truncated form of IL-2 (the mutated or modified sequence within the non-truncated portion of IL-2), then the wild-type form of that IL-2 mutant is an IL -2 similarly truncated that has a wild type sequence. For the purpose of comparing the IL-2 receptor binding affinity or the biological activity of various forms of IL-2 mutants with the corresponding wild-type IL-2 form, the term wild-type encompasses forms of IL- 2 which comprise 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 cysteine substitution at a position corresponding to IL residue 125 -2 human per alanine. In some embodiments, wild-type IL-2 for the purpose of the present invention comprises substitution of amino acid C125A (see SEQ ID NO: 3). In certain embodiments according to the invention, the wild-type IL-2 polypeptide with which the mutant IL-2 polypeptide is compared comprises the amino acid sequence of SEQ ID NO: 1. In other embodiments, the IL polypeptide -2 of the wild type with which the mutant IL-2 polypeptide is compared comprises the amino acid sequence of SEQ ID NO: 3.
[029] The term “CD25” or “IL-2 receptor α subunit” as used in the present invention, refers to any CD25 native to any vertebrate source, including mammals such as primates (eg humans) and rodents ( for example, mice and rats), unless otherwise indicated. The term covers “full length”, CD25 does not process well any form of CD25 that results from processing in the cell. The term also covers naturally occurring variants of CD25, for example, splice variants or allelic variants. In certain embodiments, CD25 is human CD25. The amino acid sequence of an exemplary human CD25 (with signal sequence, Avi-tag and His-tag) is shown in SEQ ID NO: 278.
[030] The term "high-affinity IL-2 receptor" as used in the present invention refers to the heterotrimeric form of the IL-2 receptor, consisting of the y subunit of the receptor (also known as the y subunit of the common cytokine receptor , yc, or CD132), a β receptor subunit (also known as CD122 or p70) and an α receptor subunit (also known as CD25 or p55). The term “intermediate affinity IL-2 receptor” in contrast refers to the IL-2 receptor including only the y subunit and the β subunit, without the α subunit (for a review see, for example, Olejniczak and Kasprzak, Med Sei Monit 14, RA179 to 189 (2008)).
[031] "Affinity" refers to the intensity of the total sum of non-covalent interactions between a single binding site of a molecule (for example, a receptor) and its binding partner (for example, a ligand). Unless otherwise indicated, as used in the present invention, "binding affinity" refers to the intrinsic binding affinity that reflects a 1: 1 interaction between the members of a binding pair (e.g., receptor and a linker). The affinity of a molecule X with its partner Y can generally be represented by the dissociation constant (KD), which is the ratio of dissociation and association rate constants (kdissociation and kassociation, respectively). In this way, the equivalent affinities can comprise 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 in the present invention.
[032] The affinity of the wild-type IL-2 mutant or polypeptide with various forms of the IL-2 receptor can be determined according to the method presented in the Examples by surface plasmon resonance (SPR), using standard instrumentation such as a BIAcore instrument (GE Healthcare) and receptor subunits as can be obtained by recombinant expression (see, for example, Shanafelt et al., Nature Biotechnol 18, 1,197 to 1,202 (2000)). Alternatively, the binding affinity of IL-2 mutants to different forms of the IL-2 receptor can be assessed using cell lines known to express one or the other such form of the receptor. Specific exemplary and illustrative embodiments for measuring binding affinity are described in the present invention later.
[033] “Regulatory T cell” or “Treg cell” means a specialized type of CD4 + T cell that can suppress the responses of other T cells. Treg cells are characterized by the expression of the α-subunit of the IL-2 receptor (CD25) and the forkhead box P3 (FOXP3) transcription factor (Sakaguchi, Annu Rev Imunol 22, 531 to 62 (2004)) and play a critical role in inducing and maintaining peripheral self-tolerance for antigens, including those expressed by tumors. Treg cells require IL-2 for their function and their development and induction of their suppressive characteristics.
[034] As used in the present invention, the term "effector cells" refers to a population of lymphocytes that mediate the cytotoxic effects of IL-2. Effector cells include effector T cells such as CD8 + T cytotoxic cells, NK cells, lymphokine-activated killer cells (LAK) and macrophages / monocytes.
[035] As used in the present invention, the term "antigen-binding chemical moiety" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, a chemical antigen-binding portion is able to direct the entity to which it is attached (for example, a cytokine or a second chemical antigen-binding portion) to a target site, for example, to a specific type tumor cell or tumor stroma that carries the antigenic determinant. Chemical antigen-binding moieties include antibodies and fragments thereof as further defined in the present invention. Preferred antigen-binding chemical moieties include an antigen-binding domain of an antibody, which comprises an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, the antigen-binding chemical moieties may include antibody constant regions as further defined in the present invention and known in the art. Useful heavy chain constant regions include any of the five isotypes: α, δ, ε, y, or p. Useful light chain constant regions include either of the two isotypes: K and À.
[036] "Binds specifically" means that binding is selective for the antigen and can be discriminated against for non-specific or unwanted interactions. The ability of an antigen-binding chemical moiety to bind to a specific antigenic determinant can be measured using an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to an element skilled in the art, for example, surface plasmon (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323 to 329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217 to 229 (2002)).
[037] As used in the present invention, the term "antigenic determinant" is synonymous with "antigen" and "epitope", and refers to a site (for example, a contiguous stretch of amino acid or a conformational configuration consisting of regions other than non-contiguous amino acid) in a polypeptide macromolecule to which a chemical antigen-binding portion binds, forming an antigen-antigen-binding portion. Useful antigenic determinants can be found, for example, on the surfaces of tumor cells, on the surfaces of virus-infected cells, on the surfaces of other diseased cells, free in the blood serum and / or in the extracellular matrix (ECM).
[038] As used in the present invention, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain of two or more amino acids, and does not refer to a specific length of the product. Accordingly, peptides, dipeptides, tripeptides, oligopeptides, "protein," "amino acid chain," or any other term used to refer to a chain of two or more amino acids, are included in the definition of "polypeptide, "and the term" polypeptide "can be used instead of, or interchangeably with, any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivation by known blocking / protecting groups, proteolytic cleavage, or occurring amino acid modification unnatural. A polypeptide can be derived from a natural biological source or produced by recombinant technology, but it is not necessarily translated from a designated nucleic acid sequence. This can be generated in any way, including by chemical synthesis. A polypeptide of the invention can be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more more, 1,000 or more or 2,000 or more amino acids. Polypeptides can have a defined three-dimensional structure, although they do not necessarily have such a structure. Polypeptides with a defined three-dimensional structure are called folded, and polypeptides that do not have a defined three-dimensional structure, but, preferably, can adopt a large number of different conformations, and are called unfolded.
[039] An "isolated" polypeptide or variant, or a derivative thereof, is a polypeptide that is not in its natural state. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. The recombinantly produced polypeptides and the proteins expressed in host cells are considered isolated for the purpose of the invention, since they are native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.
[040] "Percent (%) of amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence , after the alignment of the sequences and the introduction of gaps, if necessary, to achieve the maximum sequence identity percentage, and not considering any conservative substitutions as part of the sequence identity. amino acid sequence can be achieved in several ways that are included in the technique, for example, use of publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). The elements versed in the technique can determine the appropriate parameters to align the strings, including any algorithms needed to achieve maximum alignment about the full length of the strings that are compared. For the purposes of the present invention, however,% amino acid sequence identity values are generated using the ALIGN-2 sequence comparison computer program. The ALIGN-2 sequence comparison computer program was authorized by Genentech, Inc., and the source code was deposited with user documentation with the US Copyright Department, Washington DC, 20559, when it is registered under the Copyright Registration No. USTXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, USA, or can be compiled from source code. The ALIGN-2 program must be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are defined by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is used for amino acid sequence comparisons, the% amino acid sequence identity of a given A amino acid sequence for, with or against a given B amino acid sequence (which may alternatively be called a given amino acid sequence A that has or comprises a certain% amino acid sequence identity for, with or against a given amino acid sequence B) is calculated as follows: 100 times the X / Y fraction where X is the number of residues of amino acid classified as identical matches by the sequence alignment program ALIGN-2 in the program alignment of A and B, and where Y is the total number of amino acid residues in B. It will be observed that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the% identity of amino acid sequence from A to B will not be equal to the% identity of amino acid sequence those from B to A. Unless specifically stated otherwise, all% amino acid sequence identity values used in the present invention are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.
[041] The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, for example, messenger RNA (mRNA), virus-derived RNA or plasmid DNA (pDNA). A polynucleotide can comprise a conventional phosphodiester bond or an unconventional bond (for example, an amide bond, as found in peptide nucleic acids (PNA). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, for example, fragments of DNA or RNA, present in a polynucleotide.
[042] An "isolated" nucleic acid or polynucleotide molecule is a nucleic acid, DNA or RNA molecule that has been removed from its native environment. For example, a recombinant polynucleotide that encodes a therapeutic polypeptide contained in a vector is considered to be isolated for the purposes of the present invention. Additional examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified polynucleotides (partially or substantially) in solution. An isolated polynucleotide includes a polynucleotide molecule contained in cells that commonly contain the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or in a chromosomal site that is different from its natural chromosomal site. 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 strand forms. The isolated polynucleotides or nucleic acids according to the present invention further include such synthetically produced molecules. In addition, a polynucleotide or nucleic acid can be or can include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.
[043] A nucleic acid or polynucleotide that has a nucleotide sequence of at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention means that the nucleotide sequence of the polynucleotide is identical to the reference sequence except by the fact that the polynucleotide sequence can include up to five point mutations for every 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide that has a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or replaced with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence. Such changes in the reference sequence can occur at the 5 'or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, individually interposed between the residues in the reference sequence or in one or more contiguous groups within of the reference sequence. As a practical matter, if any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention this can be determined conventionally with the use of known computer programs, such as those discussed above for polypeptides (for example, ALIGN-2).
[044] The term "expression cassette" refers to a polynucleotide generated recombinantly or synthetically, with a series of specified nucleic acid elements that allow for the transcription of a particular nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plasmid 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 of the invention comprises polynucleotide sequences that encode the mutant or immunoconjugate IL-2 polypeptides of the invention or fragments thereof.
[045] The term "vector" or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule that is used to introduce and target the expression of a specific gene to which it is functionally associated in a target cell. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it was introduced. The expression vector of the present invention comprises an expression cassette. Expression vectors allow transcription of large amounts of stable mRNA. Once the expression vector is within the target cell, the ribonucleic acid or protein molecule that is encoded by the gene is produced by the cell transcription and / or translation mechanism. In one embodiment, the expression vector of the invention comprises an expression cassette comprising polynucleotide sequences that encode mutant or immunoconjugate IL-2 polypeptides of the invention or fragments thereof.
[046] The term "artificial" refers to a composition derived from a non-host or synthetic cell, for example, a chemically synthesized oligonucleotide.
[047] The terms "host cell", "host cell line," and "host cell culture" are used interchangeably and refer to the cells into which the exogenous nucleic acid was introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and the progeny derived therefrom without regard to the number of passages. The progeny may not be completely identical in nucleic acid content to a parent cell, but it may contain mutations. Mutant progeny that have the same biological function or activity as screened or selected in the originally transformed cell are included in the present invention.
[048] The term "antibody" in the present invention is used in the broadest sense and encompasses several antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies), and fragments antibody as long as they exhibit the specified antigen-binding activity.
[049] The terms "full length antibody", "whole antibody" and "whole antibody" are used interchangeably in the present invention to refer to an antibody that has a structure substantially similar to a native antibody structure or that has heavy chains containing an Fc region as defined in the present invention.
[050] An "antibody fragment" refers to a molecule other than an entire antibody that comprises a portion of an entire antibody that binds to the antigen to which the entire 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 (eg scFv), and multispecific antibodies formed from antibody fragments. For an analysis of certain antibody fragments, see Hudson et al., Nat Med 9, 129 to 134 (2003). For an analysis of scFv fragments, see, for example, Plückthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, USA, pages 269 to 315 (1994); see also WO 93/16185; and Patent No. US5,571,894 and US5,587,458. For discussion of Fab and F (ab ') 2 fragments that comprise rescue receptor binding epitope residues and that have an increased in vivo half-life, see U.S. Patent No. 5,869,046. Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, for example, EP 404.097; WO 1993/01161; Hudson et al., Nat Med 9, 129 to 134 (2003); and Hollinger et al., Proc Natl Acad Sei USA 90, 6,444 to 6,448 (1993). Tribodies and tetribodies are also described in Hudson et al., Nat Med 9, 129 to 134 (2003). Antibody fragments can be made by a variety of techniques, including, but not limited to, proteolytic digestion of an entire antibody as well as production by recombinant host cells (e.g., E. coli or phage), as described in the present invention.
[051] The term "immunoglobulin molecule" refers to a protein that has the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are linked by disulfide. From the N to C terminals, each heavy chain has a variable region (VH), also called a variable heavy domain or a variable heavy chain domain, followed by three constant domains (CH1, CH2, and CH3), also called heavy chain constant region. Similarly, from the N to C terminals, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light domain (CL), also called a region light chain constant. The heavy chain of an immunoglobulin can be assigned to one of five classes, called α (IgA), δ (IgD), ε (IgE), y (IgG), or M (IgM), some of which can be further divided into subclasses, for example, yi (IgGi), y2 (lgG2), ys (IgGs), Y4 (lgG4), ai (IgAi) and 02 (IgAa). The light chain of an immunoglobulin can be attributed to one of two types, called kappa (K) and lambda (À), based on the amino acid sequence of its constant domain. An immunoglobulin consists essentially of two Fab molecules and an Fc domain, linked through the imuπoglobuliπ hinge region.
[052] The term "antigen binding domain" refers to the part of an antibody that comprises the area that specifically binds to and is complementary to part or all of an antigen. An antigen-binding domain can be provided by, for example, one or more variable antibody domains (also called antibody variable regions). Preferably, an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
[053] The term "variable region" or "variable domain" refers to the domain of an antibody light or heavy chain that is involved in binding the antibody to the antigen. The heavy chain and light chain variable domains (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity.
[054] The term "hypervariable region" or "HVR", as used in the present invention, refers to each of the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops ("hypervariable loops" ). Generally, native four-chain antibodies comprise their HVRs; three in VH (H1, H2, H3), and in VL (L1, L2, L3). HVRs generally comprise amino acid residues from hypervariable loops and / or complementarity determining regions (CDRs), the latter being of higher sequence variability and / or involved in antigen recognition. With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable bonds. Hypervariable regions (HVRs) are also called "complementarity determining regions" (CDRs), and these terms are used interchangeably in the present invention in reference to portions of the variable region that form the antigen-binding regions. This particular region was described by Kabat et al., US Dept, of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and by Chothia et al., J Mol Biol 196: 901 to 917 (1987), in which the definitions include overlap or subsets of amino acid residues when compared to each other. However, the application of the definition to refer to a CDR of an antibody or variants thereof is intended to be included in the scope of the term as defined and used in the present invention. The appropriate amino acid residues that comprise CDRs as defined by each of the references cited above are shown below in Table 1 as a comparison. The exact residue numbers that span a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine that the residues comprise a particular CDR in view of the variable region amino acid sequence of the antibody.
1 The numbering of all CDR definitions in Table 1 is in accordance with the numbering conventions presented by Kabat et al. (see below). 2 "AbM" with a lowercase "b" as used in Table 1 refers to CDRs as defined by Oxford Molecular's "AbM" antibody modeling software.
[055] Kabat et al. also defined a numbering system for variable region sequences that is applicable to any antibody. An element of common knowledge in the art can unambiguously assign this system of "Kabat numbering" to any variable region sequence, without depending on any experimental data other than the sequence itself. As used in the present invention, "Kabat numbering" refers to the numbering system presented by Kabat et al., U.S. Dept, of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise specified, references to the numbering of specific amino acid residue positions in an antibody variable region are in accordance with the Kabat numbering system.
[056] The polypeptide sequences in the sequence listing (i.e., SEQ ID NOs: 23, 25, 27, 29, 31, 33, etc.) are not numbered according to the Kabat numbering system. However, it is quite common in the art to convert sequence numbering from the sequence listing to Kabat numbering.
[057] "Arcabouço" or "FR" refers to residues of variable domain in addition to residues of hypervariable region (HVR). The RF of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Consequently, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1 (L1) -FR2-H2 (L2) -FR3-H3 (L3) -FR4.
[058] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), for example, IgGi, IgG2, IgGs, IgG4, IgAi, and IgG2. The heavy chain constant domains that correspond to different classes of immunoglobulins are called α, δ, ε, y, and p, respectively.
[059] The term "Fc region" in the present invention is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Although the limits of the Fc region of an IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226, or Pro230, to the carboxyl terminus of the heavy chain. However, C-terminal lysine (Lys447) from the Fc region may or may not be present.
[060] A "modification-promoting heterodimerization" is a manipulation of the peptide backbone or post-translational modifications of a polypeptide, for example, an immunoglobulin heavy chain, which reduces or prevents the association of the polypeptide with an identical polypeptide for form a homodimer. A heterodimerization that promotes modification as used in the present invention particularly includes separating the modifications made to each of the two desired polypeptides to form a dimer, wherein the modifications are complementary to each other for the purpose of promoting the association of the two polypeptides. For example, a heterodimerization that promotes modification can alter the structure or charge of one or both of the desired polypeptides to form a dimer in order to make its steric association electrostatically favorable, respectively. Heterodimerization occurs between two non-identical polypeptides, such as two immunoglobulin heavy chains in which the added immunoconjugate components fused to each of the heavy chains (e.g., IL-2 polypeptide) are not the same. In the immunoconjugates of the present invention, the heterodimerization that promotes modification is in the heavy chain (s), specifically in the Fc domain, of an immunoglobulin molecule. In some embodiments, the heterodimerization that promotes modification comprises an amino acid mutation, specifically an amino acid substitution. In a particular embodiment, the modifying-promoting heterodimerization comprises a separate amino acid mutation, specifically an amino acid substitution, in each of the two immunoglobulin heavy chains.
[061] The term "effector functions" when used in reference to antibodies 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 extracellular phagocytosis (ADCP), cytokine secretion , down-regulation of cell surface receptors (for example, B cell receptor), and B cell activation.
[062] An "activating Fc receptor" is an Fc receptor that follows engagement by an Fc region of an antibody signaling events that stimulate the receptor-carrying cell to perform effector functions. Activating Fc receptors include FcyRllla (CD16a), FcyRI (CD64), FcyRlla (CD32), and FcαRI (CD89).
[063] As used in the present invention, the terms "modify, modified, modification" are considered to be any manipulation of the peptide backbone or post-translational modifications of a recombinant or naturally occurring polypeptide or fragment thereof. The modification includes modifications to the amino acid sequence, glycosylation pattern, or the side chain group of individual amino acids, as well as combinations of these approaches.
[064] As used in the present invention, the term "immunoconjugate" refers to a polypeptide molecule that includes at least one chemical portion of IL-2 and at least one chemical antigen-binding portion. In certain embodiments, the immunoconjugate comprises at least one chemical portion of IL-2, and at least two chemical portions of antigen binding. The particular immunoconjugates according to the invention essentially consist of a chemical IL-2 moiety and two antigen-binding chemical moieties joined by one or more linker sequences. The chemical antigen-binding portion can be joined to the chemical portion of IL-2 by a variety of interactions and in a variety of configurations as described in the present invention.
[065] As used in the present invention, the term "control antigen-binding chemical moiety" refers to a chemical antigen-binding moiety as it would exist free of other antigen-binding chemical moieties and effector moieties. For example, when comparing a Fab-IL2-Fab immunoconjugate of the invention with a control antigen-binding chemical portion, the control antigen-binding chemical portion is Fab-free, wherein the Fab-IL2-Fab immunoconjugate and the free Fab molecule can specifically bind to the antigen determinant.
[066] As used in the present invention, the terms "first" and "second" in relation to chemical antigen-binding moieties, etc., are used for convenience of distinction when there is more than one of each type of chemical moiety. The use of these terms is not intended to confer a specific order or orientation of the immunoconjugate unless explicitly defined in this way.
[067] An "effective amount" of an agent refers to the amount that is required to result in a physiological change in the cell or tissue to which it is administered.
[068] An "therapeutically effective amount" of an agent, for example, a pharmaceutical composition, refers to an amount effective, in dosages and time periods necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent, for example, eliminates, decreases, delays, minimizes or prevents the adverse effects of a disease.
[069] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (for example, cows, sheep, cats, dogs and horses), primates (for example, humans and non-human primates such as monkeys), rabbits and rodents (for example, mice and rats). Preferably, the individual or subject is a human.
[070] The term "pharmaceutical composition" refers to a preparation that is such that it allows the biological activity of an active ingredient contained therein to be effective, and that it does not contain additional components that are unacceptably toxic to a subject to whom the composition would be administered.
[071] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition, in addition to an active ingredient, which is non-toxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer or preservative.
[072] As used in the present invention, "treatment" (and grammatical variations of the same as "treating" or "treating") refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated. treated, and can be performed for prophylaxis or during the course of clinical pathology. Desirable treatment effects include, but are not limited to, preventing the occurrence or recurrence of disease, relieving symptoms, decreasing any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, improving or attenuation of disease status, and improved remission or prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or to slow the progression of a disease. DETAILED DESCRIPTION OF ACHIEVEMENTS
[073] The present invention aims to provide a mutant IL-2 polypeptide that has enhanced properties for immunotherapy. In particular, the invention aims to eliminate the pharmacological properties of IL-2 that contribute to toxicity, but are not essential to the efficacy of IL-2. As discussed above, different forms of the IL-2 receptor consist of different subunits and exhibit different affinities for IL-2. The intermediate affinity IL-2 receptor, which consists of the β and y receptor subunits, is expressed in resting effector cells and is sufficient for IL-2 signaling. The high-affinity IL-2 receptor, which additionally comprises the receptor's α subunit, is mainly expressed in regulatory T cells (Treg) as well as in activated effector cells in which their IL-2 coupling can promote cell-mediated immunosuppression Treg or activation-induced cell death (AICD), respectively. Thus, without sticking to the theory, the reduction or abolition of IL-2 affinity for the α-subunit of the IL-2 receptor should reduce IL-2-induced down regulation of effector cell function by regulatory T cells and the development of tumor tolerance by the IACD process. On the other hand, maintaining the affinity for the IL-2 receptor of intermediate affinity should preserve the induction of proliferation and activation of effector cells with NK and T cells by IL-2.
[074] Several IL-2 mutants already exist in the art, however, the inventors have concluded that the innovative amino acid mutations of the IL-2 polypeptide and combinations thereof are particularly suitable for giving IL-2 the desired characteristics for immunotherapy.
[075] In a first aspect, the invention provides a mutant interleukin-2 (IL-2) polypeptide that comprises an amino acid mutation that abolishes or reduces the affinity of the mutant IL-2 polypeptide for the IL receptor α subunit -2 and conserves the affinity of the mutant IL-2 polypeptide for the intermediate affinity IL-2 receptor, each compared to a wild-type IL-2 polypeptide.
[076] Mutants of human IL-2 (hlL-2) with decreased affinity for CD25 can, for example, be generated by amino acid substitution at amino acid position 35, 38, 42, 43, 45 or 72 or combinations thereof . 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, F42 , K43E, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. The particular IL-2 mutants according to the invention comprise a mutation at an amino acid position corresponding to residue 42, 45, or 72 of human IL-2, or a combination thereof. These mutants exhibit substantially similar binding affinity for the intermediate affinity IL-2 receptor, and have substantially reduced affinity for the α-subunit of the IL-2 receptor and the high affinity IL-2 receptor compared to a form of wild type of the IL-2 mutant.
[077] Other characteristics of useful mutants may include the ability to induce proliferation of T cells and / or NK cells that carry the IL-2 receptor, the ability to induce IL-2 signaling in T cells and / or NK cells that carry an IL-2 receptor, the ability to generate interferon (IFN) - Y as a secondary cytokine by NK cells, a reduced ability to induce the elaboration of secondary cytokines - particularly IL-10 and TNF-a - by mononuclear cells of peripheral blood (PBMCs), a reduced ability to activate regulatory T cells, a reduced ability to induce apoptosis in T cells, and a reduced toxicity profile in vivo.
[078] In an embodiment according to the invention, the amino acid mutation that abolishes or reduces affinity of the mutant IL-2 polypeptide for the high affinity IL-2 receptor and preserves affinity for the mutant IL-2 polypeptide for the Intermediate affinity IL-2 receptor is in a position corresponding to human IL-2 residue 72. In one embodiment, said amino acid mutation is an amino acid substitution. In one embodiment, said amino acid substitution is selected from the group of L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. In a more specific embodiment, said amino acid mutation is the substitution of amino acid L72G.
[079] In a particular aspect, the invention provides a mutant IL-2 polypeptide comprising a first and a second amino acid mutation that abolishes or reduces affinity of the mutant IL-2 polypeptide for the α-subunit of the IL-2 receptor and preserves the affinity of the mutant IL-2 polypeptide for the intermediate affinity IL-2 receptor. In one embodiment, said first amino acid mutation is in a position corresponding to residue 72 of human IL-2. In one embodiment, said first amino acid mutation is an amino acid substitution. In a specific embodiment, said first amino acid mutation is an amino acid substitution selected from the group of L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. Still in a more specific embodiment, the said amino acid substitution is L72G. Said second amino acid mutation is in a different position from said first amino acid mutation. In one embodiment, said second amino acid mutation is at a position selected from a position corresponding to residue 35, 38, 42, 43 and 45 of human IL-2. In one embodiment, said second amino acid mutation is an amino acid substitution. In a specific embodiment, said amino acid substitution is selected from the group of 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, and Y45K. In a particular embodiment, said second amino acid mutation is in a position corresponding to residue 42 or 45 of human IL-2. In a specific embodiment, said second amino acid mutation is an amino acid substitution selected from the group of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, and Y45K. In a more specific embodiment, said second amino acid mutation is the substitution of amino acid F42A or Y45A. In a more particular embodiment, said second amino acid mutation is in the position corresponding to residue 42 of human IL-2. In a specific embodiment, said second amino acid mutation is an amino acid substitution selected from the group of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, and F42K. In a more specific embodiment, said amino acid substitution is F42A. In another embodiment, said second amino acid mutation is in the position corresponding to residue 45 of human IL-2. In a specific embodiment, said second amino acid mutation is an amino acid substitution, selected from the group of Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, and Y45K. In a more specific embodiment, said amino acid substitution is Y45A. In certain embodiments, the mutant IL-2 polypeptide comprises a third amino acid mutation that abolishes or reduces affinity of the mutant IL-2 polypeptide for the α subunit of the IL-2 receptor and preserves the affinity of the mutant IL-2 polypeptide for the intermediate affinity IL-2 receptor, each compared to a wild-type IL-2 polypeptide. Said third amino acid mutation is in a different position from said first and second amino acid mutations. In one embodiment, said third amino acid mutation is in a position selected from a position corresponding to residues 35, 38, 42, 43 and 45 of human IL-2. In a preferred embodiment, said third amino acid mutation is in a position corresponding to the 42 or 45 residue of human IL-2. In one embodiment, said third amino acid mutation is in a position corresponding to residue 42 of human IL-2. In another embodiment, said third amino acid mutation is in a position corresponding to residue 45 of human IL-2. In one embodiment, said third amino acid mutation is an amino acid substitution. In a specific embodiment, said amino acid substitution is selected from the group of 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, and Y45K. In a more specific embodiment, said amino acid substitution is selected from the group of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, and Y45K. Still in a more specific embodiment, said amino acid substitution is F42A or Y45A. In one embodiment, said amino acid substitution is F42A. In another embodiment, said amino acid substitution is Y45A. In certain embodiments, the mutant IL-2 polypeptide does not comprise an amino acid mutation at the position corresponding to human IL-2 residue 38.
[080] Still in a more particular aspect of the invention, a mutant IL-2 polypeptide is provided which comprises three amino acid mutations that abolish or reduce the affinity of the mutant IL-2 polypeptide for the IL-2 receptor α subunit , but retain the affinity of the mutant IL-2 polypeptide for the intermediate affinity IL-2 receptor. In one embodiment, said three amino acid mutations are in positions corresponding to residues 42, 45 and 72 of human IL-2. In one embodiment, said three amino acid mutations are amino acid substitutions. In one embodiment, said three amino acid mutations are amino acid substitutions selected from the group of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. In a specific embodiment, said three amino acid mutations are amino acid substitutions F42A, Y45A and L72G.
[081] In certain embodiments, said 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 times, specifically at least 10 times, more specifically at least 25 times . 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 can reduce the affinity of the mutant IL-2 polypeptide to the α-subunit of the IL-2 receptor at least 30 times, at least 50 times, or at least 100 times. In one embodiment, said amino acid mutation or combination of amino acid mutations abolishes the affinity of the mutant IL-2 polypeptide for the α-subunit of the IL-2 receptor so that no binding is detectable by surface plasmon resonance as described in the present invention below.
[082] Substantially similar binding to the intermediate affinity receptor, i.e. conservation of the affinity of the mutant IL-2 polypeptide for said receptor, is achieved when the IL-2 mutant exhibits more than about 70% of the affinity in a wild-type form of the IL-2 mutant for the intermediate affinity IL-2 receptor. The IL-2 mutants of the invention can exhibit more than about 80% and even more than about 90% of such an affinity.
[083] The inventors concluded that a reduction in IL-2 affinity for the α-subunit of the IL-2 receptor in combination with the elimination of IL-2 O glycosylation results in an IL-2 protein with enhanced properties. For example, elimination of the glycosylation site O results in a more homogeneous product when the mutant IL-2 polypeptide is expressed in mammalian cells such as CHO or HEK cells.
[084] Thus, in certain embodiments, the mutant IL-2 polypeptide according to the invention comprises an additional amino acid mutation that eliminates the IL-2 glycosylation site at a position corresponding to IL-2 residue 3 human. In one embodiment, said additional amino acid mutation that eliminates the IL-2 glycosylation site at a position corresponding to human IL-2 residue 3 is an amino acid substitution. Exemplary amino acid substitutions include T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P. In a specific embodiment, said additional amino acid mutation is the replacement of amino acid T3A.
[085] In certain embodiments, the mutant IL-2 polypeptide is essentially a full-length IL-2 molecule. In certain embodiments, the mutant IL-2 polypeptide is a human IL-2 molecule. In one embodiment, the mutant IL-2 polypeptide comprises the sequence of SEQ ID NO: 1 with at least one amino acid mutation that abolishes or reduces affinity of the mutant IL-2 polypeptide for the IL-2 receptor α subunit, but it retains the affinity of the mutant IL-2 polypeptide for the intermediate affinity IL-2 receptor, compared to an IL-2 polypeptide comprising SEQ ID NO: 1 without said mutation. In another embodiment, the mutant IL-2 polypeptide comprises the sequence of SEQ ID NO: 3 with at least one amino acid mutation that abolishes or reduces the affinity of the mutant IL-2 polypeptide for the α-subunit of the IL-2 receptor 2, but retains the affinity of the mutant IL-2 polypeptide for the intermediate affinity IL-2 receptor, compared to an IL-2 polypeptide comprising SEQ ID NO: 3 without said mutation.
[086] In a specific embodiment, the mutant IL-2 polypeptide can elicit one or more of the cellular responses selected from the group consisting of: proliferation in an activated T lymphocyte cell, differentiation in an activated T lymphocyte cell, cytotoxic activity cell (CTL) proliferation in an activated B cell, differentiation in an activated B cell, proliferation in a natural killer cell (NK), differentiation in an NK cell, cytokine secretion by a T cell or an activated NK cell, and activated NK / lymphocyte (LAK) exterminating cytotoxicity.
[087] In one embodiment, the mutant IL-2 polypeptide has a reduced ability to induce IL-2 signaling in regulatory T cells, compared to a 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 a wild-type IL-2 polypeptide. In one embodiment, the mutant IL-2 polypeptide has a reduced toxicity profile in vivo, compared to a wild-type IL-2 polypeptide. In one embodiment, the mutant IL-2 polypeptide has an extended serum half-life, compared to a wild-type IL-2 polypeptide.
[088] A particular mutant IL-2 polypeptide according to the invention comprises four amino acid substitutions in positions corresponding to residues 3, 42, 45 and 72 of human IL-2. Specific amino acid substitutions are T3A, F42A, Y45A and L72G. As shown in the attached Examples, said quadruple mutant IL-2 polypeptide does not exhibit detectable binding to CD25, reduced ability to induce apoptosis in T cells, reduced ability to induce IL-2 signaling in Treg cells, and a profile of reduced toxicity in vivo. However, it retains the ability to activate IL-2 signaling in effector cells, to induce effector cell proliferation, and to generate IFN-γ as a secondary cytokine by NK cells.
[089] Furthermore, said mutant IL-2 polypeptide has additionally advantageous properties, such as reduced surface hydrophobicity, good stability and good expression productivity, as described in the Examples. Unexpectedly, said mutant IL-2 polypeptide also provides an extended serum half-life, compared to a wild-type IL-2.
[090] IL-2 mutants of the invention, in addition to those that have mutations in the IL-2 region that forms the IL-2 interface with CD25 or the glycosylation site, may also have one or more mutations in the outside amino acid sequence these regions. Such mutations in human IL-2 may provide additional advantages such as increased expression or stability. For example, cysteine at position 125 can be replaced by a neutral amino acid such as serine, alanine, threonine or valine, which produces C125S IL-2, C125A IL-2, C125T IL-2 or C125V IL-2 respectively, as described in U.S. Patent No. 4,518,584. As described therein, the N-terminal IL-2 alanine residue that produces such mutants as des-A1 C125S or des-A1 C125A can also be deleted. Alternatively or jointly, the IL-2 mutant can include a mutation whereby the methionine that normally occurs at position 104 of human wild-type IL-2 is replaced by a neutral amino acid such as alanine (see US Patent No. 5,206,344) . The resulting mutants, for example, des-A1 M104A IL-2, des-A1 M104A C125S IL-2, M104A IL-2, M104A C125A IL-2, des-A1 M104A C125A IL-2, or M104A C125S IL-2 (these and other mutants can be found in U.S. Patent No. 5,116,943 and, Weiger et al., Eur J Biochem 180, 295 to 300 (1989)) can be used in conjunction with the particular IL-2 mutations of the invention.
[091] Thus, in certain embodiments, the mutant IL-2 polypeptide according to the invention comprises an additional amino acid mutation in a position corresponding to human IL-2 residue 125. In one embodiment, said additional amino acid mutation is the substitution of amino acid C125A.
[092] The person skilled in the art will be able to determine which additional mutations can provide additional advantages for the purpose of the invention. For example, he will observe that amino acid mutations in the IL-2 sequence that reduce or abolish the affinity of IL-2 for the intermediate affinity IL-2 receptor, such as D20T, N88R or Q126D (see, for example, example, US 2007/0036752), may not be suitable to include the mutant IL-2 polypeptide according to the invention.
[093] In one embodiment, the mutant IL-2 polypeptide of the invention comprises a sequence selected from the group of SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15, and SEQ ID NO: 19. In one specific embodiment, the mutant IL-2 polypeptide of the invention comprises a sequence of SEQ ID NO: 15 or SEQ ID NO: 19. Still in a more specific embodiment, the mutant IL-2 polypeptide comprises a sequence of SEQ ID NO: 19.
[094] The mutant IL-2 polypeptides of the invention are particularly useful in the context of IL-2 fusion proteins such as immunoconjugates that carry IL-2. Such fusion proteins comprise a mutant IL-2 polypeptide of the invention fused to a non-IL-2 moiety. The non-IL-2 portion may be a synthetic or natural protein or a portion or variant thereof. Chemical portions of IL-2 include albumin, or antibody domains such as the Fc domains or antigen binding domains of imuπoglobulins.
[095] Immunoconjugates carrying IL-2 are fusion proteins that comprise a chemical antigen-binding portion and a chemical IL-2 portion. They significantly increase the effectiveness of IL-2 therapy through direct targeting of IL-2, for example, in a tumor environment. According to the invention, a chemical antigen-binding portion can be a whole antibody or immunoglobulin, or a portion or variant thereof that has a biological function such as specific antigen binding affinity.
[096] The benefits of immunoconjugate therapy are readily apparent. For example, a chemical antigen-binding portion of an immunoconjugate recognizes a specific tumor epitope and results in targeting the immunoconjugate molecule to the tumor site. Therefore, high concentrations of IL-2 can be delivered to the tumor microenvironment, thereby resulting in the activation and proliferation of a variety of immune effector cells mentioned in the present invention with the use of a much smaller dose of the immunoconjugate that would be required for unconjugated IL-2. In addition, since the application of IL-2 in the form of immunoconjugates allows for lower doses of the cytokine itself, the potential for unwanted IL-2 side effects is restricted, and the targeting of IL-2 to a specific site in the body by The use of an immunoconjugate can also result in a reduction in systemic exposure and, therefore, less side effects than those obtained with unconjugated IL-2. In addition, the increased circulating half-life of an immunoconjugate compared to unconjugated IL-2 contributes to the effectiveness of the immunoconjugate. However, this characteristic of IL-2 immunoconjugates can again aggravate the potential side effects of the IL-2 molecule: Due to the significantly longer circulating half-life of IL-2 immunoconjugate in the bloodstream than unconjugated IL-2, the likelihood for IL-2 or other portions of the fusion protein molecule to activate the components usually present in the vasculature is increased. The same concept applies to other fusion proteins that contain IL-2 fused to another chemical moiety such as Fc or albumin, resulting in an extended half-life of IL-2 in the circulation. Therefore, an immunoconjugate comprising a mutant IL-2 polypeptide according to the invention, with reduced toxicity compared to wild-type forms of IL-2, is particularly advantageous.
[097] Consequently, the invention further provides a mutant IL-2 polypeptide as described in the present invention previously, linked to at least a non-IL-2 moiety. In one embodiment, the mutant IL-2 polypeptide and the non-IL-2 moiety form a fusion protein, i.e., the mutant IL-2 polypeptide shares a peptide bond with the non-IL-2 moiety. In one embodiment, the mutant IL-2 polypeptide is linked to a first and a second chemical moiety without IL-2. In one embodiment, the mutant IL-2 polypeptide shares a carboxy or amino-terminal peptide bond with the first antigen-binding chemical moiety, and the second antigen-binding chemical moiety shares a carboxy or amino-terminal peptide bond with i) the mutant IL-2 polypeptide or ii) the first antigen-binding chemical moiety. In a specific embodiment, the mutant IL-2 polypeptide shares a carboxy terminal peptide bond with said first non-IL-2 portion and an amino terminal peptide bond with said second non-IL-2 portion. In one embodiment, said non-IL-2 portion is a bleaching portion. In a particular embodiment, said non-IL-2 moiety is a chemical antigen-binding moiety (thereby forming an immunoconjugate with the mutant IL-2 polypeptide, as described in greater detail below). In certain embodiments, the antigen-binding chemical moiety is an antibody or an antibody fragment. In one embodiment, the antigen-binding chemical moiety is a full-length antibody. In one embodiment, the antigen-binding chemical moiety is an immunoglobulin molecule, particularly an immunoglobulin molecule of the IgG class, more particularly an immunoglobulin molecule of the IgGi subclass. In such an embodiment, the mutant IL-2 polypeptide shares an amino-terminal peptide bond with one of the immunoglobulin heavy chains. In another embodiment, the antigen-binding chemical moiety is an antibody fragment. In some embodiments, said antigen-binding chemical moiety comprises an antigen-binding domain of an antibody comprising an antibody heavy chain variable region and an antibody light chain variable region. In a more specific embodiment, the antigen-binding chemical moiety is a Fab molecule or the scFv molecule. In a particular embodiment, the antigen-binding chemical moiety is a Fab molecule. In another embodiment, the antigen-binding chemical moiety is an scFv molecule. In one embodiment, said antigen-binding chemical portion is directed to an antigen presented on a tumor cell or in a tumor cell environment. In a preferred embodiment, said antigen is selected from the group of Fibroblast Activation Protein (FAP), domain A1 of Tenascin-C (TNC A1), domain A2 of Tenascin-C (TNC A2), the Extra Domain B of Fibronectin (EDB), Carcinoembryonic Antigen (CEA) and the Chondroitin Sulfate Proteoglycan associated with Melanoma (MCSP). When the mutant IL-2 polypeptide is attached to more than one antigen-binding chemical moiety, for example, a first and a second antigen-binding chemical moiety, each antigen-binding chemical moiety can be independently selected in several ways of antibodies and antibody fragments. For example, the first chemical antigen-binding portion may be a Fab molecule and the second chemical antigen-binding portion may be an scFv molecule. In a specific embodiment, each of said first and second chemical antigen-binding moieties is an scFv molecule, or each of said first and second chemical antigen-binding moieties is a Fab molecule. In a particular embodiment, each among said first and second antigen-binding chemical moieties is a Fab molecule. Likewise, when the mutant IL-2 polypeptide is attached to more than one antigen-binding chemical moiety, for example, a first and a second chemical antigen-binding portion, the antigen to which each of the chemical antigen-binding moieties is targeted can be independently selected. In one embodiment, said first and second antigen-binding chemical moieties are directed to different antigens. In another embodiment, said first and second chemical portions of antigen binding are directed to the same antigen. As described above, the antigen is particularly an antigen presented on a tumor cell or in a tumor cell environment, more particularly an antigen selected from the group of Fibroblast Activating Protein (FAP), domain Tenascin-C (TNC A1) , A2 domain of Tenascin-C (TNC A2), Extra Domain B of Fibronectin (EDB), Carcinoembryonic Antigen (CEA) and Chondroitin Sulfate Proteoglycan associated with Melanoma (MCSP). The antigen binding region can additionally incorporate any of the resources, simply or in combination, described in the present invention in relation to the antigen binding domains of immunoconjugates. IMMUNOCONJUGATES
[098] In a particular aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide comprising one or more amino acid mutations that abolish or reduce the affinity of the mutant IL-2 polypeptide for the IL receptor α subunit -2 and preserves the affinity of the mutant IL-2 polypeptide for the intermediate affinity IL-2 receptor, and at least one antigen-binding chemical moiety. In an embodiment according to the invention, the amino acid mutation that abolishes or reduces the affinity of the mutant IL-2 polypeptide for the α subunit of the IL-2 receptor and preserves the affinity of the mutant IL-2 polypeptide for the receptor of intermediate affinity IL-2 is in a position selected from a position corresponding to residues 42, 45 and 72 of human IL-2. In one embodiment, said amino acid mutation is an amino acid substitution. In one embodiment, said amino acid mutation is an amino acid substitution selected from the group of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K, more specifically an amino acid substitution selected from the group of F42A, Y45A and L72G. In one embodiment, the amino acid mutation is in a position corresponding to residue 42 of human IL-2. In a specific embodiment, said amino acid mutation is an amino acid substitution selected from the group of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, and F42. Still in a more specific embodiment, said amino acid substitution is F42A. In another embodiment, the amino acid mutation is in a position corresponding to residue 45 of human IL-2. In a specific embodiment, said amino acid mutation is an amino acid substitution selected from the group of Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, and Y45K. Still in a more specific embodiment, said amino acid substitution is Y45A. In yet another embodiment, the amino acid mutation is in a position corresponding to residue 72 of human IL-2. In a specific embodiment, said amino acid mutation is an amino acid substitution selected from the group of L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. Still in a more specific embodiment, the said amino acid substitution is L72G. In certain embodiments, the mutant IL-2 polypeptide according to the invention does not comprise an amino acid mutation at a position corresponding to human IL-2 residue 38. In a particular embodiment, the mutant IL-2 polypeptide comprised in the immunoconjugate of the invention comprises at least a first and a second amino acid mutation that abolishes or reduces affinity of the mutant IL-2 polypeptide for the IL-2 receptor α subunit and maintains affinity of the mutant IL-2 polypeptide for the intermediate affinity IL-2 receptor. In one embodiment, said first and second amino acid mutations are in two positions selected from positions corresponding to residues 42, 45 and 72 of human IL-2. In one embodiment, said first and second amino acid mutations are amino acid substitutions. In one embodiment, said first and second amino acid mutations are amino acid substitutions selected from the group of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. In a particular embodiment, said first and second amino acid mutations are amino acid substitutions selected from the group of F42A, Y45A and L72G. The mutant IL-2 polypeptide can additionally incorporate any of the resources, simply or in combination, described in the previous paragraphs in relation to the mutant IL-2 polypeptides of the invention. In one embodiment, said mutant IL-2 polypeptide shares a carbon or amino terminal peptide bond with said antigen-binding chemical portion comprised in the immunoconjugate, that is, the immunoconjugate is a fusion protein. In certain embodiments, said antigen-binding chemical moiety is an antibody or an antibody fragment. In some embodiments, said antigen-binding chemical moiety comprises an antigen-binding domain of an antibody comprising an antibody heavy chain variable region and an antibody light chain variable region. The antigen binding region can incorporate any of the resources, simply or in combination, described in the present invention above or below with respect to the antigen binding domains. IMMUNOCONJUGATE FORMATS
[099] Particularly, suitable immunoconjugate formats are described in PCT publication No. WO 2011/020783, which is incorporated by reference in its entirety by reference. Such immunoconjugates comprise at least two antigen-binding domains. Accordingly, in one embodiment, the immunoconjugate according to the present invention comprises at least a first mutant IL-2 polypeptide as described in the present invention, and at least a first and a second antigen-binding chemical moiety. In a particular embodiment, said first and second chemical antigen-binding moiety is independently selected from the group consisting of an Fv molecule, particularly an scFv molecule, and a Fab molecule. In a specific embodiment, said first IL-polypeptide 2 mutant shares a carboxy or amino-terminal peptide bond with said first antigen-binding chemical moiety and said second antigen-binding chemical moiety shares a carboxy or amino-terminal peptide bond with i) the first IL polypeptide -2 mutant or ii) the first chemical antigen-binding portion. In a particular embodiment, the immunoconjugate consists essentially of a first mutant IL-2 polypeptide and first and second chemical antigen-binding portions, joined by one or more ligand sequences. Such formats have the advantage that they bind with high affinity to the target antigen (such as a tumor antigen), but only monomerically bind to the IL-2 receptor, thereby preventing the targeting of the immunoconjugate to the IL-2 receptor that carries immune cells in places other than the target site. In a particular embodiment, a first mutant IL-2 polypeptide shares a carbon-terminal peptide bond with a first antigen-binding chemical moiety and additionally shares an amino-terminal peptide bond with a second antigen-binding chemical moiety. In another embodiment, a first antigen-binding chemical moiety shares a carboxy-terminal peptide bond with a first mutant IL-2 polypeptide, and additionally shares an amino-terminal peptide bond with a second antigen-binding chemical moiety . In another embodiment, a first antigen-binding chemical moiety shares an amino-terminal peptide bond with the first mutant IL-2 polypeptide, and additionally shares a carboxy-terminal peptide with a second antigen-binding chemical moiety. In a particular embodiment, a mutant IL-2 polypeptide shares a carboxy terminal peptide link with a first heavy chain variable region and additionally shares an amino terminal peptide link with a second heavy chain variable region. In another embodiment, a mutant IL-2 polypeptide shares a carboxy terminal peptide link with a first light chain variable region and additionally shares an amino terminal peptide link with a second light chain variable region. In another embodiment, a first light or heavy chain variable region is joined by a carboxy terminal peptide link to a first mutant IL-2 polypeptide and is additionally joined by an amino terminal peptide link to a second variable region light or heavy chain. In another embodiment, a first light or heavy chain variable region is joined by an amino-terminal peptide link to a first mutant IL-2 polypeptide and is additionally joined by a carboxy-terminal peptide link to a second variable region light or heavy chain. In one embodiment, a mutant IL-2 polypeptide shares a carboxy terminal peptide bond with a first Fab light or heavy chain and additionally shares an amino terminal peptide bond with a second Fab light or heavy chain. in another embodiment, a first Fab light or heavy chain shares a carboxy terminal peptide link with a first mutant IL-2 polypeptide and additionally shares an amino terminal peptide link with a second Fab light or heavy chain. In one embodiment, a first Fab light or heavy chain shares an amino-terminal peptide bond with a first mutant IL-2 polypeptide and additionally shares a carboxy terminal peptide bond with a second Fab light or heavy chain. In one embodiment , the immunoconjugate comprises at least one first mutant IL-2 polypeptide that shares an amino-terminal peptide bond with u one or more scFv molecules and additionally sharing a carboxy terminal peptide bond with one or more scFv molecules.
[0100] Other particularly suitable immunoconjugate formats comprise an immunoglobulin molecule as a chemical antigen-binding moiety. In such an embodiment, the immunoconjugate comprises at least one mutant IL-2 polypeptide as described in the present invention and an immunoglobulin molecule, particularly an IgG molecule, more particularly an IgGi molecule. In one embodiment, the immunoconjugate comprises no more than a mutant IL-2 polypeptide. In one embodiment, the immunoglobulin molecule is human. In one embodiment, the mutant IL-2 polypeptide shares a carbon or amino terminal peptide bond with the immunoglobulin molecule. In one embodiment, the immunoconjugate essentially consists of a mutant IL-2 polypeptide and an immunoglobulin molecule, particularly an IgG molecule, more particularly an IgGi molecule, joined by one or more linker sequences. In a specific embodiment, the mutant IL-2 polypeptide is joined at an amino-terminating amino acid to the carboxy-terminating amino acid of the immunoglobulin heavy chains. In certain embodiments, the immunoglobulin molecule comprises in the Fc domain a heterodimerization that promotes modification of two non-identical immunoglobulin heavy chains. The most extensive protein-protein interaction site between the two polypeptide chains of a human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one embodiment, said modification is in the CH3 domain of the Fc domain. In a specific embodiment, said modification is a knob-inner-hole modification, which comprises a knob modification in one of the immunoglobulin heavy chains and a hole modification in the other of one of the immunoglobulin heavy chains. Knob-into-hole technology is described, for example, in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617 to 621 (1996) and Carter, J Immunol Meth 248, 7 to 15 (2001). Generally, the method involves introducing a knob at the interface of a first polypeptide and a corresponding cavity (hole) at the interface of a second polypeptide, such that the protuberance can be positioned in the cavity in order to promote heterodimer formation. and formation of impairment heterodimer. The bulges are constructed by replacing small amino acid side chains at the interface of the first polypeptide with larger side chains (for example, tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created at the interface of the second polypeptide by replacing the large amino acid side chains with smaller ones (for example, alanine or threonine). The bulge and cavity can be made by altering the nucleic acid encoding the polypeptides, for example, by site-specific mutagenesis, or by peptide synthesis. In a specific embodiment, a knob modification comprises the T366W amino acid substitution in one of the two immunoglobulin heavy chains, and the hole modification comprises the T366S, L368A and Y407V amino acid substitutions in the other of the two immunoglobulin heavy chains. In an additionally specific embodiment, the immunoglobulin heavy chain comprising the knob modification further comprises the amino acid substitution S354C, and the immunoglobulin heavy chain comprising the knob modification further comprises the amino acid substitution Y349C. The introduction of these two cysteine residues results in the formation of a disulfide bridge between the two heavy chains, further stabilizing the dimer (Carter, J Imunol Methods 248, 7 to 15 (2001)).
[0101] In a particular embodiment, the mutant IL-2 polypeptide is joined to the carboxy terminal amino acid of the immunoglobulin heavy chain comprising the knob modification.
[0102] In an alternative embodiment, a heterodimerization that promotes modification of two non-identical polypeptide chains comprises a modification that mediates electrostatic direction effects, for example, as described in PCT publication WO 2009/089004. Generally, this method involves replacing one or more amino acid residues at the interface of the two polypeptide chains with charged amino acid residues so that homodimer formation becomes electrostatically unfavorable, but electrostatically heterodimerization is favorable.
[0103] An Fc domain gives the immunoconjugate favorable pharmacokinetic properties, including a long serum half-life that contributes to good accumulation in the target tissue and a favorable blood-tissue distribution ratio. At the same time, this can, however, lead to undesirable targeting of the immunoconjugate to cells that express Fc receptors instead of cells that carry preferred antigens. In addition, the coactivation of Fc receptor signaling pathways can lead to the release of cytokine, which, in combination with the IL-2 polypeptide and the long half-life of the immunoconjugate, results in excessive activation of cytokine receptors and serious effects collateral through systemic administration. Aligned to this, conventional IL-2 immunoconjugates have been described as being associated with infusion reactions (see, for example, King et al., J Clin Oncol 22, 4,463 to 4,473 (2004)).
[0104] Consequently, in certain embodiments, the immunoglobulin molecule comprised in the immunoconjugate according to the invention is modified to have reduced binding affinity for an Fc receptor. In such an embodiment, the immunoglobulin comprises in its Fc domain one or more amino acid mutations that reduce the binding affinity of the immunoconjugate to an Fc receptor. Typically, the same one or more amino acid mutations are present in each of the two immunoglobulin heavy chains. In one embodiment, said amino acid mutation reduces the binding affinity of the immunoconjugate to the Fc receptor 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 immunoconjugate with the Fc receptor, the combination of these amino acid mutations can reduce the binding affinity of the Fc domain to the Fc receptor by at least 10 times at least 20 times, or at least 50 times. In one embodiment, the immunoconjugate comprising a modified immunoglobulin molecule exhibits less than 20%, particularly less than 10%, more particularly less than 5% of the binding affinity for an Fc receptor as compared to an immunoconjugate comprising a molecule of unmodified immunoglobulin. In one embodiment, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is a Fey receptor, more specifically a FcyRllla, FcyRI or FcyRlla receptor. Preferably, the binding to each of these receptors is reduced. In some embodiments, binding affinity to a complement component, specifically binding affinity for C1q, is also reduced. In one embodiment, the affinity of binding to the neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, that is, the preservation of the binding affinity of the immunoglobulin to said receptor, is achieved when the immunoglobulin (or the immunoconjugate comprising said immunoglobulin) exhibits more than about 70% of the binding affinity of a unmodified form of immunoglobulin (or the immunoconjugate comprising said unmodified form of immunoglobulin) the FcRn. Immunoglobulins, or immunoconjugates that comprise said immunoglobulins, can exhibit more than about 80% and even more than about 90% of such an affinity. In one embodiment, the amino acid mutation is an amino acid substitution. In one embodiment, the immunoglobulin comprises an amino acid substitution at the P329 position of the immunoglobulin heavy chain (Kabat numbering). In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G. In one embodiment, the immunoglobulin comprises an additional amino acid substitution at a selected position of S228, E233, L234, L235, N297 and P331 of the immunoglobulin heavy chain. In a more specific embodiment, the additional amino acid substitution is S228P, E233P, L234A, L235A, L235E, N297A, N297D or P331S. In a particular embodiment, the immunoglobulin comprises amino acid substitutions at positions P329, L234 and L235 of the immunoglobulin heavy chain. In a more particular embodiment, the immunoglobulin comprises the amino acid mutations L234A, L235A and P329G (LALA P329G). This combination of amino acid substitutions almost completely abolishes the Fcy receptor binding of a human IgG molecule and therefore decreases the effector function including antibody-dependent cell-mediated cytotoxicity (ADCC).
[0105] In certain embodiments, the immunoconjugate comprises one or more proteolytic cleavage sites located between the mutant IL-2 polypeptide and the antigen-binding chemical moieties.
[0106] The components of the immunoconjugate (for example, chemical portions of mutant IL-2 antigen and / or polypeptide) can be linked directly or via various linkers, particularly peptide linkers that comprise one or more amino acids, typically about from 2 to 20 amino acids, which are described in the present invention or are known in the art. Suitable non-immunogenic linker peptides include, for example, linker peptides (G4S) n, (SG4) n or G4 (SG4) n, where n is generally a number between 1 and 10, typically between 2 and 4. CHEMICAL PORTIONS OF ANTIGEN BINDING
[0107] The antigen-binding chemical portion of the immunoconjugate of the invention is generally a polypeptide molecule that binds to a specific antigenic determinant and is capable of targeting the entity to which it is attached (for example, an IL-2 polypeptide mutant or a second antigen-binding chemical moiety) to a target site, for example, to a specific type of tumor cell or tumor stroma that carries the antigenic determinant. The immunoconjugate can bind to the antigenic determinants found, for example, on the surfaces of tumor cells, on the surfaces of cells infected by viruses, on the surfaces of other diseased cells, free in blood serum and / or in the extracellular matrix (ECM) .
[0108] Non-limiting examples of tumor antigens include MAGE, MART-1 / Melan-A, gp100, Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal associated antigen (CRC) - C017-1A / GA733, Carcinoembryonic Antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2 and PSA-3, prostate specific membrane antigen (PSMA), CD3 T cell / zeta chain receptor, MAGE family of tumor antigens (for example, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE -A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE -B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE family of tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4 , GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, family MUC, HER2 / neu, p21ras , RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin and y-catenin, p120ctn, gp1OO Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrina, Connexin 37 , lg-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papilloma virus proteins, tumor antigen Smad family, lmp-1, P1A, EBV-encoded nuclear antigen (EBNA) -1, phosphorylase brain glycogen, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2.
[0109] Non-limiting examples of viral antigens include influenza virus hemagglutinin, Epstein-Barr LMP-1 virus, hepatitis C virus E2 glycoprotein, HIV gp160, and HIV gp120.
[0110] Non-limiting examples of ECM antigens include syndecan, heparanase, integrins, osteopontin, link, cadherins, laminin, laminin-like EGF, lectin, fibronectin, notch, tenascin and matrixin.
[0111] The immunoconjugates of the invention can bind to the following specific non-limiting examples of cell surface antigens: FAP, Her2, EGFR, IGF-1R, CD2 (T cell surface antigen), CD3 (TCR-associated heteromultomer), CD22 (B cell receptor), CD23 (low affinity IgE receptor), CD30 (cytokine receptor), CD33 (myeloid cell surface antigen), CD40 (tumor necrosis factor receptor), IL-6R (receptor IL6), CD20, MCSP, and PDGFβR (platelet-derived growth factor receptor β).
[0112] In one embodiment, the immunoconjugate of the invention comprises two or more chemical antigen-binding moieties, each of which antigen-binding chemical moieties specifically binds to the same antigenic determinant. In another embodiment, the immunoconjugate of the invention comprises two or more antigen-binding chemical moieties, each of which antigen-binding chemical moieties specifically binds to different antigenic determinants.
[0113] The chemical antigen-binding portion can be any type of antibody or fragment thereof that retains specific binding for an antigenic determinant. Antibody fragments include, but are not limited to, VH fragments, VL fragments, Fab fragments, F (ab ') 2 fragments, scFv fragments, Fv fragments, minibodies, diabodies, tribodies and tetribodies (see, for example, Hudson and Souriau, Nature Med 9, 129 to 134 (2003)).
[0114] Particularly suitable antigen-binding chemical portions are described in PCT publication No. WO 2011/020783, which is incorporated by reference in its entirety by reference.
[0115] In one embodiment, the immunoconjugate comprises at least one, typically two or more chemical antigen-binding moieties that are specific to the Extra B Domain of fibronectin (EDB). In another embodiment, the immunoconjugate comprises at least one, typically two or more chemical antigen-binding moieties that can compete with monoclonal antibody L19 for binding to an EDB epitope. See, for example, PCT publication WO 2007/128563 A1 (incorporated into the present invention by reference in its entirety). In yet another embodiment, the immunoconjugate comprises a polypeptide sequence in which a first Fab heavy chain derived from the monoclonal antibody L19 shares a carboxy terminal peptide bond with a mutant IL-2 polypeptide, which in turn shares a binding of the carboxy terminal peptide with a second Fab heavy chain derived from the monoclonal antibody L19. In yet another embodiment, the immunoconjugate comprises the polypeptide sequence in which a first Fab light chain derived from the L19 monoclonal antibody shares a carboxy terminal peptide bond with a mutant IL-2 polypeptide, which in turn shares a linkage of the carboxy terminal peptide with a second Fab light chain derived from the monoclonal antibody L19. In a further embodiment, the immunoconjugate comprises a polypeptide sequence in which a first scFv derived from the L19 monoclonal antibody shares a carboxy terminal peptide bond with a mutant IL-2 polypeptide which, in turn, shares a peptide bond of carboxy terminal with a second scFv derived from the monoclonal antibody L19.
[0116] In a more specific embodiment, the immunoconjugate comprises the polypeptide sequence of SEQ ID NO: 199 or a variant thereof that retains functionality. In another embodiment, the immunoconjugate comprises a Fab light chain derived from the monoclonal antibody L19. In a more specific embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO : 201 or a variant thereof that retains functionality. In yet another embodiment, the immunoconjugate comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO : 199 and SEQ ID NO: 201 or variants thereof that retain functionality. In another specific embodiment, the polypeptides are covalently linked, for example, by a disulfide bond.
[0117] In one embodiment, the immunoconjugate of the invention comprises at least one, typically two or more chemical antigen-binding moieties that are specific to the Tenascin A1 domain (TNC-A1). In another embodiment, the immunoconjugate comprises at least one, typically two or more chemical antigen-binding moieties that can compete with monoclonal antibody F16 for binding to a TNC-A1 epitope. See, for example, PCT Publication WO 2007/128563 A1 (incorporated into the present invention by reference in its entirety). In one embodiment, the immunoconjugate comprises at least one, typically two or more chemical antigen-binding moieties that are specific for the Tenascin A1 and / or A4 domains (TNC-A1 or TNC-A4 or TNC-A1 / A4). In another embodiment, the immunoconjugate comprises a polypeptide sequence in which a first Fab heavy chain specific for the Tenascin A1 domain shares a carboxy terminal peptide bond with a mutant IL-2 polypeptide, which, in turn, shares a carboxy terminal peptide bond with a second Fab heavy chain specific for the Tenascin A1 domain. In yet another embodiment, the immunoconjugate comprises a polypeptide sequence in which a first Fab light chain specific for the Tenascin A1 domain shares a carboxy terminal peptide bond with a mutant IL-2 polypeptide which, in turn, shares a carboxy terminal peptide bond with the second Fab light chain specific for the A1 domain of Tenascin. In a further embodiment, the immunoconjugate comprises a polypeptide sequence in which a first scFv specific for the Tenascin A1 domain shares a carboxy terminal peptide bond with a mutant IL-2 polypeptide which, in turn, shares a carboxy terminal peptide with a second scFv specific for the A1 domain of Tenasciπa. In another embodiment, the immunoconjugate comprises a polypeptide sequence in which a TNC-A1 specific immunoglobulin heavy chain shares a carboxy terminal peptide bond with a mutant IL-2 polypeptide.
[0118] In a specific embodiment, the antigen-binding chemical portions of the immunoconjugate comprise a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 33 or SEQ ID NO: 35, or variants of it that retain functionality. In another specific embodiment, the antigen-binding chemical portions of the immunoconjugate comprise a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% , 99% or 100% identical to SEQ ID NO: 29 or SEQ ID NO: 31, or variants of it that retain functionality. In a more specific embodiment, the antigen-binding chemical portions of the immunoconjugate comprise a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% , 99% or 100% identical to SEQ ID NO: 33 or SEQ ID NO: 35 or variants thereof that retain functionality, and a light chain variable region sequence that is at least about 80%, 85%, 90% , 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 29 or SEQ ID NO: 31 or variants of it that retain functionality.
[0119] In another specific embodiment, the heavy chain variable region sequence of the immunoconjugate antigen-binding chemical portions is encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95% , 96%, 97%, 98%, or 99% identical to SEQ ID NO: 34 or SEQ ID NO: 36. In yet another specific embodiment, the heavy chain variable region sequence of the antigen-binding chemical portions of immunoconjugate is encoded by the polynucleotide sequence of SEQ ID NO: 34 or SEQ ID NO: 36. In another specific embodiment, the light chain variable region sequence of the immunoconjugate antigen-binding chemical portions is encoded by a polynucleotide sequence which is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 30 or SEQ ID NO: 32. In yet another specific realization , the light chain variable region sequence of the antigen-binding chemical portions of the immunoconjugate is encoded by the polynucleotide sequence of SEQ ID NO: 30 or SEQ ID NO: 32.
[0120] In a specific embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 203 or variants of it that retain functionality. In another specific embodiment, the immunoconjugate of the invention comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 205 or SEQ ID NO: 215, or variants of it that retain functionality. In yet another specific embodiment, the immunoconjugate of the invention comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 207 or SEQ ID NO: 237 or variants of it that retain functionality. In a more specific embodiment, the immunoconjugate of the present invention comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 205 and SEQ ID NO: 207 or variants thereof that retain functionality. In another specific embodiment, the immunoconjugate of the present invention comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 215 and SEQ ID NO: 237 or variants thereof that retain functionality.
[0121] In a specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99 % identical to SEQ ID NO: 204. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by the polynucleotide sequence of SEQ ID NO: 204. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 206 or SEQ ID NO: 216. Still in a another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by the polynucleotide sequence of SEQ ID NO: 206 or SEQ ID NO: 216. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 208 or SEQ ID NO: 238. In yet another embodiment, the immunoconjugate comprises a sequence of polypeptide encoded by the polynucleotide sequence of SEQ ID NO: 208 or SEQ ID NO: 238.
[0122] In one embodiment, the immunoconjugate comprises at least one, typically two or more chemical antigen-binding moieties that are specific to the A2 domain of Tenascin (TNC-A2). In another embodiment, the immunoconjugate comprises a polypeptide sequence in which a first Fab heavy chain specific for the A2 domain of Tenascin shares a carboxy terminal peptide bond with a mutant IL-2 polypeptide, which, in turn, shares a carboxy terminal peptide bond with a second Fab heavy chain specific for the A2 domain of Tenascin. In yet another embodiment, the immunoconjugate comprises a polypeptide sequence in which a first Fab light chain specific for the A2 domain of Tenascin shares a carboxy terminal peptide bond with a mutant IL-2 polypeptide, which in turn , shares a carboxy terminal peptide bond with a second Fab light chain specific for the A2 Tenascin domain. In another embodiment, the immunoconjugate comprises a polypeptide sequence in which a TNC-A2 specific immunoglobulin heavy chain shares a carboxy terminal peptide bond with a mutant IL-2 polypeptide.
[0123] In a specific embodiment, the antigen-binding chemical portions of the immunoconjugate comprise a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 27, SEQ ID NO: 159, SEQ ID NO: 163, SEQ ID NO: 167, SEQ ID NO: 171, SEQ ID NO: 175, SEQ ID NO: 179, SEQ ID NO: 183 and SEQ ID NO: 187, or variants thereof that retain functionality. In another specific embodiment, the antigen-binding chemical portions of the immunoconjugate comprise a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% , 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 23, SEQ ID NO: 25; SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 165, SEQ ID NO: 169, SEQ ID NO: 173, SEQ ID NO: 177, SEQ ID NO: 181 and SEQ ID NO: 185, or variants that retain functionality. In a more specific embodiment, the antigen-binding chemical portions of the immunoconjugate comprise a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% , 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 27, SEQ ID NO: 159, SEQ ID NO: 163, SEQ ID NO: 167, SEQ ID NO: 171, SEQ ID NO: 175, SEQ ID NO: 179, SEQ ID NO: 183 and SEQ ID NO: 187, or variants thereof that retain functionality, and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 23, SEQ ID NO: 25; SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 165, SEQ ID NO: 169, SEQ ID NO: 173, SEQ ID NO: 177, SEQ ID NO: 181 and SEQ ID NO: 185, or variants that retain functionality.
[0124] In another specific embodiment, the heavy chain variable region sequence of the immunoconjugate antigen-binding chemical portions is encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95% , 96%, 97%, 98%, or 99% identical to a sequence selected from the group of SEQ ID NO: 28, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO: 168, SEQ ID NO: 172 , SEQ ID NO: 176, SEQ ID NO: 180, SEQ ID NO: 184 and SEQ ID NO: 188. In yet another specific embodiment, the heavy chain variable region sequence of the immunoconjugate antigen-binding chemical portions is encoded by a polynucleotide sequence selected from the group of SEQ ID NO: 28, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO: 168, SEQ ID NO: 172, SEQ ID NO: 176, SEQ ID NO: 180, SEQ ID NO: 184 and SEQ ID NO: 188. In another specific embodiment, the light chain variable region sequence of the immunoconjugate antigen-binding chemical portions is encoded by a sequence polynucleotide frequency that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of SEQ ID NO: 24, SEQ ID NO : 26, SEQ ID NO: 158, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 170, SEQ ID NO: 174, SEQ ID NO: 178, SEQ ID NO: 182 and SEQ ID NO: 186 In yet another specific embodiment, the light chain variable region sequence of the immunoconjugate antigen-binding chemical portions is encoded by a polynucleotide sequence selected from the group of SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 158, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 170, SEQ ID NO: 174, SEQ ID NO: 178, SEQ ID NO: 182 and SEQ ID NO: 186.
[0125] In a specific embodiment, the immunoconjugate of the invention comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 241, SEQ ID NO: 243 and SEQ ID NO: 245, or variants thereof that retain functionality. In another specific embodiment, the immunoconjugate of the invention comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a selected sequence from the group of SEQ ID NO: 247, SEQ ID NO: 249 and SEQ ID NO: 251, or variants of it that retain functionality. In a more specific embodiment, the immunoconjugate of the present invention comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 241, SEQ ID NO: 243, and SEQ ID NO: 245 or variants thereof that retain functionality, and the polypeptide sequence that is at least about 80%, 85%, 90 %, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 247, SEQ ID NO: 249 and SEQ ID NO: 251 or variants of it that retain functionality. In another specific embodiment, the immunoconjugate of the present invention comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 241 and SEQ ID NO: 249 or SEQ ID NO: 251, or variants thereof that retain functionality. In yet another specific embodiment, the immunoconjugate of the present invention comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical SEQ ID NO: 243 and SEQ ID NO: 247 or SEQ ID NO: 249, or variants thereof that retain functionality. In another specific embodiment, the immunoconjugate of the present invention comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 245 and SEQ ID NO: 247, or variants thereof that retain functionality.
[0126] In a specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99 % identical to a sequence selected from the group of SEQ ID NO: 242, SEQ ID NO: 244 and SEQ ID NO: 246. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence selected from the group of SEQ ID NO: 242, SEQ ID NO: 244 and SEQ ID NO: 246. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90 %, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of SEQ ID NO: 248, SEQ ID NO: 250 and SEQ ID NO: 252. In yet another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence selected from the group of SEQ ID NO: 248, SEQ ID NO: 250 and SEQ ID NO: 252.
[0127] In one embodiment, the immunoconjugate comprises at least one, typically two or more chemical antigen-binding moieties that are specific for Fibroblast Activated Protein (FAP). In another embodiment, the immunoconjugate comprises a polypeptide sequence in which a FAP-specific first Fab heavy chain shares a carboxy terminal peptide bond with a mutant IL-2 polypeptide, which in turn shares a carboxy terminal peptide with a second FAP-specific Fab heavy chain. In yet another embodiment, the immunoconjugate comprises a polypeptide sequence in which a FAP-specific first Fab light chain shares a carboxy terminal peptide bond with a mutant IL-2 polypeptide, which in turn shares a bond of a carboxy terminal peptide with a second FAP-specific Fab light chain. In another embodiment, the immunoconjugate comprises a polypeptide sequence in which a FAP-specific immunoglobulin heavy chain shares a carboxy terminal peptide bond with a mutant IL-2 polypeptide.
[0128] In a specific embodiment, the antigen-binding chemical portions of the immunoconjugate comprise a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91, SEQ ID NO: 95, SEQ ID NO: 99, SEQ ID NO: 103, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: 115, SEQ ID NO: 119, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 131, SEQ ID NO: 135, SEQ ID NO: 139, SEQ ID NO: 143, SEQ ID NO: 147, SEQ ID NO: 151 and SEQ ID NO: 155, or variants that retain functionality. In another specific embodiment, the antigen-binding chemical portions of the immunoconjugate comprise a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% , 99% or 100% identical to a sequence selected from the group consisting of: SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO : 57, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89 , SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO: 101, SEQ ID NO: 105, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO. 117, SEQ ID NO: 121, SEQ ID NO: 125, SEQ ID NO: 129, SEQ ID NO: 133, SEQ ID NO: 137, SEQ ID NO: 141, SEQ ID NO: 145, SEQ ID NO: 149 and SEQ ID NO: 153, or variants of it that retain functionality. In a more specific embodiment, the antigen-binding chemical portions of the immunoconjugate comprise a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% , 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91, SEQ ID NO: 95, SEQ ID NO: 99, SEQ ID NO: 103, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: 115, SEQ ID NO: 119, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 131, SEQ ID NO: 135, SEQ ID NO: 139, SEQ ID NO: 143, SEQ ID NO: 147, SEQ ID NO: 151 and SEQ ID NO: 155, or variants thereof that retain functionality, and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a selected sequence of the group consisting of: SEQ ID NO : 37, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 69 , SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO: 101, SEQ ID NO: 105, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 117, SEQ ID NO: 121, SEQ ID NO: 125, SEQ ID NO: 129, SEQ ID NO: 133, SEQ ID NO : 137, SEQ ID NO: 141, SEQ ID NO: 145, SEQ ID NO: 149 and SEQ ID NO: 153, or variants thereof that retain functionality. In one embodiment, antigen-binding chemical portions of the immunoconjugate comprise the heavy chain variable region sequence of SEQ ID NO: 41 and the light chain variable region sequence of SEQ ID NO: 39. In one embodiment, chemical portions of antigen binding of the immunoconjugate comprises the heavy chain variable region sequence of SEQ ID NO: 51 and the light chain variable region sequence of SEQ ID NO: 49. In one embodiment, chemical antigen binding portions of the immunoconjugate comprise the heavy chain variable region sequence of SEQ ID NO: 111 and the light chain variable region sequence of SEQ ID NO: 109. In one embodiment, antigen-binding chemical portions of the immunoconjugate comprise the heavy chain variable region sequence of SEQ ID NO: 143 and the light chain variable region sequence of SEQ ID NO: 141. In one embodiment, chemical antigen-binding portions of the immunoconjugate comprise the variable region sequence d and heavy chain of SEQ ID NO: 151 and the light chain variable region sequence of SEQ ID NO: 149.
[0129] In another specific embodiment, the heavy chain variable region sequence of the immunoconjugate antigen-binding chemical portions is encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95% , 96%, 97%, 98%, or 99% identical to a sequence selected from the group consisting of: SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 92, SEQ ID NO: 96, SEQ ID NO: 100, SEQ ID NO: 104, SEQ ID NO: 108, SEQ ID NO: 112, SEQ ID NO: 116, SEQ ID NO: 120, SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 132, SEQ ID NO: 136, SEQ ID NO: 140, SEQ ID NO: 144, SEQ ID NO: 148, SEQ ID NO: 152, and SEQ ID NO: 156. In yet another specific embodiment, the heavy chain variable region sequence of the immunoconjugate antigen-binding chemical portions is encoded by a polynucleotide sequence selected from the group consisting of: SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 92, SEQ ID NO: 96, SEQ ID NO : 100, SEQ ID NO: 104, SEQ ID NO: 108, SEQ ID NO: 112, SEQ ID NO: 116, SEQ ID NO: 120, SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 132 , SEQ ID NO: 136, SEQ ID NO: 140, SEQ ID NO: 144, SEQ ID NO: 148, SEQ ID NO: 152, and SEQ ID NO: 156. In another specific embodiment, the variable region sequence of The light chain of the antigen-binding chemical portions of the immunoconjugate is encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to selected sequence from the group consisting of: SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO : 82, SEQ ID NO: 86, SEQ ID NO: 90, SEQ ID NO: 94, SEQ ID NO: 98, SEQ ID NO: 102, SEQ ID NO: 106, SEQ ID NO: 110, SEQ ID NO: 114 , SEQ ID NO: 118, SEQ ID NO: 122, SEQ ID NO: 126, SEQ ID NO: 130, SEQ ID NO: 134, SEQ ID NO: 138, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 150, and SEQ ID NO: 154. In yet another specific embodiment, the light chain variable region sequence of the antigen-binding chemical portions of the immunoconjugate is encoded by a polynucleotide sequence selected from the group consisting of: SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 90, SEQ ID NO: 94, SEQ ID NO: 98, SEQ ID NO: 102, SEQ ID NO: 106, SEQ ID NO: 110, SEQ ID NO: 114, SEQ ID NO: 118, SEQ ID NO: 122, SEQ ID NO: 126, SEQ ID NO: 130, SEQ ID NO: 134, SEQ ID NO: 138, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 150, and SEQ ID NO: 154.
[0130] In another specific embodiment, the immunoconjugate of the invention comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, and SEQ ID NO: 229, or variants thereof that retain functionality. In yet another specific embodiment, the immunoconjugate of the invention comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235 and SEQ ID NO: 239 or variants thereof that retain functionality. In a more specific embodiment, the immunoconjugate of the present invention comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 211 or SEQ ID NO: 219 or variants thereof that retain functionality, and the polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% , 99% or 100% identical to SEQ ID NO: 233 or variants of it that retain functionality. In another specific embodiment, the immunoconjugate of the present invention comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 209, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227 and SEQ ID NO: 229, or variants of it that retain functionality, and the polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 231 or variants thereof that retain functionality. In an additionally specific embodiment, the immunoconjugate of the present invention comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 213 and SEQ ID NO: 235 or variants thereof that retain functionality. In yet another specific embodiment, the immunoconjugate of the present invention comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 217 and SEQ ID NO: 239 or variants thereof that retain functionality. In yet another specific embodiment, the immunoconjugate of the present invention comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 219 and SEQ ID NO: 233 or variants thereof that retain functionality. In yet another specific embodiment, the immunoconjugate of the present invention comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 221 and SEQ ID NO: 231 or variants thereof that retain functionality. In yet another specific embodiment, the immunoconjugate of the present invention comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 223 and SEQ ID NO: 231 or variants thereof that retain functionality. In yet another specific embodiment, the immunoconjugate of the present invention comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 225 and SEQ ID NO: 231 or variants thereof that retain functionality. In yet another specific embodiment, the immunoconjugate of the present invention comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 227 and SEQ ID NO: 231 or variants thereof that retain functionality. In yet another specific embodiment, the immunoconjugate of the present invention comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 229 and SEQ ID NO: 231 or variants thereof that retain functionality. In yet another specific embodiment, the immunoconjugate of the present invention comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 211 and SEQ ID NO: 233 or variants thereof that retain functionality.
[0131] In another specific embodiment, the immunoconjugate of the invention comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 297, SEQ ID NO: 301 and SEQ ID NO: 315, or variants of it that retain functionality. In yet another specific embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a selected sequence from the group of SEQ ID NO: 299, SEQ ID NO: 303 and SEQ ID NO: 317, or variants thereof that retain functionality. In a more specific embodiment, the immunoconjugate of the present invention comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 297 or a variant thereof that retains functionality, a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 299 or a variant thereof that retains functionality, and a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 233 or a variant thereof that retains functionality. In another specific embodiment, the immunoconjugate of the present invention comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 301 or a variant thereof that retains functionality, a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical SEQ ID NO: 303 or a variant thereof that retains functionality, and a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 231 or a variant thereof that retains functionality. In yet another specific embodiment, the immunoconjugate of the present invention comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 315 or a variant thereof that retains functionality, a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 317 or a variant thereof that retains functionality, and a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 233 or a variant thereof that retains functionality.
[0132] In another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99 % identical to a sequence selected from the group of SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO : 224, SEQ ID NO: 226, SEQ ID NO: 228, and SEQ ID NO: 230. In yet another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence selected from the group of SEQ ID NO : 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228 , and SEQ ID NO: 230. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence was selected from the group of SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, and SEQ ID NO: 240. In yet another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a sequence of polynucleotide selected from the group of SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, and SEQ ID NO: 240.
[0133] In another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99 % identical to a sequence selected from the group of SEQ ID NO: 298, SEQ ID NO: 302 and SEQ ID NO: 316. In yet another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence selected from from the group of SEQ ID NO: 298, SEQ ID NO: 302 and SEQ ID NO: 316. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85 %, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of SEQ ID NO: 300, SEQ ID NO: 304 and SEQ ID NO: 318. Still in another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence and the one selected from the group of SEQ ID NO: 300, SEQ ID NO: 304 and SEQ ID NO: 318.
[0134] In one embodiment, the immunoconjugate comprises at least one, typically two or more chemical antigen-binding moieties that are specific for Melanoma Chondroitin Sulfate Proteoglycan (MCSP). In another embodiment, the immunoconjugate comprises a polypeptide sequence in which an MCSP-specific first Fab heavy chain shares a carboxy-terminal peptide bond with a mutant IL-2 polypeptide, which in turn shares a carboxy-peptide bond. terminal with a second MCSP-specific Fab heavy chain. In yet another embodiment, the immunoconjugate comprises a polypeptide sequence in which an MCSP-specific first Fab light chain shares a carboxy-terminal peptide bond with an IL-2 molecule, which in turn shares a carboxide peptide bond. terminal with a second Fab light chain specific for MCSP. In another embodiment, the immunoconjugate comprises a polypeptide sequence in which an MCSP-specific immunoglobulin heavy chain shares a carboxy-terminal peptide bond with a mutant IL-2 polypeptide.
[0135] In a specific embodiment, the antigen-binding chemical portions of the immunoconjugate comprise a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence or SEQ ID NO: 189 or SEQ ID NO: 193 or variants thereof that retain functionality. In another specific embodiment, the antigen-binding chemical portions of the immunoconjugate comprise a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence or SEQ ID NO: 191 or SEQ ID NO: 197 or variants of it that retain functionality. In a more specific embodiment, the antigen binding chemical portions of the immunoconjugate comprise a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% , 99% or 100% identical to the sequence or SEQ ID NO: 189 or SEQ ID NO: 193, or variants thereof that retain functionality, and a light chain variable region sequence that is at least about 80%, 85 %, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence or SEQ ID NO: 191 or SEQ ID NO: 197, or variants of it that retain functionality. In a more specific embodiment, the antigen binding chemical portions of the immunoconjugate comprise a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% , 99% or 100% identical to the sequence of SEQ ID NO: 189, and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98 %, 99% or 100% identical to the sequence of SEQ ID NO: 191. In another specific embodiment, the antigen-binding chemical portions of the immunoconjugate comprise a heavy chain variable region sequence that is at least about 80%, 85 %, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 193, and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 191.
[0136] In another specific embodiment, the heavy chain variable region sequence of the antigen binding chemical portions of the immunoconjugate is encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence or SEQ ID NO: 190 or SEQ ID NO: 194. In yet another specific embodiment, the heavy chain variable region sequence of the antigen-binding chemical moieties of the immunoconjugate is encoded by the polynucleotide or SEQ ID NO: 190 or SEQ ID NO: 194 sequence. In another specific embodiment, the light chain variable region sequence of the immunoconjugate antigen-binding chemical portions is encoded by a sequence of polynucleotide that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence or SEQ ID NO: 192 or SEQ ID NO: 198. Still in another specific embodiment, the light chain variable region sequence of the chemical The immunoconjugate antigen is encoded by the polynucleotide or SEQ ID NO: 192 or SEQ ID NO: 198 sequence.
[0137] In a specific embodiment, the immunoconjugate of the invention comprises the polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or SEQ ID NO: 253 or SEQ ID NO: 257, or variants thereof that retain functionality. In another specific embodiment, the immunoconjugate of the invention comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or SEQ ID NO: 255 or SEQ ID NO: 261, or variants of it that retain functionality. In a more specific embodiment, the immunoconjugate of the present invention comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical or to SEQ ID NO: 253 or SEQ ID NO: 257 or variants thereof that retain functionality, and a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98 %, 99% or 100% identical or SEQ ID NO: 255 or SEQ ID NO: 261, or variants of it that retain functionality. In another specific embodiment, the immunoconjugate of the present invention comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 253 or variants thereof that retain functionality, and a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 255 or variants of it that retain functionality. In another specific embodiment, the immunoconjugate of the present invention comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 257 or variants thereof that retain functionality, and a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 255 or variants of it that retain functionality.
[0138] In another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99 % identical to the sequence or SEQ ID NO: 254 or SEQ ID NO: 258. In yet another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by the polynucleotide or SEQ ID NO: 254 or SEQ ID NO: 258 sequence. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by the polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to sequence or SEQ ID NO: 256 or SEQ ID NO: 262. In yet another specific embodiment, the immunoconjugate comprises the polypeptide sequence encoded by the polynucleotide or SEQ ID NO: 256 or SEQ ID NO: 262 sequence.
[0139] In one embodiment, the immunoconjugate comprises at least one, typically two or more chemical antigen-binding moieties that are specific for the Carcinoembryonic Antigen (CEA).
[0140] In another embodiment, the immunoconjugate comprises a polypeptide sequence in which a CEA-specific first Fab heavy chain shares a carboxy-terminal peptide bond with a mutant IL-2 polypeptide, which in turn shares a bond carboxy-terminal peptide with a second CEA-specific Fab heavy chain. In yet another embodiment, the immunoconjugate comprises a polypeptide sequence in which a CEA-specific first Fab heavy chain shares a carboxy-terminal peptide bond with a mutant IL-2 polypeptide, which in turn shares a carboxy peptide bond -terminal with a second CEA-specific Fab heavy chain. In one embodiment, the immunoconjugate comprises a polypeptide sequence in which a heavy chain of CEA-specific immunoglobulin shares a carboxy-terminal peptide bond with a mutant IL-2 polypeptide. In a specific embodiment, the antigen binding chemical portions of the immunoconjugate comprise a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 313 or a variant thereof that retains functionality. In another specific embodiment, the antigen-binding chemical portions of the immunoconjugate comprise a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 311 or a variant thereof that retains functionality. In a more specific embodiment, the antigen binding chemical portions of the immunoconjugate comprise a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% , 99% or 100% identical to the sequence of SEQ ID NO: 313, or a variant thereof that retains functionality, and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95 %, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 311, or a variant thereof that retains functionality.
[0141] In another specific embodiment, the heavy chain variable region sequence of the immunoconjugate antigen-binding chemical portions is encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 314. In yet another specific embodiment, the heavy chain variable region sequence of the antigen-binding chemical portions of the immunoconjugate is encoded by the sequence of polynucleotide of SEQ ID NO: 314. In another specific embodiment, the light chain variable region sequence of the immunoconjugate antigen-binding chemical portions is encoded by a polynucleotide sequence that is at least about 80%, 85%, 90 %, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 312. In yet another specific embodiment, the light chain variable region sequence of the immunoconjugate antigen-binding chemical portions is encoded by the poly string nucleotide of SEQ ID NO: 312.
[0142] In another specific embodiment, the immunoconjugate of the invention comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 319, or variants thereof that retain functionality. In yet another specific embodiment, the immunoconjugate of the invention comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 321, or variants thereof that retain functionality. In yet another specific embodiment, the immunoconjugate of the invention comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence SEQ ID NO: 323, or variants thereof that retain functionality. In a more specific embodiment, the immunoconjugate of the present invention comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 319 or a variant thereof that retains functionality, a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 321 or a variant thereof that retains functionality, and a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 323 or a variant thereof that retains functionality.
[0143] In another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99 % identical to the sequence of SEQ ID NO: 320. In yet another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by the polynucleotide sequence of SEQ ID NO: 320. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 322. In yet another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by the polynucleotide sequence of SEQ ID NO: 322. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90 %, 95 %, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 324. In yet another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by the polynucleotide sequence of SEQ ID NO: 324.
[0144] The antigen-binding chemical portions of the invention include those having sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the peptide sequences shown in SEQ ID NOs 23 to 261 (odd numbers), 297 to 303 (odd numbers), 311 and 313, which include functional fragments or variants thereof. The invention also encompasses chemical antigen-binding portions comprising sequences of SEQ ID NOs 23 to 261 (odd numbers), 297 to 303 (odd numbers), 311 and 313 with conservative amino acid substitutions. POLYNUCLEOTIDS
[0145] The invention further provides isolated polynucleotides that encode a mutant IL-2 polypeptide or an immunoconjugate that comprises a mutant IL-2 polypeptide as described herein.
[0146] Polynucleotides of the invention include those that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences shown in SEQ ID NOs 2, 4, 5, 6, 8, 9, 10, 12, 13, 14, 16, 17, 18, 20, 21,22, 24 to 262 (even numbers), 293 to 296, and 298 to 324 (numbers pairs) that include functional fragments or variants thereof.
[0147] Polynucleotides encoding mutant IL-2 Polypeptides not bound to a non-IL-2 moiety are generally expressed as a single polynucleotide encoding the entire polypeptide.
[0148] In one embodiment, the present invention is directed to an isolated polynucleotide that encodes a mutant IL-2 polypeptide, wherein the polynucleotide comprises a sequence that encodes a mutant IL-2 sequence of SEQ ID NO: 7, 11 , 15 or 19. The invention also encompasses an isolated polynucleotide encoding a mutant IL-2 polypeptide, wherein the polynucleotide comprises a sequence encoding a mutant IL-2 polypeptide of SEQ ID NO: 7, 11, 15 or 19 with conservative amino acid substitutions.
[0149] In another embodiment, the invention is directed to an isolated polynucleotide that encodes a mutant IL-2 polypeptide, wherein the polynucleotide comprises a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence selected from the group of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295 and SEQ ID NO: 296. In another embodiment, the invention is directed to an isolated polynucleotide that encodes a mutant IL-2 polypeptide, wherein the polynucleotide comprises a sequence of nucleotide selected from the group of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO : 295 and SEQ ID NO: 296. In another embodiment, the invention is directed to an isolated polynucleotide that encodes an immunoconjugate or fragment thereof, wherein the polynucleotide comprises a nucleic acid sequence that is at least about 80%, 85 %, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence selected from the group of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295 and SEQ ID NO: 296. In another embodiment, the invention is directed to an isolated polynucleotide that encodes an immunoconjugate or fragment thereof, in that the polynucleotide comprises a nucleic acid sequence selected from the group of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 , SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SE Q ID NO: 22, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295 and SEQ ID NO: 296.
[0150] Polynucleotides encoding the immunoconjugates of the invention can be expressed as a single polynucleotide encoding the entire immunoconjugate or as multiple polynucleotides (e.g., two or more) that are coexpressed. Polypeptides encoded by polynucleotides that are coexpressed can be linked through, for example, disulfide bonds or other means to form a functional immunoconjugate. For example, the heavy chain portion of an antigen binding portion can be encoded by a polynucleotide separate from the immunoconjugate portion comprising the light chain portion of the antigen binding portion and the mutant IL-2 polypeptide. When coexpressed, heavy chain polypeptides will be associated with light chain polypeptides to form the antigen binding portion. Alternatively, in another example, the light chain portion of the antigen binding portion can be encoded by a polynucleotide separate from the immunoconjugate portion comprising the heavy chain portion of the antigen binding portion and the mutant IL-2 polypeptide. In one embodiment, an isolated polynucleotide of the invention encodes a fragment of an immunoconjugate that comprises a mutant IL-2 polypeptide and an antigen binding portion. In one embodiment, an isolated polynucleotide of the invention encodes the heavy chain of an antigen-binding portion and a mutant IL-2 polypeptide. In another embodiment, an isolated polynucleotide of the invention encodes the light chain of an antigen binding portion and a mutant IL-2 polypeptide.
[0151] In a specific embodiment, an isolated polynucleotide of the invention encodes a fragment of an immunoconjugate that comprises at least one mutant IL-2 polypeptide, and at least one, preferably two or more chemical antigen-binding moieties, in which one first mutant IL-2 polypeptide shares an amino or carboxy-terminal peptide bond with a first antigen-binding portion and a second antigen linkage shares an amino or carboxy-terminal peptide bond or with the first IL-2 polypeptide mutant or the first antigen binding portion. In one embodiment, the antigen-binding chemical moieties are independently selected from the group consisting of a Fv molecule, particularly a scFv molecule, and a Fab molecule. In another specific embodiment, the polynucleotide encodes the heavy chains of two of the chemical antigen-binding moieties and a mutant IL-2 polypeptide. In another specific embodiment, the polynucleotide encodes the light chains of two of the chemical antigen-binding moieties and a mutant IL-2 polypeptide. In another specific embodiment, the polynucleotide encodes a light chain from one of the antigen-binding chemical moieties, a heavy chain from a second antigen-binding moiety and a mutant IL-2 polypeptide.
[0152] In another specific embodiment, an isolated polynucleotide of the invention encodes a fragment of an immunoconjugate, wherein the polynucleotide encodes the heavy chains of two Fabs molecules and a mutant IL-2 polypeptide. In another specific embodiment, an isolated polynucleotide of the invention encodes a fragment of an immunoconjugate, wherein the polynucleotide encodes the light chains of two Fab molecules and a mutant IL-2 polypeptide. In another specific embodiment, an isolated polynucleotide of the invention encodes a fragment of an immunoconjugate, wherein the polynucleotide encodes the heavy chain of one Fab molecule, the light chain of second Fab molecule, and a mutant IL-2 polypeptide.
[0153] In one embodiment, an isolated polynucleotide of the invention encodes an immunoconjugate comprising at least one mutant IL-2 polypeptide, joined at its amino or carboxy-terminal amino acids to one or more scFv molecules.
[0154] In one embodiment, an isolated polynucleotide of the invention encodes a fragment of an immunoconjugate, wherein the polynucleotide encodes the heavy chain of an immunoglobulin molecule, particularly an IgG molecule, more particularly, an IgGi molecule, and an mutant IL-2 polypeptide. In a more specific embodiment, the isolated polynucleotide encodes the heavy chain of an immunoglobulin molecule and a mutant IL-2 polypeptide, where the mutant IL-2 polypeptide shares an amino-terminal peptide link with the immunoglobulin heavy chain .
[0155] In another embodiment, the present invention is directed to an isolated polynucleotide that encodes an immunoconjugate or fragment thereof, wherein the polynucleotide comprises a sequence that encodes a variable region sequence as shown in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 231, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, iθi i63 i65 137, i69 171 173_ 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 311 or 313. In another embodiment, the present invention is directed to an isolated polynucleotide that encodes an immunoconjugate or fragment thereof, wherein the polynucleotide comprises a sequence that encodes a polypeptide sequence as shown in SEQ ID NO: 199, 201,203, 205, 207, 209, 211,213, 215, 217, 219, 221,223, 22 5, 227, 229, 231,233, 235, 237, 239, 241,243, 245, 247, 249, 251, 253, 255, 257, 259, 261,297, 299, 301,303, 315, 317, 319, 321 or 323. In another embodiment, the invention is additionally directed to an isolated polynucleotide encoding an immunoconjugate or fragment thereof, wherein the polynucleotide comprises a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97% , 98%, or 99% identical to a nucleotide sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54 , 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104 , 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154 , 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204 , 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 25 4, 256, 258, 260, 262, 298, 300, 302, 304, 312, 314, 316, 318, 320, 322 or 324. In another embodiment, the invention is directed to an isolated polynucleotide encoding an immunoconjugate or fragment therein, wherein the polynucleotide comprises a nucleic acid sequence shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54 , 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104 , 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154 , 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204 , 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254 , 256, 258, 260, 262, 298, 300, 302, 304, 312, 314, 316, 318, 320, 322 or 324. In another embodiment, the invention is directed to an isolated polynucleotide comprising difficates an immunoconjugate or fragment thereof, wherein the polynucleotide comprises a sequence that encodes a variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 23, 25, 27, 29, 31.33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59 , 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109 , 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 231, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159 , 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197,311 or 313. In another embodiment, the invention is directed to an isolated polynucleotide encoding an immunoconjugate or fragment thereof, wherein the polynucleotide comprises the sequence encoding the polypeptide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an SEQ amino acid sequence ID NO: 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221,223, 225, 227, 229, 231,233, 235, 237, 239, 241,243, 245, 247, 249, 251,253 , 255, 257, 259, 261,297, 299, 301,303, 315, 317, 319, 321 or 323. The invention encompasses an isolated polynucleotide that encodes an immunoconjugate or fragment thereof, wherein the polynucleotide comprises a sequence that encodes the sequences of variable region of SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65 , 67, 69, 71.73, 75, 77, 79, 81.83, 85, 87, 89, 91.93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115 , 117, 119, 121, 123, 125, 127, 129, 231, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165 , 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 311 or 313 with conservative amino acid substitutions. The invention also encompasses an isolated polynucleotide that encodes an immunoconjugate of the invention or fragment thereof, wherein the polynucleotide comprises a sequence that encodes the polypeptide sequence of SEQ ID NO: 199, 201,203, 205, 207, 209, 211,213, 215, 217, 219, 221,223, 225, 227, 229, 231,233, 235, 237, 239, 241,243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 297, 299, 301, 303, 315, 317, 319, 321 or 323 with conservative amino acid substitutions.
[0156] In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, a 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. RECOMBINANT METHODS
[0157] The mutant IL-2 polypeptides of the invention can be prepared by deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods can include site-specific mutagenesis of the coding DNA sequence, PCR, gene synthesis and the like. The correct nucleotide changes can be verified, for example, by sequencing. In this regard, the native IL-2 nucleotide sequence that was described by Taniguchi et al. (Nature 302, 305 to 10 (1983)) and human nucleic acid coding IL-2 are available from public stores such as the American Type Culture Collection (Rockville MD). A sequence of native human IL-2 is shown in SEQ ID NO: 1. The substitution or insertion may involve both natural as well as unnatural amino acid residues. The nucleic acid modification includes well-known methods of chemical modification such as the addition of glycosylation sites or carbohydrate bonds and the like.
[0158] The mutant and immunoconjugate IL-2 polypeptides of the invention can be obtained, for example, by solid-state peptide synthesis or recombinant production. For recombinant production, one or more polynucleotides encoding said mutant or immunoconjugate IL-2 polypeptide (fragments), for example, as described above, is isolated and inserted into one or more vectors for cloning and / or additional expression in a cell hostess. Such a polynucleotide can be readily isolated and sequenced using conventional procedures. In one embodiment, a vector, preferably an expression vector, which comprises one or more of the polynucleotides of the invention is provided. Methods that are well known to elements skilled in the art can be used for expression vectors containing the coding sequence for a mutant or immunoconjugate IL-2 polypeptide (fragment) along with appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in v / vo recombination / genetic recombination. See, for example, the techniques described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y (1989). The expression vector can be part of a plasmid, virus, or it can be a fragment of nucleic acid. The expression vector includes an expression cassette in which the polynucleotide encoding the mutant IL-2 or the immunoconjugate (fragment) (i.e., the coding region) is cloned in operable association with a promoter and / or other control elements of transcription or translation. As used herein, a "coding region" is a portion of nucleic acid that consists of codons translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it can be considered to be part of a coding region, if present, but any flanking sequences, for example, promoters, ribosome binding sites, transcription terminators, introns, 5 'and 3' untranslated regions and the like, are not part of a coding region. Two or more coding regions can be present in a single polynucleotide construct, for example, in a single vector, or in separate polynucleotide constructs, for example, in separate (different) vectors. In addition, any vector can contain a single coding region, or can comprise two or more coding regions, for example, a vector of the present invention can encode one or more polyproteins, which are separated in a post or cotraditional way in the final proteins by means of proteolytic cleavage. In addition, a vector, polynucleotide, or nucleic acid of the invention can encode heterologous coding regions, can be fused or not fused to a first or second polynucleotide encoding the polypeptides of the invention, or a variant or derivative thereof. Heterologous coding regions include, without limitation, specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain. An operable association is when a coding region for a gene product, for example, a polypeptide, is associated with one or more regulatory sequences in order to place the expression of the gene product under the influence or control of the regulatory sequence (s) (s). Two fragments of DNA (such as a polypeptide coding region and a promoter associated with it) are "operably associated" if the induction of promoter function results in mRNA transcription encodes the desired gene product and if the nature of the link between the two DNA fragments do not interfere with the ability of regulatory expression sequences to direct expression of the gene product or interfere with the ability of the DNA model to be transcribed. Thus, a promoter region can be operably associated with a nucleic acid that encodes a polypeptide if the promoter was able to transcribe that nucleic acid. The promoter can be a cell-specific promoter that directs substantial DNA transcription only in predetermined cells. Other elements of transcription control, in addition to a promoter, for example, enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to target cell-specific transcription. Suitable promoters and other transcription control regions are disclosed in this document. A variety of transcription control regions are known to elements skilled in the art. This includes, without limitation, transcription control regions, which function in vertebrate cells, such as, but not limited to, cytomegalovirus promoter and enhancer segments (for example, the immediate early promoter, in conjunction with A-intron), simian virus 40 (for example, the early promoter), and retrovirus (such as, for example, Rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit β-globin, as well as other sequences that can control gene expression in eukaryotic cells. Additional suitable transcriptional control regions include tissue-specific promoters and enhancers as well as inducible promoters (for example, promoter-inducible tetracyclines). Similarly, a variety of translation control elements are known to those skilled in the art. This includes, but is not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly, an internal ribosome entry site, or IRES, also referred to as an ISCED sequence) . The expression cassette can also include other features such as a source of replication, and / or chromosomal integration elements such as retroviral long terminal repeats (LTRs), or inverted terminal repeats (ITRs) of adeno-associated virus (AAV).
[0159] The polynucleotide and nucleic acid coding regions of the present invention can be associated with additional coding regions that encode secretion or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention. For example, if secretion of the mutant IL-2 polypeptide is desired, the DNA encoding a signal sequence can be placed upstream of the nucleic acid encoding the mature amino acids of the mutant IL-2. The same applies to immunoconjugates of the invention or fragments thereof. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or leading secretory sequence that is cleaved from the mature protein, once the export of the growing protein chain through the rough endoplasmic reticulum has started. Elements skilled in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce a secreted or "mature" form of polypeptide. For example, human IL-2 is translated with a 20 amino acid signal sequence at the N-terminal of the polypeptide, which is subsequently cleaved to produce the mature 133 amino acid human IL-2. In certain embodiments, the native signal peptide, for example, the IL-2 signal peptide or an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative from which the sequence that retains the ability to direct secretion of the polypeptide is operably associated with it. Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, can be used. For example, the wild-type leader sequence can be replaced by the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase. Exemplary amino acid and polynucleotide sequences of signal secreting peptides are shown in SEQ ID NOs 236 to 273.
[0160] DNA encoding a short protein sequence that can be used to facilitate further purification (for example, a histidine tag) or to help label mutant or immunoconjugate IL-2, can be included inside or at the ends of the Mutant or immunoconjugate IL-2 (fragment) encoding polynucleotide.
[0161] In a further embodiment, a host cell comprising one or more polynucleotides of the invention is provided. In certain embodiments, a host cell comprising one or more vectors of the invention is provided. Polynucleotides and vectors can incorporate any of the resources, either singly or in combination, described in this document in relation to polynucleotides and vectors, respectively. In such an embodiment, a host cell comprises (for example, which has been transformed or transfected with) a vector comprising a polynucleotide encoding an amino acid sequence comprising the mutant IL-2 polypeptide of the invention. As used herein, the term "host cell" refers to any type of cellular system that can be modified to generate the mutant or immunoconjugate IL-2 polypeptides of the invention or fragments thereof. Host cells suitable for replicating and supporting expression of mutant or immunoconjugate IL-2 Polypeptides are well known in the art. Such cells can be transfected or transduced as appropriate with the particular expression vector and large amounts of vector containing cells can be grown to seed large-scale fermenters to obtain sufficient amounts of the mutant or immunoconjugate IL-2 for chemical applications. Suitable host cells include prokaryotic microorganisms, such as E. coli, or various eukaryotic cells, such as Chinese hamnster ovary (CHO) cells, insect cells, or the like. For example, polypeptides can be produced in bacteria, in particular, when glycosylation is not necessary. After expression, the polypeptide can be isolated from the bacterial cell paste in a soluble fraction and can be further purified. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cleavage or expression hosts for polypeptide coding vectors, which include strains of fungi and yeast whose glycosylation trajectories have been "humanized", resulting in the production of a polypeptide with a pattern glycosylation partially or completely human. See Gerngross, Nat Biotech 22, 1,409 to 1,414 (2004), and Li et al., Nat Biotech 24, 210 to 215 (2006). Host cells suitable for the expression of polypeptides (glycosylates) are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous strains of baculovirus have been identified so that they can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describes PLANTIBODIES ™ technology for producing antibodies in transgenic plants). Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney lineage (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 (cells TM4, as described, for example, in Mather, Biol Reprod 23, 243 to 251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), rat and buffalo 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, which include CHO-dhfr cells (llrlaub et al., Proc Natl Acad Sci USA 77, 4,216 (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, Volume 248 (BKC Lo, ed., Human Press, Totowa, NJ), pages 255 to 268 (2003). Host cells include cultured cells, for example, cultured mammalian cells, yeast cells, insect cells, bacterial cells and plant cells, to name just a few, but also cells comprised in a transgenic animal, transgenic plant or cultivated plant or tissue from animal. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese hamster ovary (CHO) cell, a human embryonic kidney cell (HEK) or lymphoid cell (for example, Y0, NSO cell , Sp20).
[0162] Standard technologies are known in the art to express foreign genes in these systems. Cells that express a fused polypeptide of mutant IL-2 to either the heavy chain or the light chain of an antigen binding domain such as an antibody, can be modified to also express the others of the antibody chains so that the expressed mutant IL-2 fused product is an antibody that has both a heavy and a light chain.
[0163] In one embodiment, a method for producing a mutant IL-2 polypeptide or an immunoconjugate according to the invention is provided, wherein the method comprises culturing a host cell that comprises a polynucleotide encoding the IL-2 polypeptide mutant or immunoconjugate, as provided herein, under conditions suitable for expression of the mutant or immunoconjugate IL-2 polypeptide, and optionally recovering the mutant or immunoconjugate IL-2 polypeptide from the host cell (or host cell culture medium).
[0164] In certain embodiments, according to the invention, the mutant IL-2 polypeptide is linked to at least a non-IL-2 moiety. A mutant IL-2 can be prepared in which the mutant IL-2 polypeptide segment is linked to one or more molecules such as a polypeptide, protein, carbohydrate, lipid, nucleic acid, polynucleotide or molecules that are combinations of these molecules (for example , glycoproteins, glycolipids, etc.). The mutant IL-2 polypeptide can also be linked to an organic chemical moiety, inorganic chemical moiety or pharmaceutical drug. As used herein, a pharmaceutical drug is a compound containing an organic portion of about 5,000 daltons or less. The mutant IL-2 polypeptide can also be linked to any biological agent that includes therapeutic compounds such as antineoplastic agents, antimicrobial agents, hormones, immunomodulators, anti-inflammatory agents and the like. Also included are radioisotopes such as those useful for imaging as well as for therapy.
[0165] The mutant IL-2 polypeptide can also be linked to multiple molecules of the same type or to more than one type of molecule. In certain embodiments, the molecule that is bound to IL-2 may confer the ability to target IL-2 to specific tissues or cells in an animal, and is referred to in this document as a "chemical targeting portion". In these embodiments, the chemical targeting portion may have an affinity for a ligand or receptor in the target tissue or cell, then directing IL-2 to the target tissue or cell. In a particular embodiment, the chemical targeting portion directs IL-2 to a tumor. Chemical targeting moieties include, for example, chemical antigen binding moieties (e.g., antibodies and fragments thereof) specific for cell surfaces or intracellular proteins, biological receptor ligands and the like. Such chemical antigen-binding moieties may be specific for tumor-associated antigens such as those described herein.
[0166] A mutant IL-2 polypeptide can be genetically fused to another polypeptide, for example, a single chain antibody, or (part of) an antibody heavy chain or light chain, or it can be chemically conjugated to another molecule. The fusion of a mutant IL-2 polypeptide in part of an antibody heavy chain is described in the Examples. A mutant IL-2 that is a fusion between a mutant IL-2 polypeptide and another polypeptide can be designed so that the IL-2 sequence is fused directly to the polypeptide or indirectly via a linker sequence. The composition and length of the binder can be determined according to methods well known in the art and can be tested for effectiveness. An example of a linker sequence between IL-2 and an antibody heavy chain is disclosed in the sequences shown, for example, in SEQ ID NOs 209, 211, 213 etc. Additional sequences can also be included to incorporate a cleavage site to separate the individual components of the fusion, if desired, for example, an endopeptidase recognition sequence. In addition, a mutant IL-2 or fusion protein thereof can also be chemically synthesized using polypeptide synthesis methods as is well known in the art (for example, Merrifield solid phase synthesis). Mutant IL-2 polypeptides can be chemically conjugated to other molecules, for example, another polypeptide, using well-known chemical conjugation methods. Bifunctional cross-linking reagents such as homofunctional or heterofunctional cross-linking reagents well known in the art can be used for this purpose. The type of cross-linking reagent to be used depends on the nature of the molecule to be coupled to IL-2 and can readily be identified by elements skilled in the art. Alternatively, or in addition, the mutant IL-2 and / or the molecule to which it is intended to be conjugated, can be chemically derived so that the two can be conjugated in a separate reaction, as is also well known in the art. .
[0167] In certain embodiments, the mutant IL-2 polypeptide is linked to one or more chemical antigen-binding portions (i.e., it is part of an immunoconjugate) that comprises at least one variable region of antibody that can bind a determinant antigenic. Variable regions can form part of and be derived from antibodies and fragments, whether naturally occurring or not. Methods for producing polyclonal antibodies and monoclonal antibodies are well known in the art (see, for example, Harlow and Lane, 'Antibodies, a laboratory manual ", Cold Spring Harbor Laboratory, 1988). Unnaturally occurring antibodies can be constructed using of solid phase peptide synthesis, can be produced recombinantly (for example, as described in U.S. Patent No. 4,186,567) or can be obtained, for example, by combinatorial screening libraries that comprise variable heavy chains and light chains variables (see, for example, U.S. Patent No. 5,969,108 by McCafferty). Immunoconjugates, chemical portions of antigen binding and methods of producing them are also described in detail in PCT publication No. WO 2011/020783, whose wholeness is hereby incorporated by reference.
[0168] Any animal antibody species, antibody fragment, antigen binding domain or variable region can be linked to a mutant IL-2 polypeptide. Non-limiting antibodies, antibody fragments, antigen binding domains or variable regions useful in the present invention can be of murine, primate or human origin. If the mutant / antibody conjugate or fusion IL-2 is intended for human use, a chimeric form of the antibody can be used so that the antibody constant regions are from a human. A humanized or completely human form of the antibody 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 a number of methods that include, but are not limited to (a) grafting non-human CDRs (eg, donor antibody) into human structure (eg, recipient antibody) and constant regions with or without residue retention of critical structure (for example, those that are important for retaining good antigen binding affinity or antibody functions), (b) grafting only from regions of non-human specificity determination (SDRs or a-CDRs; residues critical to the interaction of antibody antigen) in human structure and constant regions, or (c) transplanting the entire non-human variable domains, but "hiding" them with a human-like section by replacing surface residues. Humanized antibodies and methods for making them are reviewed, for example, in Almagro and Fransson, Front Biosci 13, 1.619 to 1.633 (2008), and are further described, for example, in Riechmann et al., Nature 332, 323 to 329 (1988); Queen et al., Proc Natl Acad Sci USA 86, 10,029 to 10,033 (1989); U.S. Patents 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Jones et al., Nature 321, 522 to 525 (1986); Morrison et al., Proc Natl Acad Sci 81,6,851 to 6,855 (1984); Morrison and Oi, Adv Immunol 44, 65 to 92 (1988); Verhoeyen et al., Science 239, 1,534 to 1,536 (1988); Padlan, Molec Immun 31 (3), 169 to 217 (1994); Kashmiri et al., Methods 36, 25 to 34 (2005) (which describes SDR graft (a-CDR)); Padlan, Mol Immunol 28, 489 to 498 (1991) (which describes "resurfacing"); Dall'Acqua et al., Methods 36, 43 60 (2005) (which describes “FR shuffling”); and Osbourn et al., Methods 36, 61 to 68 (2005) and Klimka et al., Br J cancer 83, 252 to 260 (2000) (which describes the “guided selection” approach for FR shuffling). Human antibodies and human variable regions can be produced using various techniques known in the art. Human antibodies are generally described by van Dijk and van de Winkel, Curr Opin Pharmacol 5, 368 to 74 (2001) and Lonberg, Curr Opin Immunol 20, 450 to 459 (2008). Human variable regions can form part of and be derived from human monoclonal antibodies made by the hybridoma method (see, for example, Monoclonal Antibody Production Techniques and Applications, pages 51 to 63 (Marcel Dekker, Inc., New York, 1987)). Human antibodies and human variable regions can also be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to the antigen challenge (see, for example, Lonberg, Nat Biotech 23, 1,117 to 1,125 (2005)). Human antibodies and human variable regions can also be generated by isolating Fv clone variable region sequences selected from human-derived phage display libraries (see, for example, Hoogenboom et al. In Methods in Molecular Biology 178, 1 to 37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001); and McCafferty et al., Nature 348, 552 to 554; Clackson et al., Nature 352, 624 to 628 (1991) ). The phage typically exhibits antibody fragments, either as single chain Fv (scFv) fragments or as Fab fragments. A detailed description of the preparation of chemical antigen-binding portions for immunoconjugates by phage display can be disclosed in the Examples attached to PCT Publication No. WO 2011/020783.
[0169] In certain embodiments, the chemical antigen binding portions useful in the present invention are modified to have enhanced binding affinity according to, for example, the methods disclosed in PCT publication No. WO 2011/020783 (see related examples to affinity maturity) or patent application for US Patent No. 2004/0132066, the entirety of which is hereby incorporated by reference. The ability of the immunoconjugate of the invention if bound to a specific determinant antigen can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to elements versed in the technique, for example, Surface Plasma Resonance Technique (analyzed in a BIACORE T100 system) (Liljeblad, et al., Glyco J 17, 323 to 329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217 to 229 (2002)). Competition assays can be used to identify an antibody, antibody fragment, antigen binding domain or variable domain that competes with a reference antibody to bind to a particular antigen, for example, an antibody that competes with the L19 antibody for connect to the Extra B Domain of Fibronectin (EDB). In certain embodiments, such a competing antibody is linked to the same epitope (for example, a linear or conformational epitope) that is linked by the reference antibody. Detailed exemplary methods for mapping an epitope to which an antibody is attached are provided in Morris (1996) "Epitope Mapping Protocols", in Methods in Molecular Biology, volume 66 (Humana Press, Totowa, NJ). In an exemplary competition assay, immobilized antigen (eg, EDB) is incubated in a solution that comprises a first labeled antibody that is bound to the antigen (eg, L19 antibody) and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to the antigen. The second antibody can be present in a hybridoma supernatant. As a control, the immobilized antigen is incubated in a solution that comprises the first labeled antibody, but not the second unlabeled antibody. After incubation under permissive conditions for binding the first antibody to the antigen, excess unbound antibody is removed, and the amount of label associated with the immobilized antigen is measured. If the amount of label associated with the immobilized antigen is substantially reduced in the test sample compared to the control sample, then it indicates that the second antibody is competing with the first antibody for binding to the antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).
[0170] Additional chemical modification of the mutant or immunoconjugate IL-2 mutant of the invention may be desirable. For example, problems of immunogenicity and short half-life can be improved by conjugation to substantially normal chain polymers such as polyethylene glycol (PEG) or polypropylene glycol (PPG) (see, for example, WO 87/00056).
[0171] IL-2 mutants and immunoconjugates prepared as described in this document can be purified by known techniques, 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, etc., and will be apparent to elements skilled in the art. For purification of affinity chromatography, an antibody, ligand, receptor or antigen can be used to which the mutant or immunoconjugate IL-2 polypeptide is attached. For example, an antibody that specifically binds the mutant IL-2 polypeptide can be used. For purification of affinity chromatography of immunoconjugates of the invention, a matrix with protein A or protein G can be used. For example, affinity chromatography and sequential Protein A or G size exclusion chromatography can be used to isolate an immunoconjugate essentially as described in the Examples. The purity of the mutant IL-2 polypeptides and their fusion proteins can be determined by any of a variety of well-known analytical methods including gel electrophoresis, high pressure liquid chromatography and the like. For example, the heavy chain fusion proteins expressed as described in the examples have been shown to be intact and assembled appropriately as demonstrated by reducing SDS-PAGE (see, for example, Figure 14). Two bands were resolved in approximately Mr 25,000 and Mr 60,000, corresponding to the predicted molecular weights of the light chain and heavy chain of immunoglobulin / IL-2 fusion protein. ESSAY
[0172] Mutant IL-2 polypeptides and immunoconjugates provided herein can be identified, screened, or characterized by their physical / chemical properties and / or biological activities by various assays known in the art. AFFINITY TESTS
[0173] The affinity of the mutant or wild-type IL-2 polypeptide for various forms of the IL-2 receptor can be determined according to the method presented in the Surface Plasma Resonance (SPR) Examples, using standard instrumentation as a BIAcore instrument (GE Healthcare), and receptor subunits as can be obtained by recombinant expression (see, for example, Shanafelt et al., Nature Biotechnol 18, 1,197 to 1,202 (2000)). A recombinant IL-2 receptor β / y subunit heterodimer can be generated by fusing each of the subunits to an antibody Fc domain monomer modified by knobs-into-holes technology (see, for example, U.S. Patent No. 5,731. 168) to promote heterodimerization of the appropriate Fc fusion receptor / protein subunit (see SEQ ID NOs 102 and 103). Alternatively, the binding affinity of mutant IL-2 to different forms of the IL-2 receptor can be assessed using known cell lines to express one or the other form of the receptor. A specific illustrative and exemplary embodiment for measuring binding affinity is described below and in the Examples below. According to one embodiment, KD is measured by Surface Plasma Resonance using a BIACORE® T100 machine (GE Healthcare) at 25 ° C with IL-2 receptors immobilized on CM5 chips. Soon, carboxymethylated dextran (CM5, GE Healthcare) biosensor chips are activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The recombinant IL-2 receptor is diluted with 10 mM sodium acetate, pH 5.5, to 0.5 to 30 pg / ml before injection at a flow rate of 10 pl / minute to reach approximately 200 to 1,000 (for IL-2R α subunit) or 500 to 3,000 (for IL-2R βy knobs-int-holes heterodimer) coupled protein response units (RU). After the injection of IL-2 receptor, 1 M of ethanolamine is injected to block unreacted groups. For kinetic measurements, three-fold serial dilutions of mutant or immunoconjugate IL-2 polypeptide (in the range of ~ 0.3 nM to 300 nM) are injected into HBS-EP + (GE Healthcare, 10 mM HEPES, 150 mM NaCI , 3 mM EDTA, 0.05% P20 surfactant, pH 7.4) at 25 ° C at a flow rate of approximately 30 pl / min. Association fees (kassociation) and dissociation fees (kdissociation) are calculated using a Langmuir one-to-one connection model (BIACORE ® T100 from evaluation software version 1.1.1) by simultaneously adjusting the sensorgrams of association and dissociation. The equilibrium dissociation constant (KD) is calculated as the kdissociation / kassociation ratio. See, for example, Chen et al., J Mol Biol 293, 865 to 881 (1999).
[0174] The binding of immunoconjugates of the invention to Fc receptors can be easily determined, for example, through ELISA, or through Surface Plasma Resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and Fc receptors, as can be obtained by recombinant expression. Alternatively, the binding affinity of Fc domains or immunoconjugates comprising an Fc domain for Fc receptors can be assessed with the use of known cell lines to express particular Fc receptors, such as NK cells that express the Fcyllla receptor. According to one embodiment, KD is measured by Surface Plasma Resonance using a BIACORE® T100 machine (GE Healthcare) at 25 ° C with Fc receivers immobilized on CM5 chips. Soon, carboxymethylated dextran (CM5, GE Healthcare) biosensor chips are activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The recombinant Fc receptor is diluted with 10 mM sodium acetate, pH 5.5, to 0.5 to 30 pg / ml prior to injection at a flow rate of 10 pl / minute to achieve approximately 100 to 5,000 response units (RU) of coupled protein. After the injection of the Fc receptor, 1 M of ethanolamine is injected to block unreacted groups. For kinetic measurements, three to five times serial dilutions of the immunoconjugate (in the range of ~ 0.01 nM to 300 nM) are injected into HBS-EP + (GE Healthcare, 10 mM HEPES, 150 mM NaCI, 3 mM EDTA , 0.05% of Surfactant P20, pH 7.4) at 25 ° C at a flow rate of approximately 30 to 50 pl / min. Association fees (kassociation) and dissociation fees (kdissociation) are calculated using a one-to-one connection model (BIACORE ® T100 from evaluation software version 1.1.1) by simultaneously adjusting the association sensorgrams and dissociation. The equilibrium dissociation constant (KD) is calculated as the ratio of kdissociation / kassociation. See, for example, Chen et al., J Mol Biol 293, 865 to 881 (1999). ACTIVITY TESTS
[0175] The ability of a mutant IL-2 to bind to IL-2 receptors can be indirectly measured by testing the effects of immune activation that occur downstream of the receptor binding.
[0176] In one aspect, assays are provided to identify mutant IL-2 polypeptides that have biological activity. Biological activities may include, for example, the ability to induce proliferation of IL-2 receptor support T and / or NK cells, the ability to induce IL-2 signaling on T cells and / or NK receptor support NK IL-2, the ability to generate interferon (IFN) -y as a secondary cytokine by NK cells, a reduced ability to induce elaboration of secondary cytokines, particularly IL-10 and TNF-a, by peripheral blood mononuclear cells (PBMCs) ), a reduced ability to induce apoptosis in T cells, the ability to induce tumor regression and / or enhance survival, and a reduced toxicity profile, particularly reduced vascular permeability, in vivo. Mutant IL-2 polypeptides that have such biological activity in vivo and / or in vitro are also provided.
[0177] In certain embodiments, a mutant IL-2 polypeptide of the invention is tested for such biological activity. A variety of methods are well known in the art for determining biological activities of IL-2, and details for many of these methods are also disclosed in the Examples attached thereto. The examples provide a suitable assay to test IL-2 mutants of the invention for their ability to generate IFN-γ by NK cells. Cultured NK cells are incubated with the mutant IL-2 polypeptide or immunoconjugates of the invention, and concentration of IFN-γ in the culture medium is subsequently measured by ELISA.
[0178] IL-2-induced signaling induces several signaling trajectories, and involves JAK (Janus kinase) and STAT (signal transducer and transcription activator) signaling molecules. The interaction of IL-2 with the β and y receptor subunits leads to the phosphorylation of the receptor and JAK1 and JAK3, which are associated with the β and y subunit, respectively. STAT5, then, is associated with the phosphorylated receptor and is phosphorylated by itself into a fundamental tyrosine residue. This results in the dissociation of STAT5 from the receptor, dimerization of STAT5 and translocation of the STAT5 dimers to the nucleus where they promote the transcription of target genes. The ability of mutant IL-2 Polypeptides to induce signaling through the IL-2 receptor can then be assessed, for example, by measuring STAT5 phosphorylation. Details of this method are revealed in the Examples. PBMCs are treated with mutant IL-2 polypeptides or immunoconjugates of the invention and levels of phosphorylated STAT5 are determined by flow cytometry.
[0179] The proliferation of T cells or NK cells in response to IL-2 can be measured by incubating isolated T cells or NK cells and blood with mutant IL-2 polypeptides or immunoconjugates of the invention, followed by determining the ATP content in lysates of the treated cells. Before treatment, T cells can be pre-stimulated with phytohemagglutinin (PHA-M). This assay, described in the examples, allows quantification sensitive to the number of viable cells, however there are numerous suitable alternative assays known in the art (eg [3H] - thymidine incorporation assay, CelITiter Glo ATP assays, Alamar Blue assay, assay WST-1, MTT assay).
[0180] An assay for determining apoptosis of T cells and AICD is also provided in the examples, where T cells are treated with an apoptosis-inducing antibody after incubation with the invention's mutant or immunoconjugate IL-2 polypeptides and cells Apoptotic cells are quantified by detection by phosphatidylserine / annexin exposure flow cytometry. Other assays are known in the art.
[0181] The effects of mutant IL-2 on tumor growth and survival can be evaluated in a variety of animal tumor models known in the art. For example, xenografts from human cancer cell lines can be implanted into immunodeficient mice, and treated with mutant IL-2 polypeptides or immunoconjugates of the invention, as described in the examples.
[0182] The toxicity of mutant IL-2 polypeptides and immunoconjugates of the invention in vivo can be determined based on mortality, observations in life (visible symptoms of adverse effects, for example, behavior, body weight, body temperature) and clinical pathology anatomical (for example, measurements of chemical blood values and / or histopathological analyzes).
[0183] Vascular permeability induced by IL-2 treatment can be examined in an animal vascular permeability pretreatment model. In general, the mutant or immunoconjugate IL-2 of the invention is administered to a suitable animal, for example, a mouse, and at a later time, a vascular leak reporter molecule is injected into the animal whose dissemination of the vasculature reflects the extent of permeability vascular. The vascular leak reporter molecule is preferably large enough to reveal permeability with the wild-type form of IL-2 used for pretreatment. An example of a vascular leak reporter molecule may be a whey protein such as albumin or an immunoglobulin. The vascular leak reporter molecule is preferably detectably labeled as with a radioisotope to facilitate the quantitative determination of the tissue distribution of the molecule. Vascular permeability can be measured for vessels present in any of a variety of internal body organs such as liver, lung and the like, as well as a tumor, which includes a tumor that is xenografted. The lung is a preferred organ for measuring vascular permeability of IL-2 full-length mutants. COMPOSITIONS, FORMULATIONS AND ROUTES OF ADMINISTRATION
[0184] In a further aspect, the invention provides pharmaceutical compositions comprising any of the mutant or immunoconjugate IL-2 polypeptides provided herein, for example, for use in any of the therapeutic methods below. In one embodiment, a pharmaceutical composition comprises any of the mutant or immunoconjugate IL-2 polypeptides provided herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical composition comprises any of the mutant or immunoconjugate IL-2 polypeptides provided herein and at least one additional therapeutic agent, for example, as described below.
[0185] A method is also provided to produce a mutant IL-2 polypeptide or an immunoconjugate of the invention in a form suitable for in vivo administration, the method comprising (a) obtaining a mutant or immunoconjugate IL-2 polypeptide from according to the invention, and (b) formulating the mutant or immunoconjugate IL-2 polypeptide with at least one pharmaceutically acceptable carrier, that way, a mutant or immunoconjugate IL-2 polypeptide preparation is formulated for in vivo administration.
[0186] Pharmaceutical compositions of the present invention comprise a therapeutically effective amount of one or more mutant or immunoconjugate IL-2 Polypeptides dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that are generally non-toxic to recipients at the dosages and concentrations employed, that is, they do not produce an adverse, allergic or other adverse reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition containing at least one mutant or immunoconjugate IL-2 polypeptide and optionally an additional active ingredient will be known to elements skilled in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Edition, Mack Printing Company, 1990, hereby incorporated by reference. In addition, for animal (eg, human) administration, it will be understood that preparations will meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Biological Standards office or corresponding authorities in other countries. Preferred compositions are lyophilized formulations or aqueous solutions. Exemplary IL-2 compositions are described in U.S. Patent Nos. 4,604. 377 and 4,766,106. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion medium, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption retarding agents , salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrating agents, lubricants, sweetening agents, flavoring agents, dyes, as materials and combinations thereof, as may be known to elements skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Edition, Mack Printing Company, 1990, pages 1,289 to 1,329, incorporated herein by reference). Except to the extent that any conventional vehicle is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is contemplated.
[0187] The composition may comprise different types of vehicles which depends on whether it is to be administered in solid, liquid or aerosol form, and if it needs to be sterile for such administration routes as injection, the mutant IL-2 polypeptides or immunoconjugates of the invention (and any additional therapeutic agent) can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticular, intraprostatic, intraesplênica, intrarenal, intrapleural, intratracheal, intranasal, intravitreal, intravaginal, intrarectal, intratumor , intramuscular, intraperitoneal, subcutaneous, subconjunctival, intravesicular, mucosal, intrapericardial, intraumbilical, intraocular, oral, topical, local, by inhalation (eg, aerosol inhalation), injection, infusion, continuous infusion, perfusion bath target cells located directly , through a catheter, through a wash, in creams, in lipid compositions (for example, liposomes), or by another method or any combination of those mentioned, as may be known to elements skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Edition, Mack Printing Company, 1990, incorporated herein into reference title). Parenteral administration, in particular intravenous injection, is most commonly used to deliver polypeptide molecules such as the mutant and immunoconjugate IL-2 polypeptides of the invention.
[0188] Parenteral compositions include those designed for administration by injection, for example, subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal or intraperitoneal injection. For injection, the mutant and immunoconjugate IL-2 polypeptides of the invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. The solution may contain formulation agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the mutant and immunoconjugate IL-2 polypeptides can be in powder form for constitution with a suitable vehicle, for example, sterile pyrogenic water, prior to use. Sterile injectable solutions are prepared by incorporating the IL-2 polypeptides or immunoconjugates of the invention in the required amount in the appropriate solvent with several of the other ingredients listed below, as required. Sterility can be readily accomplished, for example, by filtering through sterile filter membranes. Generally, dispersions are prepared by incorporating the various sterile active ingredients into a sterile vehicle that contains the basic dispersion medium and / or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred preparation methods are vacuum drying or lyophilization techniques that yield a powder of the active ingredient plus any desired additional ingredient from a previously filtered sterile liquid medium. themselves. The liquid medium must be adequately buffered if necessary and the liquid diluent first yielded the isotonic before the injection with sufficient saline or glucose. The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally to a safe level, for example, less than 0.5 ng / mg of protein. Suitable pharmaceutically acceptable vehicles include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants that include ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, benzyl or butyl alcohol; alkyl parabens such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; and m- cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers like polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates that include glucose, mannose, or dextrins; chelating agents such as EDTA; sugars like sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (for example, Zn protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, or the like. Optionally, the suspension can also contain stabilizers or suitable agents that increase the solubility of the compounds to allow the preparation of highly concentrated solutions. In addition, suspensions of the active compounds can be prepared as appropriate oil injection suspensions. Lipophilic solvents or suitable vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl or triglyceride supports or liposomes.
[0189] The active ingredients can be trapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly (methylmethacrylate) microcapsules, respectively, in drug delivery systems colloidal (eg, liposomes, albumin microspheres, microemulsions, nano-particles and nano-capsules) or in macroemulsions. 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 the polypeptide, the matrices being in the form of molded articles, for example, films, or microcapsules. In particular embodiments, the prolonged absorption of an injectable composition can be produced by the use in the compositions of agents that delay absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.
[0190] In addition to the compositions described previously, immunoconjugates can also be formulated as a depot preparation. Such long-acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. In this way, for example, mutant and immunoconjugate IL-2 polypeptides can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as poorly soluble derivatives, for example , as a poorly soluble salt.
[0191] Pharmaceutical compositions comprising the mutant and immunoconjugate IL-2 polypeptides of the invention can be manufactured by conventional methods of mixing, dissolving, emulsifying, encapsulating, trapping or lyophilizing. Pharmaceutical compositions can be formulated in a conventional manner with the use of one or more physiologically acceptable vehicles, diluents, excipients or auxiliaries that facilitate the processing of proteins in preparations that can be used pharmaceutically. The appropriate formulation is dependent on the route of administration chosen.
[0192] Mutant and immunoconjugate IL-2 polypeptides can be formulated into a composition in a free acid or base, neutral or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or base. They include acid addition salts, for example, those formed with the free amino groups of a protein composition, or that are formed with inorganic acids, such as hydrochloric or phosphoric acids, or organic acids such as acetic acid, oxalic, tartaric or mandelic. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or organic bases such as isopropylamine, trimethylamine, histidine or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than in the corresponding free base forms. THERAPEUTIC METHODS AND COMPOSITIONS
[0193] Any of the mutant and immunoconjugate IL-2 polypeptides provided herein can be used in therapeutic methods. The mutant and immunoconjugate IL-2 polypeptides of the invention can be used as immunotherapeutic agents, for example, in the treatment of cancer.
[0194] For use in therapeutic methods, the mutant and immunoconjugate IL-2 polypeptides of the invention can be formulated, dosed, and administered in a model consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the agent's distribution site, the method of administration, the schedule of administration, and other factors known to doctors.
[0195] The mutant and immunoconjugate IL-2 polypeptides of the invention are useful in treating disease states in which stimulation of the host's immune system is beneficial, in particular, conditions in which an enhanced cellular immune response is desirable. This can include disease states in which the host immune response is insufficient or deficient. The disease states to which the mutant or immunoconjugate IL-2 polypeptides of the invention can be administered comprise, for example, a tumor or infection in which a cellular immune response can be a critical mechanism for specific immunity. Specific disease states for which the mutant IL-2 of the present invention can be used include cancer, for example, renal cell carcinoma or melanoma; immunological deficiency, specifically in HIV positive patients, immunosuppressed patients, chronic infection and the like. The mutant or immunoconjugate IL-2 polypeptides of the invention can be administered by themselves or in any suitable pharmaceutical composition.
[0196] In one aspect, mutant IL-2 polypeptides and immunoconjugates of the invention for use as medically are provided. In additional aspects, mutant IL-2 polypeptides and immunoconjugates of the invention for use in treating a disease are provided. In certain embodiments, mutant IL-2 polypeptides and immunoconjugates of the invention for use in a treatment method are provided. In one embodiment, the invention provides a mutant IL-2 polypeptide or an immunoconjugate as described herein for use in the treatment of a disease in an individual in need thereof. In certain embodiments, the invention provides a mutant IL-2 polypeptide or an immunoconjugate for use in a method for treating an individual who has a disease comprising administering to the individual a therapeutically effective amount of the mutant IL-2 polypeptide or the immunoconjugate. In certain embodiments, the disease to be treated is a proliferative disorder. In a preferred 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, for example, an anti-cancer agent if the disease to be treated is cancer. In further embodiments, the invention provides a mutant IL-2 polypeptide or an immunoconjugate for use in stimulating the immune system. In certain embodiments, the invention provides a mutant IL-2 polypeptide or an immunoconjugate for use in a method of stimulating the immune system in an individual comprising administering to the individual an effective amount of the mutant or immunoconjugate IL-2 polypeptide to stimulate the immune system. An "individual", according to any of the above achievements, is a mammal, preferably a human. “Stimulating the immune system”, according to any of the above achievements, can include any one or more of a general increase in immune function, an increase in T cell function, an increase in B cell function, a restoration of function of lymphocytes, an increase in IL-2 receptor expression, an increase in T cell responsiveness, an increase in natural killer cell activity or lymphocin-activated killer activity (LAK) and the like.
[0197] In a further aspect, the invention provides the use of a mutant IL-2 polypeptide or an immunoconjugate of the invention in the manufacture or preparation of a medicament for the treatment of a disease in an individual who needs them. In one embodiment, the drug is for use in a method for treating a disease which comprises administering to a subject who has the disease a therapeutically effective amount of the drug. In certain embodiments, the disease to be treated is a proliferative disorder. In a preferred embodiment, the disease is cancer. In such an embodiment, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, for example, an anti-cancer agent if the disease to be treated is cancer. In an additional embodiment, the drug is for stimulating the immune system. In a further embodiment, the drug is for use in a method of stimulating the immune system in an individual which comprises administering to the individual an effective amount of the drug to stimulate the immune system. An "individual", according to any of the above embodiments, may be a mammal, preferably a human. “Stimulating the immune system”, according to any of the above achievements, may include any one or more of a general increase in immune function, an increase in T cell function, an increase in B cell function, a restoration of function of lymphocytes, an increase in IL-2 receptor expression, an increase in T cell responsiveness, an increase in natural killer cell activity or lymphokine-activated killer cell activity (LAK) and the like.
[0198] In a further aspect, the invention provides a method for treating a disease in an individual, which comprises administering to said individual a therapeutically effective amount of a mutant IL-2 polypeptide or an immunoconjugate of the invention. In one embodiment, the composition is administered to said individual, which comprises the mutant IL-2 polypeptide or the immunoconjugate of the invention in a pharmaceutically acceptable form. In certain embodiments, the disease to be treated is a proliferative disorder. In a preferred 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, for example, an anti-cancer agent, if the disease to be treated is cancer. In a further aspect, the invention provides a method for stimulating the immune system in an individual, which comprises administering to the individual an effective amount of a mutant IL-2 polypeptide or an immunoconjugate to stimulate the immune system. An "individual", according to any of the above embodiments, can be a mammal, preferably a human. “Stimulating the immune system”, according to any of the above achievements, can include any one or more of a general increase in immune function, an increase in T cell function, an increase in B cell function, a restoration of function of lymphocytes, an increase in IL-2 receptor expression, an increase in T cell responsiveness, an increase in natural killer cell activity or lymphocin-activated killer activity (LAK) and the like.
[0199] It is understood that any of the above therapeutic methods can be performed using an immunoconjugate of the invention in place, or in addition to a mutant IL-2 polypeptide.
[0200] In certain embodiments, the disease to be treated is a proliferative disorder, preferably cancer. Non-limiting examples of cancers 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, cancer of the colon, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. other cell proliferation disorders that can be treated with the use of a mutant IL-2 polypeptide or an immunoconjugate of the present invention include, but are not limited to, neoplasms located in: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovaries, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic, pelvic, skin, soft tissue, spleen, thoracic region, and urogenital system. Also included are precancerous conditions or cancer lesions and metastases. In certain embodiments, cancer is chosen from the group consisting of kidney cell cancer, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer. Similarly, other cell proliferation disorders can also be treated by the mutant and immunoconjugate IL-2 Polypeptides of the present invention. Examples of such cell proliferation disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinamias, purpura, sarcoidosis, Sézary's syndrome, Waldenstrom's macroglobulinemia, Gaucher disease, histiocytosis, and any other cell proliferative disease, neoplasm, located in an organ system listed above. In another embodiment, the disease is related to autoimmunity, transplant rejection, post-traumatic immune responses and infectious diseases (for example, HIV). More specifically, mutant and immunoconjugate IL-2 polypeptides can be used to eliminate cells involved in immune cell-mediated disorders, which include lymphoma; autoimmunity, transplant rejection, graft versus host disease, ischemia and stroke. One skilled in the art readily recognizes that in many cases, mutant IL-2 or immunoconjugate polypeptides may not provide a cure, but may only provide partial benefit. In some embodiments, a physiological change that has some benefit is also considered to be therapeutically beneficial. Thus, in some embodiments, an amount of mutant or immunoconjugate IL-2 polypeptide 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 specifically a human.
[0201] The immunoconjugates of the invention are also useful as diagnostic reagents. The binding of an immunoconjugate to a determinant antigen can be readily detected by the use of a secondary antibody specific for the IL-2 polypeptide. In one embodiment, the secondary antibody and the immunoconjugate facilitate the detection of binding of the immunoconjugate to a determinant antigen located on a cell or tissue surface.
[0202] In some embodiments, an effective amount of the mutant or immunoconjugate IL-2 polypeptides of the invention is administered to a cell. In other embodiments, a therapeutically effective amount of the mutant or immunoconjugate IL-2 polypeptides of the invention is administered to an individual for the treatment of disease.
[0203] For the prevention or treatment of disease, the appropriate dosage of a mutant or immunoconjugate IL-2 polypeptide of the invention (when used alone or in combination with one or more other additional therapeutic agent) will depend on the type of disease to be treated , the route of administration, the patient's body weight, the type of polypeptide (for example, unconjugated or immunoconjugated IL-2), the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, prior therapeutic interventions or competitors, the patient's medical history and response to the mutant or immunoconjugate IL-2 polypeptide, and the discretion of the attending physician. The physician responsible for administration, in any event, will determine the concentration of active ingredient (s) in a composition and dose (s) appropriate for the individual. Various dosage schedules that include, but are not limited to, single or multiple administrations at various time points, bolus administration, and pulse infusion are contemplated herein.
[0204] A single administration of unconjugated IL-2 can be in the range of about 50,000 IU / kg to about 1,000,000 IU / kg or more, more typically, about 600,000 IU / kg of IL-2. This can be repeated several times a day (for example, 2 to 3 x), for several days (for example, about 3 to 5 consecutive days) and then it can be repeated one or more times after a rest period (for example, example, about 7 to 14 days). Thus, a therapeutically effective amount may comprise only a single administration or many administrations over a period of time (for example, about 20 to 30 individual administrations of about 600,000 liters / kg of IL-2 each given for about one 10 to 20 days). When administered as an immunoconjugate, the therapeutic efficacy of the mutant IL-2 polypeptide may be less than that for unconjugated mutant IL-2 polypeptide.
[0205] Similarly, the immunoconjugate is properly administered to the patient at a time or in a series of treatments. Depending on the type and severity of the disease, about 1 pg / kg to 15 mg / kg (eg 0.1 mg / kg to 10 mg / kg) of immunoconjugate can be an initial candidate dosage for administration to the patient, if, for example, through one or more separate administrations, or by continuous infusion. A typical daily dosage can be in the range of about 1 pg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations for several days or more, depending on the condition, treatment will generally be continued until a desired suppression of disease symptoms occurs. An exemplary dosage of the immunoconjugate can be in the range of about 0.005 mg / kg to about 10 mg / kg. In other non-limiting examples, a dose may also comprise about 1 microgram / kg / body weight, about 5 microgram / kg / body weight, about 10 microgram / kg / body weight, about 50 microgram / kg / weight body weight, about 100 microgram / kg / body weight, about 200 microgram / kg / body weight, about 350 microgram / kg / body weight, about 500 microgram / kg / body weight, about 1 milligram / kg / weight body weight, about 5 milligram / kg / body weight, about 10 milligram / kg / body weight, about 50 milligram / kg / body weight, about 100 milligram / kg / body weight, about 200 milligram / kg / weight body weight, about 350 milligram / kg / body weight, about 500 milligram / kg / body weight, at about 1000 mg / kg / body weight or more per administration, and any of the derivable range between them. In non-limiting examples of a derivable in the range of numbers listed in this document, a range from about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 microgram / kg / body weight about 500 milligrams / kg / body weight, etc., can be administered based on the numbers described above. In this way, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 5, mg / kg or 10 mg / kg (or any combination thereof) can be administered to the patient. Such doses can be administered intermittently, for example, every week or every three weeks (for example, so that the patient receives from about two to about twenty, or for example, about six doses of the immunoconjugate). A larger initial loading dose, followed by one or more smaller doses, can be administered. However, other dosage regimens can be useful. The progress of this therapy is easily monitored by conventional techniques and trials.
[0206] The mutant and immunoconjugate IL-2 polypeptides of the invention will generally be used in an amount effective to achieve the intended purpose. For use in the treatment or prevention of a disease condition, the mutant and immunoconjugate IL-2 polypeptides of the invention, or pharmaceutical compositions thereof, are administered or applied in a therapeutically effective amount. The determination of a therapeutically effective amount is well within the capabilities of elements skilled in the art, especially in light of the detailed disclosure provided in this document.
[0207] For systemic administration, a therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays. A dose can then be formulated in animal models to achieve a circulation concentration in the range that includes ICso as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
[0208] Initial dosages can also be estimated from in vivo data, for example, animal models, using techniques that are well known. One skilled in the art can readily improve administration to humans based on animal data.
[0209] The amount and dosage range can be individually adjusted to provide plasma levels of the mutant or immunoconjugate IL-2 polypeptides that are sufficient to maintain therapeutic effect. Common patient dosages for administration by injection in the range of about 0.1 to 50 mg / kg / day, typically about 0.5 to 1 mg / kg / day. Therapeutically effective plasma levels can be achieved by administering multiple doses each day. Plasma levels can be measured, for example, by HPLC.
[0210] In cases of local administration or selective absorption, the effective local concentration of the immunoconjugates may not be related to the plasma concentration. Elements skilled in the art may improve therapeutically effective local dosages without undue experimentation.
[0211] A therapeutically effective dose of the mutant or immunoconjugate IL-2 polypeptides described herein will, in general, provide therapeutic benefit without causing substantial toxicity. The toxicity and therapeutic efficacy of a mutant or immunoconjugate IL-2 can be determined by standard pharmaceutical procedures in cell culture or experimental animals (see, for example, Examples 8 and 9). Cell culture assays and animal studies can be used to determine LDso (the lethal dose for 50% of a population) and EDso (the therapeutically effective dose in 50% of a population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the LDso / EDso ratio. Mutant and immunoconjugate IL-2 exhibiting high therapeutic indexes are preferred. In one embodiment, the mutant IL-2 polypeptide or the immunoconjugate according to the present invention exhibits a high therapeutic index. The data obtained from cell culture assays and animal studies can be used to formulate a dosage range suitable for use in humans. The dosage preferably fits into a range of circulation concentrations that includes EDso with little or no toxicity. The dosage can vary in this range depending on a variety of factors, for example, the dosage form used, the route of administration used, the condition of the individual and the like. The exact formulation, route of administration and dosage can be chosen by each doctor in view of the patient's condition. (See, for example, Fingi et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1, page 1, the entirety of which is hereby incorporated by reference).
[0212] The physician responsible for patients treated with mutant or immunoconjugate IL-2 of the invention would know how and when to terminate, discontinue, or adjust administration due to toxicity, organ dysfunction, and the like. On the other hand, the doctor in charge would also know how to adjust treatment to higher levels if the clinical response is not adequate (excluding toxicity). The magnitude of a dosage administered to manage the disorder of interest will vary with the severity of the condition being treated, the route of administration, and the like. The severity of the condition can, for example, be assessed, in part, by standard prognosis assessment methods. In addition, the dosage and perhaps the dosing frequency will also vary depending on the age, body weight, and response of the individual patient.
[0213] The maximum therapeutic dosage of a mutant or immunoconjugate IL-2 polypeptide comprising said polypeptide can be increased from that used for wild-type IL-2 or an immunoconjugate comprising wild-type IL-2, respectively. OTHER AGENTS AND TREATMENTS
[0214] The mutant IL-2 polypeptides and the immunoconjugates according to the invention can be administered in combination with one or more other agents in therapy. For example, a mutant or immunoconjugate IL-2 polypeptide of the invention can be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any agent administered to treat a symptom or disease in an individual in need of such treatment. Such an additional therapeutic agent may comprise any active ingredients suitable for the particular indication being treated, preferably those with complementary activities that do not. adversely affect one another. In certain embodiments, an additional therapeutic agent is an immunomodulatory agent, a cytostatic agent, a cell adhesion inhibitor, a cytotoxic agent, a cell apoptosis activator, or an agent that increases the sensitivity of cells to inducers In a particular embodiment, the additional therapeutic agent is an anticancer agent, for example, a microtubular disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA interleaver, an alkylating agent, a hormone therapy, a kinase inhibitor, an receptor antagonist, a tumor cell apoptosis activator, or an agent and antiangiogenic.
[0215] Such other agents are suitably present in combination in amounts that are effective for the intended purpose. The effective amount of such other agents depends on the amount of mutant or immunoconjugate IL-2 polypeptide used, the type of disorder or treatment, and other factors discussed above. The mutant and immunoconjugate IL-2 polypeptides are generally used in the same dosages and with administration routes as described in this document, or about 1 to 99% of the dosages described in this document, or in any dosage and by any route that is determined, clinically / empirically, to be appropriate.
[0216] Such combination therapies noted above encompass combined administration (wherein two or more therapeutic agents are included in the same separate compositions or compositions), and separate administration, in which case, administration of the mutant IL-2 polypeptide or The immunoconjugate of the invention can occur before, simultaneously and / or after, the administration of the additional therapeutic agent and / or adjuvant. Mutant IL-2 polypeptides and immunoconjugates of the invention can also be used in combination with radiation therapy. MANUFACTURING ITEMS
[0217] In another aspect of the invention, an article of manufacture containing materials useful for the treatment, prevention and / or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package inserted in or associated with the container. Suitable containers include, for example, pots, vials, syringes, IV solution bags, etc. Containers can be formed from a variety of materials such as glass or plastic. The container holds a composition that is alone or combined with another composition effective to treat, prevent and / or diagnose the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial that has a piercable cap with a hypodermic injection needle). At least one active agent in the composition is a mutant IL-2 polypeptide of the invention. The label or package insert indicates that the composition is used to treat the condition of choice. Furthermore, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises a mutant IL-2 polypeptide of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises an additional cytotoxic agent or otherwise therapeutic agent. The article of manufacture in that embodiment of the invention may further comprise a package insert which indicates that the compositions can be used to treat a particular condition. Alternatively or additionally, the article of manufacture may additionally comprise a second container (or third) comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution. It may additionally include other materials desirable from a user and commercial point of view, including other buffers, thinners, filters, needles and syringes.
[0218] It is understood that any of the articles of manufacture above can include an immunoconjugate of the invention in place of or in addition to a mutant IL-2 polypeptide. BRIEF DESCRIPTION OF THE FIGURES
[0219] Figure 1. Schematic representation of the immunoconjugate formats of Fab-IL-2-Fab (A) and IgG-IL-2 (B), which comprises mutant IL-2 polypeptide.
[0220] Figure 2. Purification of the naked IL-2 wild type construct. (A) Chromatogram of His tag purification for naked wild-type IL-2; (B) SDS PAGE of purified protein (8 to 12% Bis-Tris (NuPage, Invitrogen), buffer running MES).
[0221] Figure 3. Purification of the naked IL-2 wild type construct. (A) Chromatogram of the size exclusion chromatography for wild-type IL-2; (B) SDS PAGE of purified protein (8 to 12% Bis-Tris (NuPage, Invitrogen), buffer running MES).
[0222] Figure 4. Analytical size exclusion chromatography for wild-type IL-2 as determined on a Superdex 75, 10/300 GL. Cluster 1 comprises 74% of the 23 kDa species and 26% of the 20 kDa species. Cluster 2 comprises 40% of the 22 kDa species and 60% of the 20 kDa species.
[0223] Figure 5. Purification of the naked mutant quadruple IL-2 construct. (A) Chromatogram of His tag purification for mutant quadruple IL-2; (B) SDS PAGE of purified protein (8 to 12% Bis-Tris (NuPage, Invitrogen), buffer running MES).
[0224] Figure 6. Purification of the nu mutant quadruple IL-2 construct. (A) Chromatogram of the size exclusion chromatography for the mutant quadruple IL-2; (B) SDS PAGE of purified protein (8 to 12% Bis-Tris (NuPage, Invitrogen), buffer running MES).
[0225] Figure 7. Analytical size exclusion chromatography for the quadruple mutant IL-2 as determined in a Superdex 75, 10/300 GL (Cluster 2, 20 kDa).
[0226] Figure 8. Simultaneous binding to IL-2R and human FAP by Fab-IL-2-Fab based on 29B11 targeted by FAP comprising mutant or wild-type quadruple IL-2. (A) Configuration of the SPR test; (B) SPR sensogram.
[0227] Figure 9. Induction of IFN-y release by NK92 cells via FAP-targeted 4G8-based Fab-IL-2-Fab comprising mutant or wild-type IL-2, compared to Proleucine, in the solution .
[0228] Figure 10. Induction of isolated NK cell proliferation (background) by Fab-IL-2-Fab based on 4G8 targeted by FAP comprising mutant or wild-type IL-2, compared to Proleucine, in the solution.
[0229] Figure 11. Induction of proliferation of CD3 + T cells activated by Fab-IL-2-Fab based on 4G8 targeted by FAP comprising mutant or wild-type IL-2, compared to Proleucine, in the solution.
[0230] Figure 12. Induction of cell death induced by activation (AICD) of T-cells overstimulated by Fab-IL-2-Fab based on 4G8 targeted by FAP comprising mutant or wild-type IL-2, compared to Proleucine, in the solution.
[0231] Figure 13. Phospho-STAT5 FACS assay in solution with Fab-IL-2-Fab based on 4G8 targeted by FAP comprising mutant or wild-type quadruple IL-2, compared to Proleucine, in the solution. (A) regulatory T cells (CD4 + CD25 + FOXP3 +); (B) CD8 + T cells (CD3 + CD8 +); (C) CD4 + T cells (CD4 + CD25-CD127 +); (D) NK cells (CD3 'CD56 +).
[0232] Figure 14. Purification of Fab-IL-2qm-Fab immunoconjugate based on 28H1 targeted by FAP. (A) Elution profile of Protein G column. (B) Elution profile of Superdex 200 size exclusion column. (C) SDS-PAGE of 4 to 20% Novex Tris-Glycine of the final product with reduced sample and not reduced.
[0233] Figure 15. Purification of the Fab-IL-2qm-Fab immunoconjugate targeted by FAP based on 4G8. (A) Protein A. Column Elution Profile. (B) Superdex 200 size exclusion column Elution Profile. (C) NuPAGE Novex Bis-Tris Mini Gel (Invitrogen), MOPS running buffer of the final product with reduced and unreduced sample.
[0234] Figure 16. Purification of the Fab-IL-2QM-Fab immunoconjugate targeted by MHLG1 KV9 MCSP. (A) Protein A Column Elution Profile, B) Superdex 200 size exclusion column Elution Profile. C) NuPAGE Novex Bis-Tris Mini Gel, Invitrogen, MOPS running buffer of the final product with reduced sample and not reduced.
[0235] Figure 17. Target binding of Fab-IL-2-Fab constructs in human HEK293-FAP cells.
[0236] Figure 18. Target binding of Fab-IL-2-Fab constructs in human HEK293-FAP cells.
[0237] Figure 19. Binding specificity of Fab-IL-2-Fab constructs as determined in human HEK 293 DPPIV and cells transfected by HEK 293 control plasmid. The binding of a specific DPPIV antibody (CD26) is shown on the right.
[0238] Figure 20. Analysis of FAP internalization by linking Fab-IL-2-Fab constructs to FAP in GM05389 fibroblasts.
[0239] Figure 21. IL-2-induced IFN-y release through NK92 cells in the solution.
[0240] Figure 22. IL-2-induced IFN-y release through NK92 cells in the solution.
[0241] Figure 23. IL-2-induced proliferation of NK92 cells in the solution.
[0242] Figure 24. Evaluation of Fab-IL-2-Fab 28H1 clones vs. 29B11 vs. 4G8 in phosphorylation test of STAT5 with PBMCs in the solution. (A) NK cells (CD3'CD56 +); (B) CD8 + T cells (CD3 + CD8 +); (C) CD4 + T cells (CD3 + CD4 + CD25'CD127 +); (D) regulatory T cells (CD4 + CD25 + FOXP3 +).
[0243] Figure 25. Effectiveness of 4G8 Fab-IL-2 wt-Fab and 4G8 Fab-IL-2 qm-Fab immunoconjugates by FAP on human renal cell adenocarcinoma cell line ACHN.
[0244] Figure 26. Efficacy of 4G8 FAP-IL-2 qm-Fab and 28H1 Fab-IL-2 qm-Fab immunoconjugates by FAP in LLC1 of mouse Lewis lung carcinoma cell line.
[0245] Figure 27. Effectiveness of the 28H1 Fab-IL-2 wt-Fab and 28H1 Fab-IL-2 qm-Fab immunoconjugates by FAP in the LLC1 of mouse Lewis lung carcinoma cell line.
[0246] Figure 28. Low magnification (100x) of the lungs of mice treated with vehicle control (A) or 9 pg / g of IL-2 wt (B) or IL-2 qm (C). The lungs of the mice treated with 9 pg / g IL-2 wt show a vasocentric mononuclear infiltrate that moved into the alveolar spaces. Edema and bleeding is also present. Marginal infiltrate is seen in mice treated with IL-2 qm around a few vessels.
[0247] Figure 29. Upper (200x) enlargement of the lungs shown in Figure 28. The marginalization and infiltration of mononuclear cells in and around blood vessels is more severe in mice treated with IL-2 wt (A) than in mice treated with IL-2 qm (B and C).
[0248] Figure 30. Low magnification (100x) of the livers of mice treated with vehicle control (A) or 9 pg / g of IL-2 wt (B) or IL-2 qm (C). Vasocentric infiltration is seen in mice treated with IL-2 wt.
[0249] Figure 31. IFN-y secretion by NK92 cells by incubation with different wild-type (wt) and quadruple mutant (qm) preparations for 24 (A) or 48 hours (B).
[0250] Figure 32. Proliferation of NK92 cells by incubation with different wild-type (wt) and quadruple mutant (qm) preparations for 48 hours.
[0251] Figure 33. Proliferation of NK92 cells by incubation with different wild-type (wt) and quadruple mutant (qm) preparations for 48 hours.
[0252] Figure 34. Proliferation of NK cells by incubation with 28H1 IL-2 immunoconjugates targeted by different FAP or Proleucine for 4 (A), 5 (B) or 6 (C) days.
[0253] Figure 35. Proliferation of CD4 T cells by incubation with 28H1 IL-2 immunoconjugates targeted by different FAP or Proleucine for 4 (A), 5 (B) or 6 (C) days.
[0254] Figure 36. Proliferation of CD8 T cells by incubation with 28H1 IL-2 immunoconjugates targeted by different FAP or Proleucine for 4 (A), 5 (B) or 6 (C) days.
[0255] Figure 37. Proliferation of NK cells (A), CD4 T cells (B) and CD8 T cells (C) by incubation with different IL-2 immunoconjugates or Proleucine for 6 days.
[0256] Figure 38. STAT phosphorylation in NK cells (A), CD8 T cells (B), CD4 T cells (C) and regulatory T cells (D) after a 30 minute incubation with Proleucine, IL-2 type wild-type and quadruple mutant IL-2 produced on site.
[0257] Figure 39. STAT phosphorylation in NK cells (A), CD8 T cells (B), CD4 T cells (C) and regulatory T cells (D) after a 30 minute incubation with Proleucine, lgG-IL-2 which comprises wild-type IL-2 or IgG-IL-2 which comprises quadruple mutant IL-2.
[0258] Figure 40. Survival of Black 6 Mice after administration (once daily for seven days) of different dosages of IL-2 immunoconjugates comprising quadruple mutant or wild-type IL-2.
[0259] Figure 41. Serum concentrations of IL-2 immunoconjugates after a single iv administration of IgG-IL-2 constructs (A) targeted by FAP and (B) non-targeted comprising or wild-type IL-2 ( wt) or quadruple mutant (qm).
[0260] Figure 42. Serum concentrations of IL-2 immunoconjugates after a single iv administration of non-targeted Fab-IL-2-Fab constructs comprising either wild-type (wt) or quadruple mutant (qm) .
[0261] Figure 43. Purification of quadruple mutant IL-2. (A) ion chromatography of immobilized metal; (B) size exclusion chromatography; (C) SDS PAGE under non-reducing conditions (NuPAGE Novex Bis-Tris gel (Invitrogen), buffer running MES); (D) analytical size exclusion chromatography (Superdex 75 10/300 GL).
[0262] Figure 44. Proliferation of pre-activated CD8 (A) and CD4 (B) T cells after a six-day incubation with different IL-2 immunoconjugates.
[0263] Figure 45. Cell death induced by activation of CD3 + T cells after a six-day incubation with different IL-2 immunoconjugates and overnight treatment with anti-Fas antibody.
[0264] Figure 46. Purification of lgG-IL-2 quadruple mutant (qm) immunoconjugate based on 4G8 and targeted by FAP. A) Elution profile of the Protein A affinity stage chromatography. B) Elution profile of the size exclusion chromatography step. C) Analytical SDS-PAGE (NuPAGE Novex Bis-Tris Mini Gel, Invitrogen, MOPS running buffer) of the final product. D) Analytical size exclusion chromatography of the final product on a Superdex 200 column (97% monomer content).
[0265] Figure 47. Purification of IgG-IL-2 qm immunoconjugate based on 28H1 and targeted by FAP. A) Elution profile of the Protein A affinity stage chromatography. B) Elution profile of the size exclusion chromatography step. C) Analytical SDS-PAGE (reduced: NuPAGE Novex Bis-Tris Mini Gel, Invitrogen, MOPS running buffer; not reduced: NuPAGE Tris-Acetate, Invitrogen, running Tris-Acetate buffer) of the final product. D) Analytical size exclusion chromatography of the final product on a Superdex 200 column (100% monomer content).
[0266] Figure 48. Immunoconjugate of lgG-IL-2 qm based on 4G8 and targeted by FAP to human FAP expressed in stably transfected HEK 293 cells as measured by FACS, compared to the Fab-IL-2 qm construct -Fab corresponding.
[0267] Figure 49. Interferon (IFN) -y release in NK92 cells induced by 4Gg-IL-2 qm immunoconjugate based on 4G8 and targeted by FAP in the solution, compared to the Fab-IL-2 qm- construct. Fab based on 28H1.
[0268] Figure 50. Detection of phosphorylated STAT5 by FACS in different cell types after stimulation for 20 minutes with 4G8-based IgG-IL-2 qm immunoconjugate and targeted by FAP in the solution, compared to the Fab-IL- constructs 2-Fab and Fab-IL-2 qm-Fab based on 28H1 as well as Proleucine. A) NK cells (CD3'CD56 +); B) CD8 + T cells (CD3 + CD8 +); C) CD4 + T cells (CD3 + CD4 + CD25-CD127 +); D) regulatory T cells (CD4 + CD25 + FOXP3 +). EXAMPLES
[0269] The following are examples of methods and compositions of the invention. It is understood that several other achievements can be practiced, granting the general description provided above. EXAMPLE 1 GENERAL METHODS RECOMBINANT DNA TECHNIQUES
[0270] Standard methods were used to manipulate DNA as described in Sambrook et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions. General information regarding the nucleotide sequences of human immunoglobulin heavy and light chains is given in: Kabat, E.A. et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication No 91-3242. DNA SEQUENCING
[0271] DNA sequences were determined by double-stranded sequencing. GENE SYNTHESIS
[0272] The desired gene segments when required were either generated by PCR using appropriate models or were synthesized by Geneart AG (Regensburg, Germany) from synthetic oligonucleotides and PCR products by automated genetic synthesis. In cases where no exact genetic sequence was available, oligonucleotide primers were designed based on sequences from the closest homologs and the genes were isolated by RT-PCR from RNA originating from the appropriate tissue. The gene segments flanked by singularly restricted endonuclease cleavage sites were cloned into standard sequencing / cloning vectors. Plasmid DNA was purified from transformed bacteria and the concentration determined by UV spectroscopy. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing. The gene segments were designed with suitable restriction sites to allow subcloning in the respective expression vectors. All constructs were designed with a 5 'end DNA sequence that encodes a leader peptide that targets proteins for secretion in eukaryotic cells. SEQ ID NOs 263 to 273 provide leader peptides and polynucleotide sequences that encode them. PREPARATION OF FC SUBUNITY sr-IL-2R FUSIONS AND FC FUSION IL-2R SUBUNITY A
[0273] To study the binding affinity of IL-2 receptor, a tool was generated that allowed the expression of a heterodimeric IL-2 receptor; the β subunit of the IL-2 receptor was fused to an Fc molecule that was modified to heterodimerize (Fc (/ o / e)) (see SEQ ID NOs 274 and 275) using knob-into-holes ( Merchant et al., Nat Biotech. 16, 677 to 681 (1998)). The IL-2 receptor y subunit was then fused to the Fc (knob) variant (see SEQ ID NOs 276 and 277), which heterodimerized with Fc (/ O / e). The heterodimeric FC fusion protein was then used as a substrate to analyze the interaction of IL-2 / IL-2 receptor. The α-subunit of IL-2R was expressed as a monomeric chain with an AcTev dividing site and an Avi His tag (SEQ ID NOs 278 and 279). The respective IL-2R subunits were transiently expressed in HEK EBNA 293 with serum for the IL-2R βy subunit construct construct and without serum for the a subunit construct. The IL-2R βy subunit construct was purified on protein A (GE Healthcare), followed by size exclusion chromatography (GE Healthcare, Superdex 200). The α-subunit of IL-2R was purified by means of the His tag on a NiNTA column (Qiagen) followed by size exclusion chromatography (GE Healthcare, Superdex 75). PREPARATION OF IMMUNOCONJUGATES
[0274] Details on the preparation and purification of Fab-IL-2-Fab immunoconjugates, including the generation and affinity maturation of the antigen-binding chemical moieties can be found in the Examples attached to PCT Publication under N2 WO 2011/020783 , which is incorporated in this document as a reference in its entirety. As described therein, several FAP-targeted antigen binding domains were generated by phage display, including those designed 4G8, 3F2, 28H1, 29B11, 14B3, and 4B9 used in the following examples. Clone 28H1 is an affinity matured antibody based on parental clone 4G8, whereas clones 29B11, 14B3 and 4B9 are affinity matured antibodies based on parental clone 3F2. The antigen binding domain designated MHLG1 KV9 used in this document is targeted to MCSP.
[0275] The immunoconjugate sequences comprising wild-type IL-2 that have been used in the following examples can also be found in PCT Publication under N2 WO 2011/020783. The sequences corresponding to the immunoconjugates that comprise quadruple mutant IL-2 that were used in the following examples are: 4G8: SEQ ID NOs 211 and 233; 3F2: SEQ ID NOs 209 and 231; 28H1: SEQ ID NOs 219 and 233; 29B11: SEQ ID NOs 221 and 231; 14B3: SEQ ID NOs 229 and 231; 4B9: SEQ ID NOs 227 and 231; MHLG1-KV9: SEQ ID NOs 253 and 255. The DNA sequences were generated by classical molecular biology techniques and / or gene synthesis and subcloned into mammalian expression vectors (one for the light chain and one for the heavy chain / protein IL-2 fusion) under the control of an MPSV promoter and upstream of a synthetic polyA site, with each vector carrying an EBV OriP sequence. The immunoconjugates as applied in the examples below were produced by cotransfecting HEK293-EBNA cells in exponential growth with the mammalian expression vectors using calcium phosphate transfection. Alternatively, HEK293 cells growing in the suspension were transfected by polyethyleneimine (PEI) with the respective expression vectors. Alternatively, stably transfected CHO cell clusters or CHO cell clones were used for production in serum free medium. While FAP-targeted and 4G8-based Fab-IL-2 Fab constructs comprising mutant (quadruple) or wild-type IL-2 can be purified by affinity chromatography using a protein A matrix, Fab-IL-2-Fab targeted by FAP and based on affinity ripened 28H1 were purified by affinity chromatography on a small scale G protein matrix.
[0276] Briefly, 28H1 Fab-IL-2-Fab targeted by FAP, which comprises mutant (quadruple) or wild-type IL-2, was purified from cell supernatants by an affinity step (protein G) followed by chromatography size exclusion (Superdex 200, GE Healthcare). The protein G column was equilibrated in 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5, supernatant was loaded, and the column was washed with 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5. Fab-IL-2-Fab was eluted with 8.8 mM formic acid pH 3. The eluted fractions were pooled and polished by size exclusion chromatography in the final formulation buffer: 25 mM potassium phosphate, 125 mM chloride sodium, 100 mM glycine pH 6.7. The exemplary and analytical results of the purification are given below.
[0277] 3F2 Fab-IL-2-Fab or 4G8 Fab-IL-2-Fab targeted by FAP, comprising mutant (quadruple) or wild-type IL-2, were purified by a similar method consisting of an affinity step with the use of protein A followed by size exclusion chromatography (Superdex 200, GE Healthcare). The protein A column was equilibrated in 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5, supernatant was loaded, and the column was washed with 20 mM sodium phosphate, 20 mM sodium citrate, 500 mM sodium chloride pH 7.5, followed by a wash with 13.3 mM sodium phosphate, 20 mM sodium citrate, 500 mM sodium chloride pH 5.45. A third wash with 10 mM MES, 50 mM sodium chloride pH 5 was optionally performed. Fab-IL-2-Fab was eluted with 20 mM sodium citrate, 100 mM sodium chloride, 100 mM glycine, pH 3. The eluted fractions were pooled and polished by size exclusion chromatography in the final formulation buffer : 25 mM potassium phosphate, 125 mM sodium chloride, 100 mM glycine pH 6.7. The results of exemplary detailed purification procedures are given for constructs selected below.
[0278] Fg-targeted lgG-IL-2 fusion proteins were generated based on FAP antibodies 4G8, 4B9 and 28H1, in which a single quadruple mutant IL-2 (qm) was fused to the C-terminal of a heterodimeric heavy chain as shown in Figure 1B. Targeting to the tumor stroma when FAP is selectively expressed is achieved through the bivalent antibody Fab region (greed effect). Heterodimerization resulting in the presence of a single quadruple mutant IL-2 is achieved by the application of knob-inner-hole technology. In order to minimize the generation of homodimeric IgG-cytokine fusions, the cytokine was fused to the C-terminal (with deletion of the C-terminal Lys residue) of the IgG heavy chain containing knob by means of a G4- (SG4 ligand ) 2 or (G4S) 3. The antibody-cytokine fusion has IgG-like properties. To reduce FcyR effector / binding function and avoid FcR coactivation, P329G L234A L235A (LALA) mutations were introduced in the Fc domain. The sequences of these immunoconjugates are given in SEQ ID NOs 297, 299 and 233 (28H1), SEQ ID NOs 301, 303 and 231 (4B9), and SEQ ID NOs 315, 317 and 233 (4G8)). In addition, a CEA-targeted IgG-IL-2 fusion protein and a DP47GS-targeted IgG-IL-2 fusion protein in which IgG does not bind to a specified target was generated. The sequences of these immunoconjugates are given in SEQ ID NOs 305, 307 and 309 (DP47GS), and SEQ ID NOs 309, 321 and 323 (CH1A1A).
[0279] The lgG-IL-2 constructs were generated by transient expression in HEK293 EBNA cells and purified essentially as described above for the Fab-IL-2-Fab constructs. Briefly, IgG-IL-2 fusion proteins were purified by a protein A affinity step (HiTrap ProtA, GE Healthcare) equilibrated in 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5. After loading the supernatant, the column was first washed with 20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5 and subsequently washed with 13.3 mM sodium phosphate, 20 mM sodium citrate, 500 mM sodium chloride, pH 5.45. The IgG-cytokine fusion protein was eluted with 20 mM sodium citrate, 100 mM sodium chloride, 100 mM glycine, pH 3. The fractions were neutralized and pooled and purified by size exclusion chromatography (HiLoad 16 / 60 Superdex 200, GE Healthcare) in final formulation buffer: 25 mM potassium phosphate, 125 mM sodium chloride, 100 mM glycine pH 6.7. The exemplary detailed purification results and procedures are given for constructs selected below. The protein concentration of purified protein samples was determined by measuring the optical density (OD) at 280 nm, using the extinction coefficient calculated based on the amino acid sequence. The purity and molecular weight of immunoconjugates were analyzed by SDS-PAGE in the presence and absence of a reducing agent (5 mM 1,4-dithiothreitol) and axes with Coomassie blue (SimpleBlue ™ SafeStain, Invitrogen). The NuPAGE® precast gel system (Invitrogen) was used according to the manufacturer's instructions (4 to 20% Tris-glycine gels or 3 to 12% Bis-Tris). The aggregate content of immunoconjugate samples was analyzed using a Superdex 200 10 / 300GL analytical size exclusion column (GE Healthcare) in 2 mM MOPS, 150 mM NaCI, 0.02% NaNs, pH 7 , 3 and buffer running at 25 ° C. FAP BINDING AFFINITY
[0280] The FAP binding activity of the cleaved Fab fragments used in these examples as chemical portions of antigen binding was determined by surface plasmon resonance (SPR) in a Biacore machine. Soon, an anti-His antibody (Penta-His, Qiagen 34660) was immobilized on CM5 chips to capture 10 nM of cinomolgus, neurin or human FAP-His (20 s). The temperature was 25 ° C and HBS-EP was used as a buffer. The analyte concentration of Fab was 100 nM to 0.41 nM (duplicates) at a flow rate of 50 pl / minute (association: 300 s, dissociation: 600 s (4B9, 14B3, 29B11, 3F2) or 1200 s ( 28H1, 4G8), regeneration: 60 s 10 mM glycine pH 2). The fitting was performed in a 1: 1 connection model, IR = 0, Rmax = local (due to the capture format). Table 2 provides the monovalent affinities as determined by SPR. TABLE 2. AFFINITY (KD) OF FAB FRAGMENTS TARGETED BY FAP FOR FAP AS DETERMINED BY SPR.
BIOLOGICAL ACTIVITY TESTS WITH TARGETED IL-2 IMMUNOCONJUGATES
[0281] The biological activity of Fab-IL-2-Fab Immunoconjugates targeted by FAP or MCSP and IgG-IL-2 Immunoconjugates targeted by FAP, comprising mutant (quadruple) or wild-type IL-2, has been investigated in several cellular assays compared to commercially available IL-2 (Proleucine, Novartis, Chiron). IFN-r RELEASE BY NK CELLS (IN SOLUTION)
[0282] IL-2-deprived NK92 cells (100,000 cells / 96-well U-plate well) were incubated with different concentrations of IL-2 immunoconjugates, which comprise mutant (quadruple) or wild-type IL-2 , for 24 hours in NK medium (Invitrogen alpha MEM (# 22561-021) supplemented with 10% FCS, 10% horse serum, 0.1 mM 2-mercaptoethanol, 0.2 mM inositol and 0 , 02 mM folic acid). Supernatants were harvested and IFN-y release was analyzed using Becton Dickinson's human anti-IFN-y ELISA II Kit (# 550612). Proleucine (Novartis) served as a positive control for IL-2 mediated activation of cells. NK CELL PROLIFERATION
[0283] The blood of healthy volunteers was taken with syringes containing heparin and PBMCs were isolated. Untouched human NK cells were isolated from PBMCs using Miltenyi Biotec's NK II Cell Isolation Kit (# 130-091-152). The CD25 expression of the cells was verified by flow cytometry. For proliferation assays, 20,000 isolated human NK cells were incubated for 2 days in a humidified incubator at 37 ° C, 5% CO2 in the presence of different IL-2 immunoconjugates, comprising mutant (quadruple) or type IL-2 wild. Proleucine (Novartis) served as a control. After 2 days, the ATP content of cell lysates was measured using the Celegaiter Glo Cell Viability Assay (# G7571 / 2/3). The percentage of growth was calculated by adjusting the highest concentration of Proleucine to 100% proliferation and untreated cells without IL-2 stimulation to 0% proliferation. STAT5 PHOSPHORILATION TEST
[0284] The blood of healthy volunteers was taken with syringes containing heparin and PBMCs were isolated. PBMCs were treated with IL-2 immunoconjugates, which comprise mutant (quadruple) or wild-type IL-2, at the indicated concentrations or with Proleucine (Novartis) as a control. After incubating at 20 to 37 ° C, the PBMCs were fixed with pre-heated Cytofix buffer (Becton Dickinson # 554655) for 10 minutes at 37 ° C, followed by permeabilization with Phosflow Perm Buffer III (Becton Dickinson # 558050) for 30 minutes at 4 ° C. The cells were washed twice with PBS containing 0.1% BSA before FACS staining was performed using flow cytometric antibody mixtures for the detection of different cell populations and STAT5 phosphorylation. The samples were analyzed using a FACSCantoll with Becton Dickinson's HTS.
[0285] NK cells were defined as CD3'CD56 +, CD8 positive T cells were defined as CD3 + CD8 +, CD4 positive T cells were defined as CD4 + CD25CD127 + and Treg cells were defined as CD4 + CD25 + FoxP3 +. T-CELL PROLIFERATION AND AICD
[0286] The blood of healthy volunteers was taken with syringes containing heparin and PBMCs were isolated. Untouched human T cells were isolated using the Miltenyi Biotec T Cell Isolation Kit II (# 130-091-156). T cells were pre-stimulated with 1 pg / ml PHA-M (Sigma Aldrich # L8902) for 16 before adding Proleucine or Fab-IL-2-Fab immunoconjugates, which comprise mutant (quadruple) IL-2 or wild type, for cells washed for another 5 days. After 5 days, the ATP content of cell lysates was measured using the Promega Celesciter-GIo Luminescence Cell Viability Assay (# G7571 / 2/3). Relative proliferation was calculated by adjusting the highest concentration of Proleucine to 100% proliferation.
[0287] Exposure to phosphatidylserine (PS) and cell death of T cells were assayed by flow cytometric analysis (FACSCantoll, BD Biosciences) of annexin V (Anexin-V-FLUOS staining kit, Roche Applied Science) and stained cells by propidium iodide (PI). To induce activation-induced cell death (AICD), T cells were treated with an anti-Fas antibody that induces apoptosis (Millipore clone Ch11) for 16 hours after 16 hours of PHA-M and 5 days of treatment with Fab- IL-2 Fab. Anexin V staining was performed according to the manufacturer's instructions. The cells were briefly washed with Ann-V binding buffer (1x stock: 0.01 M Hepes / NaOH pH 7.4, 0.14 M NaCI, 2.5 mM CaCl2) and stained for 15 minutes in RT in the dark with Annexin V FITC (Roche). The cells were washed again in Ann-V binding buffer before adding 200 µl / well of Ann-V binding buffer containing PI (0.3 pg / ml). The cells were analyzed immediately by flow cytometry. CONNECTION TO FAP EXPRESSING CELLS
[0288] The binding of IgG-IL-2 qm and Fab-IL-2 qm-Fab targeted immunoconjugates to human FAP expressed in stably transfected HEK293 cells was measured by FACS. Shortly, 250,000 cells per well were incubated with the indicated concentration of immunoconjugates in a 96-well rounded-bottom plate, incubated for 30 minutes at 4 ° C, and washed once with PBS / 0.1% BSA. Bound immunoconjugates were detected after incubation for 30 minutes at 4 ° C with specific FITC-conjugated goat anti-human F (ab ') 2 F (ab') 2 fragment (Jackson Immuno Research Lab # 109- 096-097, working solution: 1:20 diluted in PBS / 0.1% BSA, freshly prepared) using a FACS Cantoll (FACS Diva Software). FAP INTERNALIZATION ANALYSIS BY FACS CONNECTION
[0289] For several FAP antibodies known in the art it is described that they induce the internalization of FAP by binding (described, for example, in Baum et al., J Drug Target 15, 399 to 406 (2007); Bauer et al., Journal of Clinical Oncology, 2010 ASCO Annual Meeting Proceedings (PostMeeting Edition), vol. 28 (Complement of May 20), Abstract N2 13062 (2010); Ostermann et al., Clin Cancer Res 14, 4,584 to 4,592 (2008)) . Therefore, we analyzed the internalization properties of our Fab-IL-2-Fab immunoconjugates. Briefly, GM05389 cells (human lung firoblasts) cultured in EMEM medium with 15% FCS, were detached, washed, counted, checked for viability and seeded at a density of 2x105 cells / well in 12-well plates . The next day, FAP-targeted Fab-IL-2-Fab immunoconjugates were diluted in a cold medium and allowed to bind to the cell surface for 30 minutes on ice. The excess unbound antibody was washed using cold PBS and the cells were additionally incubated in 0.5 ml of complete preheated medium at 37 ° C for the indicated periods of time. When the different time points were reached, the cells were transferred on ice, washed once with cold PBS and incubated with AffiniPure-conjugated F (ab ') 2 goat anti-human (F (ab') 2) conjugated by Secondary antibody specific FITC, Jackson Immuno Research Lab # 109-096-097, 1:20 dilution) for 30 minutes at 4 ° C. The cells were then washed twice with PBS / 0.1% BSA, transferred to a 96 well plate, centrifuged for 4 minutes at 4 ° C, 400 x g and the cell pellets were vortexed. The cells were fixed using 100 µl of 2% PFA. For FACS measurement, the cells were resuspended in 200 pl / sample of PBS / 0.1% BSA and measured with the plate protocol in FACS Cantoll (FACS Diva Software). EXAMPLE 2
[0290] Mutated versions of IL-2 were designed that comprised one or more of the following mutations (compared to the wild-type IL-2 sequence shown below in SEQ ID NO: 1): 1. T3A - O site knockout predicted glycosylation 2. F42A - IL-2 / IL-2R α interaction knockout 3. Y45A - IL-2 / IL-2R α interaction knockout 4. L72G - IL-2 / IL- α interaction knockout 2R 5. C125A - mutation described previously to avoid disulfide-connected IL-2 dimers
[0291] A mutant IL-2 polypeptide comprising all of f 1 through 4 is denoted herein as quadruple mutant IL-2 (qm). It can additionally comprise mutation 5 (see SEQ ID NO: 19).
[0292] In addition to the three F42A, Y45A and L72G mutations designed to interfere with binding to CD25, the T3A mutation was chosen to eliminate the O-glycosylation site and obtain a protein product with superior homogeneity and purity when the polypeptide or immunoconjugate IL-2 qm is expressed in eukaryotic cells such as CHO or HEK293 cells.
[0293] For purification purposes a His6 tag was introduced at the C-terminal linked via a VD sequence. For comparison, a non-mutated analog version of IL-2 was generated that only contained the C145A mutation to prevent unwanted intermolecular disulfide connections (SEQ ID NO: 3). The respective molecular weights without signal sequence was 16423 D for IL-2 nu and 16169 D for IL-2 qm nu. The mutant and wild-type quadruple IL-2 with His tag were transfected into HEK EBNA cells in serum-free medium (F17 medium). The filtered supernatant exchanged for buffer over a cross flow, before loading it into a Superflow Cartridge NiNTA (5 ml, Qiagen). The column was washed with washing buffer: 20 mM sodium phosphate, 0.5 M sodium chloride pH 7.4 and eluted with elution buffer: 20 mM sodium phosphate, 0.5 M sodium chloride 0 , 5 M imidazole pH 7.4. After loading the column was washed with 8 column volumes (CV) of wash buffer, 10 CV of 5% elution buffer (corresponds to 25 mM imidazole), then eluted with a gradient to 0.5 M imidazole . The pooled eluate was polished by size exclusion chromatography on a column of HiLoad 16/60 Superdex75 (GE Healthcare) in 2 mM MOPS, 150 mM sodium chloride, 0.02% sodium azide pH 7.3. Figure 2 shows the chromatogram of His tag purification for naked wild-type IL-2. Cluster 1 was made from fractions 78 to 85, cluster 2 from fractions 86 to 111. Figure 3 shows the chromatogram of the size exclusion chromatography for wild-type IL-2, for each cluster the fractions 12 to 14 were grouped. Figure 4 shows the analytical size exclusion chromatography for wild-type IL-2 as determined on a Superdex 75, 10/300 GL (GE Healthcare) column in 2 mM MOPS, 150 mM sodium chloride, 0, 02% sodium azide pH 7.3. Clusters 1 and 2 contained 2 proteins of ca. 22 and 20 kDa. Cluster 1 had more of the large protein, and cluster 2 had more of the small protein, putatively this difference is due to differences in O-glycosylation. The yields were ca. 0.5 mg / l supernatant for pool 1 and ca. 1.6 mg / l supernatant for pool 2. Figure 5 shows the chromatogram of the His tag purification for the mutant quadruple IL-2. Cluster 1 was made from fractions 59 to 91, cluster 2 from fractions 92 to 111. Figure 6 shows the chromatogram of the size exclusion chromatography for the mutant quadruple IL-2, here only fractions 12 to 14 from cluster 2 were maintained. Figure 7 shows the analytical size exclusion chromatography for the quadruple mutant IL-2 as determined on a Superdex 75, 10/300 GL (GE Healthcare) column in 2 mM MOPS, 150 mM sodium chloride, 0, 02% sodium azide pH 7.3. The preparation for the nu mutant quadruple IL-2 contained only a 20 kD protein. This protein has the knocked out O-glycosylation site. Aliquots of IL-2 wild-type and naked quadruple mutant were stored frozen at -80 ° C. The yields were ca 0.9 mg / l of supernatant.
[0294] A second batch of His-labeled mutant quadruple IL-2 was purified as described above by immobilized metal ion affinity chromatography (IMAC) and followed by size exclusion chromatography (SEC). The buffers used for IMAC were 50 mM Tris, 20 mM imidazole, 0.5 M NaCI pH 8 for balance and column washing, and 50 mM Tris, 0.5 M imidazole, 0.5 M NaCI pH 8 for elution. The buffer used for SEC and final formulation buffer was 20 mM histidine, 140 mM NaCI pH 6. Figure 43 shows the result of that purification. The yield was 2.3 ml / l of supernatant.
[0295] Subsequently, the affinity for the IL-2R βy heterodimer and the IL-2R α subunit were determined by surface plasmon resonance (SPR). Briefly, the ligand - or human IL-2R α subunit (Fc2) or human IL2-R (Fc3) hole y knob heterodimer - was immobilized on a CM5 chip. Subsequently, IL-2 wild-type or quadruple nude mutant (clusters 1 and 2), and Proleucine (Novartis / Chiron) were applied to the chip as analytes at 25 ° C in HBS-EP buffer in concentrations ranging from 300 nM up to 1.2 nM (1: 3 dil.). The flow rate was 30 pl / minute and the following conditions were applied by association: 180s, dissociation: 300s, and regeneration: 2 x 30s of 3M MgCl2 for hole y knob IL2-R heterodimer, 10s of 50 mM NaOH for the α-subunit of IL-2R. The 1: 1 connection was applied for fitting (1: 1 connection RI ^ O, Rmax = local for IL-2R βy, apparent KD, 1: 1 connection RI = 0, Rmax = local for IL-2R α) . Table 3 shows the respective KD values for the binding of quadruple mutant and human wild-type IL-2 as well as of Proleucine to IL-2R βy and α-subunit of IL-2R. TABLE 3. AFFINITY OF MUTANT IL-2 POLYPEPTIDES FOR IL-2R OF INTERMEDIATE AFFINITY AND THE SUBUNITY TO IL-2R

