CN114051500A - Immunoconjugates comprising interleukin-2 mutants and anti-CD 8 antibodies - Google Patents

Abstract

The present invention relates generally to immunoconjugates, and in particular to immunoconjugates comprising an interleukin-2 polypeptide mutant and an antibody that binds to CD 8. Furthermore, the invention relates to polynucleotide molecules encoding said immunoconjugates, as well as vectors and host cells comprising such polynucleotide molecules. The invention further relates to methods for producing said mutant immunoconjugates, pharmaceutical compositions comprising said mutant immunoconjugates, and uses thereof.

Description

Immunoconjugates comprising interleukin-2 mutants and anti-CD 8 antibodies

Technical Field

The present invention relates generally to immunoconjugates, and in particular to immunoconjugates comprising an interleukin-2 polypeptide mutant and an antibody that binds to CD8, said immunoconjugate being directed against CD8 only⁺The cells have strong cis-targeting effect. Furthermore, the invention relates to polynucleotide molecules encoding said immunoconjugates, as well as vectors and host cells comprising such polynucleotide molecules. The invention further relates to methods for producing said mutant immunoconjugates, pharmaceutical compositions comprising said mutant immunoconjugates, and uses thereof.

Background

Interleukin-2 (IL-2), also known as T Cell Growth Factor (TCGF), is a 15.5kDa globular glycoprotein that plays a central role in lymphocyte production, survival and homeostasis. It has a length of 133 amino acids and consists of four antiparallel amphipathic α -helices that form a quaternary structure essential for its function (Smith, Science 240,1169-76 (1988); Bazan, Science 257, 410-. The sequence of IL-2 from different species is found under NCBI RefSeq No. NP000577 (human), NP032392 (mouse), NP446288 (rat) or NP517425 (chimpanzees).

IL-2 mediates its effects by binding to the IL-2 receptor (IL-2R), which consists of up to three separate subunits, whose different associations can give rise to receptor forms with different IL-2 affinities. α (CD25), β(CD122) and γ (γ)_cCD132) subunit association results in a high affinity trimeric IL-2 receptor. The dimeric IL-2 receptor, which consists of a beta subunit and a gamma subunit, is known as the medium affinity IL-2R receptor. The alpha subunit forms a low affinity monomeric IL-2 receptor. Although the intermediate affinity dimeric IL-2 receptor binds IL-2 with approximately 100-fold lower affinity than the high affinity trimeric receptor, both dimeric and trimeric IL-2 receptor variants are able to signal upon IL-2 binding (Minami et al, Annu Rev Immunol 11,245-268 (1993)). Thus, the α -subunit CD25 is not essential for IL-2 signaling. The α -subunit confers high affinity binding to its receptor, while the β -subunits, CD122 and γ, are critical for signaling (Krieg et al, Proc Natl Acad Sci 107,11906-11 (2010)). Trimeric IL-2 receptor comprising CD25 consisting of (resting) CD4⁺Fork head frame P3(FoxP3)⁺Regulatory T (T)_reg) And (4) expressing the cells. They are also transiently induced on conventionally activated T cells, whereas in the resting state, these cells only express dimeric IL-2 receptor. T is_regCells consistently expressed the highest level of CD25 in vivo (Fontent et al, Nature Immunol 6,1142-51 (2005)).

IL-2 is synthesized predominantly by activated T cells, in particular by CD4⁺Helper T cell synthesis. It stimulates proliferation and differentiation of T cells, induces production of Cytotoxic T Lymphocytes (CTLs) and differentiation of peripheral blood lymphocytes into cytotoxic and Lymphokine Activated Killer (LAK) cells, promotes expression of cytokines and cytolytic molecules of T cells, facilitates proliferation and differentiation of B cells and immunoglobulin synthesis of B cells, and stimulates production, proliferation and activation of Natural Killer (NK) cells (e.g., as in Waldmann, Nat Rev Immunol 6, 595-.

Its ability to expand lymphocyte populations in vivo and increase the effector functions of these cells confers IL-2 anti-tumor effects, making IL-2 immunotherapy an attractive treatment option for certain metastatic cancers. Thus, high dose IL-2 therapy has been approved for patients with metastatic renal cell carcinoma and malignant melanoma.

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

The main mechanism of peripheral self-tolerance is activation-induced cell death (AICD) of IL-2-induced T cells. AICD is a process by which fully activated T cells undergo programmed cell death by engaging cell surface-expressed death receptors, such as CD95 (also known as Fas) or TNF receptors. When antigen-activated T cells expressing a high affinity IL-2 receptor (following prior exposure to IL-2) during proliferation are restimulated by antigen via the T Cell Receptor (TCR)/CD 3 complex, expression of Fas ligand (FasL) and/or Tumor Necrosis Factor (TNF) is induced, rendering the cells susceptible to Fas-mediated apoptosis. This process is IL-2 dependent (Lenardo, Nature 353,858-61(1991)) and is mediated via STAT 5. Through the process of AICD in T lymphocytes, tolerance can be established not only against self-antigens, but also against persistent antigens (such as tumor antigens) that are apparently not integral parts of the host.

In addition, IL-2 is also involved in the maintenance of peripheral CD4⁺CD25⁺Regulatory T (T)_reg) Cells (Fontent et al, Nature Immunol 6,1142-51 (2005); d' Cruz and Klein, Nature Immunol 6,1152-59 (2005); malony and Powrie, Nature Immunol 6,1171-72(2005)), which are also known as suppressor T cells. They inhibit the destruction of their (own) targets by effector T cells by cell-cell contact with the help and activation of suppressor T cells, or by the release of immunosuppressive cytokines such as IL-10 or TGF- β. T is_regDepletion of cells was shown to enhance IL-2-induced anti-tumor immunity (Imai et al, Cancer Sci 98,416-23 (2007)).

Therefore, IL-2 is not optimal for inhibiting tumor growth, because in the presence of IL-2, the generated CTLs may recognize the tumor as self and undergo AICD, or the immune response may beBy IL-2 dependent T_regAnd (4) inhibiting cells.

Another problem associated with IL-2 immunotherapy is the side effects resulting from recombinant human IL-2 treatment. Patients receiving high dose IL-2 treatment often experience severe cardiovascular, pulmonary, renal, hepatic, gastrointestinal, neurological, skin, blood and systemic adverse events that require close monitoring and hospitalization. Most of these side effects can be explained by the development of the so-called vascular (or capillary) leak syndrome (VLS), a pathological increase in vascular permeability leading to fluid extravasation in multiple organs (leading to, for example, lung and skin edema and liver cell damage) and intravascular fluid depletion (leading to a drop in blood pressure and compensatory increase in heart rate). There is no other treatment for VLS other than IL-2 elimination. Low dose IL-2 regimens have been tested in patients to avoid VLS, but at the cost of suboptimal treatment outcomes. VLS is thought to be caused by the release of pro-inflammatory cytokines such as Tumor Necrosis Factor (TNF) - α from IL-2 activated NK cells, however it has recently been shown that IL-2 induced pulmonary edema is caused by the direct binding of IL-2 to lung endothelial cells that express low to moderate levels of functional α β γ IL-2 receptor (Krieg et al, Proc Nat Acad Sci USA 107,11906-11 (2010)).

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

WO2012/107417 describes a vaccine designed to overcome the above-mentioned problems associated with IL-2 immunotherapy (toxicity induced by VLS, tumor tolerance induced by AICD, and T_regImmunosuppression by cell activation). The substitution of alanine for the phenylalanine residue at position 42, alanine for the tyrosine residue at position 45, and glycine for the leucine residue at position 72 of IL-2 substantially eliminates the binding of the IL-2 polypeptide mutant to the alpha subunit of the IL-2 receptor (CD 25).

Furthermore, for the above methods, IL-2 immunotherapy can be improved by selectively targeting IL-2 to tumors, e.g., in the form of immunoconjugates comprising antibodies that bind to antigens expressed on tumor cells. Several such immunoconjugates have been described (see, e.g., Ko et al, J Immunother (2004)27, 232-239; Klein et al, Oncoimunology (2017)6(3), e 1277306).

However, tumors may be able to evade this targeting by shedding, mutating or downregulating the target antigen of the antibody. Furthermore, in tumor microenvironments that actively exclude lymphocytes, tumor-targeted IL-2 may not be in optimal contact with effector cells such as Cytotoxic T Lymphocytes (CTLs).

Thus, there remains a need for further improvements in IL-2 immunotherapy. One approach that may avoid the tumor targeting problem is to target IL-2 directly to effector cells, in particular CTLs.

Ghasemi et al have described a fusion protein of IL-2 with a NKG2D binding protein (Ghashmemi et al, Nat Comm (2016)7,12878) for targeting IL-2 to NKG 2D-bearing cells, such as Natural Killer (NK) cells.

Characterization of the number, type, and spatial distribution of immune cells in tumor tissue can provide key information about cancer diagnosis, prognosis, therapy selection, and response to therapy. In particular, CD8⁺Cytotoxic lymphocytes have been reported to have diagnostic and prognostic significance in a variety of cancers. Antibodies that bind to CD8 are described, for example, in WO 2019/033043 a 2.

Disclosure of Invention

The present invention provides a novel method of targeting mutated forms of IL-2 with the advantageous properties of immunotherapy directly against immune effector cells such as cytotoxic T lymphocytes, but not against tumor cells. Targeting of immune effector cells is achieved by conjugating the IL-2 molecular mutants to antibodies that bind to CD 8.

The IL-2 mutants used in the present invention have been designed to overcome the problems associated with IL-2 immunotherapy, in particular toxicity induced by VLS, tumor tolerance induced by AICD, and T_regImmunosuppression caused by cell activation. In addition to avoiding tumor-targeted escape from tumors as described above, targeting the IL-2 mutant to immune effector cells may further increase CTL versus immunosuppressed T_regPreferential activation of cells. Antibody pair CD8 that binds CD8 as disclosed herein⁺The cells have strong cis-targeting effect. Thus, they do not interfere with the interaction of the T cell antigen receptor (TCR) and the peptide-bound major histocompatibility complex (pMHC). In this regard, the antibody is non-functional.

In a first aspect, the invention provides an immunoconjugate comprising a mutant interleukin-2 (IL-2) polypeptide and an antibody that binds to CD8, wherein the IL-2 polypeptide is a mutant of IL-2 polypeptide comprising the amino acid substitutions F42A, Y45A, and L72G (numbered relative to the sequence of human IL-2 as set forth in SEQ ID NO: 13).

