EA040858B1 - IMMUNOCONJUGATES CONTAINING MUTANT INTERLEUKIN-2 POLYPEPTIDES - Google Patents

Description

The technical field to which the invention belongs

The present invention relates generally to mutant interleukin-2 polypeptides. More specifically, the invention relates to mutant IL-2 polypeptides that have improved properties when used as immunotherapeutic agents. In addition, the invention relates to immunoconjugates containing said mutant IL-2 polypeptides, polynucleotide molecules encoding mutant IL-2 polypeptides or immunoconjugates, and vectors and host cells containing said polypeptide molecules. The invention also relates to methods for producing mutant IL-2 polypeptides or immunoconjugates, pharmaceutical compositions containing them, and their use.

Prerequisites for the creation of the invention

Interleukin-2 (IL-2), also referred to as T cell growth factor (TCGF), is a 15.5 kDa globular glycoprotein that plays a major role in the formation, survival, and homeostasis of lymphocytes. It contains 133 amino acids and consists of four antiparallel amphipathic α-helices that form the quaternary structure required for its functioning (Smith, Science 240, 1988, pp. 1169-1176; Bazan, Science 257, 1992, pp. 410-413) . The IL-2 sequences from various species are in the RefSeq NCBI database as No. NP000577 (human), NP032392 (mouse), NP446288 (rat) or NP517425 (chimpanzee).

IL-2 exerts its action by binding to IL-2 receptors (IL-2R), which contain up to three individual subunits, the different association of which can lead to the formation of receptor forms that differ in their affinity for IL-2. The association of the α (CD25), β (CD122) and γ (y _c , CD132) subunits results in the formation of a trimeric high affinity receptor for IL-2. The dimeric IL-2 receptor, consisting of β and γ subunits, is referred to as IL-2R with intermediate affinity. The α subunit forms a monomeric low affinity IL-2 receptor. Although the intermediate affinity dimeric IL-2 receptor binds to IL-2 with about a 100-fold lower affinity than the high affinity trimeric receptor, both dimeric and trimeric variants of the IL-2 receptor have the ability to signal after binding to IL-2 (Minami et al., Annu Rev Immunol 11, 1993, pp. 245-268). Therefore, the α-subunit, i.e. CD25 is not critical for IL-2 signaling. It provides high binding affinity for its receptor, while the β-subunit, i.e. CD122 and the γ subunit are critical for signal transduction (Krieg et al., Proc Nail Acad Sci 107, 2010, pp. 11906-11911). Trimeric IL-2 receptors, including CD25, are expressed by (resting) regulatory CD4 ⁺ , forkhead box P3 ((FoxP3) ⁺ -T cells (T _reg ). They are also transiently induced on normal activated T cells while in the resting state 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, 12005, pp. 142-151).

IL-2 is synthesized mainly by activated T cells, in particular CD4 helper T cells. It stimulates the proliferation and differentiation of T cells, induces the production of cytotoxic T lymphocytes (CTL) and differentiation of peripheral blood lymphocytes into cytotoxic cells and lymphokine-activated killer (LAK) cells, enhances the expression of cytokines and cytolytic molecules by T cells, promotes proliferation and differentiation of B cells and immunoglobulin synthesis by B cells and stimulates the formation, proliferation and activation of natural killer (NK) cells (see, for example, review by Waldmann, Nat Rev Immunol 6, 2009, pp. 595-601; Olejniczak and Kasprzak , Med Sci Monit 14, 2008, RA179-89; Malek, Annu Rev Immunol 26, 2008, pp. 453-479).

The ability of IL-2 to increase lymphocyte populations in vivo and enhance the effector functions of these cells determines the antitumor effects of IL-2, which makes IL-2-based immunotherapy an attractive treatment for certain metastatic cancers. For this reason, patients with metastatic renal cell carcinoma and malignant melanoma are allowed to be treated with high doses of IL-2.

However, IL-2 has a dual function in terms of the immune response, as it not only mediates the proliferation and activity of effector cells, but also plays a critical role in maintaining peripheral immune tolerance.

The main mechanism underlying peripheral self-tolerance is IL-2-induced activation-induced cell death (AICD) of T cells. AICD is a process in which fully activated T cells undergo programmed cell death by interacting with cell surface expressed death receptors such as CD95 (also known as Fas) or the TNF receptor. When antigen activated T cells that express the high affinity IL-2 receptor (after pre-exposure to IL-2) during proliferation are restimulated with antigen by the T cell receptor (TCR)/CD3 complex, Fas ligand (FasL) expression is induced and /or tumor necrosis factor (TNF), which makes cells susceptible to Fas-mediated apoptosis. This process is IL-2 dependent (Lenardo, Nature 353, 1991, pp. 858-861) and is mediated by STAT5. Through the AICD process,

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T-lymphocytes can develop tolerance not only to self-antigens, but also to persistent antigens, such as tumor antigens, which are obviously not a host-created component.

In addition, IL-2 is also involved in the maintenance of peripheral regulatory CD4 CD25 T cells (T _reg ) (Fontenot et al., Nature Immunol 6, 2005, pp. 1142-11451; D'Cruz and Klein, Nature Immunol 6, 2005, pp. 1152-1159; Maloy and Powrie, Nature Immunol 6, 2005, pp. 1171-1172), which are also known as suppressor T cells. They inhibit effector T cells from destroying their (self) target, either through cell-cell contact through inhibition of helper function and T cell activation, or through the release of immunosuppressive cytokines such as IL-10 or TGFβ. Treg depletion has been shown to enhance IL-2-induced antitumor immunity (Imai et al., Cancer Sci 98, 2007, pp. 416-423).

Thus, IL-2 is not an optimal agent for inhibiting tumor growth, because in the presence of IL-2, either the resulting CTLs can recognize the tumor as their own and undergo AICD, or the immune response can be inhibited by IL-2-dependent T _reg cells.

Another problem associated with IL-2 immunotherapy is the side effects of treatment with recombinant human IL-2. Serious cardiovascular, pulmonary, renal, hepatic, gastrointestinal, neurological, cutaneous, hematological, and systemic adverse events are often found in patients receiving high doses of IL-2, which require intensive monitoring and management in the patient's body. Most of these side effects can be explained by the development of the so-called vascular (or capillary) leakage syndrome (VLS), i.e. pathological increase in vascular permeability leading to fluid extravasation in many organs (causing, for example, pulmonary or skin edema and damage to liver cells) and intravascular fluid depletion (causing a drop in blood pressure and a compensatory increase in heart rate). There is no other treatment for VLS other than IL-2 withdrawal. Patients have been studied regimens based on the use of IL-2 at low doses to avoid VLS, however, the therapeutic results obtained were suboptimal. It has been suggested that VLS is caused by the release of pro-inflammatory cytokines such as tumor necrosis factor (TNF)α from activated IL-2 NK cells, however, in recent years it has been found that IL-2-induced pulmonary edema results from direct binding of IL-2 to lung endothelial cells that express low to moderate levels of functional αβγ IL2 receptors (Krieg et al., Proc Nat Acad Sci USA 107, 2010, pp. 11906-11911).

To overcome these problems associated with IL-2 immunotherapy, several approaches have been used. For example, the combination of IL-2 with certain anti-IL-2 monoclonal antibodies has been found to enhance the effects of IL-2 treatment in vivo (Kamimura et al., J Immunol 177, 2006, pp. 306-314; Boyman et al., Science 311, 2006, pp. 1924-1927). In an alternative approach, IL-2 has been mutated in various ways to reduce its toxicity and/or increase its efficacy. Neither et al (Blood 101, 2003, cc. 4853-4861, US Patent Publication No. 2003/0124678) replaced the arginine residue at position 38 of IL-2 with tryptophan to eliminate the vascular permeability activity of IL-2. Shanafelt et al (Nature Biotechnol 18, 2000, pp. 1197-1202) mutated asparagine 88 to arginine to increase T cell selectivity over NK cells. Heaton et al (Cancer Res 53, 1993, pp. 2597-2602; US No. 5229109) introduced two mutations, Arg38Ala and Phe42Lys, to reduce the secretion of pro-inflammatory cytokines from NK cells. Gillies et al (US Patent Publication No. 2007/0036752) replaced three IL-2 residues (Asp20Thr, Asn88Arg and Glnl26Asp) associated with intermediate affinity IL-2 receptor affinity to reduce VLS. Gillies et al (WO 2008/0034473) also modified IL-2 interface with CD25 by amino acid substitution Arg38Trp and Phe42Lys to reduce interaction with CD25 and activate Treg cells to increase efficiency. For the same purpose, Wittrup et al (WO 2009/061853) produced IL-2 mutants with increased affinity for CD25 but not activating the receptor, which as a result acted as antagonists. Mutations were introduced to disrupt the interaction with the β-and/or γ-subunit of the receptor.

However, none of the known IL-2 mutants was found to overcome all of the above problems associated with IL-2 immunotherapy, namely toxicity due to VLS induction, tolerance to tumor antigens due to AICD induction, and immunosuppression. caused by Treg cell activation. Thus, there remains a need in the art to further enhance the therapeutic value of IL-2 proteins.

Brief summary of the invention

The present invention is based, in particular, on the data that the problems associated with immunotherapy based on IL-2 are determined by the interaction of IL-2 with the α-subunit of the trimeric high affinity IL-2 receptor.

Thus, the first object of the invention is a mutant interleukin-2 (IL-2) polypeptide containing a first amino acid mutation that abolishes or reduces the affinity

- 2 040858 mutant IL-2 polypeptide to the high affinity IL-2 receptor and retains the affinity of the mutant IL-2 polypeptide to the IL-2 receptor with intermediate affinity, in each case compared to the wild-type IL-2 polypeptide. In one embodiment of the invention, said first amino acid mutation is at 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 consisting of L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. In a more specific embodiment of the invention, said first amino acid mutation is the L72G amino acid substitution. In some embodiments, the mutant IL-2 polypeptide contains a second amino acid mutation that abolishes or reduces the affinity of the mutant IL-2 polypeptide for the high affinity IL-2 receptor and retains the affinity of the mutant IL-2 polypeptide for the intermediate affinity IL-2 receptor, in each case compared to the wild-type IL-2 polypeptide. In one embodiment, said second mutation is at a position selected from positions corresponding to residues 35, 38, 42, 43 and 45 of human IL-2. In a particular embodiment of the invention, said second amino acid mutation is at 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 consisting of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, and F42K. In an even more specific embodiment of the invention, said second amino acid mutation is the F42A amino acid substitution. In some embodiments, the mutant interleukin-2 polypeptide contains 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 retains the affinity of the mutant IL-2 polypeptide for the intermediate affinity IL-2 receptor, in each case compared to the wild-type IL-2 polypeptide. In a specific embodiment, the mutant interleukin-2 polypeptide contains three amino acid mutations that abolish or reduce the affinity of the mutant IL-2 polypeptide for the high affinity IL-2 receptor and maintain the affinity of the mutant IL-2 polypeptide for the intermediate affinity IL-2 receptor, in each case compared to a wild-type IL-2 polypeptide, wherein the three amino acid mutations are at 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 consisting of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R and L72K. In a specific embodiment of the invention, these three amino acid mutations are the amino acid substitutions F42A, Y45A and L72G. In some embodiments, the mutant interleukin-2 polypeptide further comprises an amino acid mutation that eliminates an IL-2 glycosylation site at a position corresponding to residue 3 of human IL-2. In one embodiment, said amino acid mutation that eliminates the IL-2 Oglycosylation site at position corresponding to residue 3 of human IL-2 is an amino acid substitution selected from the group consisting of T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K and T3P. In a specific embodiment of the invention, the amino acid mutation that eliminates the IL-2 O-glycosylation site at the position corresponding to residue 3 of human IL-2 is T3A. In some embodiments, the mutant IL-2 polypeptide is typically a full length IL-2 molecule, in particular a human full length IL-2 molecule.

The invention also provides a mutant interleukin-2 polypeptide linked to a non-IL-2 fragment (non-IL-2 fragment). In some embodiments, said non-IL-2 fragment is a targeting fragment. In some embodiments, said non-IL-2 fragment is an antigen-binding fragment. In one of the embodiments of the invention, the specified antigennegative fragment is an antibody. In another embodiment, said antigen-binding fragment is an antibody fragment. In a more specific embodiment of the invention, said antigen-binding fragment is selected from a Fab molecule and an scFv molecule. In a particular embodiment of the invention, said antigen-binding fragment is a Fab molecule. In another embodiment, said antigen-binding fragment is an scFv molecule. In specific embodiments, the IL-2 mutant polypeptide is linked to a first and a second non-IL-2 fragment. In one of these embodiments, the mutant interleukin-2 polypeptide is linked by a carboxy-terminal peptide bond to said first non-IL-2 fragment and an amino-terminal peptide bond to said second non-IL-2 fragment. In one of the embodiments of the invention, the specified antigennegative fragment is an immunoglobulin molecule. In a more specific embodiment of the invention, said antigen-binding fragment is

- 3 040858 is an immunoglobulin molecule of the IgG class, in particular of the IgG1 subclass. In some embodiments of the invention, the target of the specified antigen-binding fragment is an antigen present on the tumor cell or in the environment of the tumor cell, in particular an antigen selected from the group including fibroblast activation protein (fibroblast-activating protein) (FAP), tenascin-C A1 domain (TNC A1), tenascin-C A2 domain (TNC A2), fibronectin extra domain B (EDB), carcinoembryonic antigen (CEA), and melanoma-associated chondroitin sulfate proteoglycan (MCSP).

The invention also provides an immunoconjugate containing a mutant IL-2 polypeptide as described herein and an antigen-binding fragment. In one embodiment of the immunoconjugate of the invention, the mutant IL-2 polypeptide is linked via an amino- or carboxy-terminal peptide bond to said antigen-binding moiety. In specific embodiments, the immunoconjugate comprises a first and a second antigen-binding fragment. In one of these embodiments, the mutant IL-2 polypeptide included in the immunoconjugate of the invention is linked by an amino- or carboxy-terminal peptide bond to the first antigen-binding fragment and the second antigen-binding fragment is linked by an amino- or carboxy-terminal peptide bond, or I) to the mutant IL polypeptide -2, or II) with the specified first antigennegative fragment. In one embodiment, the antigen-binding fragment included in the immunoconjugate of the invention is an antibody, in another embodiment, the antigen-binding fragment is an antibody fragment. In a particular embodiment of the invention, said antigen-binding fragment is selected from a Fab molecule and an scFv molecule. In a particular embodiment of the invention, said antigen-binding fragment is a Fab molecule. In another particular embodiment of the invention, said antigen-binding fragment is an immunoglobulin molecule. In a more specific embodiment of the invention, said antigen-binding fragment is an immunoglobulin molecule of the IgG class, in particular of the IgG1 subclass. In some embodiments of the invention, the target of said antigen-binding fragment is an antigen present on the tumor cell or in the environment of the tumor cell, in particular, an antigen selected from the group consisting of fibroblast activation protein (FAP), tenascin-C A1 domain (TNC A1), Tenascin-C A2 domain (TNC A2), fibronectin extra B domain (EDB), carcinoembryonic antigen (CEA), and melanoma-associated chondroitin sulfate proteoglycan (MCSP).

The invention also provides isolated polynucleotides encoding a mutant IL-2 polypeptide or immunoconjugate as described herein, expression vectors containing said polynucleotides, and host cells containing the polynucleotides or expression vectors.

Also provided is a method for producing a mutant IL-2 polypeptide or immunoconjugate provided in the present description, a pharmaceutical composition containing a mutant IL-2 polypeptide or an immunoconjugate provided in the present description, and a pharmaceutically acceptable carrier, and methods for using a mutant IL-2 polypeptide or immunoconjugate, presented in the present description.

In particular, the invention relates to a mutant IL-2 polypeptide or immunoconjugate provided in the present description, intended for use in the treatment of a disease in an individual who needs it. In a specific embodiment of the invention, the disease is cancer. In a specific embodiment of the invention, the individual is a human.

The invention also relates to the use of a mutant IL-2 polypeptide or immunoconjugate provided herein for the preparation of a medicament for the treatment of a disease in an individual in need thereof.

In addition, a method for treating a disease in an individual is provided, which comprises administering to said individual a therapeutically effective amount of a mutant IL-2 polypeptide or an immunoconjugate as described herein. Said disease is preferably cancer.

In addition, a method for stimulating the immune system in an individual is provided, which consists in administering to said individual in an effective amount a composition containing a mutant IL-2 polypeptide or immunoconjugate presented in the present description, in a pharmaceutically acceptable form.

Detailed Description of the Invention Definitions

The terms used in the present description have the meanings generally accepted in this field, unless otherwise specifically indicated below.

In the context of the present description, the term interleukin-2 or IL-2, unless otherwise indicated, refers to any native IL-2 from any vertebrate used as a source,

- 4 040858 including mammals such as primates (eg humans) and rodents (eg mice and rats). The concept includes unprocessed IL-2, as well as any form of IL-2 obtained as a result of processing in the cell. The term also includes naturally occurring variants of IL-2, such as splicing variants or allelic variants. By way of example, the amino acid sequence of human IL-2 is shown in SEQ ID NO: 1. Full-length human IL-2 further comprises a 20 amino acid N-terminal signal peptide having the sequence of SEQ ID NO: 272, which is absent from the mature IL molecule. -2.

The term IL-2 mutant or IL-2 mutant polypeptide as used herein is intended to refer to 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. with another molecule, for example, by fusion or chemical conjugation. The term "full length" when used with respect to IL-2 is intended to mean a mature IL-2 molecule that is of naturally occurring length. For example, full-length human IL-2 refers to a molecule that contains 133 amino acids (see, for example, SEQ ID NO: 1). The various forms of IL-2 mutants are distinguished by the presence of at least one amino acid mutation that affects the interaction of IL-2 with CD25. Such a mutation may include a substitution, deletion, truncation or modification of the wild-type amino acid residue normally located at that position. Mutations obtained by amino acid substitution are preferred. Unless otherwise indicated, an IL-2 mutant may be referred to herein as a mutant IL-2 peptide sequence, a mutant IL-2 polypeptide, a mutant IL-2 protein, or a mutant IL-2 analog.

In the context of the present description, the designation of different forms of IL-2 is made relative to the sequence presented in SEQ ID NO: 1. In the context of the present description, different designations can be used for the same mutation. For example, a mutation that results in a change from phenylalanine at position 42 to alanine can be referred to as 42A, A42, A ₄₂ , F42A, or Phe42Ala.

In the context of the present description, it is understood that the term amino acid mutation refers to amino acid substitutions, deletions, insertions and modifications. Any combination of substitution, deletion, insertion, and modification may be used to create the final construct, provided that the final construct has the desired characteristics, such as reduced ability to bind to CD25. The amino acid sequence with deletions and insertions includes amino and/or carboxy terminal deletions and insertions of amino acids. An example of a terminal deletion is the deletion of the alanine residue at position 1 of full-length human IL-2. Preferred amino acid mutations are amino acid substitutions. To change, for example, the binding characteristics of an IL-2 polypeptide, non-conservative amino acid substitutions are most preferred, ie. replacement of one amino acid with another amino acid having different structural and/or chemical properties. Preferred amino acid substitutions include replacing a hydrophobic amino acid with a hydrophilic one. Amino acid substitutions include substitution with non-naturally occurring amino acids or derivatives of the naturally occurring twenty standard amino acids (eg, 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, and the like. It is contemplated that methods of altering the amino acid side group other than genetic engineering, such as chemical modification, can also be used.

As used herein, a wild-type form of IL-2 is a form of IL-2 that is the same as a mutant IL-2 polypeptide except that, in the wild-type form, there is a wild-type amino acid at each amino acid position of the mutant IL-2 polypeptide. -2. For example, if an IL-2 mutant is full-length IL-2 (ie, IL-2 not fused or conjugated to any other molecule), then the wild-type form of that mutant is full-length native IL-2. If the IL-2 mutant is a fusion of IL-2 and another polypeptide encoded downstream of IL-2 (e.g., an antibody chain), then the wild-type form of that IL-2 mutant is IL-2 with the wild-type amino acid sequence, fused to the same downstream encoded polypeptide. In addition, if an IL-2 mutant is a truncated form of IL-2 (a mutated or modified sequence in the non-truncated portion of IL-2), then the wild-type form of that IL-2 mutant is a similarly truncated IL-2 that has the wild-type sequence . For purposes of comparing the binding affinity of the IL-2 receptor or the biological activity of different forms of IL-2 mutants and the corresponding wild-type form of IL-2, wild-type forms include forms of IL-2 containing one or more amino acid mutations that do not affect binding. IL-2 receptor versus naturally occurring native IL-2, such as replacing a cysteine at residue 125 of human IL-2 with alanine. In some embodiments, for the purposes of the present invention, the wild-type form of IL-2 contains the C125A amino acid substitution (see SEQ ID NO: 3). In some embodiments, the polypeptide

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The wild-type IL-2 to which the mutant IL-2 polypeptide is compared contains the amino acid sequence of SEQ ID NO: 1. In other embodiments, the wild-type IL-2 polypeptide to which the mutant IL-2 polypeptide is compared contains the amino acid sequence of SEQ ID NO: 3.

As used herein, CD25 or IL-2 receptor α-subunit, unless otherwise indicated, refers to any native CD25 from any vertebrate source, including mammals such as primates (eg, humans) and rodents (eg, mice and rats). The concept includes full-length unprocessed CD25, as well as any form of CD25 obtained as a result of processing in the cell. The term also includes naturally occurring variants of CD25, such as splicing variants or allelic variants. In some embodiments, the CD25 is human CD25. As an example, the amino acid sequence of human CD25 (with signal sequence, Avi tag and His tag) is shown in SEQ ID NO: 278.

In the context of the present description, the term high-affinity IL-2 receptor refers to the heterotrimeric form of the IL-2 receptor, consisting of the receptor γ subunit (which is also known as the general γ subunit of the cytokine receptor, γ ₀ , or CD 132), the receptor β subunit ( also known as CD122 or p70) and the receptor α-subunit (also known as CD25 or p55). In contrast, the term IL-2 receptor with intermediate affinity refers to an IL2 receptor that includes only the γ-subunit and the β-subunit, but does not contain the α-subunit (see, for example, a review by Olejniczak and Kasprzak, Med Sci Monit 14, 2008 , RA179-189). The concept of affinity refers to the total strength of all non-covalent interactions between an individual binding site of a molecule (eg, receptor) and its binding partner (eg, ligand). Unless otherwise indicated, as used herein, binding affinity refers to the inherent binding affinity of the components of a binding pair (eg, receptor and ligand), reflecting a 1:1 interaction. The affinity of a molecule X for its partner Y can generally be characterized by the dissociation constant (K _D ), which is the ratio of the dissociation and association reaction rate constants (k _off and k _on , respectively). Thus, equivalent affinities can correspond to different rate constants if the ratio of rate constants remains the same. Affinity can be assessed by conventional methods known in this field, including presented in the present description.

The affinity of a mutant or wild-type IL-2 polypeptide for various forms of the IL-2 receptor can be determined using the Surface Plasmon Resonance (SPR) method described in the Examples section using a standard instrumentation such as the BIAcore device (GE Healthcare) and receptor subunits that can be obtained using the method of recombinant expression (see, for example, Shanafelt and others, Nature Biotechnol 18, 2000, cc. 1197-1202). Alternatively, the binding affinity of IL-2 mutants to various forms of the IL-2 receptor can be assessed using cell lines known to be capable of expressing one or the other form of the receptor. Specific illustrative examples of options for measuring binding affinity are described below.

By regulatory T cell or Treg cell is meant a specialized type of CD4+ T cell that can suppress the responses of other T cells. Treg cells are distinguished by their ability to express the α-subunit of the IL-2 receptor (CD25) and the forkhead box transcription factor P3 (FOXP3) (Sakaguchi, Annu Rev Immunol 22, 2004, pp. 531-562), and they play a critical role in the induction and maintaining peripheral self-tolerance to antigens, including those expressed by tumors. Treg cells require IL-2 for their function and development and induction of their ability to exert an inhibitory effect.

In the context of the present description, the term effector cells refers to a population of lymphocytes that mediate the cytotoxic actions of IL-2. Effector cells are effector T cells such as cytotoxic CD8 T cells, NK cells, lymphokine-activated killer (LAK) cells, and macrophages/monocytes.

In the context of the present description, the term antigen-binding fragment refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, the antigen-binding fragment has the ability to direct the substance to which it is attached (for example, a cytokine or a second antigen-binding fragment) to a target site, for example, to a specific type of tumor cell or tumor stroma bearing the antigenic determinant. Antigen binding fragments include antibodies and fragments thereof, which will be further described below. Preferred antigen-binding fragments include the antigen-binding domain of an antibody, which contains the variable region of the heavy chain of the antibody and the variable region of the light chain of the antibody. In some embodiments, antigen-binding fragments may include antibody constant regions, which will be further described below and are known in the art. Suitable heavy chain constant regions include any of

- 6 040858 five isotypes: α, δ, ε, γ or μ. Suitable light chain constant regions include either of two isotypes: k and λ.

The term specifically binds means that the binding is selective for an antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen-binding fragment to bind to a specific antigenic determinant can be determined using an enzyme-linked immunosorbent assay (ELISA) or other techniques known to one of skill in the art, such as surface plasmon resonance technique (by performing analysis with a BIAcore device) (Liljeblad et al. ., Glyco J 17, 2000, pp. 323-329), and conventional binding assays (Heeley, Endocr Res 28, 2002, pp. 217-229).

As used herein, antigenic determinant is synonymous with antigen and epitope and refers to a site (e.g., a region of contiguous amino acids or a conformational configuration of distinct regions of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen-binding fragment binds to form a complex. antigen-binding fragment-antigen. Suitable antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, in the free state in the blood serum and/or in the extracellular matrix (ECM).

In the context of the present description, the term polypeptide refers to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (also referred to as peptide bonds). The term polypeptide refers to any chain of two or more amino acids and does not imply that the product is of a particular length. Thus, peptides, dipeptides, tripeptides, oligopeptides, protein, amino acid chain, or any other conventional term referring to a chain of two or more amino acids, all fall under the definition of polypeptide, and the term polypeptide can be used instead of or interchangeably with any of these. concepts. The term polypeptide is also intended to refer to products that carry post-expression modifications of the polypeptide, including (but not limited to) glycosylation, acetylation, phosphorylation, amidation, derivatization using known protecting/blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. The polypeptide may be obtained from a naturally occurring biological source, or may be obtained using recombinant DNA technology, and need not be translated from an engineered nucleotide sequence. It can be created in any way, including chemical synthesis. The polypeptide of the invention may be about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1000 or more or 2000 or more amino acids. The polypeptides may have a different three-dimensional structure, although they do not have to have the specified structure. Polypeptides with a defined three-dimensional structure are referred to as folded polypeptides, and polypeptides that do not have a defined three-dimensional structure but can more readily adapt to a large number of different conformations are referred to as non-folded polypeptides.

By isolated polypeptide or variant or derivative is meant a polypeptide that is not found in its natural environment. It does not require any specific level of purification. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinant polypeptides and proteins expressed in host cells are considered isolated for the purposes of the present invention if they are native or recombinant polypeptides that have been separated, fractionated, or partially or completely purified by any suitable method.

Percent (%) amino acid sequence identity relative to the reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the polypeptide reference sequence, after sequence alignment and the introduction of gaps, if necessary, to achieve the maximum percent sequence identity without any conservative substitutions. taken into account when evaluating sequence identity. Comparison to determine percent amino acid sequence identity can be performed in a variety of ways that are within the skill of the art, for example, using publicly available computer programs such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the compared sequences. However, for the purposes of the present invention, the % amino acid sequence identity value is obtained using the ALIGN-2 sequence comparison computer program. The sequence comparison computer program ALIGN-2 was developed by Genentech, Inc. and the source code is deposited with the user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under registration number U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is a publicly available program from Genentech, Inc. of South San Francisco, CA. California, or it can be compiled from source. The ALIGN-2 program can be compiled for use on the UNIX operating system, including digital UNIX V4.0D. In the ALIGN-2 program, all parameters for sequence comparison are preset and should not be changed. In situations where ALIGN-2 is used to compare amino acid sequences, the % amino acid sequence identity of a given amino acid sequence A relative to or compared to a given amino acid sequence B (which in other words can be referred to as a given amino acid sequence A that has or differs by a certain % amino acid identity sequence relative to or compared to a given amino acid sequence B) is calculated as follows:

100x quotient X/Y where X is the number of amino acid residues evaluated by the ALIGN-2 sequence comparison program as identical matches when comparing sequences A and B with said program, and where Y is the total number of amino acid residues in B. It should be obvious that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % identity of amino acid sequence A relative to amino acid sequence B should not be equal to the % identity of amino acid sequence B relative to amino acid sequence A. Unless specifically stated otherwise, in the context of this description, all % amino acid sequence identity values are obtained according to the procedure described in the last of the preceding paragraphs using the ALIGN-2 computer program.

The term polynucleotide refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), viral-derived RNA, or plasmid DNA (pDNA). The polynucleotide may contain a conventional phosphodiester bond or a non-traditional bond (eg, an amide bond, such as is present in peptide nucleic acids (PNAs)). The term nucleic acid molecule refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide.

By isolated nucleic acid or polynucleotide is meant a nucleic acid molecule, i. DNA or RNA that is separated from its native environment. For example, a recombinant polynucleotide encoding a therapeutic polypeptide included in the vector is considered to be isolated for the purposes of the present invention. Other examples of an isolated polynucleotide are recombinant polynucleotides present in heterologous host cells, or purified (partially or completely) polynucleotides in solution. An isolated polynucleotide includes a polynucleotide molecule found in cells that normally contain the polynucleotide molecule, but the polynucleotide molecule is present outside the chromosome or has a different chromosomal localization than it naturally does on the chromosome.

Isolated RNA molecules include the in vivo or in vitro derived RNA transcripts of the present invention, as well as positive, negative and double stranded forms. The isolated polynucleotides or nucleic acids of the present invention also include these synthetically produced molecules. In addition, the polynucleotide or nucleic acid may be or may include a regulatory element such as a promoter, a ribosome binding site, or a transcription terminator. By a nucleic acid or polynucleotide having/having a nucleotide sequence that is, for example, 95% identical to the nucleotide reference sequence of the present invention, it is meant that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may include up to 5 point mutations for every 100 nucleotides of the nucleotide reference sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the nucleotide reference sequence, up to 5% of the nucleotides in the reference sequence can be removed by deletion or replaced with another nucleotide, or up to 5% of the nucleotides from the total number of nucleotides in the reference sequence can be inserted into the reference sequence. These reference sequence changes can take place at positions at the 5' or 3' end of the nucleotide reference sequence, or at a different position between these end positions, and are inserted either individually between residues in the reference sequence, or they are inserted into the reference sequence. sequence as one or more contiguous groups. In practice, whether a particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence of the present invention can be decided by usually using known computer programs, such as those mentioned above for polypeptides (on

- 8 040858 example, ALIGN-2).

The term expression cassette refers to a polynucleotide obtained by recombination or synthesis, which contains a series of specific nucleotide elements that provide transcription of a particular nucleic acid in a target cell. The recombinant expression cassette can be inserted into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, a recombinant expression cassette that is part of an expression vector includes, among other sequences, a nucleotide sequence to be transcribed and a promoter. In some embodiments, the expression cassette of the invention contains polynucleotide sequences that encode mutant IL-2 polypeptides or immunoconjugates of the invention, or fragments thereof.

The term vector or expression vector is synonymous with expression construct and refers to a DNA molecule that is used to introduce and cause the expression of a particular gene to which it is operably linked in a target cell. The concept includes a vector, which is a self-replicating nucleic acid structure, as well as a vector integrated into the genome of the host cell into which it is introduced. The expression vector of the present invention contains an expression cassette. Expression vectors allow the transcription of large amounts of stable mRNA. When the expression vector is within the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cellular mechanism of transcription and/or translation. In one embodiment, the expression vector of the invention comprises an expression cassette that includes polynucleotide sequences that encode mutant IL-2 polypeptides or immunoconjugates of the invention, or fragments thereof.

The term "artificial" refers to a composition that is synthetic or not derived from a host cell, such as a chemically synthesized oligonucleotide.

As used herein, the terms host cell, host cell line, and host cell culture are used interchangeably and refer to cells into which an exogenous nucleic acid has been introduced, including progeny of said cells. Transformants and transformed host cells include the primary cells of interest as well as cultures derived from them, regardless of the number of passages. The offspring may not be strictly identical to the parent cell in the composition of nucleic acids, but may carry mutations. This concept includes mutant progeny that have the same function or biological activity as the original transformed cell selected by screening or selection.

In the context of the present description, the term antibody is used in its broadest sense and refers to various antibody structures, including (but not limited to) monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies) and antibody fragments, provided that they have the desired antigen-binding activity.

In the context of the present description, the terms full-length antibody, intact antibody and complete antibody are used interchangeably, and they refer to an antibody having a structure that is almost similar to the structure of a native antibody, or having heavy chains that contain the Fc region specified in the present description.

The term antibody fragment refers to a molecule, other than an intact antibody, that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include (but are not limited to) Fv, Fab, Fab', Fab'-SH, F(ab') ₂ , dimeric antibodies, linear antibodies, single chain antibody molecules (eg scFv) and multispecific antibodies produced from antibody fragments. For a review of some antibody fragments, see, for example, Hudson et al., Nat Med 9, 2003, ss. 129-134. For a review of scFv fragments, see, for example, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, ed. Rosenburg and Moore, Springer-Verlag, New York, 1994, pp. 269-315; see also WO 93/16185 and US Nos. 5,571,894 and 5,587,458. For a discussion of Fab- and F(ab') ₂ fragments containing rescue receptor-binding epitope residues and having an extended in vivo half-life, see US No. 5869046. Dimeric antibodies (diabodies) are antibody fragments with two antigen-binding sites, which can be bivalent or bispecific (see, for example, EP 404097; WO 1993/01161; Hudson et al., Nat Med 9, 2003, cc. 129 -134 and Hollinger et al., Proc Natl Acad Sci USA 90, 1993, pp. 6444-6448 Trimeric (triabodi) and tetrameric (tetrabodium) antibodies are also described in Hudson et al., Nat Med 9, 2003, pp. 129 -134 Antibody fragments can be created using a variety of techniques, including (but not limited to) proteolytic cleavage of intact antibodies, as well as obtained using recombinant host cells (for example, E. coli or phage), as described in the present description. .

The term immunoglobulin molecule refers to a protein having the structure of a naturally occurring antibody. For example, IgG immunoglobulins are hetero

- 9 040858 tetrameric glycoproteins with a molecular weight of approximately 150,000 Da, consisting of two light chains and two heavy chains linked by disulfide bridges. From the N-terminus to the C-terminus, each heavy chain contains a variable region (VH), also referred to as the variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3), also referred to as the constant heavy chain region. Likewise, from the N-terminus to the C-terminus, each light chain contains a variable region (VL), also referred to as the variable light domain or light chain variable domain, followed by a light chain constant domain (CL), also referred to as the constant region. light chain. An immunoglobulin heavy chain can belong to one of five classes, designated as α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which are further subclassified, such as γ1 (IgG1 ), γ ₂ (IgG ₂ ), Y3 (IgG ₃ ), Y4 (IgG4), α1 (IgA1) and α ₂ (IgA ₂ ). An immunoglobulin light chain can be one of two types, designated kappa (k) and lambda (λ), based on the amino acid sequence of its constant domain. An immunoglobulin typically consists of two Fab molecules and an Fc domain that are linked through the immunoglobulin hinge region.