[0296] The data show that the naked mutant quadruple IL-2 shows the desired behavior and has lost the binding to the α-subunit of IL-2R in which binding to IL-2R βy is retained and comparable to the respective IL-2 construct of wild type and Proleucine. The differences between clusters 1 and 2 of wild-type IL-2 can probably be attributed to differences in O-glycosylation. This variability and heterogeneity was overcome in the quadruple mutant IL-2 by introducing the T23A mutation. EXAMPLE 3
[0297] The three F42A, Y45A and L72G mutations and the T3A mutation were introduced in the Fab-IL-2-Fab format (Figure 1 A) using the anti-FAP antibody 4G8 as a model that targets the domain or as mutants unique: 1) 4G8 IL-2 T3A, 2) 4G8 IL-2 F42A, 3) 4G8 IL-2 Y45A, 4) 4G8 IL-2 L72G, or they were combined in the Fab-IL-2 mt-Fab constructs as : 5) triple mutant F42A / Y45A / L72G, or as: 6) quadruple mutant T3A / F42A / Y45A / L72G to disable the 0-glycosylation site as a cavity. The 4G-based Fab-IL-2 wt-Fab served for comparison. All constructs contained the C145A mutation to avoid disulfide-linked IL-2 dimers. The different Fab-IL 2-Fab constructs were expressed in HEK 293 cells and purified as described above by protein A and size exclusion chromatography as specified above. Subsequently, the affinity of the selected IL 2 variants for the murine and human IL 2R βy heterodimer and for the murine and human IL-2R α subunit was determined by surface plasmon resonance (SPR) (Biacore) with the use of the monomeric IL-2R α subunit and recombinant IL-2R βy heterodimer under the following conditions: The IL-2R α subunit was immobilized at two densities and the upper immobilized flow cell was used for mutants that lost binding of CD25. The following conditions were used: chemical immobilization: human IL-2R βy heterodimer 1675 RU; 5094 RU mouse IL-2R βy heterodimer; α subunit of human IL-2R 1019 RU; α subunit of human IL-2R 385 RU, α subunit of murine IL-2R 1182 RU; murine IL-2R α subunit 378 RU, temperature: 25 ° C, analytes: Fab constructs from 4G8 Fab-IL2 variants 3.1 nM at 200 nM, flows 40 pl / minute, association: 180 s, dissociation: 180 s, regeneration: 10 mM glycine pH 1.5, 60 s, 40 pl / minute. Fit: two-state reaction model (conformational change), IR = 0 Rmax = local. Results of the kinetic analysis are given in Table 4. TABLE 4. AFFINITY OF FAP-TARGETED IMMUNOCONJUGATES THAT UNDERSTAND MUTANT IL-2R POLYPEEPTIDS FOR THE INTERMEDIATE AFFINITY IL-2R AND THE IL-2R SUBUNITY (Kp)