In another aspect, the invention provides an immunoconjugate comprising a mutant interleukin-2 (IL-2) polypeptide and an antibody that binds to CD8, wherein the IL-2 polypeptide mutant is a human IL-2 molecule comprising the amino acid substitutions F42A, Y45A, and L72G (numbering relative to the sequence of human IL-2 as set forth in SEQ ID NO: 13); and wherein the antibody comprises: (a) a heavy chain variable region (VH) comprising: heavy chain complementarity determining region (HCDR)1 comprising the amino acid sequence of SEQ ID NO. 1, HCDR2 comprising the amino acid sequence of SEQ ID NO. 2, and HCDR3 comprising the amino acid sequence of SEQ ID NO. 3; and (b) a light chain variable region (VL) comprising: light chain complementarity determining region (LCDR)1 comprising the amino acid sequence of SEQ ID NO. 4, LCDR2 comprising the amino acid sequence of SEQ ID NO. 5, and LCDR3 comprising the amino acid sequence of SEQ ID NO. 6 or SEQ ID NO. 28.

In another aspect, the invention provides an immunoconjugate comprising an interleukin-2 (IL-2) polypeptide mutant and an antibody that binds to CD8, wherein the IL-2 polypeptide mutant is a human IL-2 molecule comprising the amino acid substitutions F42A, Y45A, and L72G (numbered relative to the human IL-2 sequence SEQ ID NO: 13); and wherein the antibody comprises: (a) a heavy chain variable region (VH) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No.7, and (b) a light chain variable region (VL) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 8 or SEQ ID No. 29.

In some embodiments of the immunoconjugates of the invention, the IL-2 polypeptide mutant further comprises the amino acid substitution T3A and/or the amino acid substitution C125A.

In some embodiments, the IL-2 polypeptide mutant comprises an amino acid sequence as set forth in SEQ ID NO 14.

In some embodiments, the immunoconjugate comprises no more than one IL-2 polypeptide mutant. In some such embodiments, the antibody comprises an Fc domain comprising a first subunit and a second subunit (consisting of the first subunit and the second subunit). In some such embodiments, the Fc domain is an Fc domain of the IgG class, particularly the IgG1 subclass, and/or a human Fc domain. In some embodiments, the antibody is an IgG class, particularly an IgG1 subclass immunoglobulin.

In some embodiments, wherein the immunoconjugate comprises an Fc domain comprising a modification that facilitates association of the first subunit and the second subunit of the Fc domain. In some embodiments, in the CH3 domain of the first subunit of the Fc domain, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby creating a protuberance within the CH3 domain of the first subunit that can be positioned in a cavity within the CH3 domain of the second subunit; and in the CH3 domain of the second subunit of the Fc domain, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit can be positioned. In some embodiments, the threonine residue at position 366 is replaced with a tryptophan residue in the first subunit of the Fc domain (T366W); and in the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V), and optionally the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the Kabat EU index). In some such embodiments, in the first subunit of the Fc domain, additionally, the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C), and in the second subunit of the Fc domain, additionally, the tyrosine residue at position 349 is replaced with a cysteine residue (Y349C) (numbering according to the EU index of Kabat). In some embodiments, the IL-2 polypeptide mutant is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the subunits of the Fc domain, in particular to the carboxy-terminal amino acid of the first subunit of the Fc domain, optionally via a linker peptide. 18. In some such embodiments, the linking linker peptide has the amino acid sequence of SEQ ID NO 15.

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

In some embodiments according to the invention, the immunoconjugate comprises: a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO 9 or SEQ ID NO 30, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO 10, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO 11 or SEQ ID NO 12.

In some embodiments, the immunoconjugate comprises a polypeptide comprising the amino acid sequence of SEQ ID NO 9 or SEQ ID NO 29, a polypeptide comprising the amino acid sequence of SEQ ID NO 10, and a polypeptide comprising the amino acid sequence of SEQ ID NO 11.

In some embodiments, the immunoconjugate comprises a polypeptide comprising the amino acid sequence of SEQ ID NO 9 or SEQ ID NO 29, a polypeptide comprising the amino acid sequence of SEQ ID NO 10, and a polypeptide comprising the amino acid sequence of SEQ ID NO 12.

In some embodiments, the immunoconjugate consists essentially of an IL-2 polypeptide mutant and an IgG1 immunoglobulin molecule joined by a linking sequence.

The invention further provides one or more isolated polynucleotides encoding the inventive immunoconjugates described herein, one or more vectors (particularly expression vectors) comprising said polynucleotides, and host cells comprising said polynucleotides or said vectors.

Also provided is a method of producing an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to CD8, the method comprising (a) culturing a host cell of the invention under conditions suitable for expression of the immunoconjugate, and optionally (b) recovering the immunoconjugate. The invention also provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to CD8, produced by the method.

The invention also provides a pharmaceutical composition comprising the immunoconjugate of the invention and a pharmaceutically acceptable carrier, and methods of using the immunoconjugate of the invention.

In particular, the invention comprises immunoconjugates according to the invention for use as medicaments and for the treatment of diseases. In a particular embodiment, the disease is cancer.

The invention also includes the use of an immunoconjugate according to the invention in the manufacture of a medicament for the treatment of a disease. In a particular embodiment, the disease is cancer.

Further provided is a method of treating a disease in an individual, the method comprising administering to the individual a therapeutically effective amount of a composition comprising an immunoconjugate according to the invention in a pharmaceutically acceptable form. In a particular embodiment, the disease is cancer.

Further provided is a method of stimulating the immune system of an individual, the method comprising administering to the individual an effective amount of a composition comprising an immunoconjugate according to the invention in a pharmaceutically acceptable form.

Drawings

FIG. 1A and FIG. 1B schematic representation of IgG-IL-2 immunoconjugate format comprising a mutant IL-2 polypeptide. (FIG. 1A) dual arm anti-CD 8; (FIG. 2A) one-armed anti-CD 8.

FIG. 2. determination of binding of both double-arm anti-CD 8-IL2v (CD8-IL2v TA) and single-arm anti-CD 8-IL2v (CD8-IL2v OA) to CD 8T cells in resting PBMC compared to anti-FAP-IL 2v (FAP-IL2v) by flow cytometry. Molecules were detected with a fluorescently labeled anti-human Fc specific secondary antibody. CD 8T cells were identified using CD3 and CD8 staining of PBMCs.

3A, 3B, 3C and 3D STAT5 phosphorylation in CD 8T cells (FIG. 3A), CD 4T cells (FIG. 3C), regulatory T cells (FIG. 3D) and NK cells (FIG. 3D) was determined by flow cytometry following treatment of resting PBMCs with CD8-IL2v TA, CD8-IL2v OA and FAP-IL2 v.

FIG. 4A, FIG. 4B and FIG. 4C proliferation of NK cells (FIG. 4C), CD 4T cells (FIG. 4B) and CD 8T cells (FIG. 4C) in PBMCs using CD8-IL2v TA, CD8-IL2v OA and FAP-IL2v was determined by flow cytometry.

FIG. 5A, FIG. 5B, FIG. 5C and FIG. 5D STAT5 phosphorylation in CD 8T cells (FIG. 5A), NK cells (FIG. 5B), CD 4T cells (FIG. 5C) and regulatory T cells (FIG. 5D) following treatment of resting PBMC with CD8-IL2v TA, CD8-IL2v OKT8.v11 TA and FAP-IL2v was determined by flow cytometry.

Detailed Description

Definition of

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

Unless otherwise indicated, the term "interleukin-2" or "IL-2" as used herein refers to any native IL-2 from any vertebrate source, including mammals such as primates (e.g., humans), as well as rodents (e.g., mice and rats). The term includes unprocessed IL-2 as well as any form of IL-2 produced by processing in a cell. The term also encompasses naturally occurring IL-2 variants, such as splice variants or allelic variants. The amino acid sequence of an exemplary human IL-2 is shown in SEQ ID NO 13. Unprocessed human IL-2 additionally comprises the N-terminal 20 amino acid signal peptide having the amino acid sequence shown in SEQ ID NO 19, which signal peptide is not present in the mature IL-2 molecule.

The term "IL-2 mutant" or "IL-2 polypeptide mutant" as used herein is intended to encompass any mutant form of the various forms of IL-2 molecules, including full-length IL-2, truncated forms of IL-2, and forms in which IL-2 is linked to another molecule, such as by fusion or chemical conjugation. When used with respect to IL-2, "full length" is intended to mean a mature, native-length IL-2 molecule. For example, full-length human IL-2 refers to a molecule having 133 amino acids (see, e.g., SEQ ID NO: 13). Various forms of IL-2 mutants are characterized as having at least one amino acid mutation that affects the interaction of IL-2 with CD 25. The mutation may involve a substitution, deletion, truncation or modification of the wild type amino acid residue normally located at that position. Mutants obtained by amino acid substitution are preferred. Unless otherwise indicated, IL-2 mutants may be referred to herein as IL-2 mutant peptide sequences, IL-2 polypeptide mutants, IL-2 protein mutants, or IL-2 mutant analogs.

Various forms of IL-2 are named herein with respect to the sequence shown in SEQ ID NO 13. Various names may be used herein to indicate the same mutation. For example, the mutation from phenylalanine to alanine at position 42 can be represented as 42A, A42, A₄₂F42A or Phe42 Ala.

As used herein, a "human IL-2 molecule" refers to an IL-2 molecule comprising an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, or at least about 96% identical to the human IL-2 sequence as set forth in SEQ ID NO 13. Specifically, the sequence identity is at least about 95%, more specifically at least about 96%. In particular embodiments, the human IL-2 molecule is a full-length IL-2 molecule.

The term "amino acid mutation" as used herein is meant to encompass amino acid substitutions, deletions, insertions and modifications. Any combination of substitutions, deletions, insertions and modifications may be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, such as reduced binding to CD 25. Amino acid sequence deletions and insertions include amino-terminal and/or carboxy-terminal deletions and insertions of amino acids. An example of a terminal deletion is the deletion of an alanine residue in position 1 of full-length human IL-2. Preferred amino acid mutations are amino acid substitutions. Non-conservative amino acid substitutions, i.e., the substitution of one amino acid with another having different structural and/or chemical properties, are particularly preferred for the purpose of altering the binding characteristics of, for example, an IL-2 polypeptide. Preferred amino acid substitutions include the substitution of hydrophobic amino acids with hydrophilic amino acids. Amino acid substitutions include substitutions with non-naturally occurring amino acids or with naturally occurring amino acid derivatives of twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, and the like. It is also contemplated that methods of altering amino acid side chain groups by methods other than genetic engineering, such as chemical modification, are also useful.