The term antigen-binding domain refers to the part of an antibody that contains a region that specifically binds and is complementary to part of an antigen or to a complete antigen. An antigen binding domain may be, for example, one or more antibody variable domains (also referred to as antibody variable regions). Preferably, the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

The term variable region or variable domain refers to the domain of the heavy or light chain of an antibody which/which is involved in the binding of an antibody to an antigen. The heavy chain and light chain variable domains (VH and VL, respectively) of a native antibody generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR) (see, e.g., Kindt and et al., Kuby Immunology, 6th ed., W. H. Freeman and Co., 2007, p. 91). A single VH or VL domain may be sufficient to provide antigen binding specificity.

The concept of a hypervariable region or HVR in the context of the present description refers to each of the regions of the variable domain of an antibody, the sequence of which is hypervariable, and/or which form structures in the form of loops (hypervariable loops). Typically, native four-chain antibodies contain six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically contain amino acid residues from hypervariable loops and/or from complementarity determining regions (CDRs), the latter having the most pronounced sequence variability and/or being involved in antigen recognition. In addition to CDR1 present in VH, CDRs typically contain amino acid residues that form hypervariable loops. The term hypervariable regions (HVR) also refers to complementarity determining regions (CDRs), and in the context of the present description, these terms are used interchangeably with respect to the positions of the variable region, which form antigennegative regions. This particular area is described in Kabat et al., U.S. Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest, 1983 and Chothia et al., J. Mol. Biol. 196, 1987, pp. 901-917, and these definitions refer to overlapping amino acid residues or subsets of amino acid residues when compared with each other. However, within the context of the present description, any definition of the CDR of an antibody or variants thereof is contemplated. The respective amino acid residues that make up the CDRs, as defined in each of the references cited above, are compared in Table 1 below. 1. The exact numbers of the residues that form a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art, based on the amino acid

Table 1. CDR Definitions

cab from Hotia AbM ² V _H CDR1 31-35 26-32 26-35 V _H CDR2 50-65 52-58 50-58 V _H CDR3 95-102 95-102 95-102 V _L CDR1 24-34 26-32 24-32 V _L CDR2 50-56 50-52 50-56 V _L CDR3 89-97 91-96 89-97

1Numbering of all residues included in the CDR in table. 1 is given in accordance with the nomenclature proposed by Cabot et al. (see below).

² The designation AbM with a capital letter b, used in Table. 1 refers to CDRs as defined by the Oxford Molecular Group's AbM antibody modeling program.

Cabot and co-authors also proposed a numbering system (nomenclature) for sequences

- 10 040858 variable regions, which can be used for any antibody. One of ordinary skill in the art can unambiguously apply this Cabot numbering system to any variable region sequence without having any experimental data other than knowledge of the sequence itself. As used herein, the term Cabot numbering refers to the numbering system described by Kabat et al., Sequence of Proteins of Immunological Interest, U.S. Dept. of Health and Human Services, 1983. Unless otherwise indicated, references to the numbering of positions of specific amino acid residues in the variable region of an antibody are given in accordance with the Cabot numbering system.

The numbering of polypeptide sequences in the Sequence Listing (ie, SEQ ID NOs: 23, 25, 27, 29, 31, 33, etc.) is not a Cabot numbering. However, it is within the purview of one of ordinary skill in the art to convert the sequence numbering in the Sequence Listing to Cabot numbering.

Framework regions or FR regions are regions of variable domains other than hypervariable region (HVR) residues. Variable domain FRs are typically represented by four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences are typically located in the VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The term antibody class refers to the type of constant domain or constant region characteristic of a heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG ₂ , IgG ₃ , IgG ₄ , IgA1 and IgA2. The heavy chain constant domains corresponding to the various classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively.

In the context of the present description, the term Fc-region refers to the C-terminal region of the heavy chain of an immunoglobulin, which contains at least part of the constant region. The term refers to native sequence Fc regions and variants of Fc regions. Although the border sequences of the Fc region in the IgG heavy chain may vary slightly, in general the Fc region of the human IgG heavy chain extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present.

A heterodimerization enhancing modification is a manipulation of the peptide backbone or post-translational modifications to a polypeptide, such as an immunoglobulin heavy chain, that reduces or prevents the association of the polypeptide with an identical polypeptide to form a homodimer. As used herein, a heterodimerization-enhancing modification primarily includes the various modifications made to each of the two polypeptides required to form a dimer, the modifications complementing each other so as to enhance the association of the two polypeptides. For example, a modification that enhances heterodimerization may change the structure or charge of one or both of the polypeptides required for dimer formation, so as to improve their association sterically or electrostatically, respectively. Heterodimerization occurs between two non-identical polypeptides, such as the heavy chains of two immunoglobulins, and the additional components of the immunoconjugate of the heavy chains fused to each other (eg, an IL-2 polypeptide) are not the same. In the immunoconjugates of the present invention, the heterodimerization-enhancing modification affects the heavy chain(s), in particular the Fc domain of the immunoglobulin molecule. In some embodiments, the heterodimerization enhancing modification is an amino acid mutation, in particular an amino acid substitution. In a particular embodiment of the invention, the heterodimerization enhancing modification is a single amino acid mutation, in particular an amino acid substitution, in each of the two immunoglobulin heavy chains.

The term effector functions, as used in relation to antibodies, refers to the biological activities inherent in the Fc region of an antibody, which vary depending on the isotype of the antibody. Examples of antibody effector functions are Clq binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-mediated cell-dependent cytotoxicity (ADCC), antibody-mediated cell-dependent phagocytosis (ADCP), cytokine secretion, downregulation of cell surface receptors (e.g., B-cell receptor); and activation of B cells.

An Fc activating receptor is an Fc receptor that, after interacting with the Fc region of an antibody, initiates a signaling process that stimulates the receptor-bearing cell to perform effector functions. Activating Fc receptors include FcYRIIIa (CD16a), FcyRI (CD64), FcYRIIa (CD32) and FcaRI (CD89).

As used herein, the terms design, engineered, engineered are intended to include any manipulation of the peptide backbone or post-translational modifications of a naturally occurring or recombinant polypeptide or its

- 11 040858 fragments. Engineering includes amino acid sequence modifications, glycosylation patterns, or side chain groups of individual amino acids, as well as combinations of these approaches.

As used herein, the term immunoconjugate refers to a polypeptide molecule that includes at least one IL-2 fragment and at least one antigen-binding fragment. In some embodiments, the immunoconjugate comprises at least one IL-2 fragment and at least two antigen-binding fragments. Particular immunoconjugates of the invention essentially consist of one IL-2 fragment and two antigen-binding fragments linked by one or more linker sequences. An antigen-binding fragment can be linked to an IL-2 fragment through various interactions and in a wide variety of configurations as described herein.

In the context of the present description, the concept of the control antigennegative fragment refers to the antigennegative fragment, which must be free from other antigennegative fragments and effector fragments. For example, when comparing a Fab-IL2-Fab immunoconjugate of the invention with a control antigen-binding fragment, the control antigen-binding fragment is a free Fab, and the FabIL2-Fab immunoconjugate and the free Fab molecule can both specifically bind to the same antigen determinant.

In the context of the present description, the terms first and second regarding antigen-binding fragments, etc., are used for convenience of distinction when more than one fragment of each type is present. It is implied that the use of these concepts does not define the specific order or orientation of the immunoconjugate, unless specifically indicated otherwise.

The concept of an effective amount of an agent refers to the amount necessary to provide a physiological change in the cell or tissue into which it is administered.

The concept of a therapeutically effective amount of an agent, such as a pharmaceutical composition, refers to an amount effective when used in doses and for periods of time necessary to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent, for example, eliminates, reduces, retards, minimizes, or prevents the adverse events of a disease.

The individual or subject is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as marmosets), rabbits, and rodents (e.g., mice and rats). Preferably the individual or subject is a human.

The term "pharmaceutical composition" refers to a preparation that is in such a form that it provides the biological activity of the active substance included in its composition, which should be effective and which does not contain additional components that have unacceptable toxicity for the individual to whom the composition should be administered.

A pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical composition, other than the active ingredient, that is non-toxic to an individual. Pharmaceutically acceptable carriers include, but are not limited to, a buffer, excipient, stabilizer, or preservative.

In the context of the present description, the term treatment (and its grammatical variations, such as treat or process of treatment) refers to a clinical intervention with the aim of changing the natural course of the disease in the individual being treated, and it can be carried out either for prevention or in the course of development of clinical pathology. The desired actions of treatment are (but not limited to) prevention of the onset or recurrence of the disease, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastases, reduction in the rate of disease progression, alleviation or temporary amelioration of the disease state, and remission or improvement in prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or slow the progression of a disease.

Detailed description of embodiments of the invention

The present invention is based on the task of creating a mutant IL-2 polypeptide with improved properties for immunotherapy. In particular, the invention is based on the task of eliminating the pharmacological properties of IL-2, which are involved in the manifestation of its toxicity, but are not decisive for the effectiveness of IL-2. As stated above, different forms of the IL-2 receptor are composed of different subunits and have different affinities for IL-2. The moderate affinity IL-2 receptor, consisting of the β and γ subunits of the receptor, is expressed on resting effector cells and its presence is sufficient to mediate IL-2 signaling. The high-affinity IL-2 receptor, which additionally contains the α-subunit of the receptor, is expressed

- 12 040858 mainly on regulatory T cells (T _reg ), as well as on activated effector cells, while their interaction with IL-2 can enhance T- _cell mediated immunosuppression or activation-induced cell death (AICD), respectively. Thus, without wishing to be bound by theory, it can be hypothesized that the reduction or reversal of IL-2 affinity for the α subunit of the IL-2 receptor may reduce IL-2-induced downregulation of effector cell function by regulatory T cells and the development of tumor tolerance by the process AICD. On the other hand, maintaining the affinity of the intermediate affinity IL-2 receptor supports the induction of proliferation and activation of effector cells such as NK and T cells by IL-2.

Several mutants of IL-2 are already known in the art, however, new amino acid mutations of the IL-2 polypeptide and combinations thereof have been discovered during the invention that are most advantageous for conferring IL-2 characteristics required for immunotherapy.

The first object of the invention is a mutant interleukin-2 (IL-2) polypeptide containing an 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 maintains the affinity of the mutant IL-2 polypeptide for the IL-2 receptor with intermediate affinity, in each case compared to the wild-type IL-2 polypeptide.

Mutants of human IL-2 (hIL-2) with reduced affinity for CD25 can be created, for example, by amino acid substitution of the amino acid at position 35, 38, 42, 43, 45 or 72, or combinations thereof. Examples of amino acid substitutions are K35E, K35A, R38A, R38E, R38N, R38F, R38S, R38L, R38G, R38Y, R38W, F42L, F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, K43E , Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R and L72K. Specific IL-2 mutants of the invention contain a mutation at the amino acid position corresponding to residue 42, 45, or 72 of human IL-2, or a combination thereof. These mutants have substantially the same affinity for the intermediate affinity IL-2 receptor and have greatly reduced affinity for the α-subunit of the IL-2 receptor and the high affinity IL-2 receptor compared to the wild-type form of the IL-2 mutant.

Another characteristic of promising mutants may be the ability to induce proliferation of IL-2 receptor-bearing T cells and/or NK cells, the ability to induce IL-2 signaling in IL-2 receptor-bearing T cells and/or NK cells, the ability to act on the production of interferon (IFN)-y as a secondary cytokine by NK cells, reduced ability to induce the production of secondary cytokines, primarily IL-10 and TNF-α, by peripheral blood mononuclear cells (PBMC), reduced ability to activate regular T cells, reduced the ability to induce T cell apoptosis and a reduced toxicity profile in vivo.

In one embodiment, the amino acid mutation that abolishes or reduces the affinity of the mutant IL-2 polypeptide for the high affinity IL-2 receptor and retains the affinity of the mutant IL-2 polypeptide for the intermediate affinity IL-2 receptor is at a position corresponding to residue 72 of the human IL-2. In one embodiment, the amino acid mutation is an amino acid substitution. In one embodiment, the amino acid substitution is selected from the group consisting of L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. In a more specific embodiment of the invention, the amino acid mutation is an L72G amino acid substitution.

A specific object of the invention is a mutant IL-2 polypeptide containing first and second amino acid mutations that abolish or reduce the affinity of the mutant IL-2 polypeptide for the α-subunit of the IL-2 receptor and maintain the affinity of the mutant IL-2 polypeptide for the IL-2 receptor with an intermediate affinity. In one embodiment, the first amino acid mutation is at position corresponding to residue 72 of human IL-2. In one embodiment of the invention, said first amino acid mutation is an amino acid substitution. In a particular embodiment of the invention, said first amino acid mutation is an amino acid substitution selected from the group consisting of L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. In an even more specific embodiment of the invention, the amino acid substitution is L72G. Said second amino acid mutation is in a position different from that of the first amino acid mutation. In one embodiment of the invention, 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 one embodiment of the invention, said second amino acid mutation is an amino acid substitution. In a particular embodiment, said amino acid substitution is selected from the group consisting 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 of the invention, said second amino acid mutation is at position corresponding to residue 42 or 45 of human IL-2. In a specific embodiment of the invention, the

- 13 040858 the given second amino acid mutation is an amino acid substitution selected from the group consisting of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N , Y45D, Y45R and Y45K. In a more specific embodiment of the invention, said second amino acid mutation is the F42A or Y45A amino acid substitution. In a more specific embodiment of the invention, said second amino acid mutation is at a position corresponding to residue 42 of human IL-2. In a particular embodiment of the invention, said second amino acid mutation is an amino acid substitution selected from the group consisting of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, and F42K. In a more specific embodiment of the invention, said amino acid substitution is F42A. In another embodiment, said second amino acid mutation is at position corresponding to residue 45 of human IL-2. In a particular embodiment of the invention, said second amino acid mutation is an amino acid substitution selected from the group consisting of Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, and Y45K. In a more specific embodiment of the invention, said amino acid substitution is Y45A. In some embodiments, the mutant IL-2 polypeptide contains a third 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 retains the affinity of the mutant IL-2 polypeptide for the IL-2 receptor with intermediate affinity, in each case compared to the wild-type IL-2 polypeptide. Said third amino acid mutation is in a position different from those of said first and second amino acid mutations. In one embodiment, said third 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 preferred embodiment of the invention, said third amino acid mutation is at position corresponding to residue 42 or 45 of human IL-2. In one embodiment, said third amino acid mutation is at position corresponding to residue 42 of human IL-2. In another embodiment of the invention, said third amino acid mutation is at a position corresponding to residue 45 of human IL-2. In one embodiment of the invention, said third amino acid mutation is an amino acid substitution. In a particular embodiment, said third amino acid substitution is selected from the group consisting 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 consisting of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R and Y45K. In an even more specific embodiment of the invention, said amino acid substitution is F42A or Y45A. In one embodiment of the invention, said amino acid substitution is F42A. In another embodiment of the invention, said amino acid substitution is Y45A. In some embodiments, the mutant IL-2 polypeptide does not contain an amino acid mutation at the position corresponding to residue 38 of human IL-2.

An even more specific subject of the invention is a mutant IL-2 polypeptide containing three amino acid mutations that abolish or reduce the affinity of the mutant IL-2 polypeptide for the α-subunit of the IL-2 receptor, but retain the affinity of the mutant IL-2 polypeptide for the IL-2 receptor with intermediate affinity. In one of the embodiments of the invention these three amino acid mutations are in positions corresponding to residues 42, 45 and 72 of human IL-2. In one embodiment, the three amino acid mutations are amino acid substitutions. In one embodiment, said three amino acid mutations are amino acid substitutions selected from the group consisting of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R and L72K. In a specific embodiment of the invention, these three amino acid mutations are the amino acid substitutions F42A, Y45A and L72G.

In some embodiments of the invention, these amino acid mutations reduce the affinity of the mutant IL-2 polypeptide for the α-subunit of the IL-2 receptor at least 5-fold, in particular at least 10-fold, more specifically at least 25-fold. In embodiments in which more than one amino acid mutation is present that reduces the affinity of the mutant IL-2 polypeptide for the α-subunit of the IL-2 receptor, a combination of these amino acid mutations can reduce the affinity of the mutant IL-2 polypeptide for the α-subunit of the IL-2 receptor at least 30 times, at least 50 times, or even at least 100 times. In one of the embodiments of the invention, the specified amino acid mutation or combination of amino acid mutations abolishes the affinity of the mutant IL-2 polypeptide to the α-subunit of the receptor

- 14 040858

IL-2, with the result that no binding can be detected by surface plasmon resonance, which will be described below.

It is believed that almost similar binding to the intermediate affinity receptor is achieved, i. e. maintaining the affinity of the IL-2 mutant polypeptide for said receptor, when the IL-2 mutant has an affinity greater than about 70% of the affinity of the wild-type form of the IL-2 mutant for the intermediate affinity IL-2 receptor. The IL-2 mutants of the invention may have an affinity greater than about 80% and even greater than about 90% of the indicated affinity.

In the course of the invention, it has been found that a decrease in the affinity of IL-2 for the α-subunit of the IL-2 receptor, in combination with the elimination of IL-2 O-glycosylation, makes it possible to obtain an IL-2 protein with improved properties. For example, elimination of the O-glycosylation site allows a more homogeneous product to be obtained when the IL-2 mutant polypeptide is expressed in mammalian cells such as CHO or HEK cells.

Thus, in some embodiments, the mutant IL-2 polypeptide of the invention contains an additional amino acid mutation that eliminates the IL-2 O-glycosylation site at the position corresponding to residue 3 of human IL-2. In one embodiment of the invention, said additional amino acid mutation that eliminates the IL-2 O-glycosylation site at the position corresponding to residue 3 of human IL2 is an amino acid substitution. Examples of amino acid substitutions are T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K and T3P. In a particular embodiment of the invention, said additional amino acid mutation is a T3A amino acid substitution.

In some embodiments, the mutant IL-2 polypeptide is substantially a full-length IL-2 molecule. In some 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 the affinity of the mutant IL-2 polypeptide for the α-subunit of the IL-2 receptor, but retains the affinity of the mutant polypeptide IL-2 to the IL-2 receptor with intermediate affinity compared to the IL-2 polypeptide, which contains SEQ ID NO: 1 without the indicated 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 but retains the affinity of the mutant IL-2 polypeptide to the IL-2 receptor with intermediate affinity compared to the IL-2 polypeptide, which contains SEQ ID NO: 3 without the indicated mutation.

In a particular embodiment, the mutant IL-2 polypeptide may elicit one or more cellular responses selected from the group consisting of activated T lymphocyte proliferation, activated T lymphocyte differentiation, cytotoxic T cell (CTL) activity, activated B cell proliferation, activated B cell differentiation, B cells, natural killer (NK) cell proliferation, NK cell differentiation, activated T cell or NK cell secretion, and antitumor cytotoxicity of NK/lymphokine activated killer cell (LAK).

In one embodiment, the mutant IL-2 polypeptide has a reduced ability to induce IL-2 signaling in regulatory T cells compared to the wild-type IL-2 polypeptide. In one embodiment, the mutant IL-2 polypeptide induces reduced activation-induced cell death (AICD) of T cells compared to the wild-type IL-2 polypeptide. In one embodiment, the mutant IL-2 polypeptide has a reduced in vivo toxicity profile compared to the wild-type IL2 polypeptide. In one embodiment, the mutant IL-2 polypeptide has a prolonged plasma half-life compared to the wild-type IL-2 polypeptide.

In particular, the mutant IL-2 polypeptide of the invention contains four amino acid substitutions at positions corresponding to residues 3, 42, 45 and 72 of human IL-2. Specific amino acid substitutions are T3A, F42A, Y45A and L72G. As demonstrated in the examples below, this four-mutant (four-mutant) IL-2 polypepid has no detectable level of binding to CD25, reduced ability to induce T cell apoptosis, reduced ability to induce IL-2 signaling in Treg cells, and a reduced toxicity profile. in vivo. However, it retains the ability to activate IL-2 signaling in effector cells, induce effector cell proliferation, and NK cell production of IFN-γ as a secondary cytokine.

In addition, said IL-2 mutant polypeptide has additional advantageous properties such as reduced surface hydrophobicity, good stability and high expression yield as described in the examples. Surprisingly, it was found that this mutant

- 15 040858 IL-2 polypeptide also has a prolonged serum half-life compared to

IL-2 wild type.

The IL-2 mutants of the invention, in addition to having mutations in the region of IL-2 that forms the interface between IL-2 and CD25, or in the glycosylation site, may also have one or more mutations in the amino acid sequence located outside these regions. Such additional mutations in human IL-2 may provide additional benefits such as increased levels of expression or stability. For example, the cysteine at position 125 can be replaced with a neutral amino acid such as serine, alanine, threonine, or valine to give C125S IL-2, C125A IL-2, C125T IL-2, or C125V IL-2, respectively, as described in U.S. No. 4518584. As described in this document, you can also withdraw by deletion of the N-terminal alanine residue of IL-2, obtaining a mutant such as des-A1 C125S or des-A1 C125A. Alternatively, or in addition, the IL-2 mutant may include a mutation in which the methionine normally present at position 104 of wild-type human IL-2 is changed to a neutral amino acid such as alanine (see U.S. No. 5206344). The resulting mutants are e.g. described in U.S. No. 5116943 and Weiger et al., Eur J Biochem 180, 1989, pp. 295-300) can be used in combination with specific IL-2 mutations of the invention.

Thus, in some embodiments, the mutant IL-2 polypeptide of the invention contains an additional amino acid mutation at the position corresponding to residue 125 of human IL-2. In one embodiment of the invention, said additional amino acid mutation is the C125A amino acid substitution.

It is obvious to a person skilled in the art how to determine which additional mutations may provide additional benefits for the purposes of the invention. For example, it should be apparent that amino acid mutations in the IL-2 sequence that reduce or abolish the affinity of IL-2 for an intermediate affinity IL-2 receptor, such as D20T, N88R or Q126D (see e.g. U.S. 2007/0036752) , may not be suitable for inclusion in the mutant IL-2 polypeptide of the invention.

In one embodiment, the mutant IL-2 polypeptide of the invention contains a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15, and SEQ ID NO: 19. In a specific an embodiment of the invention, the mutant IL-2 polypeptide of the invention comprises the sequence of SEQ ID NO: 15 or SEQ ID NO: 19. In an even more specific embodiment of the invention, the mutant IL-2 polypeptide of the invention comprises SEQ ID NO: 19 .

Mutant IL-2 polypeptides of the invention are most appropriately used in the context of IL-2 fusion proteins, such as IL-2-bearing immunoconjugates. Such fusion proteins contain a mutant IL-2 polypeptide of the invention fused to a non-IL-2 fragment. The He-IL-2 fragment may be a synthetic or naturally occurring protein, or a portion or variant thereof. Examples of non-IL-2 fragments are albumin, antibody domains such as Fc domains or immunoglobulin antigen binding domains.

IL-2-bearing immunoconjugates are fusion proteins containing an antigen-binding fragment and an IL-2 fragment. They significantly increase the efficacy of IL-2-based therapy by targeting IL-2, for example, to the tumor microenvironment. According to the invention, the antigen-binding fragment may be a complete antibody or an immunoglobulin, or a portion or variant thereof, having a biological function such as specific affinity for antigen binding.

The advantages of therapy using immunoconjugates are quite obvious. For example, an antigen-binding fragment of an immunoconjugate recognizes a tumor-specific epitope and this results in targeted delivery of the immunoconjugate molecule to the tumor site. This can provide high concentrations of IL-2 in the tumor microenvironment, which allows you to achieve activation and proliferation of various immune effector cells described in the present description, using a significantly lower dose of immunoconjugate compared to that required for unconjugated IL-2. In addition, the indicated use of IL-2 in the form of immunoconjugates allows to reduce the doses of the cytokine itself, thereby limiting the potential manifestation of undesirable side effects of IL-2, and the targeted transfer of IL-2 to a specific area in the body using the immunoconjugate can also lead to a decrease in systemic exposure. and as a result, reduced side effects compared to those obtained using unconjugated IL-2. In addition, the prolongation of the circulatory half-life of the immunoconjugate compared to unconjugated IL-2 is important for the efficacy of the immunoconjugate. However, this characteristic of IL-2-containing immunoconjugates may again exacerbate the potential side effects of the IL-2 molecule: since with a significantly prolonged half-life of a circulating IL-2 immunoconjugate relative to unconjugated IL-2, the likelihood that IL-2 or other components of the fusion protein molecule will activate components that

- 16 040858 are usually present in the vasculature. This also applies to other fusion proteins that contain IL-2 fused to another moiety such as Fc or albumin resulting in an extended circulatory half-life of IL-2. For this reason, the immunoconjugate containing the mutant IL-2 polypeptide of the invention has reduced toxicity compared to the forms

Wild-type IL-2 is most preferred.

Thus, the invention also relates to a mutant IL-2 polypeptide as described above, which is linked to at least one non-IL-2 fragment. In one embodiment, the mutant IL-2 polypeptide and the non-IL-2 fragment form a fusion protein, i. the mutant IL-2 polypeptide is linked by a peptide bond to a non-GE-2 fragment. In one of the embodiments of the invention, the mutant IL-2 polypeptide is linked to the first and second non-GE-2 fragment. In one embodiment, the mutant IL-2 polypeptide is linked by an amino- or carboxy-terminal peptide bond to the first antigen-binding moiety, and the second antigen-binding moiety is linked by an amino- or carboxy-terminal peptide bond to either I) the mutant IL-2 polypeptide or II) the first antigen-binding moiety. fragment. In a particular embodiment, the mutant IL-2 polypeptide is linked by a carboxy-terminal peptide bond to said first non-K-2 moiety and an amino-terminal peptide bond to said second u-P.-2 moiety. In one embodiment, said non-GE-2 moiety is a targeting moiety. In a particular embodiment of the invention, said non-K-2 fragment is an antigen-binding fragment (thereby forming an immunoconjugate with a mutant IL-2 polypeptide, which is described in more detail below). In some embodiments, the antigen-binding fragment is an antibody or antibody fragment. In one embodiment, the antigen binding fragment is a full length antibody. In one embodiment, the antigen-binding fragment is an immunoglobulin molecule, in particular an IgG class immunoglobulin molecule, more preferably an IgG1 subclass immunoglobulin molecule. In one embodiment, the mutant IL-2 polypeptide is linked by an amino-terminal peptide bond to one of the heavy chains of an immunoglobulin. In another embodiment, the antigen-binding fragment is an antibody fragment. In some embodiments, said antigen-binding fragment comprises an antibody antigen-binding domain comprising an antibody heavy chain variable region and an antibody light chain variable region. In a more specific embodiment, the antigen binding fragment is a Fab molecule or an scFv molecule. In a particular embodiment, the antigen-binding fragment is a Fab molecule. In another embodiment, the antigen binding fragment is an scFv molecule. In one embodiment, the antigen-binding fragment is directed to an antigen present on the tumor cell or in the environment of the tumor cell. In a preferred embodiment, the antigen is selected from the group consisting of fibroblast activation protein (FAP), tenascin-C A1 domain (TNC A1), tenascin-C A2 domain (TNC A2), fibronectin extra-domain B (EDB), carcinoembryonic antigen (CEA); and melanoma-associated chondroitin sulfate proteoglycan (MCSP). If the mutant IL-2 polypeptide is linked to more than one antigen-binding fragment, for example, the first and second antigen-binding fragment, then each antigen-binding fragment can be independently selected from various forms of antibodies or antibody fragments. For example, the first antigen-binding fragment may be a Fab molecule and the second antigen-binding fragment may be a scFv molecule. In a specific embodiment of the invention, each of said first and second antigen-binding fragment is a scFv molecule, or each of said first and second antigen-binding fragment is a Fab molecule. In a specific embodiment of the invention, each of said first and second antigen-binding fragment is a Fab molecule. Similarly, if the mutant IL-2 polypeptide is linked to more than one antigen-binding fragment, for example, the first and second antigen-binding fragment, then the antigen to which each antigen-binding fragment is directed can be independently selected from each other. In one of the embodiments of the invention, the specified first and second antigennegative fragments directed to different antigens. In another embodiment of the invention, said first and second antigen binding fragments are directed to the same antigen. As described above, the antigen is preferably an antigen present on the tumor cell or in the environment of the tumor cell, more preferably the antigen is selected from the group consisting of fibroblast activation protein (FAP), Tenascin-C A1 domain (TNC L1), Tenascin A2 domain -C (TNC A2), fibronectin extra domain B (EDB), carcinoembryonic antigen (CEA), and melanoma-associated chondroitin sulfate proteoglycan (MCSP). The antigen-binding region may further include any of the features, individually or in combination with each other, that are described in relation to the antigen-binding domains of immunoconjugates.

- 17 040858

Immunoconjugates

A specific object of the invention is an immunoconjugate containing a mutant IL-2 polypeptide that carries one or more amino acid mutations that abolish or reduce the affinity of the mutant IL-2 polypeptide for the α-subunit of the IL-2 receptor and retain the affinity of the mutant IL-2 polypeptide for the IL-2 receptor. 2 with intermediate affinity, and at least one antigen binding fragment. In one embodiment, the amino acid mutation that abolishes or reduces the affinity of the IL-2 mutant polypeptide for the α subunit of the IL-2 receptor and retains the IL-2 mutant polypeptide's affinity for the intermediate affinity IL-2 receptor is at a position selected from positions corresponding to residue 42, 45 and 72 of human IL-2. In one of the embodiments of the invention, the specified amino acid mutation is an amino acid substitution. In one embodiment, said amino acid mutation is an amino acid substitution selected from the group consisting of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E . In one embodiment, the amino acid mutation is at position corresponding to residue 42 of human IL-2. In a particular embodiment of the invention, said amino acid mutation is an amino acid substitution selected from the group consisting of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, and F42. And in an even more specific embodiment of the invention, the amino acid substitution is F42A. In another embodiment, the amino acid mutation is at position corresponding to residue 45 of human IL-2. In a particular embodiment, said amino acid mutation is an amino acid substitution selected from the group consisting of Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, and Y45K. In an even more specific embodiment of the invention, the amino acid substitution is Y45A. In a further embodiment of the invention, the amino acid mutation is at the position corresponding to residue 72 of human IL-2. In a particular embodiment of the invention, said amino acid mutation is an amino acid substitution selected from the group consisting of L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. In an even more specific embodiment of the invention, said amino acid substitution is L72G. In some embodiments, the mutant IL-2 polypeptide of the invention does not contain an amino acid mutation at the position corresponding to residue 38 of human IL-2. In a particular embodiment of the invention, the mutant IL-2 polypeptide included in the immunoconjugate of the invention contains at least a first and a second 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 maintains the affinity of the mutant IL-2 polypeptide for the IL-2 receptor with intermediate affinity. In one embodiment, said first and second amino acid mutations are at two positions selected from positions that correspond to residues 42, 45 and 72 of human IL-2. In one embodiment of the invention, 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 consisting of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R and L72K. In a particular embodiment of the invention, said first and second amino acid mutations are amino acid substitutions selected from the group consisting of F42A, Y45A and L72G. The mutant IL-2 polypeptide may also include any of the features, individually or in combination, described in the previous paragraphs in relation to the mutant IL-2 polypeptides of the invention. In one embodiment of the invention, said IL-2 mutant polypeptide is linked by an amino- or carboxy-terminal peptide bond to said antigen-binding moiety that is included in the immunoconjugate, i.e. The immunoconjugate is a fusion protein. In some embodiments, said antigen-binding fragment is an antibody or antibody fragment. In some embodiments, said antigen-binding fragment comprises an antibody antigen-binding domain comprising an antibody heavy chain variable region and an antibody light chain variable region. An antigen-binding region may further include any of the features, individually or in combination with each other, that are described in relation to antigen-binding domains.

Formats of immunoconjugates

The most suitable immunoconjugate formats are described in PCT publication WO 2011/020783, which is incorporated herein by reference in its entirety. Such immunoconjugates contain at least two antigen-binding domains. Thus, according to one of the options

- 18 040858 implementation of the invention the immunoconjugate proposed in the present invention contains at least the first mutant IL-2 polypeptide presented in the present description, and at least the first and second antigennegative fragments. In a specific embodiment of the invention, said first and second antigen binding fragments are independently selected from the group consisting of an Fv molecule, in particular an scFv molecule, and a Fab molecule. In a particular embodiment of the invention, said first mutant IL-2 polypeptide is linked by an amino- or carboxy-terminal peptide bond to the first antigen-binding fragment, and the second antigen-binding fragment is linked by an amino- or carboxy-terminal peptide bond to either I) the mutant IL-2 polypeptide, or II) the first antigen binding fragment. In a specific embodiment of the invention, the immunoconjugate substantially consists of a first mutant IL-2 polypeptide and first and second antigen-binding fragments linked by one or more linker sequences. These formats have the advantage that they bind with high affinity to a target antigen (such as a tumor antigen) but exhibit only monomeric binding to the IL-2 receptor, thus avoiding targeting of the immunoconjugate to IL-2 receptor-bearing immunes. cells located in areas other than the target site. In a specific embodiment of the invention, the first IL-2 mutant polypeptide is linked by a carboxy-terminal peptide bond to the first antigen-binding moiety and is further linked by an amino-terminal peptide bond to the second antigen-binding moiety. In another embodiment of the invention, the first antigen-binding fragment is linked by a carboxy-terminal peptide bond to the first IL-2 mutant polypeptide and is further linked by an amino-terminal peptide bond to the second antigen-binding fragment. In another embodiment of the invention, the first antigen-binding fragment is linked by an amino-terminal peptide bond to the first IL-2 mutant polypeptide and is further linked by a carboxy-terminal peptide bond to the second antigen-binding fragment. In a specific embodiment, the mutant IL-2 polypeptide is linked by a carboxy-terminal peptide bond to a first heavy chain variable region and is further linked by an amino-terminal peptide bond to a second heavy chain variable region. In another embodiment, the mutant IL-2 polypeptide is linked by a carboxy-terminal peptide bond to a first light chain variable region and is further linked by an amino-terminal peptide bond to a second light chain variable region. In another embodiment, the first heavy or light chain variable region is carboxy-terminally peptide-linked to the first IL-2 mutant polypeptide and further amino-terminal peptide-linked to the second heavy or light chain variable region. In another embodiment, the first heavy or light chain variable region is amino-terminally peptide-linked to the first IL-2 mutant polypeptide and further carboxy-terminally peptide-linked to the second heavy or light chain variable region. In one embodiment, the mutant IL-2 polypeptide is linked by a carboxy-terminal peptide bond to the heavy or light chain of a first Fab and is further linked by an amino-terminal peptide bond to the heavy or light chain of a second Fab. In another embodiment, the heavy or light chain of the first Fab is linked by a carboxy-terminal peptide bond to the first IL-2 mutant polypeptide and is further linked by an amino-terminal peptide bond to the heavy or light chain of the second Fab. In another embodiment, the heavy or light chain of the first Fab is linked by an amino-terminal peptide bond to the first IL-2 mutant polypeptide and is further linked by a carboxy-terminal peptide bond to the heavy or light chain of the second Fab. In one embodiment, the immunoconjugate comprises at least a first IL-2 mutant polypeptide linked by an amino-terminal peptide bond to one or more scFv molecules and further linked by a carboxy-terminal peptide bond to one or more scFv molecules.