[0298] Simultaneous binding to IL 2R βy and FAP heterodimer was shown by SPR. Briefly, the human IL 2R βy knob-into-hole construct was chemically immobilized on a CM5 chip and 10 nM of Fab-IL-2-Fab constructs were captured for 90 s. Human FAP served as an analyte in concentrations of 200 nM to 0.2 nM. The conditions were: temperature: 25 ° C, buffer: HBS-EP, flow: 30 pl / minute, association: 90 s, dissociation: 120 s. Regeneration was performed for 60 s with 10 mM glycine pH 2. The fitting was performed with a model for 1: 1 binding, RI + 0, Rmax = global. The SPR connection assay showed that the Fab-IL-2-Fab constructs, both wild-type and quadruple mutant, as well as affinity-matured FH 28H1 linker or parental 3F2 or 4G8 antibodies, were able to bind at a concentration of 10 nM simultaneously to the IL 2R βy heterodimer immobilized on the chip as well as to human FAP used as an analyte (Figure 8). The affinities determined are shown in Table 5. TABLE 5. AFFINITY OF FAP-TARGETED IMMUNOCONJUGATES, WHICH UNDERSTAND MUTANT IL-2 POLYPEPTIDS AND BIND TO INTERMEDIATE AFFINITY IL-2R, FAP (Kp)