As used herein, a "wild-type" form of IL-2 is a form of IL-2 that is otherwise identical to an IL-2 polypeptide mutant except that the wild-type form has a wild-type amino acid at each amino acid position of the IL-2 polypeptide mutant. For example, if the IL-2 mutant is full-length IL-2 (i.e., IL-2 is not fused or conjugated to any other molecule), then the wild-type form of the mutant is full-length native IL-2. If the IL-2 mutant is a fusion between IL-2 and another polypeptide encoded downstream of IL-2 (e.g., an antibody chain), then the wild-type form of the IL-2 mutant is IL-2 having the wild-type amino acid sequence fused to the same downstream polypeptide. Furthermore, if the IL-2 mutant is a truncated form of IL-2 (a mutated or modified sequence within the non-truncated portion of IL-2), then the wild-type form of the IL-2 mutant is a similarly truncated IL-2 having the wild-type sequence. For the purpose of comparing the IL-2 receptor binding affinity or biological activity of various forms of IL-2 mutants with the corresponding wild-type forms of IL-2, the term wild-type encompasses forms of IL-2 which comprise one or more amino acid mutations which do not affect IL-2 receptor binding compared to the naturally occurring native IL-2, e.g. the substitution of an alanine with a cysteine at the position corresponding to residue 125 of human IL-2. In some embodiments, the wild-type IL-2 used for the purposes of the present invention comprises the amino acid substitution C125A (see SEQ ID NO: 20). In certain embodiments according to the invention, the wild-type IL-2 polypeptide compared to the mutant IL-2 polypeptide comprises the amino acid sequence shown in SEQ ID NO 13. In other embodiments, the wild-type IL-2 polypeptide compared to the mutant IL-2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 20.

The term "CD 25" or "alpha subunit of the IL-2 receptor" as used herein, unless otherwise indicated, refers to any native CD25 from any vertebrate source, including mammals such as primates (e.g., humans), as well as rodents (e.g., mice and rats). The term includes "full-length" unprocessed CD25, as well as any form of CD25 produced by processing in a cell. The term also encompasses naturally occurring variants of CD25, such as splice variants or allelic variants. In certain embodiments, CD25 is human CD 25. The amino acid sequence of human CD25 is found, for example, in UniProt accession number P01589 (185 th edition).

The term "high affinity IL-2 receptor" as used herein refers to a heterotrimeric form of the IL-2 receptor, which consists of a receptor gamma subunit (also known as a common cytokine receptor gamma subunit, gamma)_cOr CD132, see UniProt accession number P14784 (192 th edition)), receptor beta subunit (also known as CD122 or P70, see UniProt accession number P31785 (197 th edition)), and receptor alpha subunit (also known as CD25 or P55, see UniProt accession number P01589 (185 th edition)). In contrast, the term "intermediate affinity IL-2 receptor" refers to an IL-2 receptor that contains only gamma and beta subunits, but no alpha subunits (for a review see, e.g., Olejniczak and Kasprzak, Med Sci Monit 14, RA179-189 (2008)).

"affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). As used herein, unless otherwise specified, "binding affinity" refers to an intrinsic binding affinity that reflects the member of a binding pair (e.g., antigen binding moiety and anti-antigen)Pro, or receptor and its ligand) in a 1:1 interaction. The affinity of a molecule X for its partner Y can generally be determined by the dissociation constant (K)_D) Expressed as the dissociation and association rate constants (k, respectively)_offAnd k_on) The ratio of (a) to (b). Thus, equivalent affinities may comprise different rate constants, as long as the ratio of rate constants remains the same. Affinity can be measured by well-established methods known in the art, including those described herein. A particular method of measuring affinity is Surface Plasmon Resonance (SPR).

The affinity of mutant or wild-type IL-2 polypeptides for various forms of IL-2 receptor can be determined by Surface Plasmon Resonance (SPR) according to the methods described in WO2012/107417 using standard instruments such as the BIAcore instrument (GE Healthcare) and receptor subunits such as can be obtained by recombinant expression (see, e.g., Shanafelt et al, Nature Biotechnol 18,1197-1202 (2000)). Alternatively, cell lines known to express one or the other of the forms of the receptor can be used to assess the binding affinity of the IL-2 mutants to different forms of the IL-2 receptor. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

"regulatory T cells" or "T_regBy cell "is meant a particular type of CD4 that is capable of inhibiting the response of other T cells⁺T cells. T is_regCells are characterized as expressing the alpha subunit of the IL-2 receptor (CD25) and the transcription factor forkhead box P3(FOXP3) (Sakaguchi, Annu Rev Immunol 22,531-62(2004)), and play a critical role in the induction and maintenance of peripheral self-tolerance to antigens, including those expressed by tumors. T is_regCells require IL-2 to fulfill their function and develop and induce their inhibitory characteristics.

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

By "specific binding" is meant that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antibody to bind a particular antigen (e.g., CD8) can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as Surface Plasmon Resonance (SPR) techniques (e.g., as analyzed on a BIAcore instrument) (Liljeblad et al, Glyco J17, 323-. In one embodiment, the extent of binding of an antibody to an unrelated protein is less than about 10% of the binding of the antibody to the antigen, as measured, for example, by SPR. The antibodies contained in the immunoconjugates described herein specifically bind to CD 8.

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

An "isolated" polypeptide or variant or derivative thereof means a polypeptide that is not in its natural environment. No specific level of purification is required. For example, an isolated polypeptide can be removed from its natural or native environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purposes of the present invention, as are native or recombinant polypeptides that have been isolated, fractionated or partially or substantially purified by any suitable technique.

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a reference polypeptide sequence, after aligning the candidate sequence to the reference polypeptide sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without regard to any conservative substitutions as part of the sequence identity. Alignments to determine percent amino acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA package. One skilled in the art can determine appropriate parameters for aligning the sequences, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared. However, for purposes herein, the BLOSUM50 comparison matrix was used to generate values for% amino acid sequence identity using the ggsearch program of FASTA package 36.3.8c or higher. The FASTA package is comprised of W.R.Pearson and D.J.Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85: 2444-2448; W.R.Pearson (1996) "Effective protein sequence composition" meth.enzymol.266: 227-; and Pearson et al, (1997) Genomics 46:24-36, and is publicly available from http:// fasta. bioch. virginia. edu/fasta _ www2/fasta _ down. Alternatively, sequences can be compared using a common server accessible at http:// fasta. bioch. virginia. edu/fasta _ www2/index. cgi, using the ggsearch (global protein: protein) program and default options (BLOSUM 50; open: -10; ext: -2; Ktup ═ 2) to ensure that global, rather than local, alignments are performed. The percent amino acid identity is given in the alignment header (alignment header) of the output.

The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), virally-derived RNA, or plasmid DNA (pDNA). The polynucleotide may comprise a conventional phosphodiester bond or an unconventional bond (e.g., an amide bond, such as found in Peptide Nucleic Acids (PNAs)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.

An "isolated" nucleic acid molecule or polynucleotide means a nucleic acid molecule, DNA or RNA that has been removed from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for the purposes of the present invention. Additional embodiments of the isolated polynucleotide include a recombinant polynucleotide maintained in a heterologous host cell or a purified (partially or substantially purified) polynucleotide in solution. An isolated polynucleotide includes a polynucleotide molecule that is contained in a cell that normally contains the polynucleotide molecule, but which is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location. Isolated RNA molecules include RNA transcripts of the invention, either in vivo or in vitro, as well as both positive and negative stranded forms and double stranded forms. Isolated polynucleotides or nucleic acids according to the invention also include such molecules produced synthetically. In addition, the polynucleotide or nucleic acid may be or include regulatory elements such as a promoter, ribosome binding site or transcription terminator.

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

The term "expression cassette" refers to a polynucleotide, produced recombinantly or synthetically, and a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plasmid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of the expression vector includes, among other sequences, the nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassette comprises a polynucleotide sequence encoding an immunoconjugate of the invention, or a fragment thereof.

The term "vector" or "expression vector" refers to a DNA molecule used to introduce a particular gene into a cell in operable association therewith and direct the expression of the particular gene in the cell. The term includes vectors which are self-replicating nucleic acid structures, as well as vectors which integrate into the genome of a host cell into which they have been introduced. The expression vector of the present invention comprises an expression cassette. Expression vectors allow for the transcription of a large number of stable mrnas. After the expression vector is inside the cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cellular transcription and/or translation machinery. In one embodiment, the expression vector of the invention comprises an expression cassette comprising a polynucleotide sequence encoding the immunoconjugate of the invention, or a fragment thereof.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include a primary transformed cell and progeny derived from the primary transformed cell, regardless of the number of passages. Progeny may not be completely identical to the nucleic acid content of the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. The host cell is any type of cellular system that can be used to produce the immunoconjugates of the invention. Host cells include cultured cells, for example mammalian cultured cells such as HEK cells, CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, per.c6 cells or hybridoma cells, yeast cells, insect cells and plant cells, to name a few, and also cells contained in transgenic animals, transgenic plants or cultured plants or animal tissues.

The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, monovalent antibodies (e.g., one-armed antibodies), and antibody fragments, so long as they exhibit the desired antigen binding activity, i.e., binding to CD8 (such as human CD8, cynomolgus monkey CD8, and/or rhesus monkey CD 8).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies included in the population are identical and/or bind the same epitope except for possible variant antibodies, e.g., containing naturally occurring mutations or produced during the production of a monoclonal antibody preparation, such variants typically being present in minute amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates that the characteristics of the antibody are obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods that utilize transgenic animals containing all or part of a human immunoglobulin locus, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

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

The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure that is substantially similar to a native antibody structure.

An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab '-SH, F (ab')₂Diabodies, linear antibodies, single chain antibody molecules (e.g., scFv), and single domain antibodies. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23: 1126-. For reviews of scFv fragments see, for example, Pl ü ckthun in The pharmacolgy of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.269-315 (1994); see also WO 93/16185; and U.S. patent nos. 5,571,894 and 5,587,458. For Fab fragments and F (ab') which contain salvage receptor binding epitope residues and have an extended half-life in vivo₂See U.S. Pat. No. 5,869,046 for a discussion of fragments. Diabodies are antibody fragments with two antigen binding sites, which may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993/01161; hudson et al, Nat Med 9, 129-; and Hollinger et al, Proc Natl Acad Sci USA 90, 6444-. Trisomal and tetrasomal antibodies are also described in Hudson et al, Nat Med 9,129-134 (2003). A single domain antibody is an antibody fragment comprising all or part of a heavy chain variable domain or all or part of a light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516B 1). Antibody fragments can be made by various techniquesPreparation, including but not limited to proteolytic digestion of intact antibodies and production from recombinant host cells (e.g., E.coli or phage), as described herein.

The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of about 150,000 daltons, consisting of two light chains and two heavy chains linked by disulfide bonds. From N-terminus to C-terminus, each heavy chain has a variable domain (VH) (also known as the variable heavy chain domain or heavy chain variable region) followed by three constant domains (CH1, CH2, and CH3) (also known as the heavy chain constant regions). Similarly, from N-terminus to C-terminus, each light chain has a variable domain (VL) (also known as a variable light chain domain or light chain variable region) followed by a constant light Chain (CL) domain (also known as a light chain constant region). The heavy chains of immunoglobulins can be assigned to one of the following five types: referred to as alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG) or mu (IgM), some of which may be further divided into subtypes, e.g. gamma₁(IgG₁)、γ₂(IgG₂)、γ₃(IgG₃)、γ₄(IgG₄)、α₁(IgA₁) And alpha₂(IgA₂). The light chain of an immunoglobulin can be assigned to one of two types based on the amino acid sequence of its constant domain: referred to as kappa (. kappa.) and lambda (. lamda.). An immunoglobulin consists essentially of two Fab molecules and an Fc domain connected by an immunoglobulin hinge region.