Other most suitable immunoconjugate formats contain an immunoglobulin molecule as an antigen-binding moiety. In one such embodiment, the immunoconjugate comprises at least one mutant IL-2 polypeptide as described herein and an immunoglobulin molecule, in particular an IgG molecule, more preferably an IgG1 molecule. In one of the embodiments of the invention, the immunoconjugate contains no more than one mutant IL-2 polypeptide. In one embodiment, the immunoglobulin molecule is a human immunoglobulin. In one embodiment, the IL-2 mutant polypeptide is linked by an amino- or carboxy-terminal peptide bond to an immunoglobulin molecule. In one embodiment, the immunoconjugate substantially consists of a mutant IL-2 polypeptide and an immunoglobulin molecule, in particular an IgG molecule, more preferably an IgG1 molecule, which are linked by one or more linker sequences. In a particular embodiment, the mutant IL-2 polypeptide is linked at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the immunoglobulin heavy chains. In specific embodiments of the invention, the immunoglobulin molecule contains a modification in the Fc domain that enhances the heterodimerization of two non-identical

- 19 040858 immunoglobulin heavy chains. The site of the strongest protein-protein interaction between the two polypeptide chains of the human IgG Fc domain is located in the CH3 domain of the Fc domain. Thus, in one of the embodiments of the invention, the specified modification is in the CH3 domain of the Fc domain. In a particular embodiment of the invention, said modification is a knob-in-hole modification, which includes a modification resulting in the formation of a knob in one of the immunoglobulin heavy chains and a modification resulting in the formation of a depression in the other one of the heavy chains. immunoglobulin chains. The knob-intohole technology is described, for example, in U.S. No. 5731168; U.S. No. 7695936; in Ridgway et al., Prot Eng 9, 1996, pp. 617-621 and Carter, J Immunol Meth 248, 2001, pp. 7-15). In general, the method involves introducing a bulge (protrusion) at the interface of the first polypeptide and a corresponding cavity (depression) at the interface of the second polypeptide, whereby the bulge can fit into the cavity, thus enhancing heterodimer formation and inhibiting homodimer formation. Bulges are created by replacing amino acids with small side chains at the interface of the first polypeptide with amino acids with larger side chains (eg tyrosine or tryptophan). Compensating cavities of identical or similar size to the bumps are created at the interface of the second polypeptide by replacing amino acids with large side chains with amino acids with smaller side chains (eg, alanine or threonine). The bulge and cavity can be created by altering the nucleic acid encoding the polypeptides, for example by site-directed mutagenesis or by peptide synthesis. In a specific embodiment of the invention, the overhang modification is the T366W amino acid substitution in one of the two immunoglobulin heavy chains, and the trough modification is the T366S, L368A, and Y407V amino acid substitutions in the other one of the two immunoglobulin heavy chains. . In a more specific embodiment, the immunoglobulin heavy chain containing the ridge modification further comprises the amino acid substitution S354C, and the immunoglobulin heavy chain containing the pit 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 Immunol Methods 248, 2001, pp. 7-15).

In a specific embodiment of the invention, the mutant IL-2 polypeptide is linked to the carboxy-terminal amino acid of an immunoglobulin heavy chain containing a bulge modification.

In an alternative embodiment of the invention, a modification that enhances the heterodimerization of two non-identical polypeptide chains is a modification that mediates electrostatically determined effects, such as those described in PCT publication WO 2009/089004. In general, this method involves replacing one or more amino acid residues at the interface of two polypeptide chains with charged amino acid residues, whereby homodimer formation becomes electrostatically disadvantageous and heterodimerization becomes electrostatically advantageous.

The Fc domain confers advantageous pharmacokinetic properties to the immunoconjugate, including an extended serum half-life, resulting in sufficient accumulation in the target tissue and a preferred tissue-to-blood distribution ratio. However, at the same time, this may lead to undesirable targeting of the immunoconjugate to cells expressing Fc receptors rather than to the preferred antigen-bearing cells. In addition, co-activation of the Fc receptor signaling pathways can result in the release of cytokines which, in combination with the IL-2 polypeptide and the long half-life immunoconjugate, lead to excessive activation of the cytokine receptors and lead to serious side effects when administered systemically. Accordingly, canonical IgGIL-2 containing immunoconjugates have been reported to be associated with infusion reactions (see, for example, King et al., J Clin Oncol 22, 2004, pp. 4463-4473)).

Thus, in some embodiments, the immunoglobulin molecule that is included in the immunoconjugate of the invention is designed to have a reduced binding affinity for the Fc receptor. In one such embodiment, the immunoglobulin contains in its Fc domain one or more amino acid mutations that reduce the binding affinity of the immunoconjugate to the Fc receptor. Typically, one or more of the same amino acid mutations are present in each of the two immunoglobulin heavy chains. In one of the embodiments of the invention, these amino acid mutations reduce the binding affinity of the immunoconjugate to the Fc receptor at least 2-fold, at least 5-fold, or at least 10-fold. According to embodiments of the invention in which more than one amino acid mutation occurs that reduces the binding affinity of the immunoconjugate to 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-fold, at least 20 times or at least 50 times. In one embodiment of the invention, an immunoconjugate containing

- 20 040858 engineered immunoglobulin molecule, is characterized by less than 20%, in particular less than 10%, more preferably less than 5% of the Fc receptor binding affinity characteristic of an immunoconjugate containing a non-engineered immunoglobulin molecule. In one embodiment, the Fc receptor is an activating Fc receptor. In a particular embodiment, the Fc receptor is an Fcy receptor, more particularly an FcyRIIIa, FcyRI or FcyRIIa receptor. Preferably, binding to each of these receptors is reduced. In some embodiments, the binding affinity for a component of the complement system is also reduced, in particular the binding affinity for Clq. In one embodiment, the binding affinity for the neonatal Fc receptor (FcRn) is not reduced. Virtually the same binding to FcRn, i.e. maintaining the binding affinity of an immunoglobulin for said receptor is achieved when the immunoglobulin (or an immunoconjugate containing said immunoglobulin) has an FcRn binding affinity greater than about 70% of the FcRn binding affinity of the non-engineered immunoglobulin (or the immunoconjugate containing the non-engineered immunoglobulin). Immunoglobulins or immunoconjugates containing these immunoglobulins may have an affinity of greater than about 80% and even greater than about 90% of the above affinity. In one embodiment, the amino acid mutation is an amino acid substitution. In one embodiment, the immunoglobulin contains an amino acid substitution at position P329 of the immunoglobulin heavy chain (Cabot numbering). In a more specific embodiment of the invention, the amino acid substitution is P329A or P329G, in particular P329G. In one embodiment, the immunoglobulin contains an additional amino acid substitution at a position selected from S228, E233, L234, L235, N297, and P331 of the immunoglobulin heavy chain. In a more specific embodiment of the invention, the additional amino acid substitution is S228P, E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a particular embodiment, the immunoglobulin contains amino acid substitutions at positions P329, L234 and L235 of the immunoglobulin heavy chain. In a more specific embodiment of the invention, the immunoglobulin contains the amino acid mutations L234A, L235A and P329G (LALA P329G). This combination of amino acid substitutions almost completely abolishes the binding to the Fcy receptor of the human IgG molecule and, as a result, reduces effector function, including antibody-mediated cell-dependent cytotoxicity (ADCC).

In some embodiments, the immunoconjugate contains one or more proteolytic cleavage sites located between the mutant IL-2 polypeptide and antigen-binding fragments.

Components of an immunoconjugate (e.g., antigen-binding fragments and/or mutant IL-2 polypeptide) can be linked directly or through various linkers, in particular peptide linkers containing one or more amino acids, typically about 2-20 amino acids, as described herein. or known in the art. Suitable non-immunogenic linker peptides are, for example, (G4S) _n , (SG ₄ ) _n or G ₄ (SG ₄ ) _n linker peptides, in which n is a number, typically 1 to 10, typically 2 to 4 .

Antigen binding fragments

The antigen-binding fragment of an immunoconjugate of the invention is generally a polypeptide molecule that binds to a specific antigenic determinant and has the ability to direct the substance to which it is connected (for example, a mutant IL-2 polypeptide or a second antigen-binding fragment) to a target site, for example, to a particular type of tumor cell or tumor stroma that carries the antigenic determinant. The immunoconjugate can bind to an antigenic determinant, for example, present on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, free in the serum and/or in the extracellular matrix (ECM).

Examples of tumor antigens include, but are not limited to, MAGE, MART1/Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase binding protein (ADAbp), cyclophilin b, colorectal cancer associated antigen (CRC) -C017- 1A/GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, am11, prostate antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2 and PSA-3, prostate specific membrane antigen (PSMA), T cell receptor/CD3 zeta chain, MAGE family of tumor antigens (e.g. 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), the 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, MUC family, HER2/neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin and y-catenin,

- 21 040858 p120ctn, gp100 Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig-idiotype, p15, gp75, gangliosides GM2 and GD2, viral products such as human papillomavirus proteins, Smad family of tumor antigens, lmp-1, Pia, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phospholipase, SSX-1, SSX-2 (Hom-Mel-40), SSX1, SSX-4 , SSX-5, SCP-1 and ST-7 and c-erbB-2.

Examples of viral antigens include, but are not limited to, influenza hemagglutinin, Epstein-Barr virus LMP-1, hepatitis C E2 glycoprotein, HIV gp160, and HIV gp120.

Examples of ECM antigens include, but are not limited to, syndecan, heparanase, integrins, osteopontin, link family proteins, cadherins, laminin, EGF type laminin, lectin, fibronectin, notch family proteins, tenascin, and matrixin.

The immunoconjugates of the invention can bind to the following specific cell surface antigens, including but not limited to: FAP, Her2, EGFR, IGF-1R, CD2 (T-cell surface antigen), CD3 (TCR-associated heteromultimer ), CD22 (B-cell receptor), CD23 (low affinity IgE receptor), CD30 (cytokine receptor), CD33 (myeloid cell surface antigen), CD40 (tumor necrosis factor receptor), IL-6R (IL6 receptor), CD20, MCSP and PDGFPR (platelet growth factor receptor β).

In one of the embodiments of the invention, the immunoconjugate proposed in the invention contains two or more antigennegative fragments, where each of these antigennegative fragments specifically binds to the same antigenic determinant. In another embodiment of the invention, the immunoconjugate contains two or more antigen-binding fragments, where each of these antigen-binding fragments specifically binds to different antigenic determinants.

The antigen-binding fragment may be any type of antibody or fragment thereof that retains binding specificity for the antigenic determinant. Antibody fragments include, but are not limited to, V _H fragments, V _Lα fragments, Fab fragments, F(ab') ₂ fragments, scFv fragments, Fv fragments, minibodies (minibody), dimeric antibodies, trimeric antibodies and tetrameric antibodies (see, for example, Hudson and Souriau, Nature Med 9, 2003, pp. 129-134).

The most suitable antigen-binding fragments are described in PCT publication WO 2011/020783, which is incorporated herein by reference in its entirety.

In one of the embodiments of the invention, the immunoconjugate contains at least one, usually two or more antigen-binding fragments that are specific for the fibronectin extra domain B (EDB). In another embodiment, the immunoconjugate contains at least one, typically two or more, antigen-binding fragments that can compete with the L19 monoclonal antibody for binding to the EDB epitope (see, for example, PCT publication WO 2007/128563 A1, which is fully incorporated herein by reference). In a further embodiment of the invention, the immunoconjugate comprises a polypeptide sequence in which the heavy chain of a first Fab derived from the L19 monoclonal antibody is carboxy-terminally peptide-linked to a mutant IL-2 polypeptide, which in turn is carboxy-terminal peptide-linked to the heavy chain of a second Fab derived from monoclonal antibody L19. In a further embodiment of the invention, the immunoconjugate comprises a polypeptide sequence in which the light chain of a first Fab derived from the L19 monoclonal antibody is carboxy-terminally peptide-linked to a mutant IL-2 polypeptide, which in turn is carboxy-terminal peptide-linked to the light chain of a second Fab derived from monoclonal antibody L19. In a further embodiment of the invention, the immunoconjugate comprises a polypeptide sequence in which a first scFv derived from the L19 monoclonal antibody is linked by a carboxy-terminal peptide bond to a mutant IL-2 polypeptide, which in turn is linked by a carboxy-terminal peptide bond to a second scFv derived from the L19 monoclonal antibody.

In a more specific embodiment of the invention, the immunoconjugate contains the polypeptide sequence of SEQ ID NO: 199 or a variant thereof that retains functionality. In another embodiment, the immunoconjugate comprises a light chain Fab derived from monoclonal antibody L19. In a more specific embodiment, the immunoconjugate contains 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 their options that retain functionality. In another specific embodiment of the invention, the polypeptides are covalently linked, for example, by a disulfide bridge.

In one embodiment, the immunoconjugate of the invention comprises at least one, typically two or more, antigen-binding fragments that are specific for the tenascin A1 domain (TNC-A1). In another embodiment, the immunoconjugate contains at least one, typically two or more, antigen-binding fragments that can compete with the F16 monoclonal antibody for binding to the TNC-A1 epitope (see, for example, PCT publication WO 2007 /128563 A1, which is fully incorporated into the present description by reference). In one of the embodiments of the invention, the immunoconjugate contains at least one, usually two or more antigen-binding fragments that are specific for the A1 and/or A4 domain of tenascin (TNC-A1 or TNC-A4 or TNC-A1 /A4). In another embodiment, the immunoconjugate comprises a polypeptide sequence in which the heavy chain of a first Fab specific for the Al domain of tenascin is carboxy-terminally peptide-linked to a mutant IL-2 polypeptide, which in turn is carboxy-terminal peptide-linked to the heavy chain of a second Fab, specific for the A1 domain of tenascin. In a further embodiment of the invention, the immunoconjugate comprises a polypeptide sequence in which the light chain of a first Fab specific for the Al domain of tenascin is linked by a carboxy-terminal peptide bond to a mutant IL-2 polypeptide, which in turn is linked by a carboxy-terminal peptide bond to the light chain of a second Fab, specific for the Al domain of tenascin. In a further embodiment of the invention, the immunoconjugate comprises a polypeptide sequence in which a first scFv specific for the A1 domain of tenascin is linked by a carboxy-terminal peptide bond to a mutant IL-2 polypeptide, which in turn is linked by a carboxy-terminal peptide bond to a second scFv specific for the Al domain. tenascin. In another embodiment of the invention, the immunoconjugate comprises a polypeptide sequence in which the heavy chain of an immunoglobulin specific for TNC-A1 is linked by a carboxy-terminal peptide bond to a mutant IL-2 polypeptide.

In a specific embodiment, the antigen-binding fragments 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 either SEQ ID NO: 33 or SEQ ID NO: 35 or variants that retain functionality. In another embodiment, the antigen-binding fragments 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 either SEQ ID NO: 29 or SEQ ID NO: 31 or variants thereof that retain functionality. In a more specific embodiment, the antigen-binding fragments 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 either SEQ ID NO: 33 or SEQ ID NO: 35 or functionally conserving variants thereof, and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to either SEQ ID NO: 29, or SEQ ID NO: 31 or variants thereof that retain functionality.

In another specific embodiment, the heavy chain variable region sequence of the antigen-binding fragments 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 either SEQ ID NO: 34 or SEQ ID NO: 36. In yet another specific embodiment, the heavy chain variable region sequence of the antigen binding fragments of the immunoconjugate is encoded by the polynucleotide sequence of either SEQ ID NO: 34 or SEQ ID NO: 36. In another specific embodiment, the light chain variable region sequence of the antigen binding fragments The immunoconjugate is encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to either SEQ ID NO: 30 or SEQ ID NO: 32. In yet another specific embodiment, the sequence light chain variable region and antigen-binding fragments of the immunoconjugate is encoded by the polynucleotide sequence of either SEQ ID NO: 30 or SEQ ID NO: 32.

In a particular 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 functionally retained variants thereof. In another 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 either SEQ ID NO: 205 or SEQ ID NO: 215 or variants thereof that retain functionality. In yet another specific embodiment of the invention, 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 either SEQ ID NO: 207 or SEQ ID NO: 237 or variants thereof 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 contains 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.

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 contains a polypeptide sequence encoded by the polynucleotide sequence of SEQ ID NO: 204. In another specific embodiment of the invention, the immunoconjugate contains a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80, 85, 90, 95, 96, 97%, 98%, or 99% identical to either SEQ ID NO: 206 or SEQ ID NO: 216. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by the polynucleotide sequence of either SEQ ID NO: 206 or SEQ ID NO: 216. In another specific embodiment, embodiment of the invention, the immunoconjugate contains a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to either SEQ ID NO: 208 or SEQ ID NO: 238. In yet another embodiment of the invention the immunoconjugate contains a polypeptide sequence encoded by the polynucleotide sequence of either SEQ ID NO: 208 or SEQ ID NO: 238.

In one of the embodiments of the invention, the immunoconjugate contains at least one, usually two or more antigen-binding fragments that are specific for the A2 domain of tenascin (TNC-A2). In another embodiment, the immunoconjugate comprises a polypeptide sequence in which the heavy chain of a first Fab specific for the A2 domain of tenascin is carboxy-terminally peptide bonded to a mutant IL-2 polypeptide, which is in turn carboxy-terminally peptide bonded to the heavy chain of a second Fab, specific for the A2 domain of tenascin. In a further embodiment of the invention, the immunoconjugate comprises a polypeptide sequence in which the light chain of a first Fab specific for the A2 domain of tenascin is linked by a carboxy-terminal peptide bond to a mutant IL-2 polypeptide, which in turn is linked by a carboxy-terminal peptide bond to the light chain of a second Fab, specific for the A2 domain of tenascin. In another embodiment, the immunoconjugate comprises a polypeptide sequence in which the heavy chain of an immunoglobulin specific for TNC-A2 is linked by a carboxy-terminal peptide bond to a mutant IL-2 polypeptide.

In a particular embodiment, the antigen-binding fragments 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. : 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 their variants that retain functionality. In another specific embodiment, the antigen-binding fragments 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: 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 their functional options. In a more specific embodiment, the antigen-binding fragments 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: 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 their functionally conserving variants, 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 consisting 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 thereof preserving functionality.

In another specific embodiment, the heavy chain variable region sequence of the antigen-binding fragments 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 a sequence selected from the group consisting 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 antigen-binding fragments of the immunoconjugate is encoded by a polynucleotide sequence selected from the group consisting 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 of the invention, the sequence of the variable

The light chain region of the antigen-binding fragments 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 a sequence selected from the group consisting 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 antigen-binding fragments of the immunoconjugate is encoded by a polynucleotide sequence selected from the group consisting 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 a particular 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 consisting 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 sequence selected from the group consisting of SEQ ID NO: 247, SEQ ID NO: 249 and SEQ ID NO: 251, or variants thereof that retain functionality. In a more 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 consisting of SEQ ID NO: 241, SEQ ID NO: 243, and SEQ ID NO: 245, 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 % is identical to a sequence selected from the group consisting of SEQ ID NO: 247, SEQ ID NO: 249 and SEQ ID NO: 251, 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: 241, and either SEQ ID NO: 249, or SEQ ID NO: 251, or variants thereof that retain functionality. In yet another specific embodiment of the invention, 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: 243 and or 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.

In a particular 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 consisting 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 consisting 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 consisting of SEQ ID NO: 248 , SEQ ID NO: 250 and SEQ ID NO: 252. In yet another specific embodiment of the invention, the sod immunoconjugate contains a polypeptide sequence encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO: 248, SEQ ID NO: 250 and SEQ ID NO: 252.

In one of the embodiments of the invention, the immunoconjugate contains at least one, usually two or more antigen-binding fragments specific for fibroblast-activating protein (FAP). In another embodiment, the immunoconjugate comprises a polypeptide sequence in which the heavy chain of a first FAP-specific Fab is linked by a carboxy-terminal peptide bond to a mutant IL-2 polypeptide, which in turn is linked by a carboxy-terminal peptide bond to the heavy chain of a second FAP-specific Fab. FAP. In a further embodiment of the invention, the immunoconjugate comprises a polypeptide sequence in which the light chain of a first FAP-specific Fab is linked by a carboxy-terminal peptide bond to a mutant IL-2 polypeptide, which in turn is linked by a carboxy-terminal peptide bond to the light chain of a second FAP-specific Fab. FAP. In another embodiment of the invention, the immunoconjugate comprises a polypeptide sequence in which the FAP-specific immunoglobulin heavy chain is linked by a carboxy-terminal peptide bond to a mutant IL-2 polypeptide.

- 25 040858

In a particular embodiment, the antigen-binding fragments 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. In another specific embodiment, the antigen-binding fragments 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 thereof that retain functionality. In a more specific embodiment, the antigen-binding fragments 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 functionally conserved variants thereof, 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 including 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, the antigen-binding fragments 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, the antigen-binding fragments of the immunoconjugate comprise 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, the antigen binding fragments 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, embodiment of the invention, the antigen-binding fragments 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, a the antigen-binding fragments of the immunoconjugate comprise the heavy chain variable region sequence of SEQ ID NO: 151 and the light chain variable region sequence of SEQ ID NO: 149.

In another specific embodiment, the heavy chain variable region sequence of the antigen-binding fragments 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 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. a value 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 particular embodiment,

- 26 040858 those embodiments of the invention the light chain variable region sequence of the antigen-binding fragments 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 a 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. 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 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 consisting 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 a further 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 consisting 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 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 their functionally retaining variants 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 its functionally retained variants. 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 consisting 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 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: 231 or functionally retained variants thereof. 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: 213 and SEQ ID NO: 235 or variants thereof that retain functionality. In yet another specific embodiment of the invention, 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 of the invention, 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 of the invention, 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 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 of the invention, 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 of the invention, the immunoconjugate of the present invention

- 27 040858 tenii, contains 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, preserving functionality. In yet another specific embodiment of the invention, 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.

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 consisting of SEQ ID NO: 297, SEQ ID NO: 301 and SEQ ID NO: 315, or variants thereof that retain functionality. In a further 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 consisting 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 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, functionality-retaining 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 functionality-retaining variant thereof, 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 functional variant thereof. 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: 301 or a variant thereof, functionality-retaining polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 303 or a functionality-retaining variant thereof, 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 functional variant thereof. In a further 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: 315 or a variant thereof, functionality-retaining 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 functionality-retaining variant thereof, 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 functional variant thereof.

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 consisting 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 a further specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence selected from the group consisting 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 which 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: 232, SEQ ID NO: 234, SEQ ID NO: 236, and SEQ ID NO: 240. In a further specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, and SEQ ID NO: 240.

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 consisting of SEQ ID NO: 298 , SEQ ID NO: 302 and SEQ ID NO: 316. In the following specific embodiment of the invention, the immunoconjugate contains a polypeptide sequence encoded by a polynucleotide sequence selected from the group consisting 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 consisting of SEQ ID NO: 300, SEQ ID NO: 304 and SEQ ID NO: 318. the nugate contains a polypeptide sequence encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO: 300, SEQ ID NO: 304 and SEQ ID NO: 318.

In one embodiment, the immunoconjugate comprises at least one, typically two or more, antigen-binding fragments specific for melanoma-associated chondroitin sulfate proteoglycan (MCSP). In another embodiment, the immunoconjugate comprises a polypeptide sequence in which the heavy chain of a first MCSP-specific Fab is carboxy-terminally peptide-linked to a mutant IL-2 polypeptide, which in turn is carboxy-terminal peptide-linked to the heavy chain of a second MCSP-specific Fab. MCSP. In a further embodiment of the invention, the immunoconjugate comprises a polypeptide sequence in which the light chain of a first MCSP-specific Fab is linked by a carboxy-terminal peptide bond to a mutant IL-2 polypeptide, which in turn is linked by a carboxy-terminal peptide bond to the light chain of a second Fab specific to MCSP. In another embodiment of the invention, the immunoconjugate comprises a polypeptide sequence in which the heavy chain of an immunoglobulin specific for MCSP is linked by a carboxy-terminal peptide bond to a mutant IL-2 polypeptide.

In a particular embodiment, the antigen-binding fragments 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 either SEQ ID NO: 189 or SEQ ID NO: 193 or variants thereof that retain functionality. In another specific embodiment, the antigen-binding fragments 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 either SEQ ID NO: 191 or sequences of SEQ ID NO: 197 or variants thereof that retain functionality. In a more specific embodiment, the antigen-binding fragments 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 either SEQ ID NO: 189 or sequences of 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 either the sequence of SEQ ID NO : 191, or sequences of SEQ ID NO: 197, or variants thereof that retain functionality. In a more specific embodiment, the antigen-binding fragments 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 the sequence a light chain variable region 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 fragments of the immunoconjugate comprise the sequence of the variable region a heavy chain 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 SEQ ID NO: 191.

In another specific embodiment, the heavy chain variable region sequence of the antigen-binding fragments 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 either SEQ ID NO: 190 or sequence of SEQ ID NO: 194. In another specific embodiment, the heavy chain variable region sequence of the antigen-binding fragments of the immunoconjugate is encoded by the polynucleotide sequence of either SEQ ID NO: 190 or SEQ ID NO: 194. In another specific embodiment, the variable region sequence of the light chain of the antigen-binding fragments fragments 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 either SEQ ID NO: 192 or SEQ ID NO: 198. In the following specific var In an embodiment of the invention, the sequence of the light chain variable region of the antigen-binding fragments of the immunoconjugate is encoded by the polynucleotide sequence of either SEQ ID NO: 192 or SEQ ID NO: 198.

In a particular 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 either 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 to either SEQ ID NO: 255 or SEQ ID NO: 261 or variants thereof that retain functionality. In a more 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 either SEQ ID NO: 253, or SEQ ID NO: 257 or their functionally retaining variants, and a polypeptide sequence that is at least about 80%, 85, 90, 95, 96, 97, 98, 99, or 100% identical to either SEQ ID NO: 255 , or SEQ ID NO: 261 or their variants 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: 253 or variants thereof, retaining 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 thereof that retain functionality. In another specific embodiment of the invention, 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: 257 or variants thereof, retaining 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 thereof that retain functionality.

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 either SEQ ID NO: 254 or SEQ ID NO: 258. In a further specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence of either SEQ ID NO: 254 or SEQ ID NO: 258. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to either SEQ ID NO: 256 or SEQ ID NO: 262. In a further specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence edibility of either SEQ ID NO: 256 or SEQ ID NO: 262.

In one of the embodiments of the invention, the immunoconjugate contains at least one, usually two or more antigen-binding fragments specific for carcinoembryonic antigen (CEA).

In another embodiment, the immunoconjugate comprises a polypeptide sequence in which the heavy chain of a first CEA-specific Fab is carboxy-terminally peptide-linked to a mutant IL-2 polypeptide, which in turn is carboxy-terminal peptide-linked to the heavy chain of a second CEA-specific Fab. CEA. In a further embodiment of the invention, the immunoconjugate comprises a polypeptide sequence in which the light chain of a first CEA-specific Fab is linked by a carboxy-terminal peptide bond to a mutant IL-2 polypeptide, which in turn is linked by a carboxy-terminal peptide bond to the light chain of a second Fab specific to CEA. In another embodiment of the invention, the immunoconjugate comprises a polypeptide sequence in which the heavy chain of an immunoglobulin specific for CEA is joined by a carboxy-terminal peptide bond to a mutant IL-2 polypeptide. In a particular embodiment, the antigen-binding fragments 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: 313 or a variant thereof, preserving functionality. In another specific embodiment, the antigen-binding fragments 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: 311 or a variant thereof. that preserves functionality. In a more specific embodiment, the antigen-binding fragments 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: 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 SEQ ID NO: 311 or a functional variant thereof.

In another specific embodiment, the heavy chain variable region sequence of the antigen-binding fragments 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 of SEQ ID NO: 314. In the following In a specific embodiment, the heavy chain variable region sequence of the antigen-binding fragments of the immunoconjugate is encoded by the polynucleotide sequence of SEQ ID NO: 314. In another specific embodiment, the light chain variable region sequence of the antigen-binding fragments of the immunoconjugate is encoded by a polynucleotide sequence that is at least about , 95, 96, 97, 98 or 99% identical to the SEQ sequence

- 30 040858

ID NO: 312. In a further specific embodiment, the light chain variable region sequence of the antigen-binding fragments of the immunoconjugate is encoded by the polynucleotide sequence of SEQ ID NO: 312.

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: 319 or variants thereof. preserving functionality. In a further 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: 321 or variants thereof. preserving functionality. In a further 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: 323 or variants thereof. preserving functionality. In a more specific embodiment of the invention, the immunoconjugate of the present invention contains 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 its functionally conserving variants, 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 its functionally conserving variants, and a polypeptide sequence, which is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 323 or variants thereof that retain functionality.

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 the following specific embodiment, of the invention, the immunoconjugate contains a polypeptide sequence encoded by the polynucleotide sequence of SEQ ID NO: 320. In another specific embodiment of the invention, the immunoconjugate contains 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 the following specific embodiment of the invention, the immunoconjugate contains a polypeptide sequence encoded by the polynucleotide sequence of SEQ ID NO: 322. In another specific embodiment of the invention, the immunoconjugate contains a polyp a peptide 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: 324. the sequence of SEQ ID NO: 324.

Antigen binding fragments of the invention include fragments having sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the peptide sequences set forth in SEQ ID NOs: 23-261 (odd numbers), 297303 (odd numbers), 311 and 313, including their functional fragments or variants. The invention also relates to antigen-binding fragments containing the sequences of SEQ ID NO: 23261 (odd numbers), 297-303 (odd numbers), 311 and 313 with conservative amino acid substitutions.

Polynucleotides

The invention also relates to isolated polynucleotides encoding a mutant IL-2 polypeptide or an immunoconjugate containing a mutant IL-2 polypeptide as described herein.

The 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, 24-262 (even numbers), 293-296 and 298-324 (even numbers), including their functional fragments or variants .

Polynucleotides encoding IL-2 mutant polypeptides not linked to a non-IL-2 fragment are generally expressed as an individual polynucleotide encoding the entire polypeptide.

One of the embodiments of the present invention is an isolated polynucleotide encoding a mutant IL-2 polypeptide, where the polynucleotide contains a sequence that encodes the sequence of SEQ ID NO: 7, 11, 15 or 19 of the mutant IL-2. The invention also relates to an isolated polynucleotide encoding a mutant IL-2 polypeptide, wherein the polynucleotide contains a sequence that encodes the sequence of SEQ ID NO: 7, 11, 15 or 19 of the mutant IL-2 with conservative amino acid substitutions.

Another embodiment of the invention is an isolated polynucleotide encoding a mutant IL-2 polypeptide, wherein the polynucleotide contains a sequence that is at least

- 31 040858 is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence selected from the group consisting 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. Another embodiment of the invention is an isolated polynucleotide encoding a mutant IL-2 polypeptide, wherein the polynucleotide comprises a nucleotide sequence selected from the group , comprising 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. Alternatively, implementation of the invention is an isolated polynucleotide encoding an immunoconjugate or a fragment thereof, where the polynucleotide contains a nucleotide sequence a identity that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence selected from the group consisting 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. from the group consisting 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.

The polynucleotides encoding the immunoconjugates of the invention may be expressed as an individual polynucleotide that encodes the complete immunoconjugate, or as multiple (eg, two or more) polynucleotides that are co-expressed. The polypeptides encoded by the co-expressed polynucleotides can be linked via, for example, disulfide bridges or other means to form a functional immunoconjugate. For example, the heavy chain region of the antigen-binding fragment may be encoded by a polynucleotide other than the polynucleotide encoding the portion of the immunoconjugate containing the light chain region of the antigen-binding fragment and the mutant IL-2 polypeptide. When co-expressed, the heavy chain polypeptides should be associated with the light chain polypeptides to form an antigen-binding moiety. Alternatively, in another example, the light chain region of the antigen-binding fragment may be encoded by a polynucleotide other than the polynucleotide encoding the portion of the immunoconjugate containing the heavy chain region of the antigen-binding fragment and a mutant IL-2 polypeptide. In one embodiment, the isolated polynucleotide of the invention encodes an immunoconjugate fragment containing a mutant IL-2 polypeptide and an antigen-binding fragment. In one embodiment, an isolated polynucleotide of the invention encodes a heavy chain antigen-binding fragment and a mutant IL-2 polypeptide. In another embodiment, an isolated polynucleotide of the invention encodes an antigen-binding fragment light chain and a mutant IL-2 polypeptide.

In a specific embodiment of the invention, the isolated polynucleotide of the invention encodes an immunoconjugate fragment containing at least one mutant IL-2 polypeptide and at least one, preferably two or more antigen-binding fragments, wherein the first mutant IL-2 polypeptide is combined amino - or carboxy-terminal peptide bond with the first antigen-binding fragment, and the second antigen-binding fragment is combined with an amino- or carboxy-terminal peptide bond with either the first IL2 mutant polypeptide or the first antigen-binding fragment. In one embodiment, the antigen-binding fragments are independently selected from the group consisting of an Fv molecule, in particular an scFv molecule, and a Fab molecule. In another specific embodiment of the invention, the polynucleotide encodes the heavy chains of two antigen-binding fragments and one mutant IL-2 polypeptide. In another specific embodiment of the invention, the polynucleotide encodes the light chains of two antigen-binding fragments and one mutant IL-2 polypeptide. In another specific embodiment, the polynucleotide encodes one light chain of one of the antigen-binding fragments, one heavy chain of the second antigen-binding fragment, and one mutant IL-2 polypeptide.

In another specific embodiment, the isolated polynucleotide of the invention encodes an immunoconjugate fragment, wherein the polynucleotide encodes the heavy chains of two Fab molecules and a mutant IL-2 polypeptide. In another specific embodiment, an isolated polynucleotide of the invention encodes an immunoconjugate fragment, 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 an immunoconjugate fragment, wherein the polynucleotide encodes the heavy chain of one Fab molecule, the light chain of another Fab molecule, and the mutant IL-2 polypeptide.

- 32 040858

In one embodiment, an isolated polynucleotide of the invention encodes an immunoconjugate comprising at least one mutant IL2 polypeptide linked via its amino and carboxy terminal amino acids to one or more scFv molecules.

In one embodiment, an isolated polynucleotide of the invention encodes an immunoconjugate fragment, wherein the polynucleotide encodes the heavy chain of an immunoglobulin molecule, preferably an IgG molecule, more preferably an IgG1 molecule, and a 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, wherein the mutant IL-2 polypeptide is linked by an amino-terminal peptide bond to the immunoglobulin heavy chain.

Another embodiment of the present invention is an isolated polynucleotide encoding an immunoconjugate or a fragment thereof, wherein the polynucleotide contains a sequence that encodes a variable region sequence shown in SEQ ID NOs: 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. Another embodiment of the present invention is an isolated polynucleotide encoding an immunoconjugate or fragment thereof, wherein the polynucleotide contains a sequence that encodes a polypeptide sequence shown in SEQ ID NOs: 199, 201, 203, 205 , 207, 209, 211, 213, 215, 217, 219, 221, 2 23, 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. Another embodiment of the present invention is also an isolated polynucleotide encoding an immunoconjugate, or fragment thereof, wherein the polynucleotide contains a sequence that is at least about 80, 85, 90, 95, 96, 97, 98, or 99 % identical to the 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, 254, 256, 258, 260, 262, 298, 300, 302, 304, 312, 314, 316, 318, 320, 322 or 324. of the invention is an isolated polynucleotide encoding an immunoconjugate or a fragment thereof, wherein the polynucleotide contains the nucleotide sequence shown in SEQ ID NOs: 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. Another embodiment of the present invention is an isolated polynucleotide encoding an immunoconjugate or fragment thereof, wherein the polynucleotide contains a sequence encoding a variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NOs: 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. Another embodiment of the present invention is an isolated polynucleotide encoding an immunoconjugate or its fr where the polynucleotide contains a sequence encoding a polypeptide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid 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. The invention also relates to an isolated polynucleotide encoding an immunoconjugate or a fragment thereof, wherein the polynucleotide contains a sequence that encodes the variable region sequences 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, 1 81, 183, 185, 187, 189, 191, 193, 195, 197, 311 or 313 with conservative amino acid substitutions. The invention also relates to an isolated polynucleotide encoding an immunoconjugate or a fragment thereof, where the polynucleotide contains a sequence that encodes the polypeptide sequences 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,

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301, 303, 315, 317, 319, 321, or 323 with conservative amino acid substitutions.

In some embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments of the invention, the polynucleotide proposed in the present invention is an RNA, for example, in the form of messenger RNA (mRNA). The RNA of the present invention may be single stranded or double stranded.