[0299] Taken together the SPR data showed that i) the T3A mutation does not influence the binding to CD25, ii) the three mutations F42A, Y45A and L72G do not influence the affinity for the IL 2R βy heterodimer while they reduce the affinity for CD25 in that order: wt = T3A> Y45A (ca. 5x lower)> L72G (ca. 10x lower)> F42A (ca. 33x lower); iii) the combination of the three mutations F42A, Y45A and L72G with or without the T3A mutant O-glycosylation site results in a complete loss of CD25 binding as determined under SPR conditions, iv) although the affinity of human IL-2 for murine IL-2R βy heterodimer and IL-2R α subunit is reduced by approximately a factor of 10 compared to human IL-2 receptors the selected mutations do not influence affinity for murine IL-2R βy heterodimer, but abolish binding to the murine IL-2R α subunit accordingly. This indicates that the mouse represents a valid model for studying toxicological and pharmacological effects of mutant IL-2, although in general IL-2 exhibits less toxicity in rodents than in humans.
[0300] In addition to the loss of O-glycosylation an additional advantage of the combination of the four mutations T3A, F42A, Y45A, L72G is a low surface hydrophobicity of the quadruple mutant IL-2 due to the exchange of hydrophobic residues exposed to the surface such as phenylalanine, tyrosine or leucine by alanine. An analysis of the aggregation temperature by dynamic light diffraction showed that the aggregation temperature for FAP-targeted Fab-IL-2-Fab immunoconjugates comprising mutant or wild-type quadruple IL-2 were in the same range: ca. 57 to 58 ° C for the parental 3F2 Fab-IL-2-Fab and for the affinity matured 29B11 3F2 derivative; and in the range of 62 to 63 ° C for parental Fab-IL-2- 4G8 and the affinity matured 28H1, 4B9 and 14B3 4G8 derivatives, which indicates that the combination of the four mutations had no negative impact on the stability of protein. In support of the favorable properties of the selected quadruple mutant IL-2, transient expression yields indicate that the Fab-IL-2 qm-Fab mutant quadruple can even result in higher expression yields than those observed for the respective constructs of Fab-IL-2 wt-Fab. Finally, pharmacokinetic analysis shows that both Fab-IL-2 qm-Fab and Fab-IL-2 wt-Fab based on 4G8 have comparable PK properties (see example 9 below). Based on these data and the cellular data described in example 4 below, the quadruple T3A, F42A, Y45A, L72G mutant was selected as an ideal combination of mutations to abolish IL-2 CD25 binding in the targeted Fab-IL-2-Fab immunoconjugate . EXAMPLE 4
[0301] FAP-targeted and 4G8-based Fab-IL 2-Fab immunoconjugates, which comprise wild-type IL-2 or the only 4G8 IL-2 T3A, 4G8 IL-2 F42A, 4G8 IL-2 Y45A mutants, 4G8 IL-2 L72G or its triple (F42A / Y45A / L72G) or quadruple mutant (T3A / F42A / Y45A / L72G) IL-2, were subsequently tested in cell assays compared to Proleucine as described above.
[0302] IL-2-induced IFN-y release was measured after incubating the NK92 cell line with the constructs (Figure 9). NK92 cells express CD25 on their surfaces. The results show that the Fab-IL-2-Fab immunoconjugate comprising wild-type IL-2 was less potent in inducing IFN-γ release than Proleucine as could be expected from ca. 10-fold lower affinity of Fab-IL-2 wt-Fab for the IL-2R βy heterodimer. The introduction of single mutations that interfere with CD25 binding as well as the combination of the three mutations that interfere with CD25 binding in the triple mutant IL-2 resulted in Fab-IL-2-Fab constructs that were comparable to the IL construct -2 of the wild type in terms of potency and absolute induction of IFN-y release within the error of the method. TABLE 6. IFN-r RELEASE INDUCTION OF NK CELLS BY FAB-IL-2-FAB IMMUNOCONJUGATES THAT UNDERSTAND IL-2 POLYPEPTIDES