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

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved Framework Regions (FR) and three hypervariable regions (HVRs). See, e.g., Kindt et al, Kuby Immunology, 6 th edition, w.h.freeman and co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity. As used herein, "Kabat numbering" in relation to variable region Sequences refers to the numbering system set forth by Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

As used herein, the amino acid positions of all constant regions and constant domains of the heavy and light chains are numbered according to the Kabat numbering system described in Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), and are referred to herein as "numbering according to Kabat" or "Kabat numbering". In particular, the Kabat numbering system (see Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) on pages 647 to 660) was used for the light chain constant domains Cl of the kappa and lambda isoforms, and the Kabat EU index numbering system (see pages 661 to 723) was used for the heavy chain constant domains (CH1, hinge, CH2 and CH3), which is further elucidated herein by in this case being referred to as "numbering according to the Kabat EU index".

As used herein, the term "hypervariable region" or "HVR" refers to each of the following: the antibody variable domains are hypervariable ("complementarity determining regions" or "CDRs") in sequence and/or form structurally defined loops ("hypervariable loops") and/or regions containing antigen-contacting residues ("antigen-contacting points"). Typically, an antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include:

(a) the hypervariable loops which occur at amino acid residues 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1), 53-55(H2) and 96-101(H3) (Chothia and Lesk, J.mol.biol.196:901-917 (1987));

(b) CDRs occurring at amino acid residues 24-34(L1), 50-56(L2), 89-97(L3), 31-35b (H1), 50-65(H2) and 95-102(H3) (Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991));

(c) antigen contacts occurring at amino acid residues 27c-36(L1), 46-55(L2), 89-96(L3), 30-35b (H1), 47-58(H2) and 93-101(H3) (MacCallum et al, J.mol.biol.262:732-745 (1996)); and

(d) combinations of (a), (b), and/or (c) comprising HVR amino acid residues 46-56(L2), 47-56(L2), 48-56(L2), 49-56(L2), 26-35(H1), 26-35b (H1), 49-65(H2), 93-102(H3), and 94-102 (H3).

Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domains are numbered herein according to Kabat et al, supra.

"framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FRs of a variable domain typically consist of the following four FR domains: FR1, FR2, FR3 and FR 4. Thus, the HVR and FR sequences typically occur in the following order in the VH (or VL): FR1-H1(L1) -FR2-H2(L2) -FR3-H3(L3) -FR 4.

A "humanized" antibody is a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. Such variable domains are referred to herein as "humanized variable regions". The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. In some embodiments, some FR residues in the humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., an antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity. "humanized forms" of antibodies (e.g., non-human antibodies) refer to antibodies that have undergone humanization. Other forms of "humanized antibodies" encompassed by the present invention are antibodies in which the constant regions have been otherwise modified or altered relative to the original antibody to produce the properties according to the present invention, particularly with respect to C1q binding and/or Fc receptor (FcR) binding.

A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or derived from an antibody of non-human origin using a human antibody repertoire or other human antibody coding sequences. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues. In certain embodiments, the human antibody is derived from a non-human transgenic mammal, such as a mouse, rat, or rabbit. In certain embodiments, the human antibody is derived from a hybridoma cell line. Antibodies or antibody fragments isolated from a human antibody library are also considered herein to be human antibodies or human antibody fragments.

The "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region that the heavy chain has. There are five major classes of antibodies: IgA, IgD, IgE, IgG and IgM, and some of them may be further divided into subclasses (isotypes), e.g. IgG₁、IgG₂、IgG₃、IgG₄、IgA₁And IgA₂. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively.

The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the IgG heavy chain Fc region may be slightly different, the human IgG heavy chain Fc region is generally defined as extending from Cys226 or from Pro230 to the carboxy terminus of the heavy chain. However, the antibody produced by the host cell may undergo post-translational cleavage of one or more, in particular one or two, amino acids from the C-terminus of the heavy chain. Thus, an antibody produced by a host cell by expression of a particular nucleic acid molecule encoding a full-length heavy chain may comprise the full-length heavy chain, or the antibody may comprise a cleaved variant of the full-length heavy chain (also referred to herein as a "cleaved variant heavy chain"). This may be the case where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, according to the Kabat EU index). Thus, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and lysine (K447) of the Fc region may or may not be present. If not otherwise indicated, the amino acid sequence of the heavy chain comprising the Fc domain (or a subunit of the Fc domain as defined herein) is represented herein as lacking the C-terminal glycine-lysine dipeptide. In one embodiment of the invention, a heavy chain comprising subunits of an Fc domain as specified herein is comprised in an immunoconjugate according to the invention, the heavy chain comprising an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to EU index of Kabat). In one embodiment of the invention, a heavy chain comprising a subunit of an Fc domain as specified herein is comprised in an immunoconjugate according to the invention, the heavy chain comprising an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat). The compositions of the invention, such as the pharmaceutical compositions described herein, comprise a population of immunoconjugates of the invention. The population of immunoconjugates can comprise molecules having a full-length heavy chain and molecules having a cleaved variant heavy chain. The population of immunoconjugates can consist of a mixture of molecules having full-length heavy chains and molecules having cleaved variant heavy chains, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the immunoconjugates have cleaved variant heavy chains. In one embodiment of the invention, a composition comprising a population of immunoconjugates of the invention comprises an immunoconjugate comprising a heavy chain comprising subunits of an Fc domain as specified herein and additionally a C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to EU index of Kabat). In one embodiment of the invention, a composition comprising a population of immunoconjugates of the invention comprises an immunoconjugate comprising a heavy chain comprising subunits of an Fc domain as specified herein and an additional C-terminal glycine residue (G446, numbering according to the EU index of Kabat). In one embodiment of the invention, such a composition comprises a population of immunoconjugates, the population of immunoconjugates consisting of: a molecule comprising a heavy chain comprising subunits of an Fc domain as specified herein; a molecule comprising a heavy chain comprising subunits of an Fc domain as specified herein and an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat); and molecules comprising a heavy chain comprising subunits of an Fc domain as specified herein and additionally a C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to EU index of Kabat). Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also known as EU index), as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD,1991 (see also above). As used herein, a "subunit" of an Fc domain refers to one of two polypeptides that form a dimeric Fc domain, i.e., a polypeptide comprising the C-terminal constant region of an immunoglobulin heavy chain, which is capable of stable self-association. For example, subunits of the IgG Fc domain comprise IgG CH2 and IgG CH3 constant domains.

A "modification that facilitates association of a first subunit and a second subunit of an Fc domain" is manipulation of the peptide backbone or post-translational modification of the Fc domain subunits that reduces or prevents association of a polypeptide comprising an Fc domain subunit with the same polypeptide to form a homodimer. As used herein, "association-promoting modifications" specifically include individual modifications to each of the two Fc domain subunits (i.e., the first and second subunits of the Fc domain) for which association is desired, wherein the modifications are complementary to each other to promote association of the two Fc domain subunits. For example, modifications that promote association can alter the structure or charge of one or both of the Fc domain subunits in order to make their association sterically or electrostatically favorable, respectively. Thus, (hetero) dimerization occurs between a polypeptide comprising a first Fc domain subunit and a polypeptide comprising a second Fc domain subunit, which may be different in the sense that the additional components (e.g., antigen binding portions) fused to each subunit are not identical. In some embodiments, the modifications that promote association include amino acid mutations, particularly amino acid substitutions, in the Fc domain. In a particular embodiment, the modification to facilitate association comprises a separate amino acid mutation, in particular an amino acid substitution, to each of the two subunits of the Fc domain.

The term "effector function" when used with respect to an antibody refers to those biological activities attributable to the Fc region of the antibody that vary with the antibody isotype. Examples of antibody effector functions include: c1q binding and Complement Dependent Cytotoxicity (CDC), Fc receptor binding, antibody dependent cell mediated cytotoxicity (ADCC), Antibody Dependent Cellular Phagocytosis (ADCP), cytokine secretion, immune complex mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that results in the lysis of antibody-coated target cells by immune effector cells. The target cell is a cell that specifically binds to an antibody or derivative thereof comprising an Fc region, typically through the N-terminal protein portion of the Fc region. The term "reduced ADCC" as used herein is defined as a reduction in the number of target cells lysed by the ADCC mechanism as defined above in a given time at a given antibody concentration in the medium surrounding the target cells and/or an increase in the antibody concentration necessary to achieve lysis of a given number of target cells in a given time by the ADCC mechanism in the medium surrounding the target cells. ADCC reduction is ADCC mediated by the same antibody produced by the same type of host cell but not yet engineered, relative to the same standard production, purification, formulation and storage methods (which are known to those skilled in the art) used. For example, the reduction in ADCC mediated by an antibody comprising an amino acid substitution in the Fc domain that reduces ADCC is relative to ADCC mediated by the same antibody without the amino acid substitution in the Fc domain. Suitable assays for measuring ADCC are well known in the art (see, e.g., PCT publication No. WO 2006/082515 or PCT publication No. WO 2012/130831).

An "activating Fc receptor" is an Fc receptor that: which upon engagement by the Fc domain of an antibody, triggers a signaling event that stimulates cells bearing the receptor to perform effector functions. Human activating Fc receptors include Fc γ RIIIa (CD16a), Fc γ RI (CD64), Fc γ RIIa (CD32), and Fc α RI (CD 89).

As used herein, the term "engineered, engineered" is considered to include any manipulation of the peptide backbone, or post-translational modification of naturally occurring or recombinant polypeptides or fragments thereof. Engineering includes modification of the amino acid sequence, glycosylation patterns, or side chain groups of individual amino acids, as well as combinations of these methods.

"reduced binding", e.g. reduced binding to Fc receptor or CD25, refers to a reduction in affinity for the corresponding interaction, as measured, for example, by SPR. For clarity, the term also includes reducing the affinity to zero (or below the detection limit of the analytical method), i.e. eliminating the interaction completely. Conversely, "increased binding" refers to an increase in binding affinity for the corresponding interaction.

The term "immunoconjugate" as used herein refers to a polypeptide molecule comprising at least one IL-2 molecule and at least one antibody. The IL-2 molecule can be linked to the antibody through various interactions and in various configurations as described herein. In particular embodiments, the IL-2 molecule is fused to the antibody via a peptide linker. The particular immunoconjugates according to the invention consist essentially of an IL-2 molecule(s) and an antibody(s) joined by one or more linking sequence(s).

By "fusion" is meant that the components (e.g., antibody and IL-2 molecule) are linked by peptide bonds, either directly or via one or more peptide linkers.

As used herein, the terms "first" and "second" with respect to Fc domain subunits and the like are used to facilitate distinguishing when more than one type of moiety is present. The use of these terms is not intended to confer a particular order or orientation to the immunoconjugate unless specifically stated otherwise.