Recombination methods

Mutant IL-2 polypeptides of the invention can be obtained by deletion, substitution, insertion, or modification using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis of the DNA coding sequence, PCR, gene synthesis, and the like. The correctness of nucleotide changes can be confirmed, for example, by sequencing. For this purpose, the nucleotide sequence of native IL-2 described by Taniguchi et al. cultures (Rockville, Maryland). The sequence of native human IL-2 is shown in SEQ ID NO: 1. The substitution or insertion may include naturally occurring as well as non-naturally occurring amino acid residues. Amino acid modification includes well-known chemical modification methods such as the addition of glycosylation or carbohydrate attachment sites, and the like.

Mutant IL-2 polypeptides and immunoconjugates of the invention can be obtained, for example, by solid phase peptide synthesis or by recombination. For recombinant production, one or more polynucleotides encoding a mutant IL-2 polypeptide or immunoconjugate (fragment), for example, as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Said polynucleotide is easy to isolate and sequence using conventional procedures. One embodiment of the invention is a vector, preferably an expression vector, containing one or more polynucleotides of the invention. Methods well known to those skilled in the art can be used to construct expression vectors containing the mutant IL-2 polypeptide or immunoconjugate (fragment) coding sequence along with suitable transcription/translation control signals. These methods include in vitro recombinant DNA technologies, in vivo synthesis and recombination/genetic recombination methods (see, e.g., methods described in Maniatis et al., 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 may be part of a plasmid, a virus, or may be a nucleic acid fragment. The expression vector includes an expression cassette in which a polynucleotide encoding a mutant IL-2 or an immunoconjugate (fragment) (ie, coding region) is cloned into a operable link with a promoter and/or other transcription or translation control elements. In the context of the present description, a coding region is a portion of a 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 part of the coding region if present, but any flanking sequences such as promoters, ribosome binding sites, transcription terminators, introns, 5' - and 3'-untranslated regions, etc., are not part of the coding region. Two or more coding regions may be present in an individual polynucleotide construct, eg an individual vector, or in separate polynucleotide constructs, eg separate (different) vectors. In addition, any vector may contain a single coding region or may contain two or more coding regions, for example, the vector of the present invention may encode one or more polyproteins that are post- or countertranslationally separated into final proteins by proteolytic cleavage. In addition, the vector, polynucleotide or nucleic acid of the invention may encode heterologous coding regions, either fused or not fused to the first or second polynucleotide that encodes the polypeptides of the invention, or variants or derivatives thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs such as a secretory signal peptide or a heterologous functional domain. Functional linkage occurs when the coding region of a gene product, eg a polypeptide, is associated with one or more regulatory sequences such that expression of the gene product is under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and its associated promoter) are operably linked if induction of the promoter function results in transcription of the mRNA encoding the desired gene product, and if the nature of the link between the two DNA fragments does not affect the ability to regulate expression sequences direct the expression of the gene product, or does not affect the ability of the DNA template for transcription. Thus, a promoter region must be operably linked to a nucleic acid encoding a polypeptide if the promoter is capable of transcribing the nucleic acid. The promoter may be a cell-specific promoter that allows significant DNA transcription only in pre-selected cells. Other transcription control elements besides the promoter, such as enhancers, operators, repressors, and transcription termination signals, can be operably linked to a polynucleotide to provide cell-specific transcription. Acceptable promoters and other transcriptional control regions are provided in the present description. Those skilled in the art are aware of a wide variety of transcriptional control regions. These include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as (but not limited to) promoter and enhancer segments from cytomegaloviruses (e.g., the immediate early promoter in combination with intron-A), simian virus 40 (eg early promoter); and retroviruses (eg 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 (eg, tetracycline-inducible promoters). Likewise, a wide variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to, ribosome binding sites, translation initiation codons and stop codons and elements derived from viral systems (particularly the internal ribosome entry site or IRES, also referred to as the CITE sequence). The expression cassette may also include other characteristic structures such as an origin of replication and/or chromosomal integrated elements such as long terminal repeats (LTRs) of retroviruses or inverted terminal repeats (ITRs) of adeno-associated virus (AAV).

The polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions that encode secretory or signal peptides that direct secretion of the polypeptide encoded by the polynucleotide of the present invention. For example, if secretion of a mutant IL-2 polypeptide is desired, then the DNA encoding the signal sequence can be placed upstream of the nucleic acid encoding the mature amino acids of the mutant IL-2. The same approach applies to the immunoconjugates of the invention, or fragments thereof. According to the signaling hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader that is/is cleaved from the mature protein upon initiation of export of the nascent protein chain across the rough endoplasmic reticulum. Those of ordinary skill in the art will appreciate that polypeptides secreted by vertebrate cells typically have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to form the secreted or mature form of the polypeptide. For example, human IL-2 is translated with a 20 amino acid signal sequence at the N-terminus of the polypeptide, which is then cleaved off to form mature 133 amino acid human IL-2. In some embodiments, a native signal peptide is used, such as an IL-2 signal peptide or an immunoglobulin heavy chain or light chain signal peptide, or a functional derivative of said sequence, that retains the ability to provide secretion of a polypeptide operably linked to it. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof may be used. For example, the wild-type leader can be replaced with a human tissue plasminogen activator (TPA) or mouse β-glucuronidase leader. Examples of amino acid and polynucleotide sequences of secretory signal peptides are shown in SEQ ID NOs: 236-273.

DNA encoding a short protein sequence that can be used to facilitate further purification (e.g., a histidine tag) or intended to label a mutant IL-2 or immunoconjugate can be included inside or at the ends of a polynucleotide encoding a mutant IL-2 or immunoconjugate (fragment).

An additional embodiment of the invention is a host cell containing one or more polynucleotides of the invention. Some embodiments of the invention is a host cell containing one or more vectors of the invention. Polynucleotides and vectors may have any of the features, singly or in combination, as described herein for polynucleotides and vectors, respectively. In one such embodiment, the host cell contains (e.g., transformed or transfected with) a vector containing a polynucleotide that encodes for amino acids

- 35 040858 new sequence containing the mutant IL-2 polypeptide proposed in the invention. In the context of the present description, the term host cell refers to any type of cellular system that can be designed to obtain mutant IL-2 polypeptides or immunoconjugates of the invention, and fragments thereof. Host cells suitable for replication and for maintaining the expression of IL-2 mutant polypeptides or immunoconjugates are well known in the art. Such cells can be transfected or transduced appropriately with a particular expression vector, and more cells containing the vector can be grown for use in fermenters for large scale IL-2 mutant or immunoconjugate production processes in sufficient quantities for clinical applications. Suitable host cells are prokaryotic microorganisms such as E. coli or various eukaryotic cells such as Chinese Hamster Ovary (CHO) cells, insect cells or the like. For example, polypeptides can be obtained in bacteria, in particular when there is no need for glycosylation. 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 microorganisms such as filamentous fungi or yeasts, including strains of fungi and yeasts whose glycosylation pathways have been humanized, can be used as hosts for cloning or expression of vectors that encode a polypeptide, allowing the production of a polypeptide with a partially or fully human pattern. glycosylation (see Gerngross, Nat. Biotech. 22, 2004, pp. 1409-1414 and Li et al., Nat. Biotech. 24, 2006, pp. 210-215). Host cells that can be used to express (glycosylated) polypeptides are also obtained from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells are insect cells, and plant cells can also be used. Numerous baculovirus strains and corresponding suitable insect hosts have been identified, primarily for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts (see, for example, U.S. Nos. 5959177, 6040498, 6420548, 7125978 and 6417429 (a description of the PLANTIBODIES™ technology for producing antibodies in transgenic plants). Vertebrate animal cells can also be used as hosts. For example, mammalian cell lines that are adapted to growth in suspension can be used.Other examples of suitable mammalian host cell lines are SV40-transformed monkey kidney cell line CV1 (COS-7), human embryonic kidney cell line (293 or cell line 293, subcloned for growth in suspension culture, Graham et al., J. Gen. Virol., 36, 1977, p. 59), baby hamster kidney (BHK) cells, mouse Sertoli cells (TM4 cells described for example , in Mather, Biol. soba ki (MDCK); bovine rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor cells (MMT 060562); TRI cells as described, for example, in Mather et al., Annals N.Y. Acad. Sci., 383, 1982, pp. 44-68); MRC 5 cells and FS4 cells. Other valuable mammalian host cell lines are Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Nail. Acad. Sci. USA, 77, 1980, p. 4216); and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of specific mammalian host cell lines that can be used for protein production, see, for example, Yazaki and Wu, in Methods in Molecular Biology, ed. V.K.S. Lo, Humana Press, Totowa, NJ, vol. 248, 2003, pp. 255-268. Host cells include, but are not limited to, cultured cells, such as cultured mammalian cells, yeast cells, insect cells, bacteria cells, and plant cells, as well as cells found in a transgenic animal, transgenic plant, or cultured plant or animal tissue. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a lymphoid cell (e.g., YO, NSO, Sp20 cell).

Standard techniques are known in the art for the expression of foreign genes in these systems. Cells expressing a mutant IL-2 polypeptide fused to either the heavy or light chain of an antigen-binding domain, such as an antibody, can be engineered to express other antibody chains, such as such that the expressed fusion product containing mutant IL-2 included an antibody that has both a heavy and a light chain.

One of the embodiments of the invention is a method for obtaining a mutant IL-2 polypeptide or immunoconjugate proposed in the invention, which consists in cultivating a host cell containing a polynucleotide that encodes a mutant IL-2 polypeptide or immunoconjugate presented in the present description, under conditions suitable for expressing the mutant IL-2 polypeptide or immunoconjugate, and optionally isolating the mutant IL-2 polypeptide or immunoconjugate from the host cell (or host cell culture medium).

In some embodiments, the mutant IL-2 polypeptide is linked at least

- 36 040858 with one He-IL-2 fragment. A mutant IL-2 can be produced in which a segment of the mutant IL-2 polypeptide is linked to one or more molecules, such as a polypeptide, protein, carbohydrate, lipid, nucleic acid, polynucleotide, or a molecule that contains any combination of these molecules (e.g., glycoproteins, glycolipids, etc.). The mutant IL-2 polypeptide can also be linked to an organic moiety, an inorganic moiety, or a pharmaceutical drug. In the context of the present description, a pharmaceutical drug is an organic substance containing a compound with a molecular weight of about 5000 Da or less. The mutant IL-2 polypeptide can also be combined with any biological agent, including therapeutic compounds such as antineoplastic agents, antimicrobial agents, hormones, immunomodulators, anti-inflammatory agents, and the like. It is also possible to use radioisotopes, for example those used for imaging as well as for therapy.

A mutant IL-2 polypeptide can also be coupled to multiple molecules of the same type or multiple types of molecules. In some embodiments, the molecule that is coupled to IL-2 may confer the ability to target IL-2 to specific tissues or cells in the animal body and are referred to herein as a targeting moiety. In these embodiments, the targeting moiety may have affinity for a ligand or receptor in the target tissue or cell, thereby directing IL-2 to the target tissue or cell. In a particular embodiment, the targeting moiety directs IL-2 to the tumor. Targeting moieties include, for example, antigen-binding moieties (eg, antibodies and fragments thereof) specific for cell surface or intracellular proteins, biological receptor ligands, and the like. Said antigen-binding fragments may be specific for tumor-associated antigens, such as those described above.

A mutant IL-2 polypeptide can be genetically fused to another polypeptide, such as a single chain antibody or (part of) heavy or light chains of an antibody, or can be chemically conjugated to another molecule. Fusion of a mutant IL-2 polypeptide to the heavy chain portion of an antibody is described in the Examples section. A mutant IL-2 that is a fusion component of a mutant IL-2 polypeptide and another polypeptide can be designed such that the IL-2 sequence is fused to the polypeptide directly or indirectly through a linker sequence. The composition and length of the linker can be determined using methods well known in this field, and you can evaluate its effectiveness. Examples of a linker sequence between IL-2 and an antibody heavy chain are shown in, for example, SEQ ID NOs: 209, 211, 213, etc. Additional sequences can also be included at the cleavage site to separate the individual fusion components as needed, for example an endopeptidase-recognized sequence. In addition, mutant IL-2 or a fusion protein containing it can also be chemically synthesized using polypeptide synthesis methods well known in the art (eg, Merrifield solid phase synthesis). Mutant IL-2 polypeptides can be conjugated chemically to other molecules, such as another polypeptide, using well known chemical conjugation techniques. For this purpose, bifunctional cross-linking reagents can be used, such as homofunctional and heterofunctional cross-linking reagents well known in the art. The type of crosslinker used depends on the nature of the molecule to be combined with IL-2 and can be easily identified by those skilled in the art. Alternatively or additionally, the IL-2 mutant and/or the molecule to which it is intended to be conjugated may be chemically derivatized such that both can be conjugated in a separate reaction, which is also well known in the art.

In some embodiments, a mutant IL-2 polypeptide is coupled to one or more antigen-binding fragments (ie, as part of an immunoconjugate) that contain at least an antibody variable region that is capable of binding to an antigenic determinant. The variable regions may form part of or be derived from naturally occurring or non-naturally occurring antibodies or fragments thereof. Methods for making 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). Non-naturally occurring antibodies can be generated by solid-phase peptide synthesis, can be generated by recombination techniques (e.g., as described in U.S. No. 4,186,567), or can be generated, for example, by screening combinatorial libraries containing heavy chain variable regions and light chain variable regions. (See, for example, U.S. No. 5969108 to McCafferty). Immunoconjugates, antigen-binding fragments and methods for their preparation are also described in detail in PCT publication WO 2011/020783, the entire content of which is incorporated herein by reference.

Any type of antibody, antibody fragment, antigen-binding domain, or variable region

- 37 040858 animals of animal origin can be combined with a mutant IL-2 polypeptide. Examples of antibodies, antibody fragments, antigen-binding domains or variable regions that can be used according to the present invention are (but not limited to) constructs derived from mice, primates or humans. If the mutant IL2/antibody conjugate or fusion is/is intended for use in a human, then a chimeric form of the antibody can be used in which the antibody constant regions are derived from a human antibody. Humanized or fully human form of the antibody can also be prepared using methods well known in this field (see, for example, U.S. No. 5565332 in the name of Winter). Various methods can be used to effect humanization, such as (but not limited to) (a) transplantation of non-human (e.g., from a donor antibody) CDRs into a human (e.g., recipient antibody) framework and constant regions, with or without conserving critical framework residues (e.g., residues important for maintaining good antigen-binding affinity or antibody function), (b) transplantation of non-human specificity-determining regions (SDRs or a-CDRs; residues critical for antibody-antigen interaction) into a human framework plot and constant regions, or (c) transplanting completely non-human variable domains and masking them with a resembling human segment by replacing surface residues. For a review of humanized antibodies and methods for their production, see, for example, Almagro and Fransson, Front Biosci 13, 12008, ss. 1619-1633 and are also described in, for example, Riechmann et al., Nature 332, 1988, pp. 323-329; Queen et al., Proc Natl Acad Sci USA 86, 1989, pp. 10029-10033; U.S. Nos. 5821337, 7527791, 6982321 and 7087409; Jones et al., Nature 321, 1986, pp. 522-525; Morrison et al., Proc Natl Acad Sci 81, 1984, pp. 6851-6855; Morrison and Oi, Adv Immunol 44, 1988, pp. 65-92; Verhoeyen et al., Science 239, 1988, pp. 1534-1536; Padlan, Molec Immun 31(3), 1994, pp. 169-217; Kashmiri et al., Methods 36, 2005, pp. 25-34) (description of transplantation of SDR (a-CDR)); Padlan, Mol Immunol 28, 1991, pp. 489-498 (description of recoating); Dall'Acqua et al., Methods 36, 2005, pp. 43-60 (description of FR permutation) and Osbourn et al., Methods 36, 2005, pp. 61-68, and Klimka et al., Br J Cancer 83, 2000, ss. 252-260 (describing a targeted selection approach for FR permutation). Human antibodies and human variable regions can be obtained using various techniques known in this field. Human antibodies are described generally in van Dijk and van de Winkel, Curr Opin Pharmacol 5, 2001, cc. 368-374 and Lonberg, Curr Opin Immunol 20, 2008, pp. 450-459). Human variable regions may form part of human monoclonal antibodies or may be derived from them using the hybridoma technique (see, for example, Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, 1987, cc. 51- 63). Human antibodies and human variable regions can also be generated by administering an immunogen to a transgenic animal that is modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge (see, for example, Lonberg, Nat Biotech 23 , 2005, pp. 1117-1125). Human antibodies and human variable regions can also be generated by isolating Fv clone variable region sequences selected from human phage display libraries (see, for example, Hoogenboom et al. in Methods in Molecular Biology, ed. O'Brien et al. , Human Press, Totowa, NJ, 178, 2001, pp. 1-37) and McCafferty et al., Nature 348, 552-554; Clackson et al., Nature 352, 1991, pp. 624-628). The phage typically displays antibody fragments either as single chain Fv (scFv) fragments or as Fab fragments. A detailed description of the production of antigen-binding fragments for immunoconjugates using phage display is provided in the examples attached to PCT publication WO 2011/020783.

In some embodiments, antigen-binding fragments suitable for use in accordance with the present invention are designed to have increased binding affinity, for example, using the methods described in PCT publication WO 2011/020783 (see examples regarding affinity maturation) or publication US Patent Application No. 2004/0132066, the entire content of which is incorporated herein by reference. The ability of an immunoconjugate of the invention to bind to a specific antigenic determinant can be quantified either by enzyme-linked enzyme-linked assay (ELISA) or by other methods known to one of skill in the art, such as surface plasmon resonance (using the BIACORE system). T100 system) (Liljeblad et al., Glyco J 17, 2000, pp. 323-329), and traditional binding assays (Heeley, Endocr Res 28, 2002, pp. 217-229). Competition assays can be used to identify an antibody, antibody fragment, antigen-binding domain, or variable domain that competes with a reference antibody for binding to a specific antigen, such as an antibody that competes with L19 antibody for binding to the fibronectin extra-domain B (EDB) . In some embodiments, said competing antibody binds to the same epitope (eg, linear or conformational epitope) that the reference antibody binds to. For detailed exemplary methods for mapping an epitope to which an antibody binds, see Morris, Epitope Mapping Protocols, B: Methods in Molecular Biology, Humana.

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Press, Totowa, NJ, Vol. 66, 1996. In an exemplary competitive assay, the immobilized antigen (eg, EDB) is incubated in a solution containing the first labeled antibody that binds to the antigen (eg, L19 antibody), and a second unlabeled antibody to be tested for its ability to compete with the first antibody for antigen binding. The second antibody may be present in the hybridoma supernatant. As a control, the immobilized antigen is incubated in a solution containing the first labeled antibody but not containing the second unlabeled antibody. After incubation under conditions that ensure the binding of the first antibody to the antigen, the excess of unbound antibody is removed and the amount of label associated with the immobilized antigen is estimated. If the amount of label associated with the immobilized antigen is significantly reduced in the test sample compared to the control sample, then this indicates that the second antibody competes with the first antibody for binding to the antigen (see Harlow and Lane. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, Chapter 14, 1988).

Additional modification of the IL-2 mutant or immunoconjugate of the invention may be required. For example, problems associated with immunogenicity and short half-life can be overcome by conjugation with substantially straight chain polymers such as polyethylene glycol (PEG) or polypropylene glycol (PPG) (see, for example, WO 87/00056).

IL-2 mutants and immunoconjugates obtained using the methods presented herein can be purified using techniques known in the art such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, gel filtration, and the like. . The actual conditions used to purify a particular protein depend, inter alia, on factors such as net charge, hydrophobicity, hydrophilicity, etc., and should be apparent to those skilled in the art. To purify an antibody by affinity chromatography, a ligand, receptor, or antigen to which the mutant IL-2 polypeptide or immunoconjugate binds can be used. For example, an antibody that specifically binds to a mutant IL-2 polypeptide can be used. For affinity chromatographic purification of the immunoconjugates of the invention, a protein A or protein G matrix can be used. Examples. Mutant IL-2 polypeptide purity and fusion protein content can be determined using any of a wide variety of well known analytical methods, including gel electrophoresis, high pressure liquid chromatography, and the like. For example, heavy chain-containing fusion proteins that were expressed according to the methods described in the Examples section were found to be intact and correctly assembled, as demonstrated by SDS-PAGE under reducing conditions (see, eg, FIG. 14). Two bands were separated, corresponding to approximately Mr 25,000 and Mr 60,000, which corresponded to the predicted molecular weights of the immunoglobulin/IL-2 light and heavy chain fusion protein.

Analyzes

The IL-2 mutant polypeptides and immunoconjugates provided herein can be identified, screened for, or characterized for their physical/chemical properties and/or biological activities using various assays known in the art.

Affinity Assays

The affinity of an IL-2 polypeptide, mutant or wild type, for various forms of the IL-2 receptor can be determined according to the method described in the Examples section using surface plasmon resonance (SPR) using a standard instrumentation such as the BIAcore device (GE Healthcare) , and receptor subunits, which can be obtained by recombinant expression (see, for example, Shanafelt et al., Nature Biotechnol 18, 2000, pp. 1197-1202). The heterodimer of the β/γ subunits of the recombinant IL-2 receptor can be generated by fusing each of the subunits to a knobs-into-holes modified antibody Fc domain monomer (see, for example, US No. 5,731,168) to enhance heterodimerization of fusion proteins containing the appropriate receptor subunit/Fc (see SEQ ID NOs: 102 and 103). Alternatively, the binding affinity of IL-2 mutants to various forms of the IL-2 receptor can be assessed using cell lines known to be able to express one or the other form of the receptor. A specific illustrative and exemplary embodiment for measuring binding affinity is described below in the Examples section. According to one embodiment of the invention, the K _D value was measured by surface plasmon resonance using a BIACORE® T100 device (GE Healthcare) at 25°C using IL-2 receptors immobilized on CM5 chips. In general, the method was as follows: Carboxymethylated dextran (CM5, GE Healthcare) biosensor chips were activated with N-ethyl-N'-(3dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) hydrochloride according to the supplier's instructions. Recombinant IL-2 receptor was diluted with 10 mM sodium acetate, pH 5.5 to a concentration of 0.5

- 39 040858 μg/ml before injection at a flow rate of 10 μl/min to achieve approximately 200-1000 response units (RU) of the fusion protein (for IL-2R α-subunit) or 500-3000 RU (for IL-2R βγ heterodimer, obtained by the knobs-into-holes technology). After injection of the IL-2 receptor, 1M ethanolamine was injected to block the unreacted groups. For kinetic measurements, three-fold serial dilutions of the IL-2 mutant polypeptide or immunoconjugate (range ~0.3 to 300 nM) were injected in HBS-EP+ buffer (GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05 % surfactant P20, pH 7.4) at 25° C. with a flow rate of approximately 30 μl/min. The rate of association reaction (k _on ) and dissociation reaction (k _off ) was calculated using a simple 1:1 Langmuir binding model (BIACORE® T100 Evaluation Software, version 1.1.1) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium constant of the dissociation reaction (K _D ) was calculated as the ratio k _off /k _on (see, for example, Chen et al., J Mol Biol 293, 1999, pp. 865-881).

The binding of the immunoconjugates of the invention to Fc receptors can be easily determined, for example, by ELISA or by surface plasmon resonance (SPR) using standard instrumentation, such as the BIAcore device (GE Healthcare), and Fc receptors, for example, which can be obtained using recombinant expression. Alternatively, the binding affinity of Fc domains or Fc domain-containing immunoconjugates to Fc receptors can be assessed using cell lines known to express particular Fc receptors, such as NK cells expressing the FcyIIIa receptor. According to one embodiment of the invention, the KD value was measured using the surface plasmon resonance method using a BIACORE® T100 device (GE Healthcare) at 25°C using IL-2 Fc receptors immobilized on CM5 chips. In general, the method was as follows: Carboxymethylated dextran (CM5, GE Healthcare) biosensor chips were activated with N-ethyl-N'-(3-dimethylaminopropyl)arbodiimide (EDC) and N-hydroxysuccinimide (NHS) hydrochloride according to the supplier's instructions. . The recombinant Fc receptor was diluted with 10 mM sodium acetate, pH 5.5 to a concentration of 0.5-30 μg/ml before injection at a flow rate of 10 μl/min to achieve approximately 100-5000 response units (RU) of the fusion protein. After injection of the Fc receptor, 1M ethanolamine was injected to block the unreacted groups. For kinetic measurements, 3-5-fold serial dilutions of the immunoconjugate (range ~0.1 to 300nM) were injected in HBS-EP+ buffer (GE Healthcare, 10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20, pH 7 ,4) at 25°C with a flow rate of about 30-50 µl/min. The rate of association reaction (kon) and dissociation reaction (k _off ) was calculated using a simple 1:1 Langmuir binding model (BIACORE® T100 Evaluation Software, version 1.1.1) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium constant of the dissociation reaction (KD) was calculated as the ratio koff/kon (see, for example, Chen et al., J Mol Biol 293, 1999, pp. 865-881).

Activity analyzes

The ability of mutant IL-2 to bind to IL-2 receptors can be assessed indirectly by analyzing immune activation effects that occur in direct relation to receptor binding.

One of the objects of the invention are assays designed to identify mutant IL-2 polypeptides with biological activity. Biological activities include, for example, the ability to induce proliferation of IL-2T and/or NK receptor-bearing cells, the ability to induce IL-2 signaling in IL-2T and/or NK receptor-bearing cells, the ability to influence interferon (IFN) production -y as a secondary cytokine by NK cells, decreased ability to induce production of secondary cytokines, in particular IL-10 and TNF-α, by peripheral blood mononuclear cells (PBMCs), decreased ability to induce T cell apoptosis, ability to induce tumor regression, and/or increased survival and a reduced toxicity profile, primarily reduced vascular permeability, in vivo. Also provided are mutant IL-2 polypeptides having said biological activity in vivo and/or in vitro.

In some embodiments, the biological activity of the mutant IL-2 polypeptide of the invention was evaluated. Various methods for determining the biological activity of IL-2 are widely known in the art, and many of these methods are described in detail in the Examples section below. The Examples section provides an acceptable analysis of the evaluation of IL-2 mutants of the invention for their ability to interfere with NK cell production of IFN-γ. Cultured NK cells were incubated with IL-2 mutant polypeptide or immunoconjugates of the invention, and then the concentration of IFN-γ in the culture medium was determined by ELISA.

IL-2 induced signaling induces multiple signaling pathways and includes the signaling molecules JAK (Janus kinase) and STAT (signal transducer and transcription activator). The interaction of IL-2 with the α- and γ-subunits of the receptor leads to phosphorylation of the receptor and JAK1 and JAK3, which are associated with the β- and γ-subunit, respectively. Then there is an association

- 40 040858 cyation of STAT5 with a phosphorylated receptor and its phosphorylation on a crucial tyrosine residue. This results in dissociation of STAT5 from the receptor, dimerization of STAT5, and translocation of STAT5 dimers to the nucleus, where they enhance transcription of target genes. Thus, the ability of mutant IL-2 polypeptides to induce signaling through the IL-2 receptor can be assessed, for example, by measuring STAT5 phosphorylation. Details of this method are described in the Examples section. PBMCs were treated with mutant IL-2 plypeptides or immunoconjugates of the invention and phosphorylated STAT5 levels were determined by flow cytometry.

The proliferation of T cells or NK cells in response to exposure to IL-2 can be assessed by incubating T cells or NK cells isolated from blood with mutant IL-2 polypeptides or immunoconjugates of the invention, followed by determination of the ATP content in lysate of treated cells. Before treatment, T cells can be pre-stimulated with phytohemagglutinin (PHA-M). This assay, described in the Examples section, is sensitive and quantifies viable cells, although other acceptable alternative assays are known in the art (e.g., [ ³ H]-thymidine incorporation assay, Cell Titer Glo ATP assays, assay based on Alamar Blue, WST-1 analysis, MTT analysis).

The Examples section also describes an assay designed to evaluate T cell apoptosis and AICD, in which T cells are treated with an apoptosis inducing antibody after incubation with mutant IL-2 polypeptides or immunoconjugates of the invention, and apoptotic cells are quantified based on the determination flow cytometry for phosphatidylserine/annexin deposition. Other assays are known in the art.

The effect of mutant IL-2 on tumor growth and survival can be assessed using various animal models of tumors known in the art. For example, xenografts of human cancer cell lines can be implanted in immunodeficient mice and treated with IL-2 mutant polypeptides or immunoconjugates of the invention as described in the Examples section.

Toxicity of mutant IL-2 polypeptides or immunoconjugates of the invention can be determined in vivo by mortality, by in vivo observations (assessment of visible symptoms of adverse events, such as behavior, body weight, body temperature), and by clinical and anatomical pathology ( e.g. blood chemistry and/or histopathological tests).

Vascular permeability induced by IL-2 treatment can be assessed in a pre-treated animal model of vascular permeability. In general, the IL-2 mutant or immunoconjugate of the invention is administered to an appropriate animal, eg a mouse, and the animal is later injected with a vascular leakage reporter molecule whose spread out of the vasculature reflects the degree of vascular permeability. The vascular leak reporter molecule is preferably large enough to detect permeability when using the wild-type form of IL-2 for pretreatment. An example of a vascular leak reporter molecule can be a serum protein such as albumin or immunoglobulin. The bleed reporter molecule is preferably labeled with a detectable label, such as a radioisotope, to facilitate quantification of tissue distribution of the molecule. Vascular permeability can be measured for vessels present in any of the various internal organs of the body such as the liver, lung, and the like, as well as in tumors, including xenograft tumors. The lung is the preferred organ for measuring vascular permeability using full-length IL-2 mutants.

Compositions, formulations and routes of administration

The following object of the invention are pharmaceutical compositions containing any of the mutant IL-2 polypeptides or immunoconjugates presented in the present description, for example, intended for use in any of the following therapeutic methods. In one of the embodiments of the invention, the pharmaceutical composition contains any of the mutant IL-2 polypeptides or immunoconjugates presented in the present description, and a pharmaceutically acceptable carrier. In another embodiment of the invention, the pharmaceutical composition contains any of the mutant IL-2 polypeptides or immunoconjugates presented in the present description, and at least one additional therapeutic agent, for example, listed below.

In addition, a method is provided for obtaining a mutant IL-2 polypeptide or immunoconjugate of the invention in a form suitable for in vivo administration, which consists in (a) obtaining a mutant IL-2 polypeptide or immunoconjugate of the invention, and ( b) the mutant IL-2 polypeptide or immunoconjugate is combined in a formulation with at least one pharmaceutically acceptable carrier, wherein the mutant IL-2 polypeptide or immunoconjugate is formulated to be suitable for in vivo use.

Pharmaceutical compositions of the present invention contain an effective

- 41 040858 amount of one or more mutant IL-2 polypeptides or immunoconjugates, which(s) are dissolved(s) or dispersed(s) in a pharmaceutically acceptable carrier. The terms pharmaceutically or pharmacologically acceptable refers to molecular substances and compositions that are generally non-toxic to recipients at the doses and concentrations used, i.e. do not cause harmful, allergic or other undesirable reactions when administered as needed to an animal, such as, for example, a human. The preparation of a pharmaceutical composition that contains at least one IL-2 mutant polypeptide or immunoconjugate and optionally an additional active ingredient should be apparent to those skilled in the art in light of the present disclosure, for example, from Remington's Pharmaceutical Sciences, 18th ed., ed. - in Mack Printing Company, 1990, included in the present description by reference. In addition, it is clear that preparations intended for administration to an animal (e.g., a human) must meet the standards of sterility, pyrogenicity, and general safety and purity established by the Biological Standards Division (Food and Drug Administration) of the FDA or the appropriate authority of other countries. Preferred compositions are lyophilized formulations or aqueous solutions. Examples of IL-2 compositions are given in US Nos. 4,604,377 and 4,766,106. As used herein, a pharmaceutically acceptable carrier includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonicizing agents, absorption delaying agents, salts, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, colorants and similar materials and combinations thereof, which should be known one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th ed., Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Any conventional carrier, as long as it is compatible with the active substance, can be used in therapeutic or pharmaceutical compositions.

The composition may contain various types of carriers depending on whether it is administered in solid, liquid or aerosol form and whether it is to be sterile, as is the case with administration routes such as injection. Mutant IL-2 polypeptides or immunoconjugates of the present invention (and additional therapeutic agent) can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesion, intracranial, intraarticular, intraprostate, intraspleen, intrarenal, intrapleural, intratracheal, intranasally, intravitreally, intravaginally, intrarectally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, site, by inhalation (eg, aerosol inhalation), injection , infusion, continuous infusion, localized perfusion, bathing directly in target cells, through a catheter, via lavage, in the form of creams, in lipid formulations (e.g., liposomes), or by any other method or any combination of the above routes known to one of ordinary skill in the art. given field (see, for example, Remington's Pharmaceutical Sciences, 18th ed., Mack Printing Company, 1990, incorporated herein by reference). For the introduction of polypeptide molecules, such as mutant IL-2 polypeptides and immunoconjugates proposed in the invention, the most commonly used parenteral administration, in particular intravenous injection.

Parenteral compositions include compositions formulated for administration by injection, for example, subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal, or intraperitoneal injection. For injection, mutant IL-2 polypeptides and immunoconjugates of the invention may 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 IL-2 mutant polypeptides and immunoconjugates may be in powder form, intended to be reconstituted with a suitable vehicle, such as sterile, pyrogen-free water, before use. Sterile injectable solutions are prepared by incorporating the IL-2 polypeptides or immunoconjugates of the invention in the required amount in an appropriate solvent, optionally in combination with various other ingredients listed below. Sterility can be easily achieved, for example, by filtration through sterile filter membranes. Typically, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains a basic dispersion medium and/or other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsions, the preferred methods of preparation are vacuum or freeze-drying, which allows the preparation of a powder of the active ingredient in combination with any additional ingredient required.

- 42 040858 an entom from a liquid medium previously sterilized by filtration. If necessary, the liquid medium must be suitably buffered before use and the liquid diluent first rendered isotonic with sufficient saline or glucose. The composition must be stable under the conditions of preparation and storage and protected from the contaminating action of microorganisms such as bacteria and fungi. It is customary to maintain endotoxin contamination at the minimum safe level, eg, less than 0.5 ng/mg protein. Suitable pharmaceutically acceptable carriers include, but are not limited to: buffers such as phosphate, citrate, and other organic acid buffers; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol, resorcinol; cyclohexanol; 3-pentanol and meta-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, or the like. Optionally, the suspension may also contain stabilizers or agents which increase the solubility of the compounds, thereby allowing highly concentrated solutions to be obtained. In addition, suspensions of the active compounds can be obtained in the form of appropriate oily suspensions intended for injection. Suitable lipophilic solvents or excipients include fatty fixed oils such as sesame oil, or synthetic fatty acid esters such as ethyl esters or triglycerides, or liposomes.

The active substances can be incorporated into microcapsules, for example, obtained by coacervation or interfacial polymerization methods, for example, in hydroxypropyl methylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, in liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsion. Such methods are described in Remington's Pharmaceutical Sciences, ed. A. Osol, 1980. Sustained release formulations can be prepared. Suitable examples of sustained release formulations are semi-permeable matrices of solid hydrophobic polymers comprising a polypeptide, such matrices are shaped articles such as films or microcapsules. In a specific embodiment of the invention, to achieve prolonged absorption of the injectable composition, agents that delay absorption, such as, for example, aluminum monostearate, gelatin, or combinations thereof, can be used in the composition.

In addition to the compositions described above, the immunoconjugates can also be formulated as a depot formulation. These long acting formulations may be administered by implantation (eg subcutaneous or intramuscular) or intramuscular injection. Thus, for example, IL-2 mutant polypeptides and immunoconjugates can be formulated in combination with suitable polymeric or hydrophobic materials (e.g., emulsified in a suitable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., a sparingly soluble salt.

Pharmaceutical compositions containing IL-2 mutant polypeptides and immunoconjugates of the invention can be prepared using conventional mixing, dissolution, emulsification, encapsulation, entrapment, or lyophilization processes. Pharmaceutical compositions can be formulated using conventional method using one or more physiologically acceptable carriers, diluents, excipients, or excipients that facilitate the processing of proteins to provide pharmaceutically useful formulations. The appropriate form depends on the chosen route of administration.