[0303] Subsequently, the induction of proliferation of human NK cells isolated by Fab-IL-2-Fab immunoconjugates was evaluated in a proliferation assay (Cell Titer Glo, Promega) (Figure 10). In contrast to NK92 cells, recently isolated NK cells do not express CD25 (or only very low amounts). The results show that the Fab-IL-2-Fab immunoconjugate comprising wild-type IL-2 was ca. 10 times less potent in inducing NK cell proliferation than Proleucine, as might be expected from ca. 10-fold lower affinity of the Fab-IL-2 wt-Fab immunoconjugate for the IL-2R βy heterodimer. The introduction of the single mutations that interfere with CD25 binding as well as the combination of the three mutations that interfere with CD25 in the triple mutant IL-2 resulted in Fab-IL-2-Fab constructs that were comparable to the IL-2 construct of wild type in terms of potency and absolute induction of proliferation; there was only a very small change in potency observed for the mutant Fab-IL-2-Fab triple. In a second experiment, the induction of proliferation of PHA-activated T cells was evaluated after incubation with different amounts of Proleucine and immunoconjugates of Fab-IL-2-Fab (Figure 11). As activated T cells express CD25, a marked reduction in T cell proliferation could be observed by incubation with immunoconjugates that comprise single IL-2 mutants F42A, L72G or Y45A; with F42A which shows the strongest reduction followed by L72G and Y45A, where when Fab-IL-2 wt-Fab or Fab-IL-2 (T3A) -Fab is used the activation was almost retained compared to Proleucine. These data reflect the reduction in affinity for CD25 as determined by SPR (example above). The combination of the three mutations that interfere with CD25 binding in the triple mutant IL-2 resulted in an immunoconjugate that mediated the significantly reduced induction of T cell proliferation in the solution. In line with these observations, we measured cell death of T cells as determined by Anexin V / PI staining after superstimulation induced by a first stimulus induced by a first stimulation for 16 hours with 1 pg / ml PHA, a second stimulation for 5 days with Proleucine or the respective Fab-IL-2-Fab immunoconjugates, followed by a third stimulation with 1 pg / ml PHA. In this configuration, we observed that activation-induced cell death (AICD) in over-stimulated T cells was reduced strongly with the Fab-IL-2-Fab immunoconjugates that comprise the unique IL-2 mutants F42A, L72G and Y45A that interfere with the binding of CD25, with F42A and L72G showing the strongest reduction, which was similar to the reduction achieved by combining the three mutations in the immunoconjugate comprising the triple mutant IL-2 (Figure 12). In a final set of experiments we studied the effects of Fab-IL-2 qm-Fab on the induction of STAT5 phosphorylation compared to Fab-IL-2 wt-Fab and Proleucine in human NK cells, CD4 + T cells, CD8 + T cells and Treg cells from human PBMCs (Figure 13). For NK cells and CD8 + T cells that do not show or show very low CD25 expression (meaning that IL-2R signaling is mediated through the IL-2R βy heterodimer) The results show that the Fab-IL- 2-Fab comprising wild-type IL-2 was ca. 10 times less potent in inducing STAT5 phosphorylation than Proleucine, and that Fab-IL-2 qm-Fab was comparable to the Fab-IL-2 wt-Fab construct. In CD4 + T cells, which show rapid upward regulation of CD25 by stimulation, Fab-IL-2 qm-Fab was less potent than the Fab-IL-2 wt-Fab immunoconjugate, but still showed comparable induction of IL signaling -2R in saturating concentrations. This is in contrast to Treg cells where the potency of Fab-IL-2 qm-Fab was significantly reduced compared to the immunoconjugate of Fab-IL-2 wt-Fab due to the high CD25 expression in Treg cells and the binding affinity subsequent elevation of the Fab-IL-2 wt-Fab immunoconjugate to CD25 in Treg cells. As a consequence of the abolition of CD25 binding in the Fab-IL-2 qm-Fab immunoconjugate, IL-2 signaling in Treg cells is only activated by means of the IL-2R βy heterodimer at concentrations in which IL-2R signaling it is activated in CD25 negative effector cells through the IL-2R βy heterodimer. Taken together, the quadruple mutant IL-2 described here has the ability to activate IL-2R signaling through the IL-2R βy heterodimer, but does not result in IACD in preferential stimulation of Treg cells over other effector cells. EXAMPLE 5
[0304] Based on the data described in examples 2 and 3 FAP-targeted Fab-IL-2 qm-Fab immunoconjugates matured based on clones 28H1 or 29B11 were generated and purified as described above in the general methods section. In more detail, the Fab-IL-2 qm-Fab targeted by 28H1 and targeted by FAP was purified by an affinity step (protein G) followed by size exclusion chromatography (Superdex 200). Column equilibration was performed in PBS and the supernatant from a stable CHO pool (CDCHO medium) was loaded onto a G protein column (GE Healthcare), the column was washed with PBS and the samples were subsequently eluted with 2, 5 mM HCI and the fractions were immediately neutralized with 10x PBS. Size exclusion chromatography was performed in the final formulation buffer: 25 mM sodium phosphate, 125 mM sodium chloride, 100 mM glycine pH 6.7 in a Superdex 200 column. Figure 14 shows the profiles of elution of the purification and the results of the analytical characterization of the product by SDS-PAGE (NuPAGE Novex Bis-Tris Mini Gel 4 at 20%, Invitrogen, MOPS running buffer, reduced and not reduced). Due to the low binding capacity of the 28H1 Fab fragment to protein G and additional protein A capture steps can result in higher yields.
[0305] Immunoconjugates of 4G8, 3F2 and 29B11 Fab-IL-2 qm-Fab targeted by FAP and MHLG1 KV9 Fab-IL-2 qm-Fab targeted by MCSP were purified by an affinity step (protein A) followed by chromatography size exclusion (Superdex 200). Column equilibration was performed in 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5 and supernatant was loaded onto protein A column. A first wash was performed in 20 mM sodium phosphate, 20 mM sodium sodium citrate, pH 7.5 followed by a second wash: 13.3 mM sodium phosphate, 20 mM sodium citrate, 500 mM sodium chloride, pH 5.45. The Fab-IL-2 qm-Fab immunoconjugates were eluted in 20 mM sodium citrate, 100 mM sodium chloride, 100 mM glycine pH 3. Size exclusion chromatography was performed on the final formulation buffer: 25 mM potassium phosphate, 125 mM sodium chloride, 100 mM glycine pH 6.7. Figure 15 shows the purification elution profiles and the results of the analytical characterization of the product by SDS-PAGE (NuPAGE Novex Bis-Tris Mini Gel 4 to 20%, Invitrogen, MOPS running buffer, reduced and not reduced) for 4G8 Fab-IL-2 qm-Fab and Figure 16 for the MHLG1 KV9 Fab-IL-2 qm-Fab immunoconjugate.
[0306] Fg-targeted lgG-IL-2 fusion proteins based on FAP antibodies 4G8, 4B9 and 28H1, and an IgG-IL-2 fusion protein qm not targeted by control DP47GS were generated as described above in the general method section. Figures 46 and 47 show the respective purification chromatograms and elution profiles (A, B) as well as the analytical SDS-PAGE and size exclusion chromatographies of the final purified constructs (C, D) for constructs based on 4G8 and 28H1 . The yields of transient expression were 42 mg / l for the 4G8-based IgG-IL-2 immunoconjugate and 20 mg / l for the 28H1-based IgG-IL-2 immunoconjugate.
[0307] The FAP binding activity of the IgG-IL-2 qm immunoconjugates based on anti-FAP 4G8 and 28H1 antibodies was determined by surface plasmon resonance (SPR) in a Biacore machine compared to unmodified IgG antibodies corresponding. Briefly, an anti-His antibody (Penta-His, Qiagen 34660) was immobilized on CM5 chips to capture 10 nM of His-labeled human FAP (20 s). The temperature was 25 ° C and HBS-EP was used as a buffer. The analyte concentration was 50 nM to 0.05 nM at a flow rate of 50 pl / minute (association: 300 s, dissociation: 900 s, regeneration: 60 s with 10 mM glycine pH 2). The fitting was performed based on a 1: 1 connection model, IR = 0, Rmax = local (due to the capture format). Table 7 provides the estimated apparent bivalent affinities (pM avidity) as determined by embedded SPR with 1: 1 binding RI = 0, Rmax = local. TABLE 7