An "effective amount" of an agent refers to the amount necessary to cause a physiological change in the cell or tissue to which the agent is administered.

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

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

The term "pharmaceutical composition" refers to a preparation in a form that allows the biological activity of the active ingredients contained in the preparation to be effective, and which is free of additional components having unacceptable toxicity to the subject to which the composition is to be administered.

By "pharmaceutically acceptable carrier" is meant an ingredient of the pharmaceutical composition that is not toxic to the subject, other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

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

Detailed description of the embodiments

IL-2 polypeptide mutants

The immunoconjugates according to the invention comprise the active compounds of formula (I) and (II) as active ingredients for immunotherapyIL-2 polypeptide mutants of advantageous properties. In particular, the pharmacological properties of IL-2 that contribute to toxicity but are not essential to the efficacy of IL-2 are eliminated in IL-2 polypeptide mutants. Such IL-2 polypeptide mutants are described in detail in WO2012/107417, which is incorporated herein by reference in its entirety. As mentioned above, different forms of IL-2 receptors are composed of different subunits and exhibit different IL-2 affinities. The intermediate affinity IL-2 receptor, consisting of beta and gamma receptor subunits, is expressed on resting effector cells and is sufficient for IL-2 signaling. In addition, the high affinity IL-2 receptor, which comprises the alpha subunit of this receptor, is primarily involved in regulatory T (T)_reg) On cells and on activated effector cells, wherein the engagement of the receptor with IL-2 promotes T, respectively_regCell-mediated immunosuppression or activation-induced cell death (AICD). Thus, without being bound by theory, reducing or eliminating the affinity of IL-2 for the alpha subunit of the IL-2 receptor should reduce IL-2-induced down regulation of effector cell function by regulatory T cells and the development of tumor tolerance by the AICD process. On the other hand, maintaining affinity for the medium affinity IL-2 receptor should maintain induction of proliferation and activation of IL-2 to effector cells such as NK and T cells.

The interleukin-2 (IL-2) polypeptide mutants comprised in the immunoconjugates according to the invention comprise at least one amino acid mutation that eliminates or reduces the affinity of the IL-2 polypeptide mutant for the alpha subunit of the IL-2 receptor and retains the affinity of the IL-2 polypeptide mutant for the medium affinity IL-2 receptor, each as compared to the wild-type IL-2 polypeptide.

Mutants of human IL-2(hIL-2) with reduced affinity for CD25 may be generated, for example, by amino acid substitutions at amino acid positions 35, 38, 42, 43, 45 or 72 or combinations thereof (corresponding to the amino acid sequence numbering of human IL-2 as set forth in SEQ ID NO: 13). Exemplary amino acid substitutions include K35E, K35A, R38A, R38E, R38N, R38F, R38S, R38L, R38G, R38Y, R38W, F42L, F42A, F42G, K43G, Y45G, L72G, L G, L G, and L G. Particular IL-2 mutants useful in the immunoconjugates of the invention comprise an amino acid mutation at an amino acid position corresponding to residue 42, 45 or 72, or a combination thereof, of human IL-2. In one embodiment, the amino acid mutation is an amino acid substitution selected from the group consisting of: F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R and L72K, more specifically selected from the group of F42A, Y45A and L72G. These mutants exhibit substantially similar binding affinity to the intermediate affinity IL-2 receptor and have substantially reduced affinity to the alpha subunit of the IL-2 receptor and the high affinity IL-2 receptor as compared to the wild-type form of the IL-2 mutant.

Other characteristics of useful mutants may include the ability to induce proliferation of T cells and/or NK cells bearing an IL-2 receptor, the ability to induce IL-2 signaling in T cells and/or NK cells bearing an IL-2 receptor, the ability to allow NK cells to produce Interferon (IFN) - γ as a secondary cytokine, the reduced ability to induce processing (elaboration) of secondary cytokines (in particular IL-10 and TNF- α) by Peripheral Blood Mononuclear Cells (PBMC), the reduced ability to activate regulatory T cells, the reduced ability to induce apoptosis of T cells, and the reduced in vivo toxicity profile.

Particular IL-2 polypeptide mutants useful in the present invention comprise three amino acid mutations that eliminate or reduce the affinity of the IL-2 polypeptide mutant for the alpha subunit of the IL-2 receptor, but retain the affinity of the IL-2 polypeptide mutant for the intermediate affinity IL-2 receptor. In one embodiment, the three amino acid mutations are at positions corresponding to residues 42, 45, and 72 of human IL-2. In one embodiment, the three amino acid mutations are amino acid substitutions. In one embodiment, the three amino acid mutations are amino acid substitutions selected from the group consisting of: F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. In a specific embodiment, the three amino acid mutations are the amino acid substitutions F42A, Y45A, and L72G (corresponding to the amino acid sequence numbering for human IL-2 as set forth in SEQ ID NO: 13).

In certain embodiments, the amino acid mutation reduces the affinity of the IL-2 polypeptide mutant for the alpha subunit of the IL-2 receptor by at least 5-fold, specifically at least 10-fold, more specifically at least 25-fold. In embodiments where there is more than one amino acid mutation that reduces the affinity of the mutant IL-2 polypeptide for the alpha subunit of the IL-2 receptor, the combination of these amino acid mutations can reduce the affinity of the mutant IL-2 polypeptide for the alpha subunit of the IL-2 receptor by at least 30-fold, at least 50-fold, or even at least 100-fold. In one embodiment, the amino acid mutation or combination of amino acid mutations eliminates the affinity of the IL-2 polypeptide mutant for the alpha subunit of the IL-2 receptor such that no binding is detected by surface plasmon resonance.

Substantially similar binding to the intermediate affinity receptor is achieved when the IL-2 mutant exhibits greater than about 70% of the affinity of the wild-type form of the IL-2 mutant for the intermediate affinity IL-2 receptor, i.e., retains the affinity of the IL-2 polypeptide mutant for the receptor. The IL-2 mutants of the invention may exhibit greater than about 80%, and even greater than about 90%, of such affinities.

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

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

In certain embodiments, the mutant IL-2 polypeptide is substantially a full-length IL-2 molecule. In certain embodiments, the mutant IL-2 polypeptide is a human IL-2 molecule. In one embodiment, the mutant IL-2 polypeptide comprises an amino acid sequence as set forth in SEQ ID NO. 13 having at least one amino acid mutation that eliminates or reduces the affinity of the mutant IL-2 polypeptide for the alpha subunit of the IL-2 receptor but retains the affinity of the mutant IL-2 polypeptide for the medium affinity IL-2 receptor as compared to an IL-2 polypeptide comprising an amino acid sequence as set forth in SEQ ID NO. 13 without said mutation. In another embodiment, the mutant IL-2 polypeptide comprises an amino acid sequence as set forth in SEQ ID NO. 13 having at least one amino acid mutation that eliminates or reduces the affinity of the mutant IL-2 polypeptide for the alpha subunit of the IL-2 receptor but retains the affinity of the mutant IL-2 polypeptide for the medium affinity IL-2 receptor as compared to an IL-2 polypeptide comprising an amino acid sequence as set forth in SEQ ID NO. 13 without said mutation.

In a particular embodiment, the IL-2 polypeptide mutant may elicit one or more cellular responses selected from the group consisting of: proliferation of activated T lymphocytes, differentiation of activated T lymphocytes, cytotoxic T Cell (CTL) activity, proliferation of activated B cells, differentiation of activated B cells, proliferation of Natural Killer (NK) cells, differentiation of NK cells, cytokine secretion by activated T cells or NK cells, and NK/Lymphocyte Activation Killer (LAK) anti-tumor cytotoxicity.

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

Particular IL-2 polypeptide mutants useful in the present invention comprise 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 WO2012/107417, the quadruple IL-2 polypeptide mutants show no detectable binding to CD25, reduced capacity to induce T cell apoptosis, reduced T induction_regThe ability of IL-2 signaling in cells, and reduced in vivo toxicity profiles. However, it retains the ability to activate IL-2 signaling in effector cells, induce effector cell proliferation, and produce IFN- γ as a secondary cytokine by NK cells.

Furthermore, the IL-2 polypeptide mutants have further advantageous properties, such as reduced surface hydrophobicity, good stability and good expression yield, as described in WO 2012/107417. Unexpectedly, the IL-2 polypeptide mutants also provide an extended serum half-life compared to wild-type IL-2.

The IL-2 mutants used in the present invention may have one or more mutations in amino acid sequences other than the IL-2 region forming the interface between IL-2 and CD25 or glycosylation sites, in addition to having a mutation in the IL-2 region. Such additional mutations in human IL-2 may provide additional advantages, such as enhanced expression or stability. For example, as described in U.S. patent No. 4,518,584, the cysteine at position 125 may be replaced with a neutral amino acid (such as serine, alanine, threonine, or valine) to produce C125S IL-2, C125A IL-2, C125T IL-2, or C125V IL-2, respectively. As described therein, it is also possible to delete the N-terminal alanine residue of IL-2, thereby generating mutations such as des-A1C 125S or des-A1C 125A. Alternatively or in combination, the IL-2 mutant may comprise a mutation by which the methionine normally present at position 104 of wild-type human IL-2 is replaced by a neutral amino acid such as alanine (see us patent No. 5,206,344). The resulting mutants, for example des-A1M 104A IL-2, des-A1M 104A C125S IL-2, M104A IL-2, M104A C125A IL-2, des-A1M 104A C125A IL-2, or M104A C125S IL-2 (these and other mutants can be found in U.S. Pat. No. 5,116,943 and Weiger et al, Eur J Biochem 180,295-300 (1989)) can be used in combination with a particular IL-2 mutant of the present invention.

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

The skilled person will be able to determine which further mutations may provide additional advantages for the purposes of the present invention. For example, it will be appreciated that amino acid mutations in the IL-2 sequence that reduce or eliminate the affinity of IL-2 for a medium affinity IL-2 receptor, such as D20T, N88R or Q126D (see e.g. US 2007/0036752), may not be suitable for inclusion in IL-2 polypeptide mutants according to the invention.

In one embodiment, the IL-2 polypeptide mutants comprise NO more than 12, NO more than 11, NO more than 10, NO more than 9, NO more than 8, NO more than 7, NO more than 6, or NO more than 5 amino acid mutations compared to the corresponding wild-type IL-2 sequence (e.g., the human IL-2 amino acid sequence set forth in SEQ ID NO: 13). In a particular embodiment, the IL-2 polypeptide mutant comprises NO more than 5 amino acid mutations compared to the corresponding wild-type IL-2 sequence (e.g., the human IL-2 amino acid sequence as set forth in SEQ ID NO: 13).

In one embodiment, the mutant IL-2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO. 14. In one embodiment, the IL-2 polypeptide mutant consists of the amino acid sequence shown in SEQ ID NO. 14.