Mutant IL-2 polypeptides and immunoconjugates can be included in the compositions in free acid or free base form, in neutral form or in salt form. Pharmaceutically acceptable salts are salts which substantially retain the biological activity of the free acid or free base. These include acid addition salts, for example salts formed with the free amino groups of the protein composition, or formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl group can also be obtained from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium or iron hydroxides; or organic bases such as isopropylamine, trimethylamine, histidine or procaine. Pharmaceutical salts tend to have higher solubility in aqueous and other protic solvents than the corresponding free base forms.

Methods and compositions for therapeutic use

Any of the IL-2 mutant polypeptides and immunoconjugates provided herein can be used in therapeutic methods. Mutant IL-2 polypeptides and immunoconjugates of the invention can be used as immunotherapeutic agents, for example, in the treatment of various types of cancer.

For use in therapeutic methods, the IL-2 mutant polypeptides and immunoconjugates of the invention may be formulated, dosed, and administered in accordance with good clinical practice. Factors considered in this context include the particular disease being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disease, the site of administration of the agent, the method of administration, the schedule of administration, and other factors known to medical practitioners.

Mutant IL-2 polypeptides and immunoconjugates of the invention can be used to treat disease states that benefit from stimulation of the host's immune system, in particular conditions in which an increased cellular immune response is desired. These may include disease states in which the host's immune response is unsatisfactory or deficient. The disease states in which the IL-2 mutant polypeptides and immunoconjugates of the invention can be administered are, for example, tumor or infection, in which the cellular immune response would be the critical mechanism of specific immunity. Specific disease states for which the IL-2 mutants of the present invention may be used include cancer, such as renal cell carcinoma or melanoma; immunodeficiency, in particular in HIV-positive patients, in immunosuppressed patients, chronic infection, etc. Mutant IL-2 polypeptides and immunoconjugates of the invention can be administered individually or in any suitable pharmaceutical formulation.

One of the objects of the invention are mutant IL-2 polypeptides and immunoconjugates proposed in the invention, intended for use as a drug. Further objects of the invention are IL-2 mutant polypeptides and immunoconjugates of the invention for use in the treatment of a disease. Some embodiments of the invention are mutant IL-2 polypeptides and immunoconjugates of the invention for use in a method of treatment. One embodiment of the invention is an IL-2 mutant polypeptide or immunoconjugate provided herein for use in the treatment of a disease in an individual in need thereof. In some embodiments, the invention is an IL-2 mutant polypeptide or immunoconjugate for use in a method of treating a subject who has a disease, comprising administering to the subject a therapeutically effective amount of the IL-2 mutant polypeptide or immunoconjugate. In some embodiments, the disease being treated is a proliferative disorder. In a preferred embodiment of the invention, the disease is cancer. In some embodiments, the method also includes administering to the subject in a therapeutically effective amount at least one additional therapeutic agent, such as an anticancer agent, if the disease being treated is cancer. In other embodiments, the invention is a mutant IL-2 polypeptide or immunoconjugate for use in stimulating the immune system. In some embodiments, the invention is a mutant IL-2 polypeptide or immunoconjugate for use in a method for stimulating the immune system in an individual, comprising administering to the individual an effective amount of the mutant IL-2 polypeptide or immunoconjugate to stimulate the immune system. The individual in the context of any of the above embodiments of the invention is a mammal, preferably a human. Stimulation of the immune system in the context of any of the above embodiments of the invention may include one or more indicators, such as a general increase in immune function, increase in T-cell function, increase in B-cell function, restoration of lymphocytic function, increase in expression of IL-2 receptors, increase T-cell reactivity, increased natural killer cell activity or lymphokine-activated killer cell (LAK) activity, and the like.

Another aspect of the invention is the use of a mutant IL-2 polypeptide or immunoconjugate of the invention for the manufacture or preparation of a medicament for the treatment of a disease in an individual in need thereof. In one embodiment, the drug is for use in a method for treating a disease, comprising administering to an individual who has the disease in a therapeutically effective amount of the drug. In some embodiments, the disease being treated is a proliferative disorder. In a preferred embodiment of the invention, the disease is cancer. In one of said embodiments of the invention, the method also comprises administering to the subject, in a therapeutically effective amount, at least one additional therapeutic agent, for example an anti-cancer agent, if the disease being treated is cancer. In a further embodiment of the invention, the drug is intended to stimulate the immune system. In another embodiment, the drug is for use in a method for stimulating the immune system in an individual, comprising administering to the individual an effective amount of the drug to stimulate the immune system. The individual in the context of any of the above embodiments of the invention is a mammal, preferably a human. Stimulation of the immune system in the context of any of the above embodiments of the invention may include one or more indicators, such as a general increase in immune function, increase in T-cell function, increase in B-cell function, restoration of lymphocytic function, increase in expression of IL-2 receptors, increase T-cell reactivity, increased natural killer cell activity or lymphokine-activated killer cell (LAK) activity, and the like.

Another aspect of the invention is a method for treating a disease in an individual, which comprises administering to said individual a therapeutically effective amount of an IL-2 mutant polypeptide or an immunoconjugate of the invention. In one of the embodiments of the invention, the specified individual is injected with a composition that contains a mutant IL-2 polypeptide or immunoconjugate proposed in the invention, in a pharmaceutically acceptable form. In some embodiments, the disease being treated is a proliferative disorder. In a preferred embodiment of the invention, the disease is cancer. In some embodiments, the method also includes administering to the subject in a therapeutically effective amount at least one additional therapeutic agent, such as an anticancer agent, if the disease being treated is cancer. Another aspect of the invention is a method for stimulating the immune system in an individual, comprising administering to the individual an effective amount of a mutant IL-2 polypeptide or an immunoconjugate to stimulate the immune system. The individual in the context of any of the above embodiments of the invention is a mammal, preferably a human. Stimulation of the immune system in the context of any of the above embodiments of the invention may include one or more indicators, such as a general increase in immune function, increase in T-cell function, increase in B-cell function, restoration of lymphocytic function, increase in expression of IL-2 receptors, increase T-cell reactivity, increased natural killer cell activity or lymphokine-activated killer cell (LAK) activity, and the like.

It should be obvious that any of the above therapeutic methods can be carried out using the immunoconjugate of the invention, instead of or in addition to the mutant IL-2 polypeptide.

In some embodiments, the disease being treated is a proliferative disorder, preferably cancer. Examples of cancers include (but are not limited to) bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, stomach cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer and kidney cancer. Other cell proliferation disorders that can be treated using the IL-2 mutant polypeptide or immunoconjugate of the present invention include, but are not limited to, neoplasms located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvic region, skin, soft tissue, spleen, thoracic region and urinary system. Also included within the scope of the invention are precancerous conditions or lesions and metastases of cancer. In some embodiments, the cancer is selected from the group consisting of renal cell carcinoma, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer. Similarly, other cell proliferation disorders can also be treated with the IL-2 mutant polypeptides and immunoconjugates of the present invention. Examples of such disorders of cell proliferation include, but are not limited to, hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemia, purpura, sarcoidosis, Cesari's syndrome, Waldenström's macroglobulinemia, Gaucher's disease, histocytosis, and any other disease associated with cell proliferation, in addition to neoplasms localized in the bodies mentioned above. In other embodiments, the disease is associated with autoimmunity, transplant rejection, post-traumatic immune response, and infectious diseases.

- 45 040858 (eg HIV). More specifically, mutant IL-2 polypeptides and immunoconjugates can be used to eliminate cells involved in immune cell-mediated disorders such as lymphoma; autoimmunity, graft rejection, graft-versus-host disease, ischemia, and stroke. One of skill in the art will appreciate that in many cases, IL-2 mutant polypeptides or immunoconjugates may not provide a cure, but may only provide a partial beneficial effect. In some embodiments of the invention, physiological changes that have some beneficial effect are also considered to be of therapeutic value. Thus, in some embodiments, the amount of the IL-2 mutant polypeptide or immunoconjugate that provides the physiological change is considered to be 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.

The immunoconjugates of the invention can also be used as diagnostic reagents. Binding of an immunoconjugate to an antigenic determinant can be easily detected using a secondary antibody specific for an IL-2 polypeptide. In one of the embodiments of the invention, the secondary antibody and immunoconjugate facilitate the detection of binding of the immunoconjugate to an antigenic determinant localized on the surface of a cell or tissue.

In some embodiments, the mutant IL-2 polypeptides or immunoconjugates of the invention are introduced into a cell in an effective amount. In other embodiments, the mutant IL-2 polypeptides or immunoconjugates of the invention are administered in a therapeutically effective amount to an individual for the treatment of a disease.

For the prevention or treatment of a disease, the appropriate dose of the mutant IL-2 polypeptide or immunoconjugate of the invention (when used alone or in combination with one or more other additional therapeutic agents) should depend on the type of disease being treated, the route of administration, body weight of the patient, the type of polypeptide (eg, unconjugated IL-2 or immunoconjugate), the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's medical history, and response to the IL-2 mutant polypeptide or immunoconjugate and the prescription of the attending physician. The practitioner responsible for administration, in any case, must determine the concentration of the active(their) substance(s) in the composition and the appropriate(s) dose(s) for the individual patient. Various dosing regimens include, but are not limited to, single administration or multiple administrations at different time points, bolus administration, and pulsatile infusion.

A single administration of unconjugated IL-2 may include from about 50,000 to about 1,000,000 IU/kg or more, more specifically about 600,000 IU/kg of IL-2. It can be repeated several times a day (eg, 2-3 times), for several days (eg, for about 3-5 consecutive days), and then can be repeated one or more times after a rest period (eg, about 7-14 days). Thus, a therapeutically effective amount may include only one administration or multiple administrations over a period of time (eg, about 2030 administrations to an individual of about 600,000 IU/kg of IL-2 each time, over a period of about 10-20 days). When administered in the form of an immunoconjugate, the therapeutically effective amount of the IL-2 mutant polypeptide may be lower compared to the unconjugated form of the IL-2 mutant polypeptide.

Similarly, the immunoconjugate can be administered to a patient as a single treatment or series of treatments. Depending on the type and severity of the disease, from about 1 μg/kg to about 15 mg/kg (e.g., 0.1-10 mg/kg) of the immunoconjugate may represent the initial possible dose to be administered to the patient, e.g., using one or more individual administrations. or by continuous infusion. A typical daily dose may be from about 1 μg/kg to 100 mg/kg or more, depending on the factors noted above. For repeated administrations over several days or longer, depending on the condition, treatment should generally be continued until the desired suppression of the present symptoms of the disease is achieved. As an example, the dose of the immunoconjugate may be from about 0.005 to about 10 mg/kg. In another example (but not limited to) the dose per administration can be from about 1, about 5, about 10, about 50, about 100, about 200, about 350, about 500 μg/kg of body weight, about 1, about 5, about 10, about 50, about 100, about 200, about 350, about 500 to about 1000 mg/kg/body weight or more, and be in any of the indicated ranges. As examples (but not limited to) of this range of values, from about 5 to about 100 mg/kg of body weight, from about 5 μg/kg of body weight to about 500 mg/kg of body weight, etc. can be administered. taking into account the above dose levels. Thus, a patient may be administered one or more doses of about 0.5, 2.0, 5.0, or 10 mg/kg (or any combination thereof). These 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

- 46 040858 six doses of the immunoconjugate). An initial higher loading dose may be administered followed by one or more lower doses. However, other dosing regimens may be used. The success of such therapy is easily assessed using conventional methods and assays.

Mutant IL-2 polypeptides and immunoconjugates of the invention will generally be used in an amount effective to achieve the intended purpose. When used to treat or prevent a disease state, the IL-2 mutant polypeptides and immunoconjugates of the invention, or pharmaceutical compositions thereof, are administered or used in a therapeutically effective amount. The determination of a therapeutically effective amount is within the skill of the art, primarily in light of the present detailed description of the invention.

For systemic administration, a therapeutically effective dose may first be determined by in vitro assays, eg cell culture assays. The dose can then be incorporated into an animal model form to achieve a circulating concentration in the range including the IC ₅₀ value determined in cell culture. This information can be used to more accurately determine doses that can be used in humans.

Initial doses can also be estimated from in vivo data, such as animal models, using techniques well known in the art. One of ordinary skill in the art can readily optimize use in humans based on animal data.

Dose level and interval can be adjusted individually to obtain plasma levels of mutant IL-2 polypeptides or immunoconjugates that are sufficient to maintain therapeutic effect. Usual doses intended for administration to a patient by injection are from about 0.1 to 50 mg/kg/day, typically from about 0.5 to 1 mg/kg/day. Multiple doses may be administered each day to achieve therapeutically effective plasma levels. Plasma levels can be assessed, for example, by HPLC.

In cases of topical application or selective absorption, the effective local concentration of immunoconjugates may not correspond to the plasma concentration. One skilled in the art can optimize therapeutically effective topical doses without undue experimentation.

The use of a therapeutically effective dose of the IL-2 mutant polypeptides or immunoconjugates provided herein should generally provide a therapeutic benefit without causing significant toxicity. The toxicity and therapeutic efficacy of the mutant IL-2 or immunoconjugate can be determined using standard pharmaceutical procedures in cell cultures or experimental animals (see, for example, examples 8 and 9). Cell culture assays or animal experiments can be used to determine LD ₅₀ (fatal dose in 50% of a population) and ED ₅₀ (therapeutically effective dose in 50% of a population) values. The ratio of doses characterizing toxic and therapeutic effects is referred to as the therapeutic index, which can be expressed as the ratio LD50/ED50. Mutant IL-2 and immunoconjugates having high therapeutic indices are preferred. In one embodiment, the mutant IL-2 polypeptide or immunoconjugate of the present invention has a high therapeutic index. Data from cell culture assays and animal studies can be used to determine the range of doses that can be used in humans. The dose is preferably in the range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary depending on various factors such as the dosage form used, the route of administration used, the condition of the individual, and the like. The exact formulation, route of administration, and dose may be chosen by the physician individually depending on the condition of the patient (see, for example, Fingl et al., in: The Pharmacological Basis of Therapeutics, ch. 1, 1975, p. description as reference).

It should be obvious to the physician in charge of patients receiving IL-2 mutants or immunoconjugates of the invention how and when to terminate, interrupt, or adjust administration due to toxicity, organ dysfunction, and the like. Conversely, it should be obvious to the treating physician how to adjust treatment towards higher doses if the clinical response is inadequate (preventing toxicity). The amount of dose to be administered in the treatment of the disorder of interest will vary depending on the severity of the condition being treated, the route of administration, and the like. The severity of the condition can, for example, be assessed using standard predictive evaluation methods, among other things. In addition, the dose and expected frequency of dosing should also vary depending on the age, body weight and response of the individual patient.

The maximum therapeutic dose of a mutant IL-2 polypeptide or an immunoconjugate containing said polypeptide can be increased compared to doses that use wild-type IL-2 or an immunoconjugate containing wild-type IL-2, respectively.

Other agents and treatment options

Mutant IL-2 polypeptides and immunoconjugates of the invention in the treatment

- 47 040858 can be administered in combination with one or more other agents. For example, a mutant IL-2 polypeptide or immunoconjugate of the invention may be co-administered with at least one additional therapeutic agent. The term therapeutic agent includes any agent that is administered to treat a symptom of a disease in an individual in need of such treatment. Said additional therapeutic agent may be any active substance that can be used for the particular indication to be treated, preferably with additional activities that do not adversely affect each other. In some embodiments, the additional therapeutic agent is an immunomodulator, a cytostatic agent, a cell adhesion inhibitor, a cytotoxic agent, an activator of cell apoptosis, or an agent that sensitizes cells to apoptosis inducers. In a specific embodiment, the additional therapeutic agent is an anticancer agent, for example, a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormone therapy agent, a kinase inhibitor, a receptor antagonist, a tumor cell apoptosis activator, or an anti-angiogenic agent.

These other agents may be present in combination in amounts effective for the purposes indicated. The effective amount of these other agents depends on the amount of IL-2 mutant polypeptide or immunoconjugate used, the type of disorder being treated, and other factors mentioned above. Mutant polypeptides and immunoconjugates are generally used at the same doses and using the routes of administration described herein, or at doses ranging from about 1% to about 99% of the doses described herein, or at any dose and using any route. administrations that, according to empirical/clinical data, are considered acceptable.

The combination therapies noted above include co-administration (when two or more therapeutic agents are included in the same or separate compositions) and separate administration, in which case the administration of the mutant IL-2 polypeptide or immunoconjugate of the invention can be carried out before, simultaneously and/or after administration of an additional therapeutic agent and/or adjuvant. Mutant IL-2 polypeptides and immunoconjugates of the invention can also be used in combination with radiation therapy.

Products

Another object of the invention is a product that contains products used for the treatment, prevention and/or diagnosis of the above disorders. The product is a container and a label or package insert that is placed on the container or attached to it. Acceptable containers are, for example, jars, vials, syringes, intravenous (IV) solution bags, etc. Containers can be made from various materials such as glass or plastic. The container contains a composition that, alone or in combination with another composition, is effective in treating, preventing, and/or diagnosing a condition, and may have a sterile access port (for example, the container may be an intravenous solution bag or vial fitted with a stopper that can be pierced with a hypodermic needle). At least one active ingredient 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 selected condition. In addition, the product may include (a) a first container containing the composition, where the composition contains a mutant IL-2 polypeptide of the invention; and (b) a second container containing the composition, wherein the composition contains an additional cytotoxic or other therapeutic agent. According to this embodiment of the invention, the product may contain a package insert that contains information that the compositions can be used to treat a particular condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container with a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BVI), phosphate buffered saline, Ringer's solution and dextrose solution. In addition, it may include other products required from a commercial and consumer point of view, in particular other buffers, diluents, filters, needles and syringes.

As should be obvious, any of the above products may include the immunoconjugate of the invention, instead of or in addition to the mutant IL-2 polypeptide.

Brief description of the drawings

The drawings show:

in fig. 1 is a schematic representation of the formats of Fab-IL-2-Fab (A) and IgG-IL-2 (B) immunoconjugates containing a mutant IL-2 polypeptide;

in fig. 2 shows the results of the purification of the naked (non-conjugated) wild-type IL-2 construct. (A) Chromatogram obtained after purification using His-tag, naked wild-type IL-2; (B) SDS-PAGE results of purified protein (8-12% Bis-Tris (NuPage,

- 48 040858

Invitrogen), moving buffer MES);

in fig. 3 shows the results of the purification of the wild-type naked IL-2 construct. (A) Chromatogram of wild-type IL2 obtained by gel filtration; (B) SDS-PAGE results of purified protein (8-12% Bis-Tris (NuPage, Invitrogen), MES running buffer);

in fig. 4 - results obtained by the method of analytical gel filtration of wild-type IL-2, carried out on a column of Superdex 75, 10/300 GL.

Pool 1 contained 74% 23 kD species and 26% 20 kD species, Pool 2 contained 40% 22 kD species and 60% 20 kD species;

in fig. 5 shows the results of the purification of the naked four-mutant IL-2 construct. (A) Chromatogram obtained after His-tag purification of four-mutant IL-2; (B) SDS-PAGE results, purified protein (8-12% Bis-Tris (NuPage, Invitrogen), MES running buffer);

in fig. 6 shows the results of the purification of the naked four-mutant IL-2 construct. (A) Gel filtration chromatogram of four mutant IL-2; (B) SDS-PAGE results of purified protein (8-12% Bis-Tris (NuPage, Invitrogen), MES running buffer);

in fig. 7 - results obtained by the method of analytical gel filtration of four-mutant IL-2, carried out on a column of Superdex 75, 10/300 GL (Pool 2, 20 kDa);

in fig. 8 - results showing simultaneous binding to IL-2R and human FAP of the 29B11-based Fab-IL-2-Fab construct containing wild-type or four-mutant IL-2 targeted by FAP. (A) The result of the SPR analysis; (B) SPR-sensogram;

in fig. 9 - Solution analysis results demonstrating induction of IFN-γ release by NK92 cells when exposed to a 4G8-based Fab-IL-2-Fab construct containing wild-type or four-mutant IL-2 targeted by FAP compared to proleukin;

in fig. 10 - Results of analysis in solution demonstrating the induction of proliferation of isolated NK cells (bottom) when exposed to a 4G8-based Fab-IL-2-Fab construct containing wild-type or four-mutant IL-2 targeted by FAP compared to proleukin ;

in fig. 11 - Solution assay results demonstrating induction of activated CD3 T cell proliferation by exposure to a 4G8 Fab-IL-2-Fab construct containing wild-type or four-mutant IL-2 targeted by FAP compared to proleukin;

in fig. 12 - Solution analysis results demonstrating the induction of activation-induced cell death (AICD) of overstimulated T cells when exposed to a 4G8 Fab-IL-2-Fab-based construct containing wild-type or four-mutant IL-2 that targets FAP, by comparison with proleukin;

in fig. 13 - Results of phospho-STAT5 FACS analysis in solution using a 4G8 Fab-IL-2-Fab based construct containing wild-type or four-mutant IL-2 targeting FAP compared to proleukin. (A) regulatory T cells (CD4+CD25+FOXP3+); (B) CD8 ⁺ -T cells (CD3+CD8+); (B) CD4 ⁺ T cells (CD4'CD25 CDI27'); (D) NK cells (CD3 CD56');

in fig. 14 shows purification results of a 28H1 Fab-IL-2 qm-Fab immunoconjugate targeting FAP. (A) Elution profile on a Protein G column. (B) Elution profile on a Superdex 200 gel filtration column. (C) Results of SDS-PAGE analysis (Novex Tris-glycine 4-20%) of the final product using the sample under non-reducing and reducing conditions;

in fig. 15 shows the results of the purification of the 4G8-based Fab-IL-2 qm-Fab immunoconjugate targeting FAP. (A) Elution profile on a protein A column. (B) Elution profile on a Superdex 200 gel filtration column. product using the sample under non-reducing and reducing conditions;

in fig. 16 shows purification results of the MHLG1 KV9 immunoconjugate Fab-IL2QM-Fab targeting MCSP. (A) Protein A column elution profile. (B) Superdex 200 gel filtration column elution profile. (C) NuPAGE Novex Bis-Tris minigel electrophoresis (Invitrogen), running MOPS buffer, final product using the sample under non-reducing and reducing conditions;

in fig. 17 - results showing targeted binding of Fab-IL-2-Fab constructs on human FAP-expressing HEK 293 cells;

in fig. 18 - results showing targeted binding of Fab-IL-2-Fab constructs on human FAP-expressing HEK 293 cells;

in fig. 19 - Results showing the binding specificity of the Fab-IL-2-Fab constructs as determined on human DPPIV expressing HEK 293 cells and on HEK 293 cells transfected with mimic. On the right are results showing binding of a DPPIV-specific (CD26) antibody;

in fig. 20 - results of the analysis of the internalization of FAP when binding constructs Fab-IL-2-Fab with FAP on fibroblast line GM05389;

- 49 040858 in FIG. 21 - results of analysis of IL-2 induced release of IFN-γ by NK92 cells in solution;

in fig. 22 - results of analysis of IL-2 induced release of IFN-γ by NK92 cells in solution;

in fig. 23 - results of analysis of IL-2 induced proliferation of NK92 cells in solution;

in fig. 24 shows results of comparative evaluation of Fab-IL-2-Fab based on clones 28H1, 29B11 and 4G8 by STAT5 phosphorylation solution assay using PBMC. (A) NK cells (CD3 CD56+); (B) CD8⁺-T cells (CD3+CD8+); (B) CD4⁺-T cells (CD3⁺CD4⁺CD25'CD127⁺); (D) regulatory T cells (CD4⁺CD25⁺FOXP3⁺);

in fig. 25 shows the efficacy of FAP-targeted 4G8 Fab-IL-2 wt-Fab and 4G8 Fab-IL-2 qmFab immunoconjugates on the ACHN human renal cell adenocarcinoma cell line;

in fig. 26 shows the efficacy of FAP-targeted 4G8 FAP-IL-2 qm-Fab and 28H1 Fab-IL-2 qm-Fab immunoconjugates on the mouse Lewis lung carcinoma cell line LLC1;

in fig. 27 shows the efficacy of FAP-targeted 28H1 Fab-IL-2 wt-Fab and 28H1 Fab-IL-2 qm-Fab immunoconjugates on the mouse Lewis lung carcinoma cell line LLC1;

in fig. 28 are images taken at low magnification (100x) of mouse lungs treated with vehicle (control) (A) or taken at a concentration of 9 μg/g wt IL-2 (B) or qm IL-2 (C). In the lungs of mice treated with 9 μg/g wt IL-2, a vasocentric mononuclear infiltrate is seen that has migrated into the alveolar spaces. There is swelling and hemorrhage. Mice treated with qm IL-2 showed a marginal infiltrate around several vessels;

in fig. 29 are high magnification (200x) images of the lungs shown in FIG. 28. Marginal accumulation (margination) and infiltration of mononuclear cells in and around blood vessels are more severe in mice treated with wt IL-2 (A) than mice treated with qm IL-2 (B and C);

in fig. 30 are images taken at low magnification (100x) of the liver of mice treated with vehicle (control) (A) or taken at a concentration of 9 μg/g wt IL-2 (B) or qm IL-2 (C). Mice treated with wt IL-2 show vasocentric infiltration;

in fig. 31 - results of analysis of IFN-γ secretion by NK92 cells after incubation with different preparations of IL-2 wild-type (wt) and four-mutant IL-2 (qm), for 24 (A) or 48 hours (B);

in fig. 32 shows results of NK92 cell proliferation assay after incubation with different preparations of wild-type IL-2 (wt) and four-mutant IL-2 (qm) for 48 hours;

in fig. 33 shows results of NK92 cell proliferation assay after incubation with different preparations of wild type (wt) and four mutant IL-2 (qm) IL-2 for 48 hours;

in fig. 34 shows results of NK cell proliferation assay after incubation with various FAP-targeted 28H1 IL-2 immunoconjugates or with proleukin for 4 (A), 5 (B), or 6 (C) days;

in fig. 35 shows results of CD4 T cell proliferation assay after incubation with various FAP-targeted 28H1 IL-2 immunoconjugates or with proleukin for 4 (A), 5 (B), or 6 (C) days;

in fig. 36 shows results of CD8 T cell proliferation assay after incubation with various FAP-targeted 28H1 IL-2 immunoconjugates or with proleukin for 4 (A), 5 (B), or 6 (C) days;

in fig. 37 - results of analysis of the proliferation of NK cells (A), CD4-T cells (B) and CD8-T cells (C) when incubated with various IL-2 immunoconjugates or with proleukin for 6 days;

in fig. 38 - results of the analysis of STAT phosphorylation in NK cells (A), CD8-T cells (B), CD4-T cells (C) and regulatory T cells (D) after 30-minute incubation with proleukin obtained by the company Applicant's wild-type IL-2 and four-mutant IL-2;

in fig. 39 - results of analysis of STAT phosphorylation in NK cells (A), CD8 T cells (B), CD4 T cells (C) and regulatory T cells (D) after 30 min incubation with proleukin, IgG-IL2 containing wild-type IL-2, or IgG-IL-2 containing four-mutant IL-2;

in fig. 40 Survival data for Black 6 mice after administration (once a day for seven days) of IL-2 immunoconjugates containing wild-type or four-mutant IL-2 at various doses;

in fig. 41 - serum concentration data of IL-2 immunoconjugates after a single i.v. wild-type (wt) or four-mutant (qm) IL-2;

in fig. 42 - Serum concentration data of IL-2 immunoconjugates after a single i.v.- 50 040858 administration of non-targeted Fab-IL-2-Fab constructs containing either wild-type (wt) or four-mutant (qm) IL-2;

in fig. 43 - results of purification of four mutant IL-2. (A) Chromatography with immobilized metal ion; (B) gel filtration; (B) SDS-PAGE under non-reducing conditions (NuPAGE Novex Bis-Tris gel (Invitrogen), MES running buffer); (D) analytical gel filtration (Superdex 75 10/300 GL);

in fig. 44 shows the results of a proliferation assay of pre-activated CD8-(A) and CD4(B) T cells after six days of incubation with various IL-2 immunoconjugates;

in fig. 45 shows the results of CD3 T cell death-induced activation assay after six days of incubation with various IL-2 immunoconjugates and overnight treatment with anti-Fas antibody;

in fig. 46 - Purification results of the IgG-IL-2 immunoconjugate (four mutant, (qm)) based on 4G8, which targets FAP. (A) Elution profile obtained from the Protein A affinity chromatography step. (B) Elution profile obtained from the gel filtration step. (C) Analytical SDS-PAGE analysis of the final product (NuPAGE Novex Bis-Tris Minigel (Invitrogen), MOPS running buffer. D) Analytical size exclusion of the final product on a Superdex 200 column (monomer content 97%);

in fig. 47 - Purification results of the 28H1-based IgG-IL-2 qm immunoconjugate targeting FAP. (A) Elution profile obtained from the Protein A affinity chromatography step. (B) Elution profile obtained from the gel filtration step. (B) Analysis of the final product by analytical SDS-PAGE (reducing conditions: NuPAGE Novex Bis-Tris minigel (Invitrogen), running MOPS buffer; non-reducing conditions: NuPAGE Tris-acetate (Invitrogen), running Tris-acetate buffer) . D) Analytical gel filtration of the final product on a Superdex 200 column (monomer content 100%);

fig. 48 - Results of a binding assay of the 4G8-based IgG-IL-2 qm immunoconjugate FAP-targeted to human FAP expressed in stably transfected HEK 293 cells by FACS compared to the corresponding Fab-IL-2 qm- construct. fab;

in fig. 49 - Results of an assay of interferon (IFN)-y release on NK92 cells in solution induced by a 4G8-based IgG-IL-2 qm immunoconjugate targeting FAP compared to a 28H1-based Fab-IL-2 qm-Fab construct. ;

in fig. 50 - Results of detection of phosphorylated STAT5 by FACS in various cell types after 20 min stimulation in solution with FAP-targeted 4G8-based IgG-IL-2 qm immunoconjugate, compared with constructs Fab-IL-2-Fab and Fab- IL-2 qm-Fab based on 28H1 and also with proleukin. 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

The following are examples of the methods and compositions of the invention. As should be apparent, various other embodiments of the invention may be practiced in view of the above description of the invention as a whole.

Example 1

General Methods

Recombinant DNA methods

For DNA manipulation, standard methods were used as described in Sambrook J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturer's instructions. For general information regarding the nucleotide sequences of human immunoglobulin light and heavy chains, see: Kabat E.A. et al., Sequences of Proteins of Immunological Interest, 5th Ed., NIH Publication No. 91-3242, 1991.

DNA sequencing

DNA sequences were determined by double strand sequencing.

Gene synthesis

The required gene segments were either created by PCR using appropriate templates or synthesized at Geneart AG (Regensburg, Germany) from synthetic oligonucleotides and PCR products by automated gene synthesis. In cases where the exact gene sequence was not available, oligonucleotide primers were created based on the sequences of the nearest homologues and genes were isolated by RT-PCR from RNA obtained from the corresponding tissue. Gene segments flanked by single sites recognized by restriction enzymes were cloned into standard cloning/sequencing vectors. Plasmid DNA was purified from the transformed bacteria and the concentration was determined by UV spectroscopy. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing. Created gene segments with the required restriction sites, allowing them to subclone them in the appropriate expression vectors. All constructs were created with a 5'-terminal DNA sequence,

- 51 040858 encoding a leader peptide that directs the secretion of proteins in eukaryotic cells. In SEQ

ID NO: 263-273 provides examples of leader peptides and polynucleotide sequences encoding them.

Preparation of fusions βγ-subunit of IL-2R-Fc and fusion α-subunit of IL-2R-F c

To study the binding affinity of the IL-2 receptor, a tool was created that allows the expression of a heterodimeric IL-2 receptor; The P subunit of the IL-2 receptor was fused to an Fc molecule designed to be heterodimerizable (Fc(trough)) (see SEQ ID NOS: 274 and 275) using knobs-into-holes technology (Merchant et al., Nat Biotech 16, 1998, pp. 677-68). The IL-2 receptor γ subunit was then fused to an Fc(protrusion) variant (see SEQ ID NOs: 276 and 277) heterodimerized with Fc(cavity). This heterodimeric Fc fusion containing protein was then used as a substrate for an IL-2/IL-2 receptor interaction assay. The α subunit of IL-2R was expressed as a monomeric chain carrying an AcTev cleavage site and an Avi His tag (SEQ ID NOS: 278 and 279). The corresponding IL-2R subunits were transiently expressed in EBNA 293 HEK cells with serum for the IL-2R βγ subunit construct and without serum for the α subunit construct. The IL-2R βγ subunit construct was purified on protein A (GE Healthcare) followed by gel filtration (GE Healthcare, Superdex 200). The α-subunit of IL-2R was purified using a His-tag on a NiNTA column (Qiagen) followed by gel filtration (GE Healthcare, Superdex 75).

Obtaining immunoconjugates

A more detailed description of the preparation and purification of Fab-IL-2-Fab immunoconjugates, including the generation and affinity maturation of antigen-binding fragments, is provided in the Examples section of PCT publication WO 2011/020783, which is incorporated herein by reference in its entirety. As described in that publication, various FAP-targeted antigen-binding domains were generated by phage display, including clones designated 4G8, 3F2, 28H1, 29B11, 14B3, and 4B9, which were used in the examples below. Clone 28H1 was an affinity matured antibody based on the parental clone 4G8, while clones 29B11, 14B3 and 4B9 were affinity matured antibodies based on the parental clone 3F2. The antigen-binding domain that targets MCSP is referred to herein as MHLG1 KV9.

The sequences of immunoconjugates containing wild-type IL-2, which were used in the examples below, are also presented in PCT publication WO 2011/020783. The sequences corresponding to the immunoconjugates that contained the four-mutant IL-2 used in the examples below were: for 4G8: SEQ ID NOs: 211 and 233; for 3F2: SEQ ID NOs: 209 and 231; for 28H1: SEQ ID NOs: 219 and 233; for 29B11: SEQ ID NOs: 221 and 231; for 14B3: SEQ ID NOs: 229 and 231; for 4B9: SEQ ID NOs: 227 and 231; for MHLG1-KV9: SEQ ID NOs: 253 and 255. DNA sequences were generated by gene synthesis and/or classical molecular biology techniques and subcloned into mammalian expression vectors (one for the light chain and one for the heavy chain of the IL-2 fusion protein) under control of the MPSV promoter and upstream of the polyA synthetic site, each vector carried the EBV OriP sequence. The immunoconjugates used in the examples described below were obtained by contrafection of exponential growth phase HEK293EBNA cells with mammalian expression vectors using calcium phosphate mediated transfection. Alternatively, HEK293 cells growing in suspension were transfected with polyethyleneimine (PEI) using appropriate expression vectors. Alternatively, pools of stably transfected CHO cells or clones of CHO cells were used for serum free production. While 4G8-based constructs that target FAP, such as Fab-IL-2-Fab containing wild-type or (four) mutant IL-2, can be purified by affinity chromatography using protein A as a matrix, 28H1 affinity matured constructs targeting FAP, such as Fab-IL-2-Fab, were purified by affinity chromatography using protein G as a template to obtain a laboratory scale product.

In summary, the method was as follows: a 28H1 Fab-IL-2-Fab construct targeting FAP containing wild-type or (four) mutant IL-2 was purified from supernatants by a single affinity chromatography step (Protein G) followed by gel -filtration (Superdex 200, GE Healthcare). The protein G column was equilibrated with 20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5, supernatant added, and the column 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 purified by gel filtration in final formulation buffer of the following composition: 25 mM potassium phosphate, 125 mM sodium chloride, 100 mM glycine, pH 6.7. Below are examples of purification and analytical results.