[0308] The data show that within the error of the method the affinity for human FAP is retained for the immunoconjugate based on 28H1 or only slightly decreased for the immunoconjugate based on 4G8 compared to corresponding unmodified antibodies. EXAMPLE 6
[0309] The affinity of Fab-IL-2-Fab immunoconjugates based on 29B11 and 28H1 matured in affinity and targeted by FAP, each of which comprises quadruple mutant or wild-type IL-2, and Fab-IL-2 wt-Fab based on 3F2 was determined by surface plasmon resonance (SPR) for the cinomolgus, murine and human IL-2R βy heterodimer using recombinant IL-2R βy heterodimer under the following conditions: ligand: heterodimer hole and knob β of cinomolgo, murine and human IL-2R immobilized on the CM5 chip, analyte: 28H1 or 29B11 Fab-IL-2-Fab (comprising mutant or wild-type quadruple IL-2), 3F2 Fab-IL -2-Fab (comprising wild-type IL-2), temperature: 25 ° C or 37 ° C, buffer: HBS-EP, analyte concentration: 200 nM to 2.5 nM, flow: 30 pl / minute, association: 300 s, decoupling: 300 s, regeneration: 60 s 3M MgCte, fitting: 1: 1 connection, RI ^ O, Rmax = global. The affinity of the Fab-IL-2-Fab immunoconjugate based on 29B11 and 28H1 affinity-matured and targeted by FAP, each containing quadruple mutant or wild-type IL-2, and the Fab-IL-2 wt-Fab based on 3F2 was determined by surface plasmon resonance (SPR) for the α-subunit of cinomolgus, murine and human IL-2R using the recombinant monomeric IL-2R α subunit under the following conditions: ligand: α subunit of Cinomolg, murine and human IL-2R immobilized on a CM5 chip, analyte: 28H1 or 29B11 Fab-IL-2-Fab (comprising mutant or wild-type IL-2), 3F2 Fab-IL-2-Fab ( comprising wild-type IL-2), temperature: 25 ° C or 37 ° C, buffer: HBS-EP, analyte concentration from 25 nM to 0.3 nM, flow: 30 pl / minute, association: 120 s, dissociation: 600 s, regeneration: none, fitting: 1: 1 connection, RI = 0, Rmax = global.
[0310] The results of the kinetic analysis with the IL-2R βy heterodimer are given in Table 8. TABLE 8. BINDING OF FAB-IL-2-FAB IMMUNOCONJUGATES THAT UNDERSTAND MUTANT IL-2 AND MATURE FAB IN AFFINITY FOR HETERODYMERS OF IL-2R