Immunoconjugates

An immunoconjugate as described herein comprises an IL molecule and an antibody. Such immunoconjugates significantly increase the efficacy of IL-2 therapy by directly targeting IL-2 (e.g., into the tumor microenvironment). According to the invention, the antibody comprised in the immunoconjugate may be a whole antibody or an immunoglobulin, or a part or variant thereof having a biological function, such as antigen-specific binding affinity.

The general benefits of immunoconjugate therapy are apparent. For example, the antibodies contained in the immunoconjugate recognize a tumor-specific epitope and cause targeting of the immunoconjugate molecule to the tumor site. Thus, high concentrations of IL-2 can be delivered into the tumor microenvironment, resulting in activation and proliferation of the various immune effector cells mentioned herein using much lower doses of the immunoconjugate than are required for unconjugated IL-2. Furthermore, since the application of IL-2 in the form of an immunoconjugate allows for a lower dose of the cytokine itself, the possibility of adverse side effects of IL-2 is limited, and targeting IL-2 to a specific site in the body by means of an immunoconjugate may also result in reduced systemic exposure and thus less side effects than obtained with unconjugated IL-2. Furthermore, the extended circulating half-life of the immunoconjugate compared to unconjugated IL-2 contributes to the efficacy of the immunoconjugate. However, this feature of IL-2 immunoconjugates may exacerbate the potential side effects of IL-2 molecules again: since the circulating half-life of IL-2 immunoconjugates in the bloodstream is significantly extended relative to unconjugated IL-2, the likelihood of the IL-2 or other portion of the fusion protein molecule activating components normally present in the vasculature is increased. The same problem applies to other fusion proteins containing IL-2 fused to another moiety, such as Fc or albumin, which results in an increased half-life of IL-2 in circulation. Thus, it is particularly advantageous that immunoconjugates comprising mutants of IL-2 polypeptides as described herein and in WO2012/107417 have reduced toxicity compared to the wild-type form of IL-2.

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

Accordingly, the present invention provides mutants of IL-2 polypeptides as described previously and antibodies that bind to CD 8. In one embodiment, the mutant IL-2 polypeptide and the antibody form a fusion protein, i.e., the mutant IL-2 polypeptide shares a peptide bond with the antibody. In some embodiments, the antibody comprises an Fc domain comprising a first subunit and a second subunit. In a particular embodiment, the IL-2 polypeptide mutant is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the subunits of the Fc domain, optionally via a linker peptide. In some embodiments, the antibodyThe antibody is a full-length antibody. In some embodiments, the antibody is an immunoglobulin molecule, particularly an IgG class immunoglobulin molecule, more particularly an IgG₁Subclass immunoglobulin molecules. In one such embodiment, the IL-2 polypeptide mutant shares an amino-terminal peptide bond with one of the heavy chains of the immunoglobulin heavy chain. In certain embodiments, the antibody is an antibody fragment. In some embodiments, the antibody is a Fab molecule or an scFv molecule. In one embodiment, the antibody is a Fab molecule. In another embodiment, the antibody is an scFv molecule. The immunoconjugate may also comprise more than one (one) antibody. When more than one antibody, e.g., a first antibody and a second antibody, is included in the immunoconjugate, each antibody may be independently selected from various forms of antibodies and antibody fragments. For example, the first antibody may be a Fab molecule and the second antibody may be a scFv molecule. In a specific embodiment, each of the first and second antibodies is an scFv molecule or each of the first and second antibodies is a Fab molecule. In a particular embodiment, each of the first and second antibodies is a Fab molecule. In one embodiment, each of the first and second antibodies binds to CD 8.

Immunoconjugates forms

Exemplary immunoconjugate forms are described in PCT publication No. WO 2011/020783, which is incorporated herein by reference in its entirety. These immunoconjugates comprise at least two antibodies. Thus, in one embodiment, an immunoconjugate according to the invention comprises a mutant IL-2 polypeptide as described herein, and at least a first antibody and a second antibody. In a particular embodiment, the first antibody and the second antibody are independently selected from the group consisting of: fv molecules, in particular scFv molecules; and a Fab molecule. In a particular embodiment, the mutant IL-2 polypeptide shares an amino-terminal or carboxy-terminal peptide bond with the first antibody, and the second antibody shares an amino-terminal or carboxy-terminal peptide bond with i) the mutant IL-2 polypeptide or ii) the first antibody. In a particular embodiment, the immunoconjugate consists essentially of a mutant IL-2 polypeptide and a first and a second antibody (particularly a Fab molecule) joined by one or more linking sequences. This form has the following advantages: they bind with high affinity to the target antigen (CD8), but only provide monomer binding to the IL-2 receptor, thereby avoiding targeting of the immunoconjugate to IL-2 receptor-bearing immune cells at locations other than the target site. In a particular embodiment, the IL-2 polypeptide mutant shares a carboxy-terminal peptide bond with a first antibody, in particular a first Fab molecule, and further shares an amino-terminal peptide bond with a second antibody, in particular a second Fab molecule. In another embodiment, the first antibody, in particular the first Fab molecule, shares a carboxy-terminal peptide bond with the IL-2 polypeptide mutant and further shares an amino-terminal peptide bond with the second antibody, in particular the second Fab molecule. In another embodiment, the first antibody, in particular the first Fab molecule, shares an amino terminal peptide bond with the first IL-2 polypeptide mutant and further shares a carboxy terminal peptide with the second antibody, in particular the second Fab molecule. In a particular embodiment, the IL-2 polypeptide mutant shares a carboxy-terminal peptide bond with the first heavy chain variable region and also shares an amino-terminal peptide bond with the second heavy chain variable region. In another embodiment, the IL-2 polypeptide mutant shares a carboxy-terminal peptide bond with a first light chain variable region and also shares an amino-terminal peptide bond with a second light chain variable region. In another embodiment, the first heavy or light chain variable region is joined to the IL-2 polypeptide mutant by a carboxy-terminal peptide bond and is also joined to the second heavy or light chain variable region by an amino-terminal peptide bond. In another embodiment, the first heavy or light chain variable region is joined to the IL-2 polypeptide mutant by an amino-terminal peptide bond and is also joined to the second heavy or light chain variable region by a carboxy-terminal peptide bond. In one embodiment, the IL-2 polypeptide mutant shares a carboxy-terminal peptide bond with a first Fab heavy or light chain and also shares an amino-terminal peptide bond with a second Fab heavy or light chain. In another embodiment, the first Fab heavy or light chain shares a carboxy-terminal peptide bond with the IL-2 polypeptide mutant and further shares an amino-terminal peptide bond with the second Fab heavy or light chain. In other embodiments, the first Fab heavy or light chain shares an amino-terminal peptide bond with the IL-2 polypeptide mutant and also shares a carboxy-terminal peptide bond with the second Fab heavy or light chain. In one embodiment, the immunoconjugate comprises a mutant IL-2 polypeptide sharing an amino-terminal peptide bond with one or more scFv molecules and also sharing a carboxy-terminal peptide bond with one or more scFv molecules.

However, particularly suitable forms of the immunoconjugate according to the invention comprise immunoglobulin molecules as antibodies. Such immunoconjugate forms are described in WO 2012/146628, which is incorporated herein by reference in its entirety.

Thus, in a particular embodiment, the immunoconjugate comprises a mutant IL-2 polypeptide as described herein and an immunoglobulin molecule, particularly an IgG molecule, more particularly an IgG, that binds to CD8₁A molecule. In one embodiment, the immunoconjugate comprises no more than one mutant IL-2 polypeptide. In one embodiment, the immunoglobulin molecule is human. In one embodiment, the immunoglobulin molecule comprises a human constant region, e.g., a human CH1, CH2, CH3, and/or CL domain. In one embodiment, the immunoglobulin comprises a human Fc domain, in particular human IgG₁An Fc domain. In one embodiment, the IL-2 polypeptide mutant shares an amino-terminal peptide bond or a carboxy-terminal peptide bond with the immunoglobulin molecule. In one embodiment, the immunoconjugate consists essentially of: mutant IL-2 polypeptides and immunoglobulin molecules, particularly IgG molecules, more particularly IgG molecules, joined by one or more linker sequences₁A molecule. In one embodiment, the IL-2 polypeptide mutant is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the immunoglobulin heavy chains, optionally via a linker peptide.

The IL-2 polypeptide mutants may be fused to antibodies directly or via a linker peptide comprising one or more amino acids (typically about 2-20 amino acids). Linking peptides are known in the art and described herein. Suitable non-immunogenic linker peptides include, for example, (G)₄S)_n、(SG₄)_n、(G₄S)_nOr G₄(SG₄)_nA linker peptide. "n" is typically an integer from 1 to 10, typically from 2 to 4. In one embodiment, the linker peptide is at least 5 amino acids in length, in one embodiment 5 to 100 amino acids in length, and in another embodiment 10 to 50 amino acids in length. In a particular embodiment, the linker peptide is 15 amino acids in length. In one embodiment, the linking peptide is (GxS)_nOr (GxS)_nG_mWhere G ═ glycine, S ═ serine, and (x ═ 3, n ═ 3,4, 5, or 6, and m ═ 0,1, 2, or 3) or (x ═ 4, n ═ 2,3, 4, or 5, and m ═ 0,1, 2, or 3), in one embodiment, x ═ 4 and n ═ 2 or 3, in another embodiment, x ═ 4 and n ═ 3. In a particular embodiment, the linker peptide is (G)₄S)₃(SEQ ID NO: 15). In one embodiment, the linker peptide has (or consists of) the amino acid sequence of SEQ ID NO: 15.

In a particular embodiment, the immunoconjugate comprises a mutated IL-2 molecule and an immunoglobulin molecule, particularly an IgG, that binds to CD8₁Subclass immunoglobulin molecules, wherein the mutant IL-2 molecule is fused at its amino terminal amino acid to the carboxy terminal amino acid of one of the immunoglobulin heavy chains through a linking peptide as set forth in SEQ ID NO: 15.

In a particular embodiment, the immunoconjugate comprises a mutant IL-2 molecule and an antibody that binds to CD8, wherein said antibody comprises an Fc domain comprising a first subunit and a second subunit, in particular a human IgG₁An Fc domain, and the mutant IL-2 molecule is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the subunits of the Fc domain by a linking peptide as set forth in SEQ ID NO: 15.

CD8 antibody

The antibodies comprised in the immunoconjugates of the invention bind to CD8, in particular human CD8, and are capable of directing the IL-2 polypeptide mutant to a target site expressing CD8, in particular to T cells expressing CD8, such as tumor-associated T cells.

Suitable CD8 antibodies that can be used in the immunoconjugates of the invention are described in PCT publication WO 2019/033043 a2, the entire contents of which are incorporated herein by reference.