FAP-targeted constructs such as 3F2 Fab-IL-2-Fab or 4G8 Fab-IL-2-Fab containing wild-type or (four) mutant IL-2 were purified by a similar method, including

- 52 040858 one purification step on protein A followed by gel filtration (Superdex 200, GE Healthcare). The protein A column was equilibrated using 20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5, supernatant was added and the column was washed with 20 mM sodium phosphate, 20 mM sodium citrate, 500 mM sodium chloride, pH 7.5, and then washed 13.3 mM sodium phosphate, 20 mM sodium citrate, 500 mM sodium chloride, pH 5.45. Optionally, a third wash was performed with 10 mM MES, 50 mM sodium chloride, pH 5. 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 purified by gel filtration in final formulation buffer of the following composition: 25 mM potassium phosphate, 125 mM sodium chloride, 100 mM glycine, pH 6.7. Examples of cleaning procedures and results for selected constructs listed below are presented in more detail below.

FAP-targeted IgG-IL-2 qm fusion proteins were generated from anti-FAP antibodies 4G8, 4B9, and 28H1, in which one individual four-mutant (qm) IL-2 was fused to the C-terminus of one heterodimeric heavy chain as shown in fig. 1B. To achieve targeted transfer to the tumor stroma, in which FAP is selectively expressed, the Fab region of the bivalent antibody (avidity phenomenon) was used. Knob-into-hole technology was used to achieve heterodimerization in the presence of individual four-mutant IL-2. In order to minimize the creation of homodimeric IgG-cytokine fusions, the cytokine was fused to the C-terminus (with deletion of the C-terminal Lys residue) of the IgG heavy chain overhang via a G ₄ -(SG ₄ ) ₂ - or (G ₄ S) ₃ -linker. The antibody-cytokine fusion had IgG-like properties. P329G L234A L235A (LALA) mutations were introduced into the Fc domain to reduce FcyR binding/effector function and prevent FcR coactivation. The sequences of these immunoconjugates are shown 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 fusion protein such as IgG-IL-2 and a DP47GS control non-targeted IgG-IL-2 qm fusion protein in which IgG does not bind to a specific target was created. The sequences of these immunoconjugates are shown in SEQ ID NOs: 305, 307 and 309 (DP47GS) and SEQ ID NOs: 319, 321 and 323 (CH1A1A).

The IgG-IL-2 constructs were generated by transient expression in HEK293 EBNA cells and purified generally as described above for the Fab-IL-2-Fab constructs. In general, the method was as follows: IgG-IL-2 fusion proteins were purified using one step protein A affinity chromatography (HiTrap ProtA, GE Healthcare), equilibrated using 20 mM sodium phosphate, 20 mM sodium citrate, pH 7, 5. After adding the supernatant, the column was first washed with 20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5, and then 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. Fractions were neutralized and pooled and purified by gel filtration (HiLoad 16/60 Superdex 200, GE Healthcare) in final buffer for product having the following composition: 25 mm potassium phosphate, 125 mm sodium chloride, 100 mm glycine, pH 6.7. The following are examples of results from cleaning procedures and results for selected constructs listed below.

The protein concentration in the purified protein samples was determined by measuring the optical density (OD) at 280 nm using the molar extinction coefficient calculated from the amino acid sequence. The purity and molecular weight of the immunoconjugates were analyzed by SDS-PAGE with or without a reducing agent (5 mM 1,4-dithiothreitol) and stained with Coomassie Brilliant Blue (SimpleBlue™ SafeStain, Invitrogen). NuPAGE® PreCast Gel System (Invitrogen) was used according to the manufacturer's instructions (4-20% Tris-Glycine gels or 3-12% Bis-Tris). Aggregate content in immunoconjugate samples was analyzed using a Superdex 200 10/300GL analytical gel filtration column (GE Healthcare) in running buffer containing 2 mM MOPS, 150 mM NaCl, 0.02% NaN ₃ , pH 7.3, at 25°C.

FAP binding affinity

The FAP binding capacity of the cleaved Fab fragments used as antigen binding fragments in these examples was determined based on surface plasmon resonance (SPR) using a Biacore device. In general, the method was as follows: an anti-His antibody (PentaHis, Qiagen 34660) was immobilized on CM5 chips to capture 10 nM of human, murine, or cynomolgus FAP-His (20 sec). The temperature was 25° C. and HBS-EP was used as a buffer. The concentration of the analyzed Fab ranged from 100 to 0.41 nM (with duplication), the flow rate was 50 μl/min (association: 300 s, dissociation: 600 s (4B9, 14B3, 29B11, 3F2) or 1200 s (28H1, 4G8) , regeneration: 60 s, 10 mM glycine pH 2). The fit was based on a 1:1 binding model, RI=0, Rmax=local value (as the capture format was used). Table 2 presents data on the affinity of monovalent components according to SPR data.

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Table 2. Affinity (K _D ) of FAP-targeted Fab fragments for FAP according to SPR

K _D in nM Human FAP Fap monkey cynomolgus Mouse FAP 4B9 Fab 0.3 0.31 0.23 0.24 5 5.2 14VZ Fab 0.47 0.47 0.61 0.59 4.7 4.7 29B11 Fab 0.19 0.19 0.21 0.2 1.3 1.2 3F2 Fab 6 6 4.7 5.3 8.9 9.5 28H1 Fab 2.6 2.6 3.7 3.7 0.13 0.18 4G8 Fab 53 (48 steady state) 51 (48 steady state) 33 (33 steady state) 35 (34 steady state) 0.07 0.07

Bioactivity Assays of Specifically Targeted (Targeted) IL-2 Immunoconjugates Biological activity of Fab-IL-2-Fab immunoconjugates targeting FAP or MCSP and IgG-IL-2 immunoconjugates targeting FAP containing wild-type or (four ) mutant IL-2 was tested using several cellular assays in comparison to commercially available IL-2 (Proleukin, Novartis, Chiron).

Release of IFN-γ by NK cells (in solution)

IL-2 deficient NK92 cells (100,000 cells/well in a 96-well U-bottom plate) were incubated with various concentrations of IL-2 immunoconjugates containing wild-type or (four) mutant IL-2 for 24 h in NK medium (MEM alpha from Invitrogen (#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-γ release was analyzed using ELISA kit II containing anti-human IFN-γ antibody, Becton Dickinson (No. 550612). Proleukin (Novartis) served as a positive control for IL-2 mediated cell activation.

Proliferation of NK cells

Received blood samples from healthy volunteers containing heparin syringes and isolated PBMC. Intact human NK cells were isolated from PBMCs using Miltenyi Biotec Human NK Isolation Kit II (No. 130-091-152). Expressing CD25 cells were assessed using flow cytometry. For proliferation assay, 20,000 isolated human NK cells were incubated for 2 days in a humid chamber at 37°C, 5% CO2 in the presence of various IL-2 immunoconjugates containing wild-type or (four) mutant IL-2. Proleukin (Novartis) served as control. After 2 days, the ATP content of the cell lysates was measured using a CellTiter-Glo fluorescent cell viability assay from Promega (No. G7571/2/3). Growth percentage was calculated by taking proliferation at the highest proleukin concentration as 100% and proliferation of untreated unstimulated IL-2 cells as 0%.

STAT5 phosphorylation assay

Received blood samples from healthy volunteers containing heparin syringes and isolated PBMC. PBMCs were treated with IL-2 immunoconjugates containing wild-type or (four) mutant IL-2 at the indicated concentrations, or with proleukin (Novartis) as a control. After a 20-minute incubation at 37°C, PBMCs were fixed with pre-warmed Cytofix buffer (Becton Dickinson, no. 558050) for 30 min at 4°C. Cells were washed twice with PBS containing 0.1% BSA before performing FACS staining, which used mixtures of antibodies for flow cytometry to detect various cell populations and phosphorylate STAT5. Samples were analyzed using a FACSCantoII with HTS from Becton Dickinson.

NK cells were defined as CD3-CD56+, CDS-positive T cells were defined as CD3+CD8+, CD4 positive T cells were defined as CD4+CD25-CD127+ and Treg cells were defined as CD4⁺CD25⁺FoxP3⁺.

T cell proliferation and AICD

Received blood samples from healthy volunteers containing heparin syringes and isolated PBMC. Intact T cells were isolated using the Pan T Cell Isolation Kit II from Miltenyi Biotec (No. 130-091-156). T cells were prestimulated with 1 μg/ml PHA-M (Sigma Aldrich, no. LS902) for 16 hours before adding proleukin or Fab-IL-2-Fab immunoconjugates containing wild type or (four) mutant IL-2 to the washed cells in

- 54 040858 for 5 more days. After 5 days, the ATP content of the cell lysates was measured using a CellTiter-Glo fluorescent cell viability assay from Promega (No. G7571/2/3). Relative proliferation was calculated by taking proliferation at the highest proleukin concentration as 100%.

Phosphatidylserine (PS) exposure and T cell death were analyzed by flow cytometry (FACSCantoII, BD Biosciences) using cells stained with annexin V (Annexin-V-FLUOS staining kit, Roche Applied Science) and propidinium iodide (PI ). To induce activation-induced cell death (AICD), T cells were treated with an apoptosis-inducing anti-Fas antibody (Millipore, clone Ch11) for 16 hours after 16 hours of PHA-M and 5 days of Fab-IL-2-Fab immunoconjugates. . Annexin V (Ann-V) staining was performed according to the manufacturer's instructions. In general, the method was as follows: cells were washed with Ann-V binding buffer (1x stock solution: 0.01 M Hepes/NaOH, pH 7.4, 0.14 M NaCl, 2.5 mM CaCl ₂ ) and stained for 15 min at CT using the VFITC annexation complex (Roche). Cells were washed again with Ann-V binding buffer before adding 200 μl/well of Ann-V binding buffer containing PI (0.3 μg/ml). Cells were immediately analyzed by flow cytometry.

Binding to FAP Expressing Cells

The binding of FAP-targeted IgG-IL-2 qm and Fab-IL-2 qm-Fab immunoconjugates to human FAP expressed on stably transfected HEK293 cells was assessed by FACS. In general, the method was as follows: 250,000 cells per well were incubated with immunoconjugates taken at the indicated concentrations in a round bottom 96-well plate for 30 min at 4°C and washed once with PBS/0.1% BSA. Bound immunoconjugates were determined after incubation for 30 min at 4°C with FITC-conjugated F(ab') ₂ -fragment of goat antibody specific for F(ab') ₂ , AffiniPure (Jackson Immuno Research Lab, no. 109-096 -097, working solution: 1:20 dilution in PBS/0.1% BSA, freshly prepared) using a FACS CantoII device (FACS Diva software).

FACS Analysis of FAP Internalization on Binding

Several anti-FAP antibodies known in the art are known to induce FAP internalization upon binding (described, for example, in Baum et al., J Drug Target 15, 2007, pp. 399-406; Bauer et al., Journal of Clinical Oncology, ASCO Annual Meeting Proceedings (post-conference edition), May 28, 2010 (updated May 20), Abstract #13062 (2010); Ostermann et al., Clin Cancer Res 14, 2008, pp. 4584-4592). For this reason, the ability to internalize the Fab-IL-2-Fab immunoconjugates of the invention was analyzed in the course of the invention. In general, the method was as follows: GM05389 cells (human lung fibroblasts) that were cultured in EMEM medium supplemented with 15% FCS were isolated, washed, counted, analyzed for viability, and plated at a density of 2x105 cells/well in 12-well plates. . The next day, Fab-IL-2-Fab immunoconjugates targeting FAP were diluted with cold medium and allowed to bind to the cell surface for 30 min on ice. Excess unbound antibody was washed off with cold PBS and cells were further incubated in 0.5 ml of complete prewarmed medium at 37° C. for the indicated times. After various time points, the cells were transferred to ice, washed once with cold PBS, and incubated with a secondary antibody (FITC-conjugated F(ab') ₂ fragment of a goat antibody specific for F(ab') ₂ , AffiniPure (Jackson Immuno Research Lab , no. 109-096-097, dilution 1:20) for 30 min at 4° C. The cells were then washed twice with PBS/0.1% BSA, transferred to a 96-well plate, centrifuged (400 xg) for 4 min at 4°C and the cell debris was resuspended by vigorous agitation Cells were fixed using 100 µl 2% PFA (paraformaldehyde) For FACS evaluation, cells were resuspended using 200 µl PBS/0.1% BSA/sample and measured with using the plate protocol for the FACS CantoII device (FACS Diva software) Example 2

The invention has developed mutant versions of IL-2 that contain one or more of the following mutations (compared to the wild-type IL-2 sequence shown in SEQ ID NO: 1):

1. T3A provides shutdown of the predicted O-glycosylation site;

2. F42A provides shutdown of IL-2/IL-2Ra interaction;

3. Y45A provides shutdown of IL-2/IL-2Ra interaction;

4. L72G provides shutdown of IL-2/IL-2Ra interaction;

5. C125A a previously described mutation preventing disulfide bonding of IL-2 dimers.

A mutant IL-2 polypeptide containing all mutations 1-4 is designated in the context of the present description as four-mutant (qm) IL-2. It may also contain mutation 5 (see SEQ ID NO: 19).

In addition to the three F42A, Y45A and L72G mutations designed to affect CD25 binding, the T3A-55 040858 mutation is chosen to eliminate the O-glycosylation site and produce a protein product with higher homogeneity and purity when the IL-2qm polypeptide or immunoconjugate is expressed in eukaryotic cells such as CHO or HEK293 cells.

For the purpose of purification, the His6 tag was introduced at the C-terminus, connecting through the VD sequence. For comparison, a non-mutated analogous version of IL-2 was created, including only the C145A mutation, thereby preventing the formation of unwanted intermolecular disulfide bridges (SEQ ID NO: 3). The corresponding molecular weights without the signal sequence were 16423 Da for naked IL-2 and 16169 Da for naked IL-2 qm. Wild-type IL-2 and His-tagged four-mutant IL-2 were transfected into HEK EBNA cells in serum-free medium (F17 medium). The filtered supernatant was buffer exchanged by cross-flow before it was added to a NiNTA Superflow cartridge (5 ml, Qiagen). The column was washed with wash 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 (corresponding to 25 mM imidazole), then eluted using an imidazole gradient up to 0.5 M. The pooled eluate was finally purified by gel filtered on a HiLoad 16/60 column with Superdex75 (GE Healthcare) in 2 mM MOPS, 150 mM sodium chloride, 0.02% sodium azide, pH 7.3. In FIG. 2 shows the chromatogram obtained after His-tag purification of naked wild-type IL-2. Pool 1 was obtained from fractions 78-85, pool 2 from fractions 86-111. In FIG. 3 shows a chromatogram obtained after gel filtration of wild-type IL-2, fractions 12-14 are pooled in each pool. In FIG. 4 shows the result of analytical gel filtration of wild-type IL-2 on a Superdex 75, 10/300 GL column (GE Healthcare) in 2 mM mOpS, 150 mM sodium chloride, 0.02% sodium azide, pH 7.3. Pool 1 and 2 included two proteins with a molecular weight of approximately 22 and 20 kDa. Pool 1 included more protein with a larger size, and pool 2 included more protein with a smaller size, presumably this difference is due to differences in O-glycosylation. Yields were about 0.5 mg/l supernatant per pool 1 and about 1.6 mg/l supernatant per pool 2. FIG. 5 shows the chromatogram obtained after purification using the His-tag of four mutant IL-2. Pool 1 consisted of fractions 59-91, pool 2 of fractions 92-111. In FIG. 6 shows the chromatogram obtained after gel filtration of four mutant IL-2, while retaining only pool 2 containing fractions 12-14. In FIG. 7 shows the result of analytical gel filtration of four mutant IL-2 on a Superdex 75, 10/300 GL column (GE Healthcare) in 2 mM MOPS, 150 mM sodium chloride, 0.02% sodium azide, pH 7.3. The naked four-mutant IL-2 preparation included only one 20 kDa protein. The O-glycosylation site was switched off in this protein. Aliquots of naked wild-type and four-mutant IL-2 were stored frozen at -80°C. Yields were approximately 0.9 mg/l supernatant.

A second batch of His-tagged four-mutant IL-2 was purified as described above by immobilized metal affinity chromatography (IMAC) and then by size exclusion chromatography (SEC). The IMAC buffers used were equilibration and column wash buffer containing 50 mM Tris, 20 mM imidazole, 0.5 M NaCl, pH 8, and elution buffer containing 50 mM Tris, 0.5 M imidazole, 0.5 M NaCl, pH 8. The SEC used and final product buffer included 20 mM histidine, 140 mM NaCl, pH 6. FIG. 43 shows the results of this purification. The yield was 2.3 ml/l supernatant.

The affinity for the IL-2R βγ heterodimer and the IL-2R α-subunit was then determined by surface plasmon resonance (SPR). In general, the method was as follows: the ligand - either the α-subunit of human IL-2R (Fc2) or the heterodimer of IL2-R, including β as a ledge, γ as a trough (Fc3) - was immobilized on a CM5 chip. Wild-type naked IL-2 (pools 1 and 2) or four-mutant IL-2 and proleukin (Novartis/Chiron) were then loaded onto the chip as analytes at 25° C. in HBS-EP buffer at concentrations of 300 to 1, 2 nM (1:3 dilution). The flow rate was 30 μl/min and the following conditions were used: association 180 s, dissociation: 300 s and regeneration: 2 x 30 s 3 M MgCl ₂ for IL2-Re heterodimer ledge, γ dimple, 10 s 50 mM NaOH for α-subunit IL-2R. A 1:1 binding model was used for approximation (binding 1:1, RI^0, Rmax=local value for IL-2R βγ, apparent K _D value, binding 1:1, RI=0, Rmax=local value for IL-2Ra ). in table. 3 shows the corresponding K _D values characterizing the binding of wild-type human IL-2R and four-mutant IL-2, as well as proleukin, to IL-2R βγ and the α-subunit of IL-2R.

- 56 040858

Table 3. Affinity of mutant IL-2 polypeptides for IL-2R with intermediate affinity and _____________________α-subunit of IL-2R_____________________

Kd in nM T = 25°С Hu IL-2R βγ (kinetics) Hu IL-2R a (kinetics) Hu IL-2R a (steady state) "naked" IL-2 wt, 5.6 17.4 30.3 pool 1 5 16.6 23.9 "naked" IL-2 wt, 2.8 10.6 19.7 pool 2 1.8 10 17.6 "naked" IL-2 qm 2.7 2 no binding no binding proleukin 2.4 7.5 19 2.8 12.5 17.8

The data obtained demonstrate that naked four-mutant IL-2 has the desired behavior and loses the ability to bind to the α-subunit of IL-2R, while binding to IL-2R βγ is retained and is comparable to the binding of the corresponding wild-type IL-2 construct and proleukin. The differences between wild-type IL-2 pools 1 and 2 appear to be related to differences in O-glycosylation. This variability and heterogeneity can be overcome in four mutant IL-2R by introducing the T23A mutation.

Example 3

Three F42A, Y45A and L72G mutations and a T3A mutation were introduced into the Fab-IL-2-Fab format (Fig. 1A) using the 4G8 anti-FAP antibody as a targeting domain model or as a single mutation construct: 1) 4G8 IL -2 T3A, 2) 4G8 IL-2 F42A, 3) 4G8 IL-2 Y45A, 4) 4G8 IL-2 L72G, or they were also combined in Fab-IL-2 mt-Fab constructs in the form of: 5) three F42A mutations /Y45A/L72G, or 6) four 2T3A/F42A/Y45A/L72G mutations to inactivate the glycosylation site. For comparison, the Fab-IL-2 wt-Fab construct based on 4G8 was used. All constructs contained the C145A mutation, which prevents the formation of disulfide-linked IL-2 dimers. Various Fab-IL-2-Fab constructs were expressed in HEK 293 cells and purified according to the above method using Protein A and gel filtration as described above. The affinity of the selected IL 2 variants for the human and mouse IL2R βγ heterodimer and for the human and mouse IL-2R α-subunit was then determined by surface plasmon resonance (SPR) (Biacore) using the recombinant IL-2R βγ heterodimer and the monomer containing IL-2R α-subunit, under the following conditions: IL-2R α-subunit was immobilized using two immobilization densities, a higher immobilization flow cell was used for mutants that lost the ability to bind to CD25. The following conditions were used: chemical immobilization: heterodimer of human IL-2R βγ - 1675 RU; mouse IL-2R βγ heterodimer - 5094 RU; α-subunit of human IL-2R-1019 RU; α-subunit of human IL-2R-385 RU, α-subunit of mouse IL-2R - 1182 RU; Mouse IL-2R α-subunit - 378 RU, temperature: 25°C, analytes: Fab-IL-2 variants containing Fab-IL-2 constructs based on 4G8-ot 3.1 to 200 nM, flow rate - 40 μl/min, association : 180 s, dissociation: 180 s, regeneration: 10 mM glycine, pH 1.5: 60 s, 40 µl/min. Approximation: two-state reaction model (conformational change), RI = 0 Rmax = local value. The results of the kinetic analysis are presented in table. 4.

Table 4 Affinity of FAP-targeted immunoconjugates containing IL-2 mutant polypeptides for intermediate affinity IL-2R and IL-2R α-subunit (KD)

Fab-IL-2-Fab construction Hu IL-2R βγ Hu IL-2R a Mu IL-2R βγ Mi IL-2R a 4G8 IL-2 wt 3.8nM 4.5nM 45.6nM 29nM 4G8 IL-2 TZA 1.6nM 4.9nM 15.6nM 15nM 4G8 IL-2 F42A 4.7nM 149nM 57nM ZbZnM 4G8 IL-2 Y45A 3.9nM 22.5nM 41.8nM 369nM 4G8 IL-2 L72G ND 45,ZnM ND ND 4G8 IL-2 trimutant F42A/Y45A/L72G 5.6nM no binding 68.8nM ND 4G8 IL-2 four-mutant T3A/F42A/Y45A/L72G 5.2nM no binding 56.2nM no binding

Co-binding to the IL-2R βγ heterodimer and FAP was demonstrated by SPR. In general, the method was as follows: a knob-into-hole human IL-2R βγ construct was chemically immobilized on a CM5 chip and a 10 nM Fab-IL-2-Fab construct was captured for 90 seconds. Human FAP served as the analyte and was used at concentrations from 200 to 0.2 nM. The following conditions were used: temperature: 25° C., buffer: HBS-EP, flow rate: 30 μl/min, association: 90 s, dissociation: 120 s. Regeneration was performed for 60 s using 10 mM glycine, pH 2. Fitting was performed using a 1:1 binding model, RI^0, Rmax = global value. SPR bridging analysis demonstrated that Fab-IL-2-Fab constructs, both wild-type and four-mutant, as well as the FAP-affinity maturated binding clone 28H1 or its parent antibody 3F2 or 4G8 at a concentration of 10 nM were able to bind simultaneously to the IL 2R βγ heterodimer immobilized on the chip, as well as to the human FAP used as the analyzed substance (Fig. 8). The results of determining the affinity are presented in table.5.

Table 5. FAP Affinity (KD) of FAP-Targeted Immunoconjugates Containing Mutant IL-2 Polypeptides and Binding to IL-2R with Intermediate Affinity

Fab-IL-2-Fab construction kd 4G8 Fab-IL-2 wt-Fab 5.0hM 4G8 Fab-IL-2 qm-Fab 5.6hM 29В11 Fab-IL-2 wt-Fab 0.32hM 29В11 Fab-IL-2 qm-Fab 0.89hM 3F2 Fab-IL-2 wt-Fab 1.2hM

Taken together, the SPR data demonstrate that i) the T3A mutation does not affect CD25 binding, ii) the three F42A, Y45A, and L72G mutations do not affect affinity for the IL-2R βγ heterodimer, but they reduce affinity for CD25 as follows : wt = T3A > Y45A (approximately 5 times lower) > L72G (approximately 10 times lower) > F42A (approximately 33 times lower); iii) the combination of the three mutations F42A, Y45A, and L72G, with or without the T3A O-glycosylation site-influencing mutation, results in a complete loss of CD25 binding ability when measured under SPR conditions, iv) although the affinity of human IL-2 for the mouse IL heterodimer -2R βγ and α-subunit of IL-2R is reduced approximately 10-fold compared to human IL-2R receptors, the selected mutations do not affect affinity for the mouse IL-2R βγ heterodimer, but abolish binding to the α-subunit of mouse IL-2R, respectively. This result indicates that mice are a suitable model for studying the pharmacological and toxicological effects of IL-2 mutants, although IL-2 is generally less toxic to rodents than to humans.

In addition to the loss of the O-glycosylation site, one of the additional advantages of the combination of the four mutations T3A, F42A, Y45A, L72G is the reduction in the surface hydrophobicity of the four mutant IL-2 due to the replacement of surface-exposed hydrophobic residues such as phenylalanine, tyrosine, or leucine with alanine. Dynamic light scattering aggregation temperature analysis demonstrated that the aggregation temperature of FAP-targeted Fab-IL-2-Fab immunoconjugates containing wild-type or four-mutant IL-2 was in the same range: about 57-58°C for Fab- IL-2-Fab based on the parent 3F2 and for the affinity matured 3F2 derivative 29B11; and in the range of 62-63°C for Fab-IL-2-Fab based on parental 4G8 and 4G8 derivatives with affinity matured 28H1, 4B9 and 14B3 4G8, indicating that the combination of four mutations does not adversely affect protein stability . In support of the preferred properties of the selected four IL-2 mutations, it was found that the yield after transient expression of the immunoconjugate in the four-mutant Fab-IL-2 qm-Fab format may even exceed the expression levels found with the respective Fab-IL-2 wt-Fab constructs. Finally, pharmacokinetic analysis (PK) demonstrated that the 4G8-based constructs of both Fab-IL-2 qm-Fab and Fab-IL-2 wt-Fab had comparable PK properties (see Example 9 below). Based on these data and data from cell-based assays as described in Example 4 below, four mutations T3A, F42A, Y45A, L72G were selected as the ideal combination of mutations to abolish CD25 binding to IL-2 in a targeted Fab-IL immunoconjugate. -2-Fab.

Example 4

4G8-based Fab-IL 2-Fab immunoconjugates targeting FAP containing wild-type IL-2 or having a single mutation 4G8 IL-2 T3A, 4G8 IL-2 F42A, 4G8 IL-2 Y45A, 4G8 IL-2 L72G, or IL-2 having respectively three mutations (F42A/Y45A/L72G) or four mutations (T3A/F42A/Y45A/L72G) was sequentially tested using the cell-based assays described above in comparison to proleukin.

IL-2 induced IFN-γ release was assessed after incubation of NK92 NK cells with the indicated constructs (FIG. 9). NK92 cells express CD25 on their surface. The results demonstrated that the Fab-IL-2-Fab immunoconjugate containing wild-type IL-2 was less effective than proleukin in inducing IFN-γ release, which is expected given the approximately 10-fold lower affinity of Fab-IL-2. 2 wt-Fab to the IL-2R βγ heterodimer. The introduction of individual mutations affecting CD25 binding, as well as a combination of three mutations affecting CD25 binding, into trimutant IL-2 resulted in FabIL-2-Fab constructs that were comparable, within method error, to a construct containing wild-type IL2. type, in terms of efficiency and absolute induction of IFN-γ release.

- 58 040858

Table 6. Induction of IFN-γ release by NK cells with Fab-IL-2-Fab immunoconjugates containing mutant IL-2 polypeptides

Design _EC50 [nM] proleukin 4.1 4G8 Fab-IL 2 wt-Fab 23.0 4G8Fab-IL-2(T3A)-Fab 16.2 4G8 Fab-IL-2 (F42A)-Fab 15.4 4G8 Fab-IL-2(Y45A)-Fab 20.9 4G8 Fab-IL-2 (L72G)-Fab 16.3 4G8 Fab-IL-2 (trimutant 42/45/72)-Fab 24.4

The induction of proliferation of the isolated human NK cells by the Fab-IL2-Fab immunoconjugates was then assessed based on a proliferation assay (Cell Titer Glo, Promega) (FIG. 10). Unlike NK92 cells, freshly isolated NK cells do not express CD25 (or express very little). The results demonstrated that the Fab-IL-2-Fab immunoconjugate containing wild-type IL-2 was approximately 10-fold lower in potency in inducing NK cell proliferation than proleukin, which is expected given the approximately 10-fold lower affinity of the Fab-IL-2 wt-Fab immunoconjugate for the IL-2R βγ heterodimer. The introduction of individual mutations affecting CD25 binding, as well as a combination of three mutations affecting CD25 binding, into trimutant IL-2 resulted in Fab-IL-2-Fab constructs that were comparable, within method error, to a construct containing wild-type IL-2, in terms of efficacy and absolute proliferation induction; only a very small shift in potency was found for the tri-mutant Fab-IL-2-Fab. In a second experiment, the induction of proliferation of PHA-activated T cells was evaluated after incubation with various amounts of proleukin and Fab-IL-2-Fab immunoconjugates (FIG. 11). Since activated T cells express CD25, a marked decrease in T cell proliferation was detected when incubated with immunoconjugates containing IL-2 with one of the F42A, L72G, or Y45A mutations; in the case of F42A, the most pronounced decrease was demonstrated, followed by L72G and Y45A mutations in descending order of action, while when using Fab-IL-2 wt-Fab or Fab-IL-2 (T3A)-Fab, activation remained almost the same level, as with the use of proleukin. These data indicate a decrease in affinity for CD25 as assessed by SPR (see example described above). The combination of three mutations affecting CD25 binding in trimutant IL-2 results in an immunoconjugate that mediates a significant reduction in the induction of T cell proliferation in solution. In the context of these results, the invention assessed cell death of T cells using annexin V/PI staining followed by overstimulation induced by a first stimulation, which was carried out by a 16-hour treatment with 1 μg/ml PHA, a second stimulation by treatment for 5 days with Proleukin or, respectively, Fab-IL-2-Fab immunoconjugates, followed by a third stimulation by treatment with 1 μg/ml PHA. Using this process, it was found that activation-induced cell death (AICD) in overstimulated T cells was significantly reduced when using Fab-IL-2-Fab immunoconjugates containing IL-2 with individual F42A, L72G and Y45A mutations affecting CD25 binding, however, when using F42A and L72G, the strongest decrease was found, which was similar to the decrease achieved when using three mutations in an immunoconjugate containing a three-mutant IL-2 (Fig. 12). In a recent series of experiments, the effects of Fab-IL-2 qm-Fab on the induction of STAT5 phosphorylation were examined in comparison to Fab-IL-2 wt-Fab and proleukin in human NK cells, CD4 T cells, CD8 T cells, and Tregs. -cells from human PBMCs (Fig. 13). For NK cells and CD8 T cells, which are characterized by no or very low expression of CD25 (meaning that IL-2R signaling is mediated by the IL-2R βγ heterodimer), the Fab-IL-2-Fab format containing wild-type IL-2 was about 10 times less efficient than proleukin in inducing STAT5 phosphorylation and that Fab-IL-2 qm-Fab was comparable to Fab-IL-2 wt-Fab construct. In the case of CD4 T cells, which are characterized by rapid upregulation of CD25 upon stimulation, the FabIL-2 qm-Fab immunoconjugate was less effective than the Fab-IL-2 wt-Fab, but still had a comparable ability to induce IL- 2R when applied at saturating concentrations. This is in contrast to the data obtained for Treg cells, for which the efficiency of Fab-IL-2 qm-Fab was significantly lower compared to the efficiency of the Fab-IL-2 wt-Fab immunoconjugate, due to the high level of expression on CD25 Treg cells. and, as a consequence, high binding affinity of the Fab-IL-2 wt-Fab immunoconjugate to CD25 on Treg cells. As a result of the abolition of the ability to bind to CD25 in the Fab-IL-2 qm-Fab immunoconjugate, IL-2 signaling in Treg cells is activated only through the IL-2R βγ heterodimer when used at concentrations at which IL-2R signaling activates CD25- negative effector cells through the IL-2R βγ heterodimer. In general, four mutant IL-2 as described herein is found to have the ability to activate IL-2R signaling via the IL-2R βγ heterodimer, but does not result in either AICD or preferential stimulation of Treg cells by compared to other effector cells.

Example 5

Based on the data described in Examples 2 and 3, affinity matured immunoconjugates (affinity matured Fab-containing immunoconjugates) of Fab-IL-2 qm-Fabs targeting FAP based on clones 28H1 or 29B11 were generated and purified using methods described in General Methods In general, a 28H1-based Fab-IL-2 qm-Fab targeting FAP was purified using a single affinity chromatography step (Protein G) followed by gel filtration (Superdex 200). PBS was used to equilibrate the column and stable CHO cell pool supernatant (CDCHO medium) was applied to a protein G column (GE Healthcare), the column was washed with PBS and then the samples were eluted with 2.5 mM HCl and the fractions were immediately neutralized with 10x PBS. Gel filtration was performed in final product buffer having the following composition: 25 mM sodium phosphate, 125 mM sodium chloride, 100 mM glycine, pH 6.7 on a Superdex 200 column. FIG. 14 shows the elution profiles after purification and the results of the analytical characterization of the product by SDS-PAGE (4-20% NuPAGE Novex bis-Tris mini-gel, Invitrogen, MOPS running buffer, reducing and non-reducing conditions). Given the low binding capacity of the 28H1 Fab fragment to protein G and protein A, the use of additional capture steps can lead to higher product yields.

Immunoconjugates Fab-IL-2 qm-Fab based on clones 4G8, 3F2 and 29B11 targeting FAP and Fab-IL-2 qm-Fab based on clone MHLG1 KV9 targeting MCSP were purified by a single affinity chromatography step (protein A) followed by gel filtration (Superdex 200). 20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5 was used to equilibrate the column, and the supernatant was applied to the protein A column. a second washing step was carried out using: 13.3 mM sodium phosphate, 20 mM sodium citrate, 500 mM sodium chloride, pH 5.45. Fab-IL-2 qm-Fab immunoconjugates were eluted with 20 mM sodium citrate, 100 mM sodium chloride, 100 mM glycine, pH 3. Gel filtration was performed in final product buffer having the following composition: 25 mM potassium phosphate, 125 mM sodium chloride, 100 mM glycine, pH 6.7. In FIG. 15 shows the elution profiles after purification and the results of the analytical characterization of the product by SDS-PAGE (4-20% NuPAGE Novex bis-Tris mini-gel, Invitrogen, MOPS running buffer, reducing and non-reducing conditions) of the 4G8 Fab-IL-2 immunoconjugate. qm-Fab, in Fig. 16 - immunoconjugate MHLG1 KV9 Fab-IL-2 qm-Fab.

FAP-targeted IgG-IL-2 qm fusion proteins based on anti-FAP antibodies 4G8, 4B9 and 28H1 and a DP47GS control IgG-IL-2 qm omnidirectional protein were generated according to the methods described above in the General Methods section. In FIG. 46 and 47 respectively show the chromatograms and elution profiles obtained during purification (A, B), as well as the results of analytical SDS-PAGE and gel filtration of the final purified constructs (C, D) of immunoconjugates based on 4G8 and 28H1. Yields after transient expression were 42 mg/l for the 4G8-based IgGIL-2 qm immunoconjugate and 20 mg/l for the 28H1-based IgG-IL-2 qm immunoconjugate.

The FAP binding capacity of the 4G8 and 28H1 anti-FAP IgG-IL-2 qm immunoconjugates was determined by surface plasmon resonance (SPR) using the Biacore device in comparison to the corresponding unmodified IgG type antibodies. In general, the method was as follows: an anti-His antibody (Penta-His, Qiagen, no. 34660) was immobilized on CM5 chips to capture 10 nM His-labeled human FAP (20 s). The temperature was 25° C. and HBS-EP was used as a buffer. The analyte concentrations ranged from 50 to 0.05 nM, the flow rate was 50 μl/min (association: 300 s, dissociation: 900 s, regeneration: 60 with 10 mM glycine, pH 2). The fit was based on a 1:1 binding model, RI = 0, Rmax = local value (because the capture format was used). In table. 7 shows estimated apparent affinity values (pM, avidity) determined by SPR, approximated using a 1:1 binding model, RI = 0, Rmax = local value.