[0311] While the affinity of human IL-2 for the human IL-2R βy heterodimer is described to be around 1 nM, Fab-IL-2-Fab immunoconjugates (which comprise mutant or quadruple IL-2 wild type) both have a reduced affinity between 6 and 10 nM, and as shown for naked IL-2 above the affinity for murine IL-2R is around 10 times weaker than for human IL-2R and cinomolgo.
[0312] The results of the kinetic analysis with the IL-2R α subunit are given in Table 9. Under the chosen conditions there is no detectable binding of the immunoconjugates comprising the quadruple mutant IL-2 to the human IL-2R α subunit , murine or cine. TABLE 9. LINK OF FAB-IL-2-FAB IMMUNOCONJUGATES THAT UNDERSTAND MUTANT IL-2 AND FAB MATURED IN AFFINITY FOR THE IL-2RS SUBUNITY


[0313] The affinity of MHLG1-KV9 Fab-IL-2-Fab immunoconjugates targeted by MCSP, comprising the mutant or wild-type quadruple IL-2, was determined by surface plasmon resonance (SPR) for the heterodimer of Human IL-2R βy using recombinant IL-2R βy heterodimer under the following conditions: Human IL-2R hole y knob β heterodimer was immobilized on a CM5 chip (1600 RU). MHLG1-KV9 Fab-IL-2 wt-Fab and Fab-IL-2 qm-Fab were used as an analyte at 25 ° C in HBS-P buffer. The analyte concentration was 300 nM to 0.4 nM (1: 3 dil.) For IL-2R βy at a flow rate of 30 pl / minute (association time 180 s, dissociation time 300 s). Regeneration was performed for 2x30 s with 3M MgCIs for IL-2R βy. The data were fitted using a 1: 1 link, RI ^ O, Rmax = site for IL-2R βy.
[0314] The affinity of MHLG1-KV9 Fab-IL-2-Fab immunoconjugates targeted by MCSP, comprising mutant or wild-type quadruple IL-2, was determined by surface plasmon resonance (SPR) for the α subunit of human IL-2R with the use of recombinant monomeric IL-2R α subunit under the following conditions: human IL-2R α subunit was immobilized on a CM5 chip (190 RU). MHLG1-KV9 Fab-IL-2 wt-Fab and Fab-IL-2 qm-Fab were used as an analyte at 25 ° C in HBS-P buffer. Analyte concentration was 33.3 nM to 0.4 nM (1: 3 dil.) For IL-2R α at m flow of 30 pl / minute (association time 180 s, dissociation time 300 s). Regeneration was done for 10 s with 50 mM NaOH for IL-2R a. The data were fitted using a 1: 1 link, RI = 0, Rmax = global for IL-2R a.
[0315] The results of the kinetic analysis with the IL-2R βy heterodimer are given in Table 10. TABLE 10

[0316] The data confirm that the MHLG1- KV9 Fab-IL-2 qm-Fab immunoconjugate targeted by MCSP has retained affinity for the IL-2R βy receptor, while the binding affinity for CD25 is abolished compared to the immunoconjugate. comprising wild-type IL-2.
[0317] Subsequently, the affinity of the 4G8 and 28H1-based IgG-IL-2 qm immunoconjugates for the IL-2R βy heterodimer and the IL-2R α subunit was determined by surface plasmon resonance (SPR) in comparison direct to the Fab-IL-2 qm-Fab immunoconjugate format. Briefly, the ligands - either the human IL-2R α subunit or the human IL-2R βy heterodimer - were immobilized on a CM5 chip. Subsequently, 4G8 and 28H1-based IgG-IL-2 qm immunoconjugates or 4G8 and 28H1-based Fab-IL-2 qm-Fab immunoconjugates were applied to the chip as analytes at 25 ° C in HBS-EP buffer in concentrations ranging from 300 nM to 1.2 nM (1: 3 dil.). The flow rate was 30 pl / minute and the following conditions were applied by association: 180s, dissociation: 300 s, and regeneration: 2 x 30 s with 3 M MgCl2 for IL-2R βy heterodimer, 10 s with 50 mM NaOH for the α-subunit of IL-2R. 1: 1 connection was applied for fitting (1: 1 connection RI ^ O, Rmax = local for IL-2R βy, apparent KD, 1: 1 connection RI = 0, Rmax = local for IL-2R α). The respective KD values are given in Table 11. TABLE 11

[0318] The data show that IgG-IL-2 qm immunoconjugates based on 4G8 and 28H1 bind with at least as good an affinity as Fab-IL-2 qm-Fab immunoconjugates for the IL-2R βy heterodimer, whereas they do not bind to the α-subunit of IL-2R due to the introduction of mutations that interfere with CD25 binding. In comparison to the Fab-IL-2 qm-Fab immunoconjugates corresponding to the affinity of the IgG-IL-2 fusion proteins seems to be slightly accentuated within the error of the method. EXAMPLE 7
[0319] In a first set of experiments we confirmed that FAP-targeted Fab-IL-2-Fab immunoconjugates comprising either wild-type or mutant IL-2 were able to bind to FAP expressing HEK 293-FAP cells human by FACS (Figure 17) and the quadruple IL-2 mutation did not impact binding to cells that express FAP (Figure 18). TABLE 12. BINDING FAB-IL-2-FAB IMMUNOCONJUGATES TO HEK CELLS THAT EXPRESS FAP


[0320] In particular, these binding experiments showed that FAP ligands matured in affinity 28H1, 29B11, 14B3 and 4B9 as Fab-IL-2 qm-Fab showed absolute superior binding to HEK 293-FAP target cells compared to immunoconjugates of Fab-IL-2 Fab based on parental FAP ligands 3F2 (29B11, 14B3, 4B9) and 4G8 (28H1) (Figure 17), while retaining high specificity and no binding to DPPIV-transfected HEK 293 cells , a homologue close to FAP, or cells transfected by HEK 293 control plasmid. For comparison, the mouse anti-human CD26-PE DPPIV antibody clone M-A261 (BD Biosciences, # 555437) was used as a positive control (Figure 19). Analysis of the internalization properties showed that binding of Fab-IL-2-Fab immunoconjugates does not result in the induction of FAP internalization (Figure 20).
[0321] In an additional experiment, the binding of 4G8-based lgG-IL-2 qm and Fab-IL-2 qm-Fab immunoconjugates to human FAP expressed in stably transfected HEK293 cells was measured by FACS. The results are shown in Figure 48. The data shows that the IgG-IL-2 qm immunoconjugate binds to FAP expressing cells with an EC50 value of 0.9 nM, comparable to that of the Fab-IL-2 qm construct -Fab based on corresponding 4G8 (0.7 nM).
[0322] Affinity-matured anti-FAP Fab-IL-2-Fab immunoconjugates comprising wild-type IL-2 or the quadruple mutant were subsequently tested in cell assays compared to Proleucine as described in the examples above.
[0323] IL-2-induced IFN-y release was measured in the supernatant by ELISA after incubating the NK92 cell line with these immunoconjugates (Figure 21) for 24 hours. NK92 cells express CD25 on their surface. The results show that the Fab-IL-2-Fab immunoconjugate comprising wild-type IL-2 was less potent in inducing IFN-γ release than Proleucine as could be expected from ca. 10-fold lower affinity of the Fab-IL-2 wt-Fab immunoconjugate for the IL-2R βy heterodimer. The Fab-IL-2 qm-Fab immunoconjugates were somewhat comparable to the respective wild-type construct for a clone selected in terms of potency and absolute induction of IFN-y release despite the fact that NK92 cells express some CD25 . It could, however, be noted that 29B11 Fab-IL-2 qm-Fab induced less cytokine release compared to 29B11 Fab-IL-2 wt-Fab as well as constructs 28H1 and 4G8, for which it had only a small change observed potency for Fab-IL-2 qm-Fab over Fab-IL-2 wt-Fab.
[0324] Additionally, the MHLG1-KV9-based Fab-IL-2 qm-Fab immunoconjugate was compared to the MCSP-targeted Fab-IL-2 qm-Fab immunoconjugates based on 28H1 and 29B11 in the IFN- y in NK92 cells. Figure 22 shows that Fab-IL-2 qm-Fab based on MHLG1-KV9 and targeted by MCSP is somewhat comparable in inducing IFN-y release for FAP-targeted Fab-IL-2 qm-Fab immunoconjugates .
[0325] Subsequently, the induction of proliferation of NK92 cells by IL-2 over a period of 3 days was evaluated in a proliferation assay by measuring ATP using CelITiter Glo (Promega) (Figure 23). Since NK92 cells express low amounts of CD25, the difference between Fab-IL-2-Fab immunoconjugates that comprise wild-type IL-2 and immunoconjugates that comprise quadruple mutant IL-2 could be detected in the proliferation assay, however , under saturation conditions, both achieved similar absolute induction of proliferation.
[0326] In an additional experiment we studied the effects of the FAP-directed Fab-IL-2 qm-Fab immunoconjugate and matured to 28H1 affinity in inducing phosphorylation of STAT5 compared to 28H1 Fab-IL-2 wt-Fab and Proleucine in human NK cells, CD4 + T cells, CD8 + T cells and Treg cells of human PBMCs (Figure 24). For NK cells and CD8 + T cells, which show no or very low CD25 expression (meaning that IL-2R signaling is mediated via the IL-2R βy heterodimer), the results showed that the Fab-IL- immunoconjugate 2-Fab comprising wild-type IL-2 was ca. > 10 times less potent in inducing IFN-y release than Proleucine, and that the Fab-IL-2 qm-Fab immunoconjugate was only slightly less potent than the Fab-IL-2 wt-Fab construct. In CD4 + T cells that show rapid upward regulation of CD25 upon stimulation, Fab-IL-2 qm-Fab was significantly less potent than the Fab-IL-2 wt-Fab immunoconjugate, but still showed comparable induction of IL signaling -2R in saturation concentrations. This is in contrast to Treg cells, in which the potency of Fab-IL-2 qm-Fab has been significantly reduced compared to the Fab-IL-2 wt-Fab construct due to expression in Treg cells and binding affinity subsequent elevation of the Fab-IL-2 wt-Fab construct to CD25 in Treg cells. As a consequence of the abolition of CD25 binding in the Fab-IL-2 qm-Fab immunoconjugate, IL-2 signaling in Treg cells is only achieved by means of the IL-2R βy heterodimer at concentrations in which IL- 2R is activated in CD25 negative effector cells through the IL-2R βy heterodimer. The respective p50 EC50 values are given in Table 13. TABLE 13. IFN-r RELEASE INDICATION OF NK CELLS BY 28H1 FAB-IL-2-FAB IMPACTED FAP TARGETS THAT UNDERSTAND MUTANT IL-2 POLYPEPTIDES

[0327] In another set of experiments, the biological activity of lgG-IL-2 qm and Fab-IL-2 qm-Fab immunoconjugates based on 4G8 and targeted by FAP was investigated in several cell assays.
[0328] Immunoconjugates of 28H1-based Fab-IL-2 qm-Fab and 4G8-based lgG-IL-2 qm were studied for the induction of IFN-y release by NK92 cells as induced by activation of IL-2R βy signaling. Figure 49 shows that the Ig8-IL-2 qm immunoconjugate based on 4G8 and targeted by FAP was equally effective in inducing IFN-y release as the affinity-matured 28H1-based Fab-IL-2 qm-Fab immunoconjugate .
[0329] We also studied the effects of the Ig8-IL-2 qm immunoconjugate based on 4G8 and targeted by FAP on the induction of STAT5 phosphorylation compared to the immunoconjugates of Fab-IL-2 wt-Fab and Fab-IL-2 qH-Fab based on 28H1 as well as Proleucine on human NK cells, CD4 + T cells, CD8 + T cells and Treg cells of human PBMCs. The results of these experiments are shown in Figure 50. For NK cells and CD8 + T cells the 4G8-based IgG-IL-2 qm immunoconjugate was <10 less potent in inducing STAT5 phosphorylation than Proleucine, but slightly more potent than immunoconjugates of Fab-IL-2 wt-Fab and Fab-IL-2 qm-Fab based on 28H1. In CD4 + T cells, the 4G8-based IgG-IL-2 qm immunoconjugate was less potent than the 28H1 Fab-IL-2 wt-Fab immunoconjugate, but slightly more potent than the 28H1 Fab-IL-2 qm-Fab immunoconjugate , and also showed induction of IL-2R signaling at saturation concentrations comparable to Proleucine and 28H1 Fab-IL-2 wt-Fab. This is in contrast to Treg cells in which the potency of the immunoglobulins of 28H1 Fab-IL-2 qm-Fab and lgG-IL-2 qm based on 4G8 was significantly reduced compared to the immunoconjugate of Fab-IL-2 wt- Fab.
[0330] Taken together the quadruple mutant IL-2 described here has the ability to activate IL-2R signaling through IL-2R βy heterodimer similar to wild-type IL-2, but does not result in preferential simulation of Treg cells over other effector cells. EXAMPLE 8
[0331] The antitumor effects of FAP-targeted Fab-IL-2 qm-Fab immunoconjugates were evaluated in vivo compared to FAP-targeted Fab-IL-2 wt-Fab immunoconjugates in ACHN xenograft and syngeneic models of LLC1. All FAP-targeted Fab-IL-2 Fab immunoconjugates (comprising mutant or wild-type quadruple IL-2) recognize murine FAP as well as murine IL-2R. While the ACHN xenograft model in SCID human FcyRIII transgenic mice is strongly positive for FAP in IHC, it is an immunocompromised model and can only reflect immune effector mechanisms mediated by NK cells and / or macrophages / monocytes, but lacks immunity mediated by T cell and therefore cannot reflect AICD or effects mediated by Treg cells. The syngeneic LLC1 model in contrast in completely immunocompetent mice may reflect adaptive T cell-mediated immune effector mechanisms as well, but shows relatively low FAP expression in murine stroma. Each of these models, therefore, partially reflects the situation as found in human tumors. ACHN KIDNEY CELL CARCINOMA XENOFEST MODEL
[0332] Immunoconjugates of 4G8 Fab-IL-2 wt-Fab and 4G8 Fab-IL-2 qm-Fab targeted by FAP were tested using an ACHN human renal cell adenocarcinoma cell line, injected intrarenally into transgenic mice of human SCID FcyRIII. ACHN cells were originally obtained from ATCC (American Type Culture Collection) and after expansion deposited in the internal cell bank Glycart. ACHN cells were cultured in DMEM containing 10% FCS, at 37 ° C in an atmosphere saturated with water in 5% CO2. The in vitro passage 18 was used for intrarenal injection, with a viability of 98.4%. A small incision (2 cm) was made in the direct flank and peritoneal wall of anesthetized SCID mice. A 50 µl cell suspension (1x106 ACHN cells in AimV medium) was injected 2 mm subcapsularly into the kidney. Skin and peritoneal wall wounds were closed with staples. The female SCID-FcyRIII mice (GLYCART-RCC), aged 8 to 9 weeks at the beginning of the experiment (reproduced in RCC, Switzerland) were kept under specific pathogen-free conditions with daily cycles of 12 hours of light / 12 hours darkness according to compromised guidelines (GV- Solas; Felasa; TierschG). The experimental study protocol was reviewed and approved by the local government (P 2008016). Upon arrival, the animals were kept for a week to get used to the new environment and for observation. Continuous health monitoring was carried out on a regular basis. The mice were injected intrarenally on Study Day 0 1x106 ACHN cells and were randomized and weighed. One week after tumor cell injection, mice were injected iv with 4G8 Fab-IL-2 wt-Fab and 4G8 Fab-IL-2 qm-Fab three times a week three weeks. All mice were injected iv with 200 pl of the appropriate solution. The mice in the vehicle group were injected with PBS and the 4G8 Fab-IL-2 wt-Fab or 4G8 Fab-IL-2 qm-Fab immunoconjugate treatment groups. To obtain the appropriate amount of immunoconjugate per 200 pl, the stock solutions were diluted with PBS when needed. Figure 25 shows that both 4G8 Fab-IL-2 wt-Fab and 4G8 Fab-IL-2 qm-Fab immunoconjugates mediated superior efficacy in terms of marked average survival compared to the vehicle group with an advantage for 4G8 Fab -IL-2 wt-Fab over on the 4G8 Fab-IL-2 qm-Fab immunoconjugate in terms of effectiveness. TABLE 14-A
SINGENIC MODEL LEWIS LLC1 LUNG CARCINOMA
[0333] Immunoconjugates of 4G8 Fab-IL-2 qm-Fab and 28H1 Fab-IL-2 qm-Fab targeted by FAP were tested using mouse Lewis lung carcinoma cell line LLC1, injected by iv in the Black 6 mice. Lewis lung carcinoma cells from LLC1 were originally obtained from the ATCC and after expansion deposited in the internal cell bank Glycart. The tumor cell line was cultured routinely in DMEM containing 10% FCS (Gibco) at 37 ° C in an atmosphere saturated with water in 5% CO2. Passage 10 was used for transplantation, at a 97.9% viability. 2x105 cells per animal were injected iv into the tail vein in 200 µl of Aim V cell culture medium (Gibco). Black 6 mice (Charles River, Germany), aged 8 to 9 weeks at the beginning of the experiment, were kept under specific pathogen-free conditions with daily cycles of 12 hours of light / 12 hours of darkness according to compromised guidelines (GV- Soles; Felasa; TierschG). The experimental study protocol was reviewed and approved by the local government (P 2008016). Upon arrival, the animals were kept for a week to get used to the new environment and for observation. Continuous health monitoring was carried out on a regular basis. The mice were injected iv on study day 0 with 2x105 LLC1 cells, and were randomized and weighed. One week after tumor cell injection, mice were injected iv with 4G8 Fab-IL-2 qm-Fab or 28H1 Fab-IL-2 qm-Fab, three times a week for three weeks. All mice were injected iv with 200 pl of the appropriate solution. The mice in the vehicle group were injected with PBS and the treatment group with the 4G8 Fab-IL-2 qm-Fab or 28H1 Fab-IL-2 qm-Fab constructs. To obtain the appropriate amount of immunoconjugate per 200 µl, the stock solutions were diluted with PBS when necessary. Figure 26 shows that the 4G8 Fab-IL-2 qm-Fab or the 28H1 Fab-IL-2 qm-Fab constructs matured in affinity mediated superior effectiveness in terms of marked average survival compared to the vehicle group. TABLE 14-B


[0334] In another experiment, the 28H1 Fab-IL-2 wt-Fab and 28H1 Fab-IL-2 qm-Fab immunoconjugates were tested on the same LLC1 of mouse Lewis lung carcinoma cell line, injected by iv in Black 6 mice. Passage 9 was used for transplantation, at a viability of 94.5%. 2x105 cells per animal were injected iv into the tail vein in 200 µl of Aim V cell culture medium (Gibco). The mice were injected iv on study day 0 with 2x105 LLC1 cells, and were randomized and weighed. One week after tumor cell injection, mice were injected iv with 28H1 Fab-IL-2 wt-Fab or 28H1 Fab-IL-2 qm-Fab, three times a week for three weeks. All mice were injected iv with 200 pl of the appropriate solution. The mice in the vehicle group were injected with PBS and the treatment group with the 28H1 Fab-IL-2 wt-Fab or 28H1 Fab-IL-2 qm-Fab constructs. To obtain the appropriate amount of immunoconjugate per 200 µl, the stock solutions were diluted with PBS when necessary. Figure 27 shows that the 28H1 Fab-IL-2 wt-Fab and 28H1 Fab-IL-2 qm-Fab immunoconjugates mediated superior effectiveness in terms of average survival compared to the vehicle group with a slight advantage for 28H1 Fab- IL-2 wt-Fab on the 28H1 Fab-IL-2 qm-Fab immunoconjugate in terms of effectiveness. TABLE 14-C

EXAMPLE 9
[0335] Fab-IL-2 qm-Fab based on 4G8 and targeted by FAP was subsequently compared to the immunoconjugate of Fab-IL-2 wt-Fab based on 4G8 and targeted by FAP in a toxicokinetic and intravenous toxicity study of seven days in Black 6 mice. Table 15 shows the toxicity study design and toxicokinetic studies. TABLE 15. STUDY DESIGN


[0336] The purpose of this study was to characterize and compare the toxicity and toxicokinetic profiles of wild type interleukin-2 (IL-2) (wt) 4G8 Fab-IL2-Fab targeted by FAP and mutant quadruple IL-2 (qm) of 4G8 Fab-IL2-Fab targeted by FAP after daily intravenous administration to male mice that do not carry tumor for 7 days. For this study, 5 groups of 5 male mice / group were administered intravenously 0 (vehicle control), 4.5 or 9 pg / g / day of IL-2 wt, or 4.5 or 9 pg / g / day of IL-2 qm. An additional 4 groups of 6 male mice / group were administered 4.5 or 9 pg / g / day of IL-2 wt, or 4.5 or 9 pg / g / day of IL-2 qm in order to assess toxicokinetics . The study duration was changed from 7 days to 5 days due to the clinical signs observed in animals given 4.5 and 9 pg / g / day of IL-2 wt. The toxicity assessment was based on mortality, lifetime observations, body weight, and anatomical and clinical pathology. Blood was collected at various time points from animals in the toxicokinetic groups for toxicokinetic analysis. Toxicokinetic data showed that mice treated with IL-2 wt or IL-2 qm had measurable plasma levels until the last bleeding moment, which indicates that the mice were exposed to the respective compounds for the duration of the treatment. The AUC0-inf values for Day 1 suggest comparable exposure of IL-2 wt and IL-2 qm at both dosage levels. Sparse samples were taken on Day 5 and showed plasma concentrations equivalent to Day 1, suggesting that no accumulation occurred after 5 days of dosing both compounds. In more detail the following observations were noted. TOXICOCINETICS
[0337] Table 16 summarizes the average plasma toxicokinetic parameters for FAP-targeted 4G8 Fab-IL-2 qm-Fab and FAP-targeted 4G8 Fab-IL-2 wt-Fab as determined by WinNonLin Version 5.2.1 and a Commercial kappa specific ELISA (Human Kappa ELISA Quantification Set, Betil Laboratories). TABLE 16
* TK parameters were calculated in WinNonin Version 5.2.1 using non-compartmental analysis.
[0338] The individual serum concentrations are given below:


[0339] These data data show that both 4G8 Fab-IL-2 qm-Fab and 4G8 Fab-IL-2 wt-Fab show comparable pharmacokinetic properties with slightly higher exposure for 4G8 Fab-IL-2 qm-Fab . MORTALITY
[0340] In group 9 pg / g of 4G8 Fab-IL-2 wt-Fab targeted by FAP, treatment-related mortality occurred in one animal prior to necropsy on Day 5. Hypoactivity, cold skin, and bowed posture were observed before of death. This animal probably died due to a combination of cellular infiltration into the lung that was accompanied with edema and hemorrhage and marked bone marrow necrosis. Mortality is summarized in Table 17. TABLE 17. MORTALITY DAY 5

CLINICAL OBSERVATIONS
[0341] Observations of hypoactivity, cold skin, and bowed posture were observed in animals given 4.5 and 9 pg / g / day of IL-2 wt. The clinical observations were summarized in Table 18. TABLE 18. CLINICAL OBSERVATIONS, DAY 5