The immunoconjugates of the invention can comprise two or more (two or more) antibodies that can bind to the same or different antigens. However, in particular embodiments, each of these antibodies binds to CD 8. In one embodiment, the antibodies comprised in the immunoconjugate of the invention are monospecific. In a particular embodiment, the immunoconjugate comprises a single monospecific antibody, in particular a monospecific immunoglobulin molecule.

The antibody may be any type of antibody or fragment thereof that retains specific binding to CD8, particularly human CD 8. Antibody fragments include, but are not limited to, Fv molecules, scFv molecules, Fab molecules, and F (ab')₂A molecule. However, in particular embodiments, the antibody is a full length antibody. In some embodiments, the antibody comprises an Fc domain comprising a first subunit and a second subunit. In some embodiments, the antibody is an immunoglobulin, particularly an IgG class, more particularly an IgG₁Subclass immunoglobulin.

In some embodiments, the antibody is a monoclonal antibody.

Functional characteristics

The anti-CD 8 antibodies provided herein have one or more of the following characteristics: (a) antibodies do not inhibit or stimulate CD8⁺Activation of T cells; (b) antibodies do not induce CD8⁺T cell proliferation; (c) the antibody does not induce IFN γ production; (d) the antibody specifically binds to human CD 8; (e) the antibody specifically binds rhesus monkey CD 8; (f) the antibody specifically binds cynomolgus monkey CD 8; (g) antibodies do not bind CD4⁺A cell; (g) antibodies do not bind CD3^-A cell; (ii) a And (h) the antibody does not deplete CD8 from circulation⁺T cells. Such characteristics can be assessed using well known methods, for example the method used in PCT application WO 2019/033043 a 2.

anti-CD 8 antibodies are antibodies that bind to CD8 with sufficient affinity and specificity. In certain embodiments, the anti-CD 8 antibody is administered at about 1 μ M, 100nM, 50nM, 40nM, 30nM, 20nM, 10nM, 5nM, 1nM, 0.5nM, 0.1nM, 0.05nM, or 0.001nM (e.g., 10 nM)^-8M or less, and (c) is,for example from 10^-8M to 10^-13M, e.g. from 10^-9M to 10^-13M), including any range between these values, binds human CD 8. In certain embodiments, the anti-CD 8 antibody is administered at about 1 μ M, 100nM, 50nM, 40nM, 30nM, 20nM, 10nM, 5nM, 1nM, 0.5nM, 0.1nM, 0.05nM, or 0.001nM (e.g., 10 nM)^-8M or less, e.g. from 10^-8M to 10^-13M, e.g. from 10^-9M to 10^-13M), including any range between these values, binds to rhesus monkey CD 8. In certain embodiments, the anti-CD 8 antibody is administered at 1 μ M, 100nM, 50nM, 40nM, 30nM, 20nM, 10nM, 5nM, 1nM, 0.5nM, 0.1nM, 0.05nM, or 0.001nM (e.g., 10 nM)^-8M or less, e.g. from 10^-8M to 10^-13M, e.g. from 10^-9M to 10^-13M) (including any range between these values) binds to cynomolgus monkey CD 8.

In certain embodiments, the anti-CD 8 antibody (a) is administered at about 1 μ M, 100nM, 50nM, 40nM, 30nM, 20nM, 10nM, 5nM, 1nM, 0.5nM, 0.1nM, 0.05nM, or 0.001nM (e.g., 10 nM)^-8M or less, e.g. from 10^-8M to 10^-13M, e.g. from 10^-9M to 10^-13M) (including any range between these values) binds human CD8 at (b) about 1. mu.M, 100nM, 50nM, 40nM, 30nM, 20nM, 10nM, 5nM, 1nM, 0.5nM, 1.1nM, 0.05nM or 0.001nM (e.g., 10 nM)^-8M or less, e.g. from 10^-8M to 10^-13M, e.g. from 10^-9M to 10^-13M) (including any range between these values) and (c) at about 1. mu.M, 100nM, 50nM, 40nM, 30nM, 20nM, 10nM, 5nM, 1nM, 0.5nM, 0.1nM, 0.05nM, or 0.001nM (e.g., 10nM, 5nM, 1nM, 0.5nM, or 0.001nM^-8M or less, e.g. from 10^-8M to 10^-13M, e.g. from 10^-9M to 10^-13M) (including any range between these values) binds to cynomolgus monkey CD 8. Kn of the anti-CD 8 antibodies provided herein to human CD8, rhesus monkey CD8, and/or cynomolgus monkey CD8 can be determined by any method known in the art including, but not limited to, for example, ELISA, Fluorescence Activated Cell Sorting (FACS) analysis, Radioimmunoprecipitation (RIA), and surface, among othersPlasmon Resonance (SPR). In certain embodiments, the Kn of the anti-CD 8 antibodies provided herein to human CD8, rhesus monkey CD8, and/or cynomolgus monkey CD8 is determined via SPR. In certain embodiments, the Kn of an anti-CD 8 antibody provided herein to human CD8, rhesus monkey CD8, and/or cynomolgus monkey CD8 is determined via fcacs.

In certain embodiments, the anti-CD 8 antibodies provided herein do not bind (e.g., specifically bind) to mouse CD 8. In certain embodiments, the anti-CD 8 antibody does not bind (e.g., specifically binds) to rat CD 8. In certain embodiments, the anti-CDS antibody does not bind (e.g., specifically bind) to mouse CD8 or rat CD8, e.g., as determined via SPR and/or FACS.

In some embodiments, the antibody comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO. 1, HCDR2 comprising the amino acid sequence of SEQ ID NO. 2, HCDR3 comprising the amino acid sequence of SEQ ID NO. 3, LCDR1 comprising the amino acid sequence of SEQ ID NO. 4, LCDR2 comprising the amino acid sequence of SEQ ID NO. 5, and LCDR3 comprising the amino acid sequence of SEQ ID NO. 6.

In some embodiments, the antibody comprises: (a) a heavy chain variable region (VH) comprising: HCDR1 comprising the amino acid sequence of SEQ ID NO. 1, HCDR2 comprising the amino acid sequence of SEQ ID NO. 2, HCDR3 comprising the amino acid sequence of SEQ ID NO. 3; and (b) a light chain variable region (VL) comprising: LCDR1 comprising the amino acid sequence of SEQ ID NO. 4, LCDR2 comprising the amino acid sequence of SEQ ID NO. 5, and LCDR3 comprising the amino acid sequence of SEQ ID NO. 6. In some embodiments, the heavy and/or light chain variable region is a humanized variable region. In some embodiments, the heavy and/or light chain variable region comprises a human Framework Region (FR).

In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID No. 7. In some embodiments, the antibody comprises a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody comprises: (a) a heavy chain variable region (VH) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No.7, and (b) a light chain variable region (VL) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 8.

In one particular embodiment, the antibody comprises: (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 7; and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 8.

In some embodiments, the antibody comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO. 1, HCDR2 comprising the amino acid sequence of SEQ ID NO. 2, HCDR3 comprising the amino acid sequence of SEQ ID NO. 3, LCDR1 comprising the amino acid sequence of SEQ ID NO. 4, LCDR2 comprising the amino acid sequence of SEQ ID NO. 5, and LCDR3 comprising the amino acid sequence of SEQ ID NO. 28.

In some embodiments, the antibody comprises: (a) a heavy chain variable region (VH) comprising: HCDR1 comprising the amino acid sequence of SEQ ID NO. 1, HCDR2 comprising the amino acid sequence of SEQ ID NO. 2, HCDR3 comprising the amino acid sequence of SEQ ID NO. 3; and (b) a light chain variable region (VL) comprising: LCDR1 comprising the amino acid sequence of SEQ ID NO. 4, LCDR2 comprising the amino acid sequence of SEQ ID NO. 5, and LCDR3 comprising the amino acid sequence of SEQ ID NO. 28. In some embodiments, the heavy and/or light chain variable region is a humanized variable region. In some embodiments, the heavy and/or light chain variable region comprises a human Framework Region (FR).

In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID No. 7. In some embodiments, the antibody comprises a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 29. In some embodiments, the antibody comprises: (a) a heavy chain variable region (VH) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7, and (b) a light chain variable region (VL) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 29.

In one particular embodiment, the antibody comprises: (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 7; and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 29.

In some embodiments, the antibody is a humanized antibody. In one embodiment, the antibody is an immunoglobulin molecule comprising a human constant region, in particular an immunoglobulin molecule of the IgG class comprising human CH1, CH2, CH3 and/or CL domains. Exemplary sequences of human constant domains are given in SEQ ID NO:22 and SEQ ID NO:23 (human kappa and lambda CL domains, respectively) and SEQ ID NO:24 (human IgG1 heavy chain constant domains CH1-CH2-CH 3). In some embodiments, the antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO 22 or SEQ ID NO 23, particularly the amino acid sequence of SEQ ID NO 24. In some embodiments, the antibody comprises a heavy chain variable region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 24. In particular, the heavy chain constant region may comprise an amino acid mutation in the Fc domain as described herein.

Fc domains

In particular embodiments, the antibody comprised in the immunoconjugate according to the invention comprises an Fc domain comprising a first subunit and a second subunit. The Fc domain of an antibody consists of a pair of polypeptide chains comprising the heavy chain domains of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin g (IgG) molecule is a dimer, each subunit of which comprises a CH2 and CH3 IgG heavy chain constant domain. The two subunits of the Fc domain are capable of stably associating with each other. In one embodiment, the immunoconjugate of the invention comprises no more than one Fc domain.

In one embodiment, the Fc domain of the antibody comprised in the immunoconjugate is an IgG Fc domain. In a particular embodiment, the Fc domain is IgG₁An Fc domain. In another embodiment, the Fc domain is IgG₄An Fc domain. In a more specific embodiment, the Fc domain is IgG₄An Fc domain comprising an amino acid substitution at position S228 (Kabat EU index numbering), in particular the amino acid substitution S228P. The amino acid substitution reduces IgG₄In vivo Fab arm exchange of antibodies (see Stubenrauch et al, Drug Metabolism and Disposition 38,84-91 (2010)). In another specific embodiment, the Fc domain is a human Fc domain. In an even more particular embodiment, the Fc domain is a human IgG₁An Fc domain. Human IgG₁An exemplary sequence of the Fc region is given as SEQ ID NO 21.

Fc domain modification to promote heterodimerization

The immunoconjugates according to the invention comprise mutants of an IL-2 polypeptide, in particular single (not more than one) IL-2 polypeptide, which IL-2 polypeptide mutants are fused to one or the other of said two subunits of an Fc domain, whereby the two subunits of said Fc domain are typically comprised in two non-identical polypeptide chains. Recombinant co-expression and subsequent dimerization of these polypeptides results in several possible combinations of the two polypeptides. In order to improve the yield and purity of immunoconjugates in recombinant production, it would therefore be advantageous to introduce modifications in the Fc domain of the antibody that promote association of the desired polypeptide.

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

There are several methods of modifying the CH3 domain of the Fc domain to carry out heterodimerization, which are described in detail in, for example, WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, in all such methods, the CH3 domain of the first subunit of Fc domains and the CH3 domain of the second subunit of Fc domains are engineered in a complementary manner such that each CH3 domain (or heavy chain comprising it) can no longer homodimerize with itself, but is forced to heterodimerize with the other CH3 domain that is complementarily engineered (such that the first and second CH3 domains heterodimerize and do not form homodimers between the two first or second CH3 domains).

In a particular embodiment, the modification that facilitates association of the first and second subunits of the Fc domain is a so-called "knob-into-hole" modification comprising a "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other of the two subunits of the Fc domain.

Mortar and pestle construction techniques are described, for example, in US 5,731,168; US 7,695,936; ridgway et al, Prot Eng 9, 617. sup. 621(1996) and Carter, J Immunol Meth 248,7-15 (2001). In general, the method involves introducing a bulge ("protuberance") at the interface of a first polypeptide and a corresponding cavity ("hole") in the interface of a second polypeptide, such that the bulge can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. The bulge is constructed by substituting a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). Compensatory cavities having the same or similar size as the bulge are created in the interface of the second polypeptide by substituting a larger amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

Thus, in a particular embodiment, in the CH3 domain of the first subunit of the Fc domain of the antibody comprised in the immunoconjugate, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby creating a protuberance within the CH3 domain of the first subunit that is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.

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

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

The projections and cavities can be made by altering the nucleic acid encoding the polypeptide, for example by site-specific mutagenesis or by peptide synthesis.

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

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

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

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

In a particular embodiment, the IL-2 polypeptide mutant is fused (optionally via a linker peptide) to the first subunit of the Fc domain (comprising the "overhang" modification). Without wishing to be bound by theory, fusion of the IL-2 polypeptide mutant with the overhang-containing subunit of the Fc domain will (further) minimize the production of an immunoconjugate comprising two IL-2 polypeptide mutants (steric hindrance of the two overhang-containing polypeptides).

Other CH3 modification techniques for carrying out heterodimerization are contemplated as alternatives according to the present invention and are described in, for example, WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO 2013/096291.

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

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

In another embodiment, the antibody comprised in the immunoconjugate of the invention comprises the amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations Y349 2, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, or the antibody comprises the amino acid mutations Y349C, T366W in the CH3 domain of the first subunit of the Fc domain and the amino acid mutations S354C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and additionally the amino acid mutation R39409; K370E in the CH3 domain of the first subunit of the Fc domain, and the amino acid mutation D399K; E357K in the CH3 domain of the second subunit of the Fc domain (all numbering according to the Kabat EU index).

In one embodiment, the heterodimerization method described in WO 2013/157953 is used instead. In one embodiment, the first CH3 domain comprises the amino acid mutation T366K and the second CH3 domain comprises the amino acid mutation L351D (numbering according to the Kabat EU index). In another embodiment, the first CH3 domain comprises the additional amino acid mutation L351K. In another embodiment, the second CH3 domain further comprises an amino acid mutation (numbering according to the Kabat EU index) selected from the group consisting of Y349E, Y349D and L368E (preferably L368E).

In one embodiment, the heterodimerization method described in WO 2012/058768 is used instead. In one embodiment, the first CH3 domain comprises the amino acid mutation L351Y, Y407A and the second CH3 domain comprises the amino acid mutation T366A, K409F. In another embodiment, the second CH3 domain comprises a further amino acid mutation at position T411, D399, S400, F405, N390 or K392, e.g. selected from: a) T411N, T411R, T411Q, T411K, T411D, T411E or T411W, b) D399R, D399W, D399Y or D399K, c) S400E, S400D, S400R or S400K, D) F405I, F405M, F405T, F405S, F405V or F405W, e) N390R, N390K or N390D, F) K392V, K392M, K392R, K392L, K392F or K392E (numbering according to the Kabat index EU). In another embodiment, the first CH3 domain comprises the amino acid mutation L351Y, Y407A and the second CH3 domain comprises the amino acid mutation T366V, K409F. In another embodiment, the first CH3 domain comprises the amino acid mutation Y407A and the second CH3 domain comprises the amino acid mutations T366A, K409F. In another embodiment, the second CH3 domain further comprises the amino acid mutations K392E, T411E, D399R and S400R (numbering according to the EU index of Kabat).

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

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

In one embodiment, the antibody or Fc domain thereof comprised in the immunoconjugate is an IgG₂Subclass, and alternatively use the heterodimerization method described in WO 2010/129304.

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

In a further embodiment, the heterodimerization process described in WO 2007/147901 is used instead. In one embodiment, the first CH3 domain comprises the amino acid mutations K253E, D282K and K322D, and the second CH3 domain comprises the amino acid mutations D239K, E240K and K292D (numbering according to the EU index of Kabat).

In yet another embodiment, the heterodimerization process described in WO 2007/110205 may alternatively be used.

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

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

The Fc domain confers advantageous pharmacokinetic properties to the immunoconjugate, including a long serum half-life that contributes to good accumulation in the target tissue and a favorable tissue-to-blood partition ratio. At the same time, however, it may lead to undesired targeting of the immunoconjugate to cells expressing Fc receptors, rather than the preferred antigen-bearing cells. Furthermore, co-activation of the Fc receptor signaling pathway can lead to cytokine release, which, in combination with the long half-life of the IL-2 polypeptide and immunoconjugate, leads to over-activation and severe side effects on cytokine receptors after systemic administration. In line with this, conventional IgG-IL-2 immunoconjugates have been described in connection with infusion reactions (see, e.g., King et al, JClin Oncol 22,4463-4473 (2004)).

Thus, in particular embodiments, the IgG is naturally associated with₁The Fc domain of the antibody comprised in the immunoconjugate according to the invention exhibits a reduced binding affinity to the Fc receptor and/or a reduction in the Fc domain compared to the Fc domainThe effector function of (1). In one such embodiment, the Fc domain (or antibody comprising the Fc domain) exhibits identity to a native IgG₁Fc domain (or comprising native IgG)₁Fc domain antibody) to an Fc receptor, and/or to a native IgG, by less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5%₁Fc domain (or comprising native IgG)₁Fc domain antibody) to less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of effector function. In one embodiment, the Fc domain (or antibody comprising said Fc domain) does not substantially bind to an Fc receptor and/or induce effector function. In a particular embodiment, the Fc receptor is an fey receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a particular embodiment, the Fc receptor is an activating human Fc γ receptor, more particularly human Fc γ RIIIa, Fc γ RI or Fc γ RIIa, most particularly human Fc γ RIIIa. In one embodiment, the effector function is one or more effector functions selected from the group of CDC, ADCC, ADCP and cytokine secretion. In a particular embodiment, the effector function is ADCC. In one embodiment, the IgG is naturally associated with₁Fc domain domains exhibit substantially similar binding affinities for neonatal Fc receptor (FcRn) compared to Fc domain domains. When the Fc domain (or an antibody comprising said Fc domain) exhibits native IgG₁Fc domain (or comprising native IgG)₁Fc domain antibody) binding affinity for FcRn of greater than about 70%, particularly greater than about 80%, more particularly greater than about 90%, substantially similar binding to FcRn is achieved.

In certain embodiments, the Fc domain is engineered to have reduced binding affinity to an Fc receptor and/or reduced effector function as compared to a non-engineered Fc domain. In particular embodiments, the Fc domain of the antibody included in the immunoconjugate comprises one or more amino acid mutations that reduce the binding affinity of the Fc domain to an Fc receptor and/or effector function. Typically, the same amino acid mutation or mutations are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to the Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In embodiments where there is more than one amino acid mutation that reduces the binding affinity of the Fc domain 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-fold, or even at least 50-fold. In one embodiment, an antibody comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, more particularly less than 5% of the binding affinity to an Fc receptor as compared to an antibody comprising a non-engineered Fc domain. In a particular embodiment, the Fc receptor is an fey receptor. In some embodiments, the Fc receptor is a human Fc receptor. In some embodiments, the Fc receptor is an activating Fc receptor. In a particular embodiment, the Fc receptor is an activating human Fc γ receptor, more particularly human Fc γ RIIIa, Fc γ RI or Fc γ RIIa, most particularly human Fc γ RIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments, the binding affinity to complement components, particularly to C1q, is also reduced. In one embodiment, the binding affinity for neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn is achieved when the Fc domain (or antibody comprising the Fc domain) exhibits greater than about 70% of the binding affinity of the non-engineered form of the Fc domain (or antibody comprising the non-engineered form of the Fc domain) for FcRn, i.e., the binding affinity of the Fc domain for the receptor is retained. The Fc domain or antibodies comprised in the immunoconjugates of the invention comprising said Fc domain may exhibit greater than about 80% and even greater than about 90% of such affinity. In certain embodiments, the Fc domain of the antibody included in the immunoconjugate is engineered to have reduced effector function as compared to a non-engineered Fc domain. Reduced effector function may include, but is not limited to, one or more of the following: reduced Complement Dependent Cytotoxicity (CDC), reduced antibody dependent cell-mediated cytotoxicity (ADCC), reduced Antibody Dependent Cellular Phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced apoptosis, reduced cross-linking of target-bound antibodies, reduced dendritic cell maturation, or reduced T-cell sensitization. In one embodiment, the reduced effector function is a reduced effector function selected from one or more of the group of reduced CDC, reduced ADCC, reduced ADCP and reduced cytokine secretion. In a particular embodiment, the reduced effector function is reduced ADCC. In one embodiment, the reduced ADCC is less than 20% of the ADCC induced by the non-engineered Fc domain (or an antibody comprising a non-engineered Fc domain).

In one embodiment, the amino acid mutation that reduces the binding affinity and/or effector function of the Fc domain to an Fc receptor is an amino acid substitution. In one embodiment, the Fc domain comprises an amino acid substitution (numbering according to the Kabat EU index) at a position selected from the group of E233, L234, L235, N297, P331 and P329. In a more particular embodiment, the Fc domain comprises an amino acid substitution (numbering according to the Kabat EU index) at a position selected from the group of L234, L235 and P329. In some embodiments, the Fc domain comprises amino acid substitutions L234A and L235A (numbering according to the Kabat EU index). In one such embodiment, the Fc domain is an IgG₁Fc domain, in particular human IgG₁An Fc domain. In one embodiment, the Fc domain comprises an amino acid substitution at position P329. In a more particular embodiment, the amino acid substitution is P329A or P329G, in particular P329G (numbering according to the EU index of Kabat). In one embodiment, the Fc domain comprises an amino acid substitution at position P329, and a further amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (numbering according to the Kabat EU index). In a more specific embodiment, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In particular embodiments, the Fc domain comprises amino acid substitutions (numbering according to the Kabat EU index) at positions P329, L234 and L235. In a more specific embodiment, the Fc domain comprises the amino acid mutations L234A, L235A, and P329G ("P329G LALA", "PGLALA", or "lalapc"). Specifically, in particular embodiments