Table 7

K _D [pM] hu fap 4G8 IgG-IL-2 qm 100 4G8 IgG 50 28H1 IgG-IL-2 qm 175 28H1 IgG 200

The data obtained indicate that, within the error of the method, the affinity for human FAP is maintained in the 28H1-based immunoconjugate or slightly reduced in the 4G8-based immunoconjugate compared to the corresponding unmodified antibodies.

Example 6

Affinity matured Fab-IL-2-Fab immunoconjugates based on 28H1 and 29B11 targeting FAP, each containing wild-type or four-mutant IL-2,

- 60 040858 and 3F2 based wt-Fab-IL-2 Fab-IL-2 were determined by surface plasmon resonance (SPR) against human, mouse or cynomolgus IL-2R βγ heterodimer using recombinant IL-2R βγ heterodimer under the following conditions: ligand: IL-2R heterodimer Pprotrusion in the cavity of humans, mice and monkeys Cynomolgus immobilized on a CM5 chip, analyzed substance: 28H1 or 29B11 Fab-IL-2-Fab (containing wild-type or four-mutant IL-2), 3F2 Fab-IL-2-Fab (containing wild-type IL-2), temperature: 25 or 37°C, buffer: HBS-EP, analyte concentrations: 200 to 2.5 nM, flow rate: 30 µl/min, association: 300 s, dissociation: 300 s, regeneration: 60 s, 3M MgCl ₂ , approximation: 1:1 binding model, RIr0, Rmax = global value. The affinity of the 28H1 and 29B11 affinity matured Fab-IL-2-Fab immunoconjugates targeting FAP, each containing wild-type or four-mutant IL-2, and the 3F2-based wt-Fab-IL2 Fab-IL2 were determined by surface resonance. plasmon (SPR) against human, mice or cynomolgus α-subunit IL-2R using recombinant monomeric IL-2R α-subunit under the following conditions: ligand: human, mouse and cynomolgus α-subunit IL-2R immobilized with CM5- chip, analyte: 28H1 or 29B11 Fab-IL-2Fab (containing wild-type or mutant IL-2), 3F2 Fab-IL-2-Fab (containing wild-type IL-2), temperature: 25°C or 37°C , buffer: HBS-EP, analyte concentrations: 25 to 0.3 nM, flow rate: 30 µl/min, association: 120 s, dissociation: 600 s, regeneration: none, approximation: 1:1 binding model, RI r 0, Rmax = global value.

The results of the kinetic analysis using the heterodimer IL-2R βγ are presented in table. 8. Table 8. Binding of immunoconjugates containing affinity matured Fab and mutant

IL-2, with IL-2R βγ heterodimers

K _D in nM Hu IL-2R βγ (25°C) Hu IL-2R βγ (37°C) Cyno IL-2R βγ (25°C) Cyno IL-2R βγ (37°C) Mu IL-2R βγ (25°C) Mu IL-2R βγ (37°C) 28H1 Fab-IL-2 9.7 19 11.5 29.2 112 186 wt-Fab 9 22 11.6 30.4 79 219 28H1 Fab-IL-2 7.5 14.3 8.9 21.3 66 142 qm-Fab 6.9 14.7 8.4 21.2 54 106 29B11 Fab-IL- 6.5 9.5 6.9 14 93 71 2 wt-Fab 5.7 12.4 6.7 19 74 74 29B11 Fab-IL- 7.2 13.1 7.8 16.7 60 44 2 qm-Fab 7.4 13 8.4 18.1 63 42 3F2 Fab-IL-2 5 ND 6.4 ND 40 ND wt-Fab 4.8 6.1 40

It was found that while the affinity of human IL-2 for human IL-2R βγ dimer was approximately 1 nM, the Fab-IL-2-Fab immunoconjugates (containing wild-type or four-mutant IL-2) both had reduced affinity (from 6 to 10 nM) and, as described above, the affinity of naked IL-2 for mouse IL-2R was about 10 times weaker than for human and cynomolgus IL-2R.

The results of the kinetic analysis using the α-subunit of IL-2R are presented in table. 9. Under the selected conditions, there is no detectable binding of immunoconjugates containing four-mutant IL-2R to the α-subunit of human, mouse, or cynomolgus IL-2R.

Table 9. Binding of Fab-IL-2-Fab immunoconjugates containing affinity matured Fab and IL-2 mutant to IL-2R α-subunit

Kq to hM Hu IL-2R a (25°C) HuIL-2Rα (37°C) Cyno IL-2Rα (25°С) Cyno IL-2R a (37°C) Mu IL-2Rα (25°С) Mu IL-2R a (37°C) 28H1 Fab-IL-2 16 28.8 16 36.5 43.3 67.5 wt-Fab 16.2 28.2 16.2 35.6 44 61.1 28H1 Fab-IL-2 qm-Fab No binding No binding no binding no binding no binding No binding 29B11 Fab-IL-2 5 7.6 4.8 7.3 P,4 13.3 wt-Fab 4.6 7.7 4.3 7.4 9.6 13.8 29B11 Fab-IL-2 qm-Fab No binding No binding no binding no binding no binding No binding 3F2 Fab-IL-2 wt-Fab 5.7 6.1 ND 5 5.4 ND 12.3 12.1 ND

The affinity of MHLG1-KV9 Fab-IL-2-Fab immunoconjugates targeting MCSP containing wild-type or four-mutant IL-2 was determined by surface plasmon resonance (SPR) for the human IL-2R βγ heterodimer using a recombinant heterodimer IL-2R βγ under the following conditions: human IL-2R β protrusion γ trough immobilized on a CM5 chip (1600 RU). MHLG1-KV9 Fab-IL-2 wt-Fab and Fab-IL-2 qm-Fab were used as test substances at 25° C. in HBS-P buffer. The concentrations of the analyzed substances in the study in relation to IL-2R βγ ranged from 300 to 0.4 nM (1:3 dilution), the flow rate was 30 μl/min (association duration 180 s, dissociation duration 300 s). Regeneration in the case of IL-2R βγ was carried out using 2x30 with 3 M MgCl ₂ . The data were approximated based on a 1:1 binding model, RI A 0, Rmax = local value in the case of IL-2R βγ.

The affinity of MHLG1-KV9 Fab-IL-2-Fab immunoconjugates targeting MCSP containing wild-type or four-mutant IL-2 was determined by surface plasmon resonance (SPR) for the human IL-2R α-subunit using recombinant monomeric α-subunit of IL-2R under the following conditions: human α-subunit of IL-2R immobilized on the CM5 chip (190 RU). MHLG1-KV9 Fab-IL-2 wt-Fab and Fab-IL-2 qm-Fab were used as test substances at 25° C. in HBS-P buffer. The concentrations of the analyzed substances in the study in relation to IL-2Rα ranged from 33.3 to 0.4 nM (dilution 1:3), flow rate 30 μl/min, (association duration 180 s, dissociation duration 300 s). Regeneration in the case of IL-2R α was carried out for 10 s using 50mM NaOH. Data were approximated based on a 1:1 binding model, RI A 0, Rmax = global value for IL-2R α.

The results of the kinetic analysis using the heterodimer IL-2R βγ are presented in table. 10.

Table 10

K _D in nM T = 25°С Hu IL 2R βγ (kinetics) Hu IL 2R a (kinetics) Hu IL 2R a (stationary state) MHLG1-KV9 Fab-IL-2 wt-Fab 8.6 9.8 8.8 10.1 6.8 10.9 MHLG1-KV9 Fab-IL 2 qm-Fab 7.3 10.7 no binding no binding

The data confirm that the MHLG1-KV9 Fab-IL-2 qm-Fab immunoconjugate targeted by MCSP retained affinity for the IL-2R βγ receptor while the binding affinity for CD25 was lost compared to the immunoconjugate containing IL-2 wild type.

Next, the affinity of 4G8 and 28H1 based IgG-IL-2 qm immunoconjugates for IL-2R βγ heterodimer and IL-2R α-subunit was determined by surface plasmon resonance (SPR) by direct comparison with the Fab-IL-2 qm-Fab format. immunoconjugate. In general, the method was as follows: ligands, either the α-subunit of human IL-2R or the heterodimer of human IL2R βγ-immobilized on the CM5 chip. Then, IgG-IL-2 qm immunoconjugates based on 4G8 and 28H1, or Fab-IL-2 qm-Fab immunoconjugates based on 4G8 and 28H1 were applied to the chip as analyzed substances at 25°C in HBS-EP buffer at concentrations from 300 up to 1.2 nM (1:3 dilution). The flow rate was 30 μl/min and the following conditions were used: association: 180 s, dissociation: 300 s and regeneration: 2 x 30 s using 3M MgCl ₂ in the case of IL-2R βγ heterodimer, 10 s using 50 mM NaOH in the case of α -IL-2R subunits. A 1:1 binding model was used for approximation (binding 1:1, RI A 0, Rmax = local value for IL-2R βγ, apparent value of K _D , binding RI = 0, Rmax = local value for IL-2Rα). The corresponding values of K _D are presented in table. eleven.

Table 11

Apparent value of K _D [nM] Hu IL-2R βγ Hu IL-2R a 4G8 IgG-IL-2 qm 5.9 no binding 4G8 Fab-IL-2 qm-Fab 10.4 no binding 28H1 IgG-IL-2 qm 6.2 no binding 28H1 Fab-IL-2 qm-Fab 11.4 no binding

The data demonstrate that the 4G8 and 28H1 based IgG-IL-2 qm immunoconjugates bound at least as high affinity as the Fab-IL-2 qm-Fab immunoconjugates to the IL-2R βγ heterodimer, while they did not bind to the α-subunit of IL-2R due to the introduction of mutations that affect binding to CD25. Compared to the corresponding Fab-IL-2 qm-Fab immunoconjugates, the affinity of the IgG-IL-2 qm fusion proteins appears to be slightly increased within the method error.

Example 7

At the time of the invention, it was confirmed in a first set of experiments by FACS evaluation that Fab-IL-2-Fab immunoconjugates targeting FAP containing either wild-type or mutant IL-2 had the ability to bind to the human FAP-expressing cell line HEK 293-FAP (FIG. 17) and that the presence of four mutations in IL-2 did not affect binding to FAP-expressing cells (FIG. 18).

- 62 040858

Table 12. Binding of Fab-IL-2-Fab immunoconjugates to FAP-expressing HEK cells

EU ₅₀ values (nM) 28H1 Fab-IL-2-Fab 0.64 28H1 Fab-IL-2 qm-Fab 0.70 29В11 Fab-IL-2-Fab 0.66 29В11 Fab-IL-2 qm-Fab 0.85 4G8 Fab-IL-2-Fab 0.65

In particular, in these binding experiments, it was found that the affinity matured Fab-IL-2 qm-Fab 28H1, 29B11, 14B3 and 4B9 binding agents had increased absolute binding capacity to HEK 293-FAP target cells by compared with Fab-IL-2-Fab immunoconjugates based on parental FAP binding agents 3F2 (29B11, 14B3, 4B9) and 4G8 (28H1) (Fig. 17), while maintaining high specificity and lack of binding to HEK 293 cells transfected with DPPIV, a close homologue of FAP, or HEK 293 cells transfected with a mimic. Mouse anti-human CD26-PE DPPIV, clone M-A261 (BD Biosciences, no. 555437) was used as a positive control (FIG. 19). Internalization capability analysis demonstrated that binding of Fab-IL-2-Fab immunoconjugates did not induce FAP internalization (FIG. 20).

In another experiment, FACS evaluated the binding of 4G8-based IgG-IL-2 qm and Fab-IL-2 qm-Fab immunoconjugates targeted by FAP to human FAP expressed on stably transfected HEK293 cells. The results are shown in FIG. 48. The data demonstrate that the IgG-IL-2 qm immunoconjugate binds to FAP-expressing cells with an EC ₅₀ value of 0.9 nM, comparable to that of the 4G8-based Fab-IL-2 qm-Fab construct (0 .7 nM).

Then, affinity matured Fab-IL-2-Fab immunoconjugates targeting FAP containing wild-type IL-2 or four-mutant IL-2 were tested using cellular assays versus proleukin according to the methods described above in the examples.

The IL-2-induced release of IFN-γ in the supernatant was assessed by ELISA after incubation of NK92 NK cells with the indicated immunoconjugates (FIG. 21) for 24 hours. NK92 cells express CD25 on their surface. The results demonstrated that the FabIL-2-Fab immunoconjugate, which contained wild-type IL-2, had less ability to induce IFN-γ release compared to proleukin, which is expected given the approximately 10-fold lower affinity of the Fab-IL-2 immunoconjugate. wt-Fab to IL-2R βγ heterodimer. The FabIL-2 qm-Fab immunoconjugates were found to be virtually comparable to the corresponding wild-type construct of the selected clone in terms of efficacy and absolute induction of IFN-γ release, despite the fact that NK92 cells express some CD25. However, it was found that the 29B11 Fab-IL-2 qm-Fab construct induced a lower level of cytokine release compared to the 29B11 Fab-IL-2 wt-Fab constructs, as well as 28H1- and 4G8-constructs, for which only a small shift in efficiency was found. Fab-IL-2 qm-Fab relative to Fab-IL-2 wt-Fab.

In addition, an MHLG1-KV9 based Fab-IL-2 qm-Fab immunoconjugate targeting MCSP was compared with 28H1 and 29B11 based Fab-IL-2 qm-Fab immunoconjugates using an IFN-γ release assay on NK92 cells. . As shown in FIG. 22, the MHLG1-KV9 based Fab-IL-2 qm-Fab immunoconjugate targeting MCSP is nearly comparable in its ability to induce IFN-γ release to Fab-IL-2 qm-Fab immunoconjugates targeting FAP.

The induction of proliferation of NK92 cells by IL-2 was then evaluated for 3 days using an ATP-based proliferation assay with CellTiter Glo (Promega) (FIG. 23). Given that NK92 cells express low levels of CD25, the difference between Fab-IL-2-Fab immunoconjugates containing wild-type IL-2 and immunoconjugates containing four-mutant IL-2 can be detected using a proliferation assay, but under conditions saturation with both to achieve a similar absolute induction of proliferation.

The following experiment evaluated the effect of FAP-targeted 28H1 affinity matured Fab-IL-2 qm-Fab immunoconjugate on the induction of STAT5 phosphorylation versus 28H1 Fab-IL-2 wt-Fab and Proleukin using human NK cells. , CD4 ⁺ T cells, CD8 ⁺ T cells, and T _reg cells from human PBMCs (FIG. 24). For NK cells and CD8 ⁺ -T cells, which are characterized by the absence or very low level of expression of CD25 (i.e., IL-2R signaling occurs through the IL-2R βγ heterodimer), the results demonstrated that the Fab-IL-2- The Fab containing wild-type IL-2 had approximately >10-fold lower ability to induce IFN-γ release compared to proleukin and that the Fab-IL-2 qm-Fab immunoconjugate was only slightly more effective than the Fab-IL- 2 wt-Fab. Rapid upregulation of CD25 has been demonstrated in CD4+ T cells upon stimulation, immunoconjugation

- 63 040858 gat Fab-IL-2 qm-Fab was significantly less effective than Fab-IL-2 wt-Fab, but still retained a comparable ability to induce IL-2R signaling when applied at saturating concentrations. This is in contrast to the effect found with T^ cells, for which the efficacy of Fab-IL-2 qm-Fab was significantly lower than that of the Fab-IL-2 wt-Fab construct due to the high level of CD25 expression on T^ cells. and the resulting high binding affinity of the Fab-IL-2 wt-Fab construct to CD25 on T^ cells. As a result of the loss of the ability to bind to CD25 in the Fab-IL-2 qm-Fab immunoconjugate, IL-2 signaling in T _-cells is only activated via the IL-2R βγ heterodimer when used at concentrations at which IL-2R signaling is activated at CD25-negative effector cells through the IL-2R βγ heterodimer. The corresponding values of the EU ₅₀ (in pM) are presented in table. 13.

Table 13. Induction of IFN-γ release from NK cells by 28H1-based Fab-IL-2-Fab immunoconjugates targeting FAP containing mutant IL-2 polypeptides

EU ₅₀ [pM] NK cells CO8 ⁺ -T cells CO4 ⁺ -T cells t - ¹ reg cells proleukin 222 1071 92 1 28H1 Fab-IL-2 wt-Fab 3319 14458 3626 15 28H1 Fab-IL-2 qm-Fab 3474 20583 70712 19719

In another series of experiments, the biological activity of 4G8 IgG-IL-2 qm and Fab-IL-2 qm-Fab based immunoconjugates targeting FAP was studied using several cellular assays.

4G8 IgG-IL-2 qm and 28H1 Fab-IL-2 qm-Fab immunoconjugates targeting FAP were investigated for induction of IFN-γ release by NK92 cells as induced by activation by IL-2R βγ signaling. In FIG. 49 demonstrates that the 4G8-based IgG-IL-2 qm immunoconjugate targeting FAP was as effective in inducing IFN-γ release as the 28H1-based Fab-IL-2 qm-Fab affinity matured immunoconjugate.

The invention also studied the effect of the 4G8-based IgG-IL-2 qm immunoconjugate, which targets FAP, on the induction of STAT5 phosphorylation compared to the 28H1-based Fab-IL-2 qm-Fab and Fab-IL-2 qm-Fab immunoconjugates. , as well as proleukin using human NK cells, CD4 ⁺ T cells, CD8 ⁺ T cells and T ^ cells from human PBMC. The results of these experiments are shown in Fig. 50. For NK cells and CD8 ⁺ T cells, the 4G8-based IgG-IL-2 qm immunoconjugate was found to have approximately >10-fold lower ability to induce STAT5 phosphorylation compared to proleukin, but slightly higher potency than Fab-IL-2 wt-Fab and Fab-IL-2 qm-Fab immunoconjugates based on 28H1. For CD4 ⁺ T cells, the 4G8-based IgG-IL-2 qm immunoconjugate was less effective than the 28H1-based Fab-IL-2 wt-Fab immunoconjugate, but not slightly more effective than the Fab-IL-2 qm- immunoconjugate. 28H1-based Fab, this immunoconjugate was also shown to induce IL-2R signaling when applied at saturating concentrations, comparable to that of proleukin and 28H1-based Fab-IL-2 wt-Fab. This is in contrast to the effect found when using Treg cells, for which the efficacy of the 4G8-based IgG-IL-2 qm immunoconjugate and the 28H1-based Fab-IL-2 qm-Fab immunoconjugate was found to be slightly lower compared to the Fab-IL-2 immunoconjugate. wt-Fab.

Taken together, these data demonstrate that four-mutant IL-2 as described herein has the ability to activate IL-2R signaling through the IL-2R βγ heterodimer, similar to that of wild-type IL-2, but does not result in more preferential stimulation of T _reg cells compared to other effector cells.

Example 8

The antitumor activity of FAP-targeted Fab-IL-2 qm-Fab immunoconjugates was evaluated in vivo compared to FAP-targeted Fab-IL-2 wt-Fab immunoconjugates in ACHN xenograft and LLC1 syngeneic models. All Fab-IL-2-Fab immunoconjugates targeting FAP (containing wild-type or four-mutant IL-2) recognized murine FAP as well as murine IL-2R. Since the ACHN xenograft models generated in SCID mice transgenic for human FcyRIII showed a pronounced positive reaction for FAP when assessed by immunohistochemistry (IHC), this model is a model with impaired immunological reactivity and can only reflect immune effector mechanisms, mediated by NK cells and/or macrophages/monocytes, but lacking T cell mediated immunity and therefore this model cannot reflect AICD or T _reg mediated actions. In contrast to the above model, the syngeneic LLC1 model generated in fully immunocompetent mice may also reflect adaptive T-cell mediated immune effector mechanisms, but has a very low level of FAP expression in the mouse stroma. Thus, each of these models is partially

- 64 040858 reflects the situation characteristic of human tumors.

Model created using xenograft of renal cell carcinoma line ACHN

FAP-targeted 4G8 Fab-IL-2 wt-Fab and 4G8 Fab-IL-2 qm-Fab immunoconjugates were tested using ACHN human renal cell adenocarcinoma cells injected into the kidneys of SCID mice transgenic for human FcyRIII. ACHN cells were originally obtained from ATCC (American Type Culture Collection) and, after expansion, were deposited in Glycart's own (internal) cell bank. ACHN cells were cultured in DMEM containing 10% FCS at 37°C in a water vapor-saturated atmosphere with 5% CO2. Passage 18 obtained in vitro was used for intrarenal injection, its viability was 98.4%. A small incision (2 cm) was made in the right flank and abdominal wall of anesthetized SCID mice. Subcapsularly, 50 μl of a suspension (1x10 ACHN cells in AimV medium) (2mM) was injected into the kidney. Wounds on the skin and the abdominal wall were closed with staples. Female SCID-FcyRIII mice (GLYCART-RCC strain), which were 8-9 weeks old at the start of the experiment (inseminated by RCC, Switzerland), were housed under specific pathogen-free conditions with daily cycles of 12 h light/12 h dark according to accepted guidelines. (GV-Solas; Felasa; TierschG). The pilot study protocol was reviewed and approved by the local authorities (R 2008016). After delivery, the animals were kept for one week to acclimatize to the new environment and for examination. Health monitoring was carried out regularly. Mice were injected into the kidney on experiment day 0 with 1x10 ACHN cells, randomly divided into groups and weighed. 1 week after tumor cell injection, mice were injected with i.v. 4G8 Fab-IL-2 wt-Fab and 4G8 FabIL-2 qm-Fab three times a week for three weeks. All mice were injected with i.v. 200 µl of the corresponding solution. Mice from the vehicle treated group were injected with PBS, mice from the treatment groups were injected with the 4G8 Fab-IL-2 wt-Fab or 4G8 Fab-IL-2 qm-Fab immunoconjugate. To obtain the appropriate amount of immunoconjugate per 200 μl, the stock solutions were diluted, if necessary, with PBS. In FIG. 25 demonstrates both 4G8 Fab-IL-2 wt-Fab and 4G8 Fab-IL-2 qm-Fab immunoconjugate mediated high potency based on criteria such as increased median survival compared to the vehicle treated group, with the efficacy of the 4G8 immunoconjugate Fab-IL-2 wt-Fab was found to be superior to the 4G8 Fab-IL-2 qm-Fab immunoconjugate.

Table 14-A

Compound Dose Buffer Composition Concentration (mg/ml) 4G8 Fab-IL-2-Fab wt, i.e. FAP 4G8 wt 20 mcg 25mM potassium phosphate, 125mM NaCl, 100mM glycine, pl 6.7 1.45 4G8 Fab-IL-2-Fab is four mutant, i.e. FAP 4G8 qm 20 mcg 25mM potassium phosphate, 125mM NaCl, 100mM glycine, pl 6.7 4.25

Syngeneic model of Lewis lung carcinoma LLC1

FAP-targeted 4G8 Fab-IL-2 qm-Fab and 28H1 Fab-IL-2 qm-Fab immunoconjugates were tested using the mouse Lewis lung carcinoma cell line LLC1, which was created by i.v. injection into Black 6 mice. Lewis lung carcinoma LLC1 cells were obtained from ATCC and, after expansion, were deposited in an internal Glycart cell bank. The tumor cell line was cultured according to conventional methods in DMEM containing 10% FCS (Gibco) at 37°C in a water vapor-saturated atmosphere with 5% CO2. Passage 10 was used for transplantation, its viability was 97.9%. 2x10 cells per animal were injected with i.v. into the tail vein in 200 μl of Aim V cell culture medium (Gibco). Black 6 mice (Charles River, Germany), which were 8–9 weeks old at the beginning of the experiment, were kept under specific pathogen-free conditions with daily cycles of 12 h light/12 h dark according to accepted guidelines (GV-Solas; Felasa; TierschG ). The pilot study protocol was reviewed and approved by the local authorities (R 2008016). After delivery, the animals were kept for one week to acclimatize to the new environment and for examination. Health status was regularly monitored. Mice were injected i.v.B day 0 with 2x105 LLC1 cells, randomly divided into groups and weighed. 1 week after tumor cell injection, mice were injected with i.v. 4G8 Fab-IL-2 qm-Fab or 28H1 Fab-IL-2 qm-Fab three times a week for three weeks. All mice were injected with i.v. 200 µl of the corresponding solution. Mice from the vehicle treated group were injected with PBS, mice from the treatment groups were injected with the 4G8 Fab-IL-2 qm-Fab or 28H1 Fab-IL-2 qm-Fab construct. To obtain the appropriate amount of immunoconjugate per 200 μl, the stock solutions were diluted, if necessary, with PBS. In FIG.

- 65 040858 demonstrated that the 4G8 Fab-IL-2 qm-Fab construct or the 28H1 Fab-IL-2 qm-Fab affinity matured construct was superior in terms of criteria such as increased median survival when compared to the vehicle treated group.

Table 14-B

Compound Dose Buffer Composition Concentration (mg/ml) 28H1 Fab-IL-2-Fab is four mutant, i.e. FAP 28Н1 qm 30 mcg 25mM potassium phosphate, 125mM NaCl, 100mM glycine, pH 6.7 2.74 4G8 Fab-IL-2-Fab is four mutant, i.e. FAP 4G8 qm 30 mcg 25mM potassium phosphate, 125mM NaCl, 100mM glycine, pH 6.7 4.25

In another experiment, FAP-targeted 28H1 Fab-IL-2 wt-Fab and 28H1 Fab-IL-2 qmFab immunoconjugates were tested using the same murine Lewis lung carcinoma model created with the LLC1 cell line, which was produced by iv injection mice of the Black 6 line. Passage 9 was used for transplantation, its viability was 94.5%. 2x10 ⁵ cells per animal were injected iv into the tail vein in 200 μl of Aim V cell culture medium (Gibco). Mice were injected on day 0 IVB with 2x10 ⁵ LLd cells, randomly divided into groups and weighed. 1 week after tumor cell injection, mice were injected with iv 28H1 Fab-IL-2 wt-Fab or 28H1

Fab-IL-2 qm-Fab three times a week for three weeks. All mice were injected with i.v. 200 µl of the corresponding solution. Mice from the vehicle treated group were injected with PBS, mice from the treatment groups were injected with the 28H1 Fab-IL-2 wt-Fab or 28H1 Fab-IL-2 qm-Fab construct. To obtain the appropriate amount of immunoconjugate per 200 μl, the stock solutions were diluted, if necessary, with PBS. In FIG. 27 demonstrates that the 28H1 Fab-IL-2 wt-Fab and 28H1 Fab-IL-2 qm-Fab immunoconjugates were superior in terms of improved median survival compared to the vehicle-treated group, while the efficacy of the 28H1 Fab-IL-2 qm-Fab immunoconjugate was -IL-2 wt-Fab was slightly higher than the immunoconjugate 28H1 Fab-IL-2 qm-Fab.

Table 14-B

Compound Dose Buffer Composition Concentration (mg/ml) 28H1 Fab-IL-2-Fab four mutant = FAP 28H1 qm 45 mcg 25mM potassium phosphate, 125mM NaCl, IOOmM glycine, pH 6.7 2.74 28H1 Fab-IL-2-Fab wt = FAP 28H1 wt 45 mcg 25mM potassium phosphate, 125mM NaCl, IOOmM glycine, pH 6.7 1.66

Example 9

A seven-day comparative study was then carried out between a 4G8-based Fab-IL-2 qm-Fab immunoconjugate targeting FAP and a 4G8-based Fab-IL-2 wt-Fab immunoconjugate targeting FAP to assess intravenous toxicity and toxicokinetic analysis using mice of the Black 6 line in table.15 presents the experimental design for evaluation of toxicity and toxicokinetic analysis.

Table 15 Experiment plan

Group Type Dose [µg/g] Purpose 1 D-PFR 0 control 2 4G8 Fab-IL-2 wt-Fab 4.5 titrimetric toxicity analysis 3 9.0 4 4G8 Fab-IL-2 qm-Fab 4.5 5 9.0

6 4G8 Fab-IL-2 wt-Fab 4.5 toxicokinetic analysis 7 9.0 8 4G8 Fab-IL-2 qm-Fab 4.5 9 9.0

The aim of this study was to characterize and compare toxicity profiles and perform a toxicokinetic analysis of a 4G8 Fab-IL2-Fab immunoconjugate containing wild-type interleukin-2 (IL-2) targeting FAP and a G48 Fab-IL-2-Fab immunoconjugate, containing a four-mutant IL-2 (qm) targeting FAP after daily intravenous administration to tumor-free male mice for 7 days. For this study, five groups of 5 male mice/group were injected intravenously with 0 (using vehicle as control), 4.5 or 9 µg/g/day wt IL-2 or 4.5 or 9 µg/g/day qm IL -2. An additional four groups of mice of 6 male mice/group were injected with 4.5 or 9 μg/g/day wt IL-2 or 4.5 or 9 μg/g/day qm IL-2 to

- 66 040858 toxicokinetic analyzes. The duration of the study varied from 7 to 5 days depending on the clinical signs found in animals treated with 4.5 and 9 μg/g/day wt IL-2. Assessment of toxicity was based on mortality, lifetime follow-up, body weight, and clinical and anatomical pathology. Animals at different time points were taken blood samples in groups intended for toxicokinetic analysis, for the implementation of toxicokinetic analysis. Toxicokinetic analysis data demonstrated that mice treated with wt IL-2 or qm IL-2 had detectable plasma levels up to the time of the last blood sample, indicating that mice were exposed to the respective compounds during treatment. The AUC _0-inf values obtained on day 1 suggest comparable wt IL-2 and qm IL-2 exposure at both doses. Several samples were taken on day 5 and showed plasma concentrations equivalent to those found on day 1, suggesting no accumulation of any compound after 5 days of dosing. More specifically, the following data was obtained.

Toxicokinetics (TK)

In table. 16 summarizes the average plasma toxicokinetic parameters for 4G8 Fab-IL-2 qmFab, which targets FAP, and 4G8 Fab-IL-2 wt-Fab, which targets FAP, obtained using the WinNonLin program, version 5.2.1 and supplied to sale of kappa-specific ELISA (ELISA kit for quantification of the human kappa chain, Bethyl Laboratories).

Table 16

Parameter Units Group 6, 4G8-FAP IL-2 wt Group 7, 4G8-FAP IL-2 wt Group 8, 4G8-FAP mutant IL-2 Group 9, 4G8-FAP mutant IL-2 Stakh ng/ml 47198 97986 60639 146416 Stax/dose (ng/ml) (mcg/g) 0.011 0.011 0.0135 0.016 AUC nghh/ml 331747 747499 355030 926683 AUC/dose nghh/ml(mcg/g) 0.074 0.083 0.079 0.103 Tl/2z h 3.6 3.11 4, 3 3.12 starting dose µg/g 4.5 9 4.5 9 route of administration IV IV IV IV

TC parameters were calculated using the WinNonLin program, version 5.2.1, using non-compartmental analysis

Individual serum concentrations are presented below:

Group (dose) Blood draw day Time (h) Animals Conc, in serum (ng/ml) Average conc. (ng/ml) Group 6 (4.5 µg/g) 4G6 FabIL2-Fab WT 1 1 26 27 64241 30155 47198 1 5.5 28 29 14693 16875 15784 1 24 thirty 31 318 520 419 5 5.5 29 thirty 31 13061 13620 13325 13335 Group 7 (9 µg/g) 4G6 FabIL2-Fab WT 1 1 32 33 101208 94764 97986 1 5.5 34 35 35766 32359 34062 1 24 36 37 573 588 580 5 5.5 32 33 35 36 31779 51143 53409 13562 37473 Group 8 (4.5 µg/g) 4G6 FabIL2-Fab mutant 1 1 38 39 73326 47953 60639 1 5.5 40 41 12168 14371 13269 1 24 42 43 494 487 490 5 5.5 40 41 6561 15352 10957 5 24 38 39 42 43 608 543 1298 437 721 Group 6 (9 µg/g) 4G6 FabIL2-Fab mutant 1 1 44 45 162970 129862 146416 1 5.5 46 47 20475 29125 24800 1 24 48 49 478 509 493

- 67 040858

Group (dose) Blood draw day Time (h) Animals Conc, in serum (ng/ml) Average conc. (ng/ml) 5 5.5 46 47 20504 75557 48031 5 24 44 45 48 49 634 796 661 719 703

These data demonstrate that both 4G8 Fab-IL-2 qm-Fab and 4G8 Fab-IL-2 wt-Fab had comparable pharmacokinetic properties, but slightly higher exposure occurred for 4G8 Fab-IL-2 qm-Fab.

Mortality

In the group treated with 4G8 Fab-IL-2 wt-Fab targeting FAP at 9 μg/g, one animal died as a result of treatment on day 5. Before death, he was found to have decreased activity, cold skin and hunched pose. This animal probably died as a result of a combination of cellular infiltration into the lung, which was accompanied by edema and hemorrhage, and marked necrosis of the bone marrow. Mortality data are summarized in Table 17.

Table 17 Mortality on Day 5

Group Type Dose [µg/g] Detected doom Serious toxicity/killing * * Total 1 D-PFR 0 0/5 0/5 0/5 2 4G8 Fab-IL-2 wt- 4.5 0/5 5/5 5/5 3 fab 9 1/5* 4/5 4/5 4 4G8 Fab-IL-2qm- 4.5 0/5 0/5 0/5 5 fab 9 0/5 0/5 0/5

6 4G8 Fab-IL-2 wtFab 4.5 1/6 5/6 6/6 7 9 2/6 4/6 6/6 8 4G8 Fab-IL-2 qmFab 4.5 0/6 0/6 0/6 9 9 0/6 0/6 0/6

*prior to necropsy **study was scheduled to run for 7 days, but all mice treated with wild-type IL-2 immunoconjugate developed lesions by day 5 and were sacrificed because they were not likely to survive

Clinical examinations

Signs such as decreased activity, cold skin and hunched posture were found in animals treated with wt IL-2 at doses of 4.5 and 9 μg/g/day. Data from clinical examinations are summarized in Table. 18.

Table 18. Clinical Examinations on Day 5

Group Type Dose [µg/g] hunched posture Reduced activity Cold to the touch body 1 D-PFR 0 0/5 0/5 0/5 2 4G8 Fab-IL-2 wt- 4.5 4/5 4/5 5/5 3 fab 9 5/5 5/5 5/5 4 4G8 Fab-IL-2qm- 4.5 0/5 0/5 0/5 5 fab 9 0/5 0/5 0/5

6 4G8 Fab-IL-2 wtFab 4.5 6/6 2/6 2/6 7 9 6/6 5/6 6/6 8 4G8 Fab-IL-2 qmFab 4.5 0/6 0/6 0/6 9 9 0/6 0/6 0/6

Body weight

A moderate decrease in body weight was found after 5 days of treatment in animals treated with wt IL-2 at a dose of 4.5 or 9 μg/g/day (9% and 11%, respectively). A slight decrease in body weight was found after 5 days of treatment in animals treated with qm IL-2 at a dose of 4.5 or 9 μg/g/day (2% and 1%, respectively). A moderate (9%) decrease in body weight was also found in the vehicle treated control group after 5 days of treatment. However, the reduction percentage should be 5% if the dead animal is excluded (animal #3). The decrease in body weight in the vehicle-treated group may be related to stress.

Hematology

Decreased platelet counts were found in animals treated with 4.5 (~4.5-fold) and 9 μg/g/day (~11-fold) 4G8 Fab-IL-2 wt-Fab, which correlated with a decrease in

- 68 040858 megakaryocytes in the bone marrow, as well as with systemic wasting effects (fibrin) in the spleen and lung of these animals (see section Histopathology, below). These data suggest that the reduced platelet count is likely due to the combined effects of uptake and decreased production/crowding in the bone marrow due to increased production of lymphocytes/myeloid cells due to direct or indirect effects of IL-2.

Hematological data suggest a non-strict relationship with compound administration, demonstrating an absolute reduction in lymphocyte counts when treated with 4.5 (~5-fold) and 9 μg/g (~3-fold) 4G8 Fab-IL-2 wtFab compared to with an average amount in the treated filler group. These data do not show a clear dose-dependence, but may be considered secondary, related to effects associated with stress found in in vivo studies, or an increased pharmacological effect of the compound (lymphocytes migrate into tissues). No treatment-related hematological changes were found in response to administration of 4G8 Fab-IL-2 qm-Fab. Several individual hematological data were statistically different from the corresponding data for the controls. However, these data are insufficient to suggest that they indicate pathological changes.

Macroscopic pathology and histopathology

Treatment-related macroscopic signs, including an enlarged spleen, were found in 5/5 and 4/5 mice from the groups that were treated with 4G8 Fab-IL-2 wt-Fab at doses of 4.5 and 9 μg/g, respectively, and in 1/ 5 mice from groups treated with 4G8 Fab-IL-2 qm-Fab at both doses of 4.5 and 9 µg/g.

Treatment-related histopathological features were found in groups treated with 4G8 Fab-IL-2 wt-Fab at 4.5 and 9 µg/g and 4G8 Fab-IL-2 qm-Fab at 4.5 and 9 µg/g , in lung, bone marrow, liver, spleen, and thymus, they differed in frequency of occurrence, severity of stage, or nature of the changes, as will be described below.

Treatment-related histopathological features in the lung included mild to severe mononuclear cell infiltration, which was found in 5/5 mice from groups treated with 4.5 and 9 μg/g 4G8 Fab-IL-2 wt-Fab, and marginal infiltration in 5/5 mice from groups treated with 4G8 Fab-IL-2 qm-Fab at doses of 4.5 and 9 μg/g. Mononuclear cell infiltration includes lymphocyte infiltration (some of which were found to have cytoplasmic granules) as well as reactive macrophages. These cells were found to often have a vasocentric distribution, often being marginalized within vessels in the lung. It was also established that these cells also surrounded the vessel, but in more serious cases their distribution was more diffuse. Marginal to mild hemorrhage was found in 5/5 mice from the groups treated with 4G8 Fab-IL-2 wt-Fab at doses of 4.5 and 9 µg/g and marginal in 2/5 mice from the group treated with 4G8 Fab -IL-2 qm-Fab at 9 µg/g. Although hemorrhage was most often perivascular, in more severe cases it was found in the alveolar space. Mild to moderate edema was found in 5/5 mice from the groups treated with 4G8 Fab-IL-2 wt-Fab at doses of 4.5 and 9 µg/g and marginal in 5/5 mice from the group treated with 4G8 Fab -IL-2 qm-Fab at 9 µg/g. Although the edema was often perivascular, in more severe cases it is also found in the alveolar space. Marginal cell degeneration and karyorrhexis were found in 2/5 and 5/5 mice from the groups treated with 4.5 and 9 μg/g 4G8 Fab-IL-2 wt-Fab, respectively, and these included degeneration of infiltrating or reactive lymphocytes. MSB dye-selected animals were found to be positive for fibrin in the lungs of animals from groups treated with 4G8 Fab-IL-2 wt-Fab at both doses of 4.5 and 9 µg/g, which correlated with a decrease in the number of platelets found in these animals .

Treatment-related signs in bone marrow, including an increase from marginal to mild total marrow cell saturation, were found in 5/5 mice and 2/5 mice from both 4G8 Fab-IL-2 wt-Fab and 4G8 Fab treated groups. -IL-2 qm-Fab at a dose of 4.5 μg/g, and 5/5 mice and 2/5 mice from the groups treated with each of the indicated constructs at a dose of 9 μg/g, respectively. This was characterized by an increase from marginal to moderate levels of lymphocytic-myelocytic hyperplasia in these groups, which was confirmed, in particular, by an increase in the number of CD3-positive T cells in the bone marrow and sinuses (in particular, T-lymphocytes, which was confirmed by immunohistochemistry using rap-T-cell marker CD3 in selected animals). The increase in CD3 positive T cells was moderate in both 4G8 Fab-IL-2 wt-Fab treated groups and marginal to weak in both 4G8 Fab-IL-2 qm-Fab treated groups. A marginal to weak decrease in megakaryocytes was found in 2/5 of the mice treated with 4.5 µg/g 4G8 Fab-IL-2 wt-Fab and 5/5 in mice treated with the same construct at 9 µg/g. d, a marginal to moderate decrease in erythroid progenitors was found in 3/5 mice from the group treated with 4.5 μg/g 4G8 Fab-IL-2 wt-Fab and in 5/5 mice treated with the same construct at a dose of 9 mcg/g. Bone marrow necrosis was found in 1/5 mice in the 4G8 Fab-IL-2 wt-Fab group treated at 4.5 µg/mg (minimum) and in 5/5 mice (mild to severe) treated with the same construct at a dose of 9 mcg / g. A reduced number of megakaryocytes in the bone marrow has been correlated with a reduced platelet count, which may be a direct result of bone marrow congestion in

- 69 040858 as a result of an increase in lymphocytes/myeloid progenitors and/or bone marrow necrosis and/or platelet depletion due to inflammation in various tissues (see data for spleen and lung). The decrease in erythroid precursors found in the bone marrow did not correlate with peripheral blood hematology findings, probably due to time-related factors (bone marrow studies were performed prior to peripheral blood evaluation) and the longer half-life of peripheral erythrocytes (compared to platelets). The mechanism of bone marrow necrosis in the bone marrow may be secondary, resulting from marked overcrowding of the brain cavity (due to the production and growth of lymphocytes/myeloid cells), systemic or local release of cytokines from proliferating cell types, a factor associated with local effects of hypoxia , or other pharmacological actions of the compound.

Treatment-related signs in the liver, including the presence of mild to moderate primary vasocentric mononuclear cell infiltration and marginal to moderate individual cell necrosis, were found in 5/5 mice treated with 4G8 Fab-IL-2 wt-Fab at doses of 4.5 and 9 mcg/mg. Marginal necrosis of individual cells was found in 2/5 and 4/5 mice treated with 4G8 Fab-IL-2 qm-Fab at doses of 4.5 and 9 μg/mg, respectively. The mononuclear infiltrate consisted mainly of lymphocytes (particularly T-lymphocytes, as confirmed by immunohistochemistry using the pan-T-cell marker CD3, performed on selected animals), it is most often characterized by a vasocentric location, and very rarely a location in central and portal vessels. Animals selected for immunohistochemistry were stained with F4/80 and found to have increased numbers and size of (activated) macrophages/Kupffer cells in hepatic sinusoids in groups treated with 4G8 Fab-IL-2 wt-Fab and 4G8 Fab-IL-2 qmFab at a dose of 9 mcg/mg.

Treatment-related signs in the spleen, including the presence of moderate to severe lymphoid hyperplasia/infiltration and mild to moderate macrophage hyperplasia/infiltration, were found in 5/5 mice in groups treated with 4G8 Fab-IL-2 wt-Fab at doses of 4, 5 and 9 µg/mg, and mild to moderate lymphoid hyperplasia/infiltration and marginal to mild macrophage hyperplasia/infiltration in 5/5 mice in 4G8 Fab-IL-2 qm-Fab treated groups at doses of 4.5 and 9 mcg/mg.

Immunohistochemical study performed using mice treated with 4G8 Fab-IL-2 wt-Fab and 4G8 Fab-IL-2 qm-Fab at 9 µg/mg using the CD3 pan-T cell marker as well as the F4 macrophage marker /80 showed different pictures. When using 4G8 FabIL-2 wt-Fab at a dose of 9 μg/mg, the pattern of immunoreactivity of T cells and macrophages was preserved primarily in areas of the red pulp, since the architecture of the primary follicles was altered by lymphocytolysis and necrosis (as described below). Using 4G8 Fab-IL-2 qm-Fab at 9 µg/mg, the specific staining showed a pattern similar to that found in vehicle-treated control mice, but with extension into the periarterial lymphocytic membranes (PALS) of the white pulp of the T cell population and the presence of larger expanded area of red pulp. T-cell and macrophage-positive staining was also found within the red pulp, similar in pattern to the staining pattern in the control vehicle-treated group, but expanded. These results correlate with the data of macroscopic studies of the enlarged spleen. Marginal necrosis was found in 3/5 mice and marginal to mild in 5/5 mice in the 4G8 Fab-IL-2 wt-Fab treated groups at doses of 4.5 and 9 μg/mg, respectively. Necrosis was generally localized around the area of primordial follicles, and when stained with MSB, selected animals tested positive for fibrin in groups treated with 4G8 Fab-IL2 wt-Fab at both doses of 4.5 and 9 μg/mg, which correlated , in particular with the reduced platelet content found in these animals. Lymphocytolysis was found in groups treated with 4G8 FabIL-2 wt-Fab at 4.5 μg/g (minimal to mild) and 9 μg/g (moderate to severe).

Treatment-related signs in the thymus, including a minimal to low lymphocyte count in the 4G8 Fab-IL-2 wt-Fab treated group at both 4.5 and 9 µg/mg and 4G8 Fab-IL-2 treated qm-Fab at a dose of 4.5 µg/mg. Thymic cortex and medulla were not examined separately in 4G8 Fab-IL-2 wt-Fab treated groups, however immunohistochemistry using a rap-T cell marker (CD3) performed on selected animals treated with 4G8 Fab-IL-2 wt-Fab 2 wt-Fab at 9 µg/mg and 4G8 Fab-IL-2 qm-Fab at 9 µg/mg showed strong positive staining of most cells in the thymus. The increased lymphocyte content in the thymus is considered as a direct pharmacological action of both compounds, in which IL-2 induced the proliferation of lymphocytes migrating into the thymus (T cells) from the bone marrow for additional differentiation and clonal expansion. This occurred in all groups except for the group treated with 4G8 Fab-IL-2 qm-Fab at 9 μg/mg, which probably had a transient effect. Lymphocytolysis was weak in the 4G8 Fab-IL-2 wt-Fab treated group at 4 µg/mg and moderate to severe in the 4G8 Fab-IL-2 wt-Fab treated group at 9 µg/mg. Moderate depletion of lymphoid elements was found in groups treated with 4G8 Fab-IL-70 040858 wt-Fab at doses of both 4.5 and 9 μg/ml. Because these results are more significant in groups treated with 4G8 Fab-IL-2 wt-Fab at doses of 4.5 and 9 µg/mg, these animals were classified as agonizing on day 5 and mild to severe lymphocytolysis and moderate lymphoid depletion may be associated with this lifetime factor (stress-related activities due to poor physical condition).

Histopathological evidence of a non-strict relationship between compound administration and liver manifestations includes marginal mixed cell infiltration/activation (lymphocytes and macrophages) found as a small lesion/microgranulomatosis randomly located in the liver of 5/5 mice from the 4G8 Fab-IL-treated groups. 2 qm-Fab at both doses of 4.5 and 9 mcg/mg. This marginal change was also found in the vehicle-treated control group, but less frequently and less severely. Marginal to mild gastric glandular enlargement and atrophy was found in 5/5 mice, and marginal ileal villous atrophy was found in 3/5 mice treated with 4G8 Fab-IL-2 wt-Fab at 9 μg/mg. This fact is most likely related to the poor physical condition found in lifetime studies in these mice, reflected by reduced body weight, primarily in the group treated with 4G8 Fab-IL-2 wt-Fab at 9 μg/mg.

Injection site signs, including mixed cell infiltration, perivascular edema, and myodegeneration, were found in both vehicle-treated controls and 4G8 Fab-IL-2 wt-Fab 9 µg/mg and 4G8 Fab-IL treated groups. -2 qm-Fab at 9 mcg/mg. Epidermal necrosis was found in one animal. These signs are not related to the handling(s) itself, but to daily i.v. injections and tail manipulation. Another animal showed macrophage infiltration of skeletal muscle (detected on a histological section of lung tissue) associated with myodegeneration and myoregeneration, probably due to chronic injury and not associated with treatment. Marginal lymphoid depletion was found in 3/5 and 4/5 mice in the 4G8 Fab-IL-2 qm-Fab treated groups at doses of 4.5 and 9 µg/mg, respectively, most likely due to normal physiological changes found in the thymus mice with increasing age (also found with similar occurrence and severity in 4/5 mice from the vehicle-treated control group of animals).

In general, daily intravenous administration of 4G8 Fab-IL-2 wt-Fab or 4G8 Fab-IL-2 qm-Fab at doses of 4.5 or 9 µg/g/day for up to 5 days to male mice resulted in similar processing-related histological features for both compounds. However, the changes were greater and more severe with the FAP-targeted 4G8 Fab-IL-2 wt-Fab immunoconjugate in the lung (FIGS. 28 and 29) (mononuclear infiltration with lymphocytes and reactive macrophages, hemorrhage and edema), in the bone brain (lymphomyelohyperplasia and increased and increased cell saturation), liver (Fig. 30) (necrosis of individual cells, increase in the number and activation of Kupffer cells/macrophages), spleen (marked enlargement, infiltration/hyperplasia of macrophages and lymphocytes) and thymus ( elevated lymphocytes). In addition, mortality, lymphocytolysis, necrosis or degeneration of cells in the lung, spleen, bone marrow and thymus, as well as a decrease in the number of megakaryocytes and erythrocytes in the bone marrow and a decrease in platelets in the peripheral blood, were found only in animals treated with wt IL-2. On the basis of clinical and anatomical features, as well as clinical observations and comparable systemic exposure of both compounds, it can be concluded that qm IL-2 under the conditions of this study had significantly less pronounced systemic toxicity after administration of 5 doses than wt IL- 2.

Example 10

Induction of wild-type IL-2 and four-mutant IL-2 secretion of IFN-γ by NK cells NK-92 cells were kept in minimal medium for 2 hours before seeding at 100,000 cells/well in a 96-well flat bottom plate. The IL-2 constructs were added titrimetrically to seeded NK-92 cells. After 24 or 48 hours, the plates were centrifuged before collecting the supernatants to quantify human IFN-γ using a commercially available IFN-γ ELISA kit (BD no. 550612).

Two different inventive wild-type IL-2 preparations (probably slightly different in their O-glycosylation profiles see example 2), commercially available wild-type IL-2 (proleukin) and inventive four-mutant IL-2 were tested. IL-2 (first batch).

In FIG. 31 demonstrates that four-mutant IL-2 is as effective as commercially available (proleukin) or inventive wild-type IL-2 in inducing NK cell secretion of IFN-γ within 24 (A) or 48 hours ( B).

Example 11 Induction of wild-type IL-2 and four-mutant IL-2 NK cell proliferation

NK-92 cells were kept in minimal medium for 2 hours before seeding at 100,000 cells/well in a 96-well flat bottom plate. IL-2 constructs were added titrimetrically to

- 71 040858 seeded NK-92 cells. After 48 hours, ATP content was assessed to determine the number of viable cells using the CellTiter-Glo Luminescent Cell Viability Assay from Promega according to the manufacturer's instructions.

The same IL-2 formulations were tested as in Example 10.

In FIG. 32 demonstrates that all tested molecules had the ability to induce NK cell proliferation. When used at low concentrations (<0.01M), four-mutant IL-2 was slightly less potent than inventive wild-type IL-2, and all inventive formulations were less potent than commercially available wild-type IL-2 type (proleukin).

In the second experiment, the following preparations of IL-2 were tested: wild-type IL-2 (pool 2), four-mutant IL-2 (first and second batch).

In FIG. 33 demonstrates that all tested molecules had approximately the same ability to induce NK cell proliferation, with the two mutant IL-2 preparations being only marginally less potent than wild-type IL-2 preparations at the lowest concentrations.

Example 12.

Induction of Human PBMC Proliferation with Immunoconjugates Containing Wild-Type or Four-Mutant IL-2

Peripheral blood mononuclear cells (PBMCs) were generated using Histopaque-1077 (Sigma Diagnostics Inc., St. Louis, MO, USA). In general, the method was as follows: samples of venous blood from healthy volunteers were taken into heparinized syringes. Blood was diluted 2:1 with PBS free of calcium and magnesium and layered on Histopaque-1077. The gradient was centrifuged at 450 x g for 30 min at room temperature (RT) without interruption. The PBMC-containing interphase was harvested and washed three times with PBS (350 x g followed by centrifugation at 300 x g for 10 min at RT).

The PBMCs were then labeled with 40nM CFSE (carboxyfluorescein succinimidyl ester) for 15 min at 37°C. Cells were washed with 20 ml of medium before isolating labeled PBMCs for 30 min at 37°C. Cells were washed, counted, and 100,000 cells were plated in 96-well U-bottom plates. Pre-diluted proleukin (commercially available wild-type IL-2) or IL2 immunoconjugates were added titrimetrically to seeded cells, which were incubated for the indicated times. After 4-6 days, cells were washed, stained for appropriate cell surface markers, and analyzed by FACS using a BD FACSCanto II device. NK cells were defined as CD37CD56+, CD4 T cells as CD3+/CD8 ⁻ , and CD8 T cells as CD3+/CD8+.

In FIG. 34 shows NK cell proliferation data after incubation with various FAP-targeted 28H1 IL-2 immunoconjugates for 4 (A), 5 (B), or 6 (C) days. All tested constructs induced NK cell proliferation in a concentration dependent manner. Proleukin appeared to be more effective than the immunoconjugates at lower concentrations, however, this difference was not found at higher concentrations. At earlier time points (day 4), the IgG-IL2 constructs were slightly more efficient than the Fab-IL2-Fab constructs. At later time points (Day 6), the efficacy of all constructs was comparable, with the Fab-IL2 qm-Fab construct having poor efficacy at low concentrations.

In FIG. 35 shows CD4 T cell proliferation data after incubation with various FAP-targeted 28H1 IL-2 immunoconjugates for 4 (A), 5 (B), or 6 (C) days. All constructs tested induced CD4 T cell proliferation in a concentration dependent manner. Proleukin proved to be more effective than immunoconjugates, and immunoconjugates containing wild-type IL-2 were slightly more effective than those containing four-mutant IL-2. As in the case of NK cells, the Fab-IL2 qm-Fab construct had the least activity. Most likely, the proliferating CD4 T cells are partially regulatory T cells, at least in the case of constructs containing wild-type IL-2.

In FIG. 36 shows CD8 T cell proliferation data after incubation with various FAP-targeted 28H1 IL-2 immunoconjugates for 4 (A), 5 (B), or 6 (C) days. All tested constructs induced CD8-T cell proliferation in a concentration dependent manner. Proleukin proved to be more effective than immunoconjugates, and immunoconjugates containing wild-type IL-2 were slightly more effective than those containing four-mutant IL-2. As with NK cells and CD4 T cells, the Fab-IL2 qm-Fab construct was the least active.

In FIG. 37 shows the results of another experiment comparing 28H1 IgG-IL-2 targeted by FAP containing either wild-type or four-mutant IL-2 and proleukin. The duration of incubation was 6 days. As demonstrated on

- 72 040858 drawing, all three containing IL-2 constructs induced the proliferation of NK cells (A) and CD8 T cells (B) depending on the dose with the same efficiency. Against CD4-T cells (B), the IgG-IL2 qm immunoconjugate had lower activity, especially when used at moderate concentrations, which may be due to the lack of activity against CD25-positive (including regulatory) T cells, which represent a subpopulation CD4 T cells.

Example 13 Activation of effector cells by wild-type and four-mutant IL-2 (pSTAT5 assay)

PBMCs were prepared according to the method described above. 500,000 PBMC/well were plated in 96-well U-bottom plates and incubated for 45 min at 37° C. in RPMI medium containing 10% FCS and 1% glutamax (Gibco). PBMCs were then incubated with proleukin derived from inventive wild-type IL-2 or four-mutant IL-2 at the indicated concentrations for 20 minutes at 37° C. to induce STAT5 phosphorylation. The cells were then immediately fixed (BD, Cytofix buffer) for 10 min at 37°C and washed once, followed by a permeation step (BD, Buffer III Phosflow Perm) for 30 min at 4°C. Cells were then washed with PBS/0.1% BSA and stained with antibody mixtures used for FACS to detect NK cells (CD37CD56+), CD8 ⁺ -T cells (CD3'CD87n CD4 ⁺ -T cells (CD3 ⁺ /CD4 ⁺ /CD257CD127 ⁺ ) or Treg cells (CD4 ⁺ /CD25 ⁺ /CD1277FoxP3 ⁺ ) as well as pSTAT5 for 30 min at CT in the dark Cells were washed twice with PBS/0.1% BSA and resuspended in 2% PFA prior to analysis by flow cytometry (BD , FACSCanto II device).

In FIG. 38 shows data on STAT phosphorylation in NK cells (A), CD8 T cells (B), CD4 T cells (C) and regulatory T cells (D) after 30 minutes of incubation with proleukin obtained during the creation of the invention IL -2 wild type (pool 2) and four mutant IL-2 (lot 1). All three IL-2 preparations were equally effective in inducing STAT phosphorylation in NK cells as well as CD8 T cells. In CD4 T cells, and even more so in regulatory T cells, four-mutant IL-2 was less active than preparations containing wild-type IL-2.

Example 14 Activation of effector cells with IgG-IL-2 immunoconjugate containing wild-type IL-2 and four-mutant IL-2 (pSTAT5 assay)

The experimental conditions were as described above (see Example 13).

In FIG. 39 shows data on STAT phosphorylation in NK cells (A), CD8 T cells (B), CD4 T cells (C) and regulatory T cells (D) after 30 min incubation with proleukin, IgG-IL-2 containing wild-type IL-2, or IgG-IL-2 containing four-mutant IL-2. In all cell types, proleukin was more effective in inducing STAT phosphorylation than IgG-IL-2 immunoconjugates. IgG-IL-2 constructs containing wild-type IL-2 and four-mutant IL-2 were equally effective in NK cells as well as CD8 T cells. In CD4 T cells, and even more so in regulatory T cells, four-mutant IgG-IL-2 had lower activity than an IgG-IL-2 immunoconjugate containing wild-type IL-2.

Example 15 Maximum Tolerated Doses (MTD) of Fab-IL2 wt-Fab and Fab-IL2 qm-Fab Immunoconjugates Targeting FAP

The effects of increasing doses of FAP-targeting Fab-IL2-Fab immunoconjugates containing either wild-type (wt) or four-mutant (qm) IL-2 were evaluated in tumor-free Black 6 immunocompetent mice.

Black 6 female mice (Charles River, Germany), which were 8–9 weeks old at the beginning of the experiment, were kept under special pathogen-free conditions with daily cycles of 12 h light/12 h dark according to accepted guidelines (GV-Solas; Felasa; TierschG). The pilot study protocol was reviewed and approved by the local authorities (R 2008016). After delivery, the animals were kept for one week to acclimatize to the new environment and for examination. During the entire period of time, health status was regularly monitored.

Mice were injected with i.v. once a day for 7 days 4G8 Fab-IL2 wt-Fab at doses of 60, 80 and 100 µg/mouse or 4G8 Fab-IL2 qm-Fab at doses of 100, 200, 400, 600 and 1000 µg/mouse. All mice were injected with i.v. 200 µl of the appropriate solution. To obtain the appropriate amount of immunoconjugate per 200 μl, stock solutions were diluted with PBS if necessary.

In FIG. 40 demonstrated that the MTD (maximum tolerated dose) of Fab-IL2 qm-Fab was 10 times higher than Fab-IL2 wt-Fab, namely, it was 600 μg/mouse when Fab-IL2 qm-Fab was administered daily for 7 days compared to 60 µg/mouse with wtFab Fab-IL2 administered daily for 7 days.

- 73 040858

Table 19

Compound Dose Buffer Composition Concentration (mg/ml) 4G8 Fab-IL2 wt-Fab 60.80, 100 mcg 25mM potassium phosphate, 125mM NaCl, 100mM glycine, pH 6.7 3.32 (mother solution) 4G8 Fab-IL2 qm-Fab 100.200, 400.600, 1000 mcg 25mM potassium phosphate, 125mM NaCl, 100mM glycine, pH 6.7 4.25 (mother solution)

Example 16 Single Dose Pharmacokinetic Characteristics of IgG-IL2 wt and qm Immunoconjugates Targeting FAP and Non-Targeted IgG-IL2 wt and qm Immunoconjugates

A single dose pharmacokinetic (PK) study of FAP-targeted IgG-IL2 immunocojugates containing either wild-type or four-mutant IL-2 and non-targeted IgG-IL-2 immunoconjugates containing either wild-type or four-mutant IL-2 was performed on tumor-free immunocompetent mice of line 129.

Female mice of line 129 (Harlan, UK), which were 8-9 weeks old at the beginning of the experiment, were kept under special pathogen-free conditions with daily cycles of 12 h light/12 h dark according to accepted guidelines (GV-Solas; Felasa; TierschG) . The pilot study protocol was reviewed and approved by the local authorities (R 2008016). After delivery, the animals were kept for one week to acclimatize to the new environment and for examination. During the entire period of time, health status was regularly monitored.

Mice were injected with i.v. FAP-targeted immunoconjugates such as 28H1 IgG-IL2 wt (2.5 mg/kg) or 28H1 IgG-IL2 qm (5 mg/kg), or non-targeted DP47GS IgG-IL2 wt immunoconjugates (5 µg/kg) or DP47GS IgG-IL2 qm (5 mg/kg). All mice were injected with i.v. 200 µl of the corresponding solution. To obtain the appropriate amount of immunoconjugate per 200 μl, stock solutions were diluted with PBS if necessary.

Mice were bled at the following time points: 1, 8, 24, 48, 72, 96 hours and every 2 days thereafter for 3 weeks. Serum was extracted and stored at -20°C until the ELISA analysis. Serum concentrations of the immunoconjugates were determined using ELISA to quantify the antibody contained in the IL-2-containing immunoconjugate (Roche Penzberg). Absorbance was determined by measuring at a wavelength of 405 nm and a reference wavelength of 492 nm (adjustable VersaMax type microplate reader, Molecular Devices).

In FIG. 41 shows the results of a pharmacokinetic study of these IL-2 immunoconjugates. Both the FAP-targeted constructs (A) and non-targeted (B) IgG-IL2 qm constructs had longer serum half-life (about 30 hours) compared to the corresponding IgG-IL2 wt constructs (about 15 hours).

Table 20

Compound Dose Buffer Composition Concentration (mg/ml) 28Hl-IgG- IL2wt 2.5 mg/kg 20mM histidine, 140mM NaCl, pH 6.0 3.84 (mother solution) 28Hl-IgG- IL2 qm 5 mg/kg 20mM histidine, 140mM NaCl, pH 6.0 2.42 (mother solution) DP47GS- IgG-IL2wt 5 mg/kg 20mM histidine, 140mM NaCl, pH 6.0 3.74 (mother solution) DP47GS- IgG-IL2QM 5 mg/kg 20mM histidine, 140mM NaCl, pH 6.0 5.87 (mother solution)

Example 17 Single Dose Pharmacokinetic Characteristics of Non-Targeted Fab-IL2 wt-Fab and Fab-IL2 qm-Fab Immunoconjugates

A single dose pharmacokinetic (PK) study of non-targeted Fab-IL2-Fab immunocojugates containing either wild-type or four-mutant IL-2 was performed in tumor-free 129 immunocompetent mice.

Female mice of line 129 (Harlan, UK), which were 8–9 weeks old at the start of the experiment, were kept under special pathogen-free conditions with daily cycles of 12 h light/12 h dark according to accepted guidelines (GV-Solas; Felasa; TierschG) . The pilot study protocol was reviewed and approved by the local authorities (R 2008016). After delivery, the animals were kept for one week to acclimatize to the new environment and for examination. During the entire period, health status was regularly monitored.

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Claims (17)

Mice were injected with i.v. immunoconjugates DP47GS Fab-IL2 wt-Fab at a dose of 65 nmol/kg or DP47GS Fab-IL2 qm-Fab at a dose of 65 nM/kg. All mice were injected with i.v. 200 µl of the corresponding solution. To obtain the appropriate amount of immunoconjugate per 200 μl, stock solutions were diluted with PBS if necessary. Mice were bled at the following time points: 0.5, 1, 3, 8, 24, 48, 72, 96 hours and every 2 days thereafter for 3 weeks. Serum was extracted and stored at -20°C until the ELISA analysis. Serum concentrations of the immunoconjugates were determined using ELISA to quantify the antibody contained in the IL-2-containing immunoconjugate (Roche Penzberg). Absorbance was determined by measuring at a wavelength of 405 nm and a reference wavelength of 492 nm (adjustable VersaMax type microplate reader, Molecular Devices). In FIG. 42 shows the results of a pharmacokinetic study of these IL-2 immunoconjugates. The Fab-IL2-Fab wt and qm constructs had a serum half-life of approximately 3-4 hours. The difference in serum half-life between constructs containing wild-type IL-2 or four-mutant IL-2 was less pronounced for Fab-IL2 -Fab constructs than for IgG-like immunoconjugates, which themselves had a longer half-life. Table 21 Compound Dose Buffer Composition Concentration (mg/ml) DP47GS Fab-IL2 wt- Fab 6 5 nM/kg 100 M glycine, 125 mM NaCI, 25 mM KH ₂ PO ₄ , pH 6.7 3.84 (stock solution) DP47GS Fab-IL2 qm-Fab 6 5 nM/kg 100mM glycine, 125mM NaCl, 25mM KH ₂ PO ₄ , pH 6.7 2.42 (stock solution) Example 18 Activation of Induced Cell Death of Activated IL-2 PBMCs PBMC freshly isolated from the body of healthy donors were preactivated overnight with PHA-M at a concentration of 1 μg/ml in RPMI1640 medium containing 10% FCS and 1% glutamine. After pre-activation, PBMCs were harvested, labeled with 40nM CFSE in PBS, and plated in 96-well plates at 100,000 cells/well. Preactivated PBMCs were stimulated using various concentrations of IL-2 immunoconjugates (4B9 IgG-IL-2 wt, 4B9 IgG-IL-2 qm, 4B9 Fab-IL-2 wt-Fab and 4B9 Fab-IL-2 qm-Fab) . Six days after treatment with IL-2, the PBMCs were treated overnight with an activating anti-Fas antibody at a concentration of 0.5 μg/ml. After 6 days, the proliferation of CD4-(CD3 ⁺ CD8 ^- ) and CD8 ^- (CD3 ⁺ CD8 ⁺ ) T cells was analyzed using CFSE dilutions. The percentage of live T cells after treatment with antibody to Fas was determined by setting a discrimination window on CD3 ⁺ - annexation of V-negative live cells. As shown in FIG. 44, all constructs induced the proliferation of pre-activated T cells. When used at low concentrations, constructs containing wild-type IL-2 (wt) were more active than constructs containing IL-2 qm. IgG-IL-2 wt, Fab-IL2 wt-Fab and proleukin had similar activity. The Fab-IL-2 qm-Fab construct had slightly lower activity than IgG-IL-2 qm. Constructs containing wild-type IL-2 had higher activity against CD4 T cells than against CD8 T cells, most likely due to the activation of regulatory T cells. Constructs containing four-mutant IL-2 had similar activity against CD8 ^- and CD4 ^- T cells. As shown in FIG. 45, T cells stimulated with high concentrations of wild-type IL-2 were more sensitive to anti-Fas antibody-induced apoptosis than T cells treated with four-mutant IL-2. Although the invention has been described above in sufficient detail with the aid of illustrations and examples provided for purposes of better understanding, the description and examples should not be construed as limiting the scope of the invention. All patent and scientific publications cited in this specification are incorporated herein by reference in their entirety. CLAIM 1. An immunoconjugate containing a mutant IL-2 polypeptide and an antigen-binding fragment, wherein said mutant IL-2 polypeptide is a human IL-2 molecule containing amino acid substitutions F42A, Y45A and L72G (numbering corresponds to the sequence of human IL-2 SEQ ID NO: 1 ); and wherein said antigen-binding fragment is an immunoglobulin molecule of the IgG1 subclass specific for fibroblast-activating protein (FAP) comprising (i) the sequence of SEQ ID NO: 41 of the heavy chain variable region and the sequence - 75 040858 SEQ ID NO: 39 light chain variable region; (ii) the sequence of SEQ ID NO: 51 of the heavy chain variable region and the sequence SEQ ID NO: 49 light chain variable region; (iii) the sequence of SEQ ID NO: 111 of the heavy chain variable region and the sequence SEQ ID NO: 109 light chain variable region; (iv) the heavy chain variable region SEQ ID NO: 143 and the light chain variable region SEQ ID NO: 141; or (iv) the heavy chain variable region SEQ ID NO: 151 and the light chain variable region SEQ ID NO: 149. 2. The immunoconjugate of claim 1, wherein said IL-2 mutant polypeptide further comprises T3A amino acid substitutions and/or C125A amino acid substitutions. 3. An immunoconjugate according to claim 1 or 2, wherein said mutant IL-2 polypeptide contains the sequence of SEQ ID NO: 19. 4. An immunoconjugate according to any one of claims 1 to 3, wherein said mutant IL-2 polypeptide is connected by its amino-terminal amino acid to the carboxy-terminal amino acid of one of the immunoglobulin heavy chains. 5. An immunoconjugate according to any one of claims 1 to 4, wherein the immunoconjugate contains no more than one mutant IL-2 polypeptide. 6. An immunoconjugate according to any one of claims 1 to 5, wherein the IgG1 subclass immunoglobulin molecule contains a modification in the Fc domain that promotes heterodimerization of two non-identical immunoglobulin heavy chains. 7. The immunoconjugate according to claim 6, where the modification is a knob-intohole type modification (protrusion-hollow), containing a protrusion modification in one of the immunoglobulin heavy chains and a cavity modification in the other immunoglobulin heavy chain, where the protrusion modification contains the T366W amino acid substitution in one of the two immunoglobulin heavy chains, and the trough modification contains the amino acid substitutions T366S, L368A and Y407V in the other one of the two immunoglobulin heavy chains. 8. The immunoconjugate of claim 7, wherein the immunoglobulin heavy chain containing the ridge modification further comprises the S354C amino acid substitution, and the immunoglobulin heavy chain containing the trough modification further comprises the Y349C amino acid substitution. 9. An immunoconjugate according to any one of claims 1 to 8, wherein the IgG1 molecule contains in its Fc domain one or more amino acid mutations that reduce the binding affinity of the immunoconjugate for the FcyRIIIa, FcyRI or FcyRIIa receptor. 10. The immunoconjugate of claim 9, wherein the IgG1 molecule contains the amino acid mutations L234A, L235A and 3329G. 11. An immunoconjugate according to any one of claims 1 to 10, comprising (i) a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 297, a polypeptide sequence that is at least about 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 299, 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; (ii) a polypeptide sequence that is at least about 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 301, a polypeptide sequence that is at least about 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 303, 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 (iii) a polypeptide sequence that is at least about 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 315, a polypeptide sequence that is at least about 80, 85 , 90, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 317, a polypeptide sequence that is at least about 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical SEQ ID NO: 233. 12. An immunoconjugate according to any one of claims 1 to 11, wherein said FAP-specific IgG1 immunoglobulin molecule comprises the heavy chain variable region of SEQ ID NO: 111 and the light chain variable region of SEQ ID NO: 109. 13. An immunoconjugate according to any one of claims 1 to 12, comprising the polypeptide sequence of SEQ ID NO: 301, the polypeptide sequence of SEQ ID NO: 303, and the polypeptide sequence of SEQ ID NO: 231. 14. An immunoconjugate according to any one of claims 1 to 13, essentially consisting of a mutant IL-2 polypeptide and an IgG1 molecule linked by a linker sequence. 15. An isolated polynucleotide encoding an immunoconjugate according to any one of claims 1-14. 16. A host cell containing a polynucleotide according to claim 15. 17. A method for producing an immunoconjugate according to any one of claims 1-14, containing a mutant polypeptide -