BODY WEIGHT
[0342] A moderate decrease in body weight was observed after 5 days of treatment in animals given 4.5 and 9 (9% and 11%, respectively) pg / g / day of IL-2 wt. A slight decrease in body weight was observed after 5 days of treatment in animals given 4.5 and 9 (2% and 1%, respectively) pg / g / day of IL-2 qm. A moderate decrease (9%) in body weight was also seen in vehicle controls after 5 days of treatment. However, the percentage of decrease would be 5% if a potential atypical case (Animal # 3) was excluded. The loss of body weight in the vehicle group may have been attributed to stress. HEMATOLOGY
[0343] A reduced platelet count was seen in animals given 4.5 (~ 4.5 times) and 9 pg / g / day (~ 11 times) 4G8 Fab-IL-2 wt-Fab, which correlated with megakaryocytes reduced in the bone marrow as well as systemic consumer effects (fibrin) in the spleen and lung of these animals (see section on Histopathology below). These observations indicate that the reduced platelets were probably due to the combined effects of consumption and decreased bone marrow production / agglomeration due to increased lymphocyte / myeloid cell production as a direct or indirect effect of IL-2.
[0344] Hematological observations of uncertain relationship to make up the administration consisted of decreases in absolute lymphocyte count with 4G8 Fab-IL-2 wt-Fab by 4.5 (~ 5 times) and 9 pg / g (~ 3 times) compared to the average value of the vehicle control group. These observations lacked dependence on clear dosage, but could be considered secondary to the effects associated with stress observed in the observations of life or exaggerated pharmacology of the compound (lymphocytes migrating to tissues). There were no treatment-related hematological changes attributed to the administration of 4G8 Fab-IL-2 qm-Fab. Few isolated hematological observations were statistically different from their respective controls. However, these observations were of insufficient magnitude to suggest pathological relevance. HISTOPATHOLOGY AND GROSS PATHOLOGY
[0345] Crude treatment-related observations included an enlarged spleen seen in 5/5 and 4/5 of the 4.5 and 9 pg / g mice in 4G8 Fab-IL-2 wt-Fab groups, respectively, and in 1 / 5 in both 4.5 and 9 pg / g of 4G8 Fab-IL-2 qm-Fab treatment groups.
[0346] Treatment-related histopathological observations were present in groups given 4.5 and 9 pg / g of 4G8 Fab-IL-2 wt-Fab and 4.5 and 9 pg / g of 4G8 Fab-IL-2 qm- Fab in the lung, bone marrow, liver, spleen, and thymus, with differences in incidence, severity rating or nature of the changes, as reported below.
[0347] Histopathological observations related to treatment in the lung consisted of mononuclear infiltration found mild to marked in 5/5 of the mice in groups 4,5 and 9 pg / g of 4G8 Fab-IL-2 wt-Fab and in margin in 5/5 of the mice in the groups of 4.5 and 9 pg / g of 4G8 Fab-IL-2 qm-Fab. Mononuclear infiltration consisted of lymphocytes (some of which were observed to have cytoplasmic granules) as well as reactive macrophages. It was observed more frequently that these cells had vasocentric patterns, often with marginalization observed within the vessels in the lung. It was also observed that these cells surrounded the vessels, but in more severe cases, the pattern was more diffuse. Hemorrhage was seen extreme to light in 5/5 of the mice in groups 4,5 and 9 pg / g of 4G8 Fab-IL-2 wt-Fab and marginally in 2/5 of the mice in group 9 pg / g of 4G8 Fab-IL-2 qm-Fab. Although hemorrhage was more frequently perivascular, in more severe cases, it was observed in alveolar spaces. A mild to moderate edema was seen in 5/5 of the mice in groups 4.5 and 9 pg / g of 4G8 Fab-IL-2 wt-Fab and marginally in 5/5 of the mice in group 9 pg / g of 4G8 Fab-IL-2 qm- Fab. Although edema has been seen with perivascular frequency, in more severe cases, it has also been seen in alveolar spaces. Marginal cell degeneration and cariorhexis were observed in 2/5 and 5/5 of the mice in groups 4,5 and 9 pg / g of 4G8 Fab-IL-2 wt-Fab, respectively and consisted of degeneration of reactive or infiltrating leukocytes. The animals selected with MSB stains were positive for the fibrin found within the lungs of animals in both groups 4.5 and 9 pg / g of 4G8 Fab-IL-2 wt-Fab that correlate in part with the reduced platelets observed in these animals.
[0348] Treatment-related changes in bone marrow included marginal to mild increased general marrow cellularity in 5/5 of the mice and 2/5 of the mice of both 4.5 and 5/5 of the mice and 2/5 of the mice. mice from both groups 9 pg / g of 4G8 Fab-IL-2 wt-Fab and 4G8 Fab-IL-2 qm-Fab, respectively. This was characterized by the increased marginal lymphocyte-myelocyte hyperplasia for these groups that was supported, in part, by the increased numbers of positive CD3 T cells within the medulla and paranasal sinuses (specifically T lymphocytes, confirmed by immunohistochemistry with the CD3 pan-T cell made in selected animals). The increase in CD3 positive T cell was moderate in both 4G8 Fab-IL-2 wt-Fab and marginal to light groups in both 4G8 Fab-IL-2 qm-Fab groups. There were decreases in marginal to mild in megakaryocytes in 2/5 of the mice in 4.5 and 5/5 of the mice in the 9 pg / g groups of 4G8 Fab-IL-2 wt-Fab and decreases in marginal to moderate in the precursors of erythroid were observed in 3/5 of the mice in 4.5 and 5/5 of the mice in groups 9 pg / g of 4G8 Fab-IL-2 wt-Fab. Bone marrow necrosis was observed in 1/5 of the mice in 4.5 (minimum) and 5/5 of the mice in groups 9 pg / g of (mild to marked) 4G8 Fab-IL-2 wt-Fab. The reduced number of megakaryocytes in the bone marrow correlated with decreased platelets, which could be due to the direct agglomeration of the bone marrow by enlarged lymphocytes / myeloid precursors and / or bone marrow necrosis, and / or platelet consumption due to inflammation in several tissues (see spleen and lung). The reduced erythroid precursors in the bone marrow, do not correlate with the observations of peripheral blood hematology due to the temporal effects (seen in the bone marrow before the peripheral blood) and the longer half-life of peripheral erythrocytes (compared to platelets). The mechanism of bone marrow necrosis in the bone marrow may be secondary due to the evident agglomeration of the marrow cavity (due to the production and growth of lymphocytes / myeloid cells), local or systemic release of cytokines from the proliferating cell types, possibly in local effects of hypoxia or other pharmacological effects of the compound.
[0349] Treatment-related observations in the liver consisted of first mild to moderate vasocentric mononuclear cell infiltrate and marginal to light single cell necrosis in 5/5 of the mice in groups 4.5 and 9 pg / g of 4G8 Fab-IL -2 wt-Fab. Marginal single cell necrosis was seen in 2/5 and 4/5 of the mice in the 4.5 and 9 pg / g group of 4G8 Fab-IL-2 qm-Fabs, respectively. Mononuclear infiltrates consisted primarily of lymphocytes (specifically T-lymphocytes, confirmed by immunohistochemistry with the pan-T cell marker CD3 made in selected animals) that were most frequently seen vasocentricly as well as marginally within the central and portal vessels. The animals selected for immunohistochemical staining for F4 / 80 showed increased (activated) size and numbers of macrophages / Kupffer cells throughout the hepatic sinusoids in the 9 pg / g groups of 4G8 Fab-IL-2 wt-Fab and 4G8 Fab -IL-2 qm-Fab.
[0350] Observations related to treatment in the spleen consisted of moderate to marked lymphoid infiltration / hyperplasia and mild to moderate macrophage infiltration / hyperplasia in 5/5 of the mice in groups 4.5 and 9 pg / g of 4G8 Fab -IL-2 wt-Fab and mild to moderate lymphoid infiltration / hyperplasia with marginal to light macrophage infiltration / hyperplasia in 5/5 of the mice in groups 4.5 and 9 pg / g of 4G8 Fab-IL-2 qm-Fab. Immunohistochemistry for 9 pg / g of 4G8 Fab-IL-2 wt-Fab and 4G8 Fab-IL-2 qm-Fab showed different patterns with the use of the pan-T cell marker CD3, as well as the macrophage marker F4 / 80. For 9 pg / g of 4G8 Fab-IL-2 wt-Fab, the pattern of macrophage and T cell immunoreactivity remained primarily within the areas of red pulp, and the architecture of the primary follicles was altered by lymphocytolysis and necrosis (described below) ). For 9 pg / g of 4G8 Fab-IL-2 qm-Fab, special spots showed a pattern similar to that of vehicle control, but with expansion of white pulp with periarteriolar lymphoid sheath (PALS), by a T cell population and a larger expanded red pulp area. Macrophage and T cell positivity was also evident within the red pulp, with a pattern similar to the vehicle control group, but expanded. These observations correlate with the gross observations of the enlarged spleen. Necrosis was observed marginally in 3/5 of the mice and marginally and slightly in 5/5 of the mice in groups 4.5 and 9 pg / g of 4G8 Fab-IL-2 wt-Fab, respectively. Necrosis was generally located around the area of primary follicles and animals using the MSB stain was positive for fibrin in both groups 4.5 and 9 pg / g of 4G8 Fab-IL-2 wt-Fab that correlate in part with the reduced platelets seen in these animals. Lymphocytolysis was seen in the 4.5 pg / g (minimal to light) and 9 pg / g (moderate to marked) groups 4G8 Fab-IL-2 wt-Fab.
[0351] Treatment-related observations in the thymus included minimal to mild increases in lymphocytes in both 4.5 and 9 pg / g 4G8 Fab-IL-2 wt-Fab groups and 4.5 ug / g 4G8 Fab -IL-2 qm-Fab. The cortex and medulla were not evident individually, in groups 4G8 Fab-IL-2 wt-Fab, but immunohistochemistry for the pan-T cell marker (CD3) in animals selected in groups 9 pg / g of 4G8 Fab -IL-2 wt-Fab and 9 pg / g of 4G8 Fab-IL-2 qm-Fab showed strong positivity for most cells within the thymus. The enlarged lymphocytes in the thymus were considered to be a direct pharmacological effect of both compounds in which IL-2-induced proliferation of lymphocytes that migrate to the bone marrow thymus (T cells) for further differentiation and clonal expansion. This occurred in all groups except for 9 pg / g of 4G8 Fab-IL-2 qm-Fab, which is probably a temporal effect. Lymphocytolysis was mild in the 4.5 pg / g group of 4G8 Fab-IL-2 wt-Fab, and was moderate to marked in the 9 pg / g group of 4G8 Fab-IL-2 wt-Fab. Moderate lymphoid depletion was observed in both groups 4.5 and 9 pg / g of 4G8 Fab-IL-2 wt-Fab. While these observations appear more robust in groups 4.5 and 9 pg / g of 4G8 Fab-IL-2 wt-Fab, these animals were described as dying on Day 5, and mild to marked lymphocytolysis as well as moderate lymphoid depletion may be related to this observation in life (effects related to stress due to poor physical condition).
[0352] Histopathology observations of uncertain relationship to compound administration to the liver consisted of a marginal mixed cell activation / infiltrate (lymphocytes and macrophages) observed as small foci / microgranulomas randomly scattered throughout the liver in 5/5 of the mice in both 4.5 and 9 pg / g groups of 4G8 Fab-IL-2 qm-Fab. This marginal change was also seen in the vehicle control group, but with less incidence and severity. Stomach glandular dilatation and atrophy was seen marginally to slightly in 5/5 of the mice and villous ileal atrophy was seen marginally in 3/5 of the mice in the 9 pg / g group of 4G8 Fab-IL-2 wt-Fab. This finding is probably attributed to the poor physical condition seen in these mice such as reduced body weight, especially in the 9 pg / g group of 4G8 Fab-IL-2 wt-Fab observed in observations in life.
[0353] Injection site observations included mixed cell infiltrate, perivascular edema, and myodegeneration that was also seen in vehicle control, groups 9 pg / g of 4G8 Fab-IL-2 wt-Fab and 9 pg / g of 4G8 Fab-IL-2 qm-Fab. One animal had epidermal necrosis. These observations were not attributed to the treatment (s) itself, but to i.v. injection and tail handling. Another animal had macrophage infiltration of skeletal muscle (seen in the histology section of lung tissue) associated with myodegeneration and myoregeneration probably due to a chronic injury and was not assigned to treatment. Lymphoid depletion was observed in 3/5 and 4/5 of mice in groups 4.5 and 9 pg / g of 4G8 Fab-IL-2 qm-Fab, respectively and was probably attributed to physiological changes seen in the thymus as mice get older (also seen in similar incidence, 4/5 of mice, and severity in vehicle control animals).
[0354] In short, daily intravenous administration of 4G8 Fab-IL-2 wt-Fab or 4G8 Fab-IL-2 qm-Fab in doses of 4.5 or 9 pg / g / day for up to 5 days in male mice resulted in histological observations related to similar treatment with both compounds. However, the observations were generally more prevalent and more severe 4G8 Fab-IL-2 wt-Fab targeted by FAP in the lung (Figures 28 and 29) (mononuclear infiltration consisting of reactive lymphocytes and macrophages, hemorrhage, and edema), bone marrow (lympho-myelo-hyperplasia and increased cellularity), liver (Figure 30) (single cell necrosis, increase in Kupffer cell / macrophage in number and activation), spleen (grossly enlarged, macrophage and lymphocyte infiltration / hyperplasia) and thymus (enlarged lymphocytes). In addition, mortality, lymphocytolysis, necrosis or cell degeneration in the lung, spleen, bone marrow, and thymus, as well as reduced megakaryocytes and erythrocytes in bone marrow and reduced platelets in peripheral blood were seen in animals given IL-2 wt. Based on anatomical and clinical pathological observations, as well as clinical observations, and the comparable systemic exposure of both compounds, IL-2 qm under the conditions of that study exhibited markedly less systemic toxicity after 5 dosages than IL-2 wt. EXAMPLE 10 INDUCTION OF NK CELL IFN-r secretion by IL-2 MUTANT QUADRUPLE AND WILD TYPE
[0355] NK-92 cells were deprived of food for 2 hours before sowing 100,000 cells / well in a 96-well F-bottom plate. The IL-2 constructs were titrated on the seeded NK-92 cells. After 24 hours or 48 hours, the plates were centrifuged before collecting the supernatants to determine the amount of human IFN-y using a commercial IFN-y ELISA (BD # 550612).
[0356] Two preparations produced on-site other than wild-type IL-2 (probably differing slightly in their O-glycosylation profiles, see Example 2), a commercially available wild-type IL-2 (Proleucine) and quadruple IL-2 mutant prepared on site (first batch) were tested.
[0357] Figure 31 shows that the mutant quadruple IL-2 is equally potent as commercially obtained (Proleucine) or wild-type IL-2 produced on site in inducing IFN-y secretion by NK cells by 24 (A) or 48 hours (B). EXAMPLE 11 INDUCTION OF NK CELL PROLIFERATION BY IL-2 MUTANT QUADRUPLE AND WILD TYPE
[0358] NK-92 cells were deprived of food for 2 hours before sowing 10,000 cells / well in clear 96-well black F-bottom plates. The IL-2 constructs were titrated on the seeded NK-92 cells. After 48 hours, the ATP content was measured to determine the number of viable cells using the Promega Celesciter-Glo Luminescent Cell Viability Assay Kit according to the manufacturer's instructions.
[0359] The same IL-2 preparations as in Example 10 were tested.
[0360] Figure 32 shows that all the tested molecules were able to induce proliferation of NK cells. At low concentrations (<0.01 nM) quadruple mutant IL-2 was slightly less active than wild-type IL-2 produced on site, and all preparations produced on-site were less active than wild-type IL-2 commercially obtained (Proleucine).
[0361] In a second experiment, the following IL-2 preparations were tested: wild-type IL-2 (cluster 2), quadruple mutant IL-2 (first and second batch).
[0362] Figure 33 shows that all tested molecules were similarly active in inducing NK cell proliferation, with the two mutant IL-2 preparations being only minimally less active than wild-type IL-2 preparations. in the lowest concentrations. EXAMPLE 12 PROLIFERATION INDUCTION OF HUMAN PBMC BY IMMUNOCONJUGATES CONCERNING IL-2 MUTANT QUADRUPLE OR WILD TYPE
[0363] Peripheral blood mononuclear cells (PBMC) were prepared using Histopaque-1077 (Sigma Diagnostics Inc., St. Louis, MO, USA). Soon, venous blood from healthy volunteers was drawn in heparinized syringes. The blood was diluted 2: 1 with PBS free of magnesium and calcium, and layered over Histopaque-1077. The gradient was centrifuged at 450 x g for 30 minutes at room temperature (RT) without pauses. The interphase containing the PBMCs was collected and washed three times with PBS (350 x g followed by 300 x g for 10 minutes in RT).
[0364] Subsequently, PBMCs were labeled with 40 nM CFSE (succinimidyl ester carboxyfluorecein ester) for 15 minutes at 37 ° C. The cells were washed with 20 ml of medium before recovering the labeled PBMCs for 30 minutes at 37 ° C. The cells were washed, counted and 100,000 cells were seeded in 96-well U-bottom plates. Pre-diluted Proleucine (commercially available wild-type IL-2) or IL-2 immunoconjugates were titrated on the seeded cells that were incubated at the indicated time points. After 4 to 6 days, the cells were washed, marked for appropriate cell surface markers, and analyzed by FACS using a BD FACSCantoll. NK cells were defined as CD37CD56 +, CD4 T cells as CD37CD8 'and CD8 T cells as CD37CD8 +
[0365] Figure 34 shows the proliferation of NK cells after incubation with 28H1 IL-2 immunoconjugates targeted by different FAP for 4 (A), 5 (B) or 6 (C) days. All tested constructs induced NK cell proliferation in a concentration-dependent manner. Proleucine was more effective than immunoconjugates at lower concentrations, this difference no longer existed at higher concentrations, however. At the previous time points (day 4), the IgG-IL2 constructs appeared slightly more potent than the Fab-IL2-Fab constructs. At later time points (day 6), all constructs had comparable efficacy, with the Fab-IL2 qm-Fab construct being less potent at low concentrations.
[0366] Figure 35 shows the proliferation of CD4 T cells after incubation with 28H1 IL-2 immunoconjugates targeted by different FAP for 4 (A), 5 (B) or 6 (C) days. All constructs tested induced CD4 T cell proliferation in a concentration-dependent manner. Proleucine had a higher activity than immunoconjugates, and immunoconjugates that comprise wild-type IL-2 were slightly more potent than those that comprise quadruple mutant IL-2. As for NK cells, the Fab-IL2 qm-Fab construct had the least activity. Proliferating CD4 T cells are more likely to be partially regulatory T cells, at least for wild-type IL-2 constructs.
[0367] Figure 36 shows the proliferation of CD8 T cells after incubation with 28H1 IL-2 immunoconjugates targeted by different FAP for 4 (A), 5 (B) or 6 (C) days. All constructs tested induced CD8 T cell proliferation in a concentration-dependent manner. Proleucine had a higher activity than immunoconjugates, and immunoconjugates that comprise wild-type IL-2 were slightly more potent than those that comprise quadruple mutant IL-2. As for NK cells and CD4 T cells, the Fab-IL2 qm-Fab construct had the least activity.
[0368] Figure 37 represents the results of another experiment, in which 28H1 IgG-IL-2 targeted by FAP, comprising either mutant or wild-type quadruple IL-2, and Proleucine were compared. The incubation time was 6 days. As shown in the figure, all three IL-2 constructs induce proliferation of NK cell (A) and CD8 T cell (C) in a dose dependent manner with similar potency. For CD4 (B) T cells, the IgG-lL2 qm immunoconjugate had less activity, particularly at medium concentrations, which may be due to a lack of activity on CD25-positive T cells (including regulators) which are a subset of cells T CD4. EXAMPLE 13 EFFECTIVE CELL ACTIVATION BY IL-2 QUADRUPLE MUTANT AND WILD TYPE (PSTAT5 TEST)
[0369] PBMCs were prepared as described above. 500,000 PBMCs / well were seeded in 96-well U-bottom plates and rested for 45 minutes at 37 ° C in RPMI medium containing 10% FCS and 1% Glutamax (Gibco). After that, PBMCs were incubated with Proleucine, wild-type IL-2 or quadruple mutant IL-2 produced at the concentrations indicated for 20 minutes at 37 ° C to induce STAT5 phosphorylation. Subsequently, the cells were fixed immediately (BD Cytofix Buffer) for 10 minutes at 37 ° C and washed once, followed by a permeabilization step (BD Phosflow Perm Buffer III) for 30 minutes at 4 ° C. After that, the cells were washed with PBS / 0.1% BSA and labeled with mixtures of FACS antibodies for the detection of NK cells (CD37CD56 +), CD8 + T cells (CD3 + / CD8 +), CD4 + T cells (CD3 + / CD4 + / CD257CD127 +) or Treg cells (CD4 + / CD25 + / CD1277FoxP3 +), as well as pSTAT5 for 30 minutes in RT in the dark. The cells were washed twice with PBS / 0.1% BSA and resuspended in 2% PFA before flow cytometric analysis (BD FACSCantoll).
[0370] Figure 38 shows phosphorylation of STAT in NK cells (A), CD8 T cells (B), CD4 T cells (C) and regulatory T cells (D) after a 30 minute incubation with Proleucine, IL-2 wild-type (cluster 2) and quadruple mutant IL-2 (lot 1) produced on site. All three IL-2 preparations were equally potent in inducing phosphorylation of STAT in NK as well as CD8 T cells. In CD4 T cells and even more in regulatory T cells, the quadruple mutant IL-2 had less activity than that of wild-type IL-2 preparations. EXAMPLE 14 EFFECTIVE CELL ACTIVATION BY IGG-IL-2 QUADRUPLE MUTANT AND WILD TYPE (PSTAT5 TEST)
[0371] The experimental conditions were as described above (see Example 13).
[0372] Figure 39 shows phosphorylation of STAT in NK cells (A), CD8 T cells (B), CD4 T cells (C) and regulatory T cells (D) after a 30 minute incubation with Proleucine, IgG-IL -2 which comprises wild-type IL-2 or IgG-IL-2 which comprises quadruple mutant IL-2. And all cell types Proleucine was more potent in inducing STAT phosphorylation than in IgG-IL-2 immunoconjugates. The IgG-IL-2 quadruple and wild-type mutant constructs were equally potent in NK as well as CD8 T cells. In CD4 T cells and even more in regulatory T cells, the quadruple mutant IgG-IL-2 had less activity than that of the wild-type IgG-IL-2 immunoconjugate. EXAMPLE 15 MAXIMUM TOLERATED DOSAGE (BAT) OF FAB-IL2 WT-FAB AND FAB-IL2 QM-FAB IMMUNOCONJUGATES
[0373] Increasing dosages of FAP-targeted Fab-IL2-Fab immunoconjugates, comprising either quadrant mutant (qm) or wild-type (wt) IL-2, were tested in tumor-free immunocompetent Black 6 mice.
[0374] Black 6 female mice (Charles River, Germany), aged 8 to 9 weeks at the beginning of the experiment, were kept under specific pathogen-free conditions with daily cycles of 12 hours of light / 12 hours of darkness according to committed guidelines (GV-Solas; Felasa; TierschG). The experimental study protocol was reviewed and approved by the local government (P 2008016). Upon arrival, the animals were kept for a week to get used to the new environment and for observation. Continuous health monitoring was carried out on a regular basis.
[0375] The mice were injected iv once daily for 7 days with 4G8 Fab-IL2 wt-Fab in dosages of 60, 80 and 100 pg / mouse or 4G8 Fab-IL2 qm-Fab in dosages of 100, 200, 400, 600 and 1,000 pg / mouse. All mice were injected i.v. with 200 pl of the appropriate solution. To obtain the appropriate amount of immunoconjugate per 200 pl, the stock solutions were diluted with PBS as needed.
[0376] Figure 40 shows that the BAT (maximum tolerated dosage) for Fab-IL2 qm-Fab is 10 times greater than for Fab-IL2 wt-Fab, namely 600 pg / mouse daily for 7 days for Fab-IL2 qm-Fab versus 60 pg / mouse daily for 7 days for Fab-IL2 wt-Fab. TABLE 19
EXAMPLE 16 PHARMACOKINETICS OF A SINGLE DOSAGE OF IGG-IL2 WT AND QM NOT TARGETED OR TARGETED BY FAP
[0377] A single-dose pharmacokinetics (PK) study was performed on 129 tumor-free immunocompetent mice for FAP-targeted lgG-IL2 immunoconjugates comprising either mutant or wild-type quadruple IL-2 and lgG-IL2 immunoconjugates non-targeted that comprise either mutant or wild-type quadruple IL-2.
[0378] 129 female mice (Harlan, UK), aged 8 to 9 weeks at the beginning of the experiment, were kept under specific pathogen-free conditions with daily cycles of 12 hours of light / 12 hours of darkness according to guidelines compromised (GV-Solas; Felasa; TierschG). The experimental study protocol was reviewed and approved by the local government (P 2008016). Upon arrival, the animals were kept for a week to get used to the new environment and for observation. Continuous health monitoring was carried out on a regular basis.
[0379] The mice were injected iv once 28H1 lgG-IL2 wt targeted by FAP (2.5 mg / kg) or 28H1 lgG-IL2 qm (5 mg / kg), or DP47GS IgG-IL2 wt not targeted (5 mg / kg) or DP47GS lgG-IL2 qm (5 mg / kg). All mice were injected i.v. with 200 pl of the appropriate solution. To obtain the appropriate amount of immunoconjugate per 200 pl, the stock solutions were diluted with PBS as needed.
[0380] The mice were bled at 1, 8, 24, 48, 72, 96 hours; and thereafter every 2 days for 3 weeks. The sera were extracted and stored at -20 ° C until ELISA analysis. Serum immunoconjugate concentrations were determined using an ELISA to quantify the IL2 immunoconjugate antibody (Roche-Penzberg). Absorption was measured using a measuring wavelength of 405 nm and a reference wavelength of 492 nm (VersaMax calibrable microplate reader, Molecular Devices).
[0381] Figure 41 shows the pharmacokinetics of these IL-2 immunoconjugates. Both FAP-targeted IgG-IL2 (A) constructs (B) and non-targeted (B) have a longer serum half-life (approximately 30 hours) than the corresponding IgG-IL2 wt constructs (approximately 15 hours). TABLE 20
EXAMPLE 17 PHARMACOKINETICS OF A SINGLE DOSE OF FAB-IL2 WT-FAB AND FAB-IL2 QM- FAB NOT TARGETED
[0382] A single-dose pharmacokinetics (PK) study was performed on 129 tumor-free immunocompetent mice for non-targeted Fab-IL2-Fab immunoconjugates that comprise either wild-type or mutant quadruple IL-2.
[0383] 129 female mice (Harlan, UK), 8 to 9 weeks old at the beginning of the experiment, were kept under specific pathogen-free conditions with daily cycles of 12 hours of light / 12 hours of darkness according to guidelines compromised (GV-Solas; Felasa; TierschG). The experimental study protocol was reviewed and approved by the local government (P 2008016). Upon arrival, the animals were kept for a week to get used to the new environment and for observation. Continuous health monitoring was carried out on a regular basis.
[0384] The mice were injected i.v. once with DP47GS Fab-IL2 wt-Fab at a dosage of 65 nmol / kg or DP47GS Fab-IL2 qm-Fab at a dosage of 65 nM / kg. All mice were injected i.v. with 200 pl of the appropriate solution. To obtain the appropriate amount of immunoconjugate per 200 pl, the stock solutions were diluted with PBS as needed.
[0385] The mice were bled at 0.5, 1, 3, 8, 24, 48, 72, 96 hours and thereafter every 2 days for 3 weeks. The sera were extracted and stored at -20 ° C until ELISA analysis. Serum immunoconjugate concentrations were determined using an ELISA to quantify the IL2 immunoconjugate antibody (Roche-Penzberg). Absorption was measured using a measuring wavelength of 405 nm and a reference wavelength of 492 nm (VersaMax calibrable microplate reader, Molecular Devices).
[0386] Figure 42 shows the pharmacokinetics of these IL-2 immunoconjugates. The Fab-IL2-Fab wt and qm constructs have an approximate serum half-life of 3 to 4 h. The difference in serum half-life between constructs that comprise quadruple mutant or wild-type IL-2 is less pronounced for Fab-IL2-Fab constructs than for IgG-like immunoconjugates, which alone have longer half-lives. . TABLE 21
EXAMPLE 18 CELL DEATH INDUCED BY ACTIVATION OF IL-2-ACTIVATED PBMCS
[0387] PBMCs recently isolated from healthy donors were pre-activated overnight with PHA-M at 1 pg / ml in RPMI1640 with 10% FCS and 1% Glutamine. After pre-activation, PBMCs were harvested, labeled with 40 nM CFSE in PBS, and seeded in 96 well plates in 100,000 cells / well. Pre-activated PBMCs were stimulated with different concentrations of IL-2 immunoconjugates (4B9 lgG-IL-2 wt, 4B9 lgG-IL-2 qm, 4B9 Fab-IL-2 wt-Fab, and 4B9 Fab-IL-2 qm-Fab). After six days of IL-2 treatment, PBMCs were treated with 0.5 pg / ml of anti-Fas activating antibody overnight. The proliferation of CD4 (CD3 + CD8_) and CD8 (CD3 + CD8 +) T cells was analyzed after six days by dilution of CFSE. The percentage of live T cells after anti-Fas treatment was determined by monitoring CD3 + Anexin V negative living cells.
[0388] As shown in Figure 44, all proliferations induced by pre-activated T cell construct. At low concentrations, constructs that comprise wild-type IL-2 wt were more active than constructs that comprise IL-2 qm. IgG-IL-2 wt, Fab-IL-2 wt-Fab and Proleucine had similar activity. Fab-IL-2 qm-Fab was slightly less active than IgG-IL-2 qm. Constructs that comprise wild-type IL-2 were more active in CD4 T cells than in CD8 T cells, most likely due to the activation of regulatory T cells. Constructs that comprise quadruple mutant IL-2 were similarly active on CD8 and CD4 T cells.
[0389] As shown in Figure 45, T cells stimulated with high concentrations of wild-type IL-2 are more sensitive to anti-Fas-induced apoptosis than T cells treated with mutant quadruple IL-2.
[0390] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited in this document are expressly incorporated by reference in their entirety.

权利要求:
Claims (24)
[0001]
1. MUTANT INTERLEUCIN-2 (IL-2) POLYPEPTIDE, characterized by comprising three amino acid mutations that abolish or reduce the affinity of the mutant IL-2 polypeptide to the high-affinity IL-2 receptor and preserve the affinity of the polypeptide of mutant IL-2 to the intermediate affinity IL-2 receptor, each compared to a wild-type IL-2 polypeptide, wherein said three amino acid mutations are in positions corresponding to residue 42, 45 and 72 of Human IL-2 (SEQ ID NO: 1).
[0002]
2. POLYPEPTIDE, according to claim 1, characterized in that said three amino acid mutations are amino acid substitutions selected from the group of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R and L72K.
[0003]
POLYPEPTIDE, according to any one of claims 1 to 2, characterized in that said amino acid mutation at the position corresponding to the human IL-2 residue 72 is the amino acid substitution L72G.
[0004]
4. POLYPEPTIDE, according to any one of claims 1 to 3, characterized in that said amino acid mutation in the position corresponding to human IL-2 residue 42 is the amino acid substitution F42A.
[0005]
POLYPEPTIDE, according to any one of claims 1 to 4, characterized in that said amino acid mutation in the position corresponding to residue 45 of human IL-2 is the substitution of amino acid Y45A.
[0006]
6. POLYPEPTIDE, according to any one of claims 1 to 5, characterized in that said three amino acid mutations are amino acid substitutions F42A, Y45A and L72G.
[0007]
7. POLYPEPTIDE, according to any one of claims 1 to 6, characterized in that it further comprises an amino acid mutation that eliminates the IL-2 O-glycosylation site at a position corresponding to residue 3 of human IL-2.
[0008]
8. POLYPEPTIDE, according to claim 7, characterized by the said amino acid mutation that eliminates the IL-2 glycosylation site in a position corresponding to residue 3 of human IL-2 being an amino acid substitution selected from the group of T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K and T3P.
[0009]
9. POLYPEPTIDE, according to any one of claims 7 to 8, characterized in that said amino acid mutation that eliminates the IL-2 O-glycosylation site in a position corresponding to residue 3 of human IL-2 is the amino acid substitution T3A.
[0010]
10. POLYPEPTIDE according to any one of claims 1 to 9, characterized in that said mutant IL-2 polypeptide is a human IL-2 molecule.
[0011]
11. POLYPEPTIDE according to any one of claims 1 to 10, characterized in that said mutant IL-2 polypeptide comprises the sequence of SEQ ID NO: 19.
[0012]
12. POLYPEPTIDE, according to any one of claims 1 to 11, characterized in that said mutant IL-2 polypeptide is linked to a non-IL-2 moiety.
[0013]
13. IMMUNOCONJUGATE, characterized in that it comprises a mutant IL-2 polypeptide, as defined in any one of claims 1 to 12, and an antigen binding portion.
[0014]
14. IMMUNOCONJUGATE, according to claim 13, characterized in that said antigen-binding chemical portion is an antibody or an antibody fragment.
[0015]
IMMUNOCONJUGATE, according to claim 13, characterized in that said antigen-binding chemical portion is selected from a Fab molecule and a scFv molecule.
[0016]
16. IMMUNOCONJUGATE, according to claim 13, characterized in that said antigen-binding chemical portion is an immunoglobulin molecule, particularly an IgG molecule.
[0017]
17. IMMUNOCONJUGATE, according to any one of claims 13 to 16, characterized in that said antigen-binding chemical portion is directed to an antigen presented in a tumor cell or in a tumor cell environment.
[0018]
18. ISOLATED POLYNUCLEOTIDE, characterized in that it is SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22, in which it encodes the mutant IL-2 polypeptide, as defined in any one of claims 1 to 12, or the immunoconjugate, as defined in any one of claims 13 to 17.
[0019]
19. MICRO-ORGANISM HOSTING CELL, characterized by comprising the polynucleotide, as defined in claim 18.
[0020]
20. METHOD OF PRODUCTION OF A MUTANT IL-2 POLYPEPTIDE, which comprises three amino acid mutations that abolish or reduce the affinity of the mutant IL-2 polypeptide to the high affinity IL-2 receptor and preserve the affinity of the polypeptide of Mutant IL-2 for the intermediate affinity IL-2 receptor, each compared to a wild-type IL-2 polypeptide, wherein said three amino acid mutations are in positions corresponding to IL residues 42, 45 and 72 -2 human (SEQ ID NO: 1), or an immunoconjugate thereof, characterized in that it comprises culturing the host cell, as defined in claim 19, under conditions suitable for the expression of the mutant IL-2 polypeptide or the immunoconjugate.
[0021]
21. PHARMACEUTICAL COMPOSITION, characterized in that it comprises the mutant IL-2 polypeptide, as defined in any of claims 1 to 12, or immunoconjugate, as defined in any of claims 13 to 17, and a pharmaceutically acceptable carrier.
[0022]
22. USE OF THE MUTANT IL-2 POLYPEPTIDE, as defined in any of claims 1 to 12, or of the immunoconjugate, as defined in any of claims 13 to 17, characterized in that it is for the manufacture of a medicament to treat a disease in an individual in need of it.
[0023]
23. USE, according to claim 222, characterized by said disease being cancer.
[0024]
24. USE OF THE MUTANT IL-2 POLYPEEPTIDE, as defined in any of claims 1 to 12, or immunoconjugate, as defined in any of claims 13 to 17, characterized in that it is in a pharmaceutically acceptable form in the manufacture of a medicament for stimulate an individual's immune system.

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法律状态:
2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-03-27| B15K| Others concerning applications: alteration of classification|Ipc: C07K 16/40 (2006.01), A61K 38/20 (2006.01), A61K 4 |

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2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-03-19| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-11-12| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-04-07| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2020-07-28| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-08-18| B09X| Republication of the decision to grant [chapter 9.1.3 patent gazette]|

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2020-11-17| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 07/02/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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EP11153964|2011-02-10|

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EP11153964.9|2011-02-10|

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EP11164237.7|2011-04-29|

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EP11164237|2011-04-29|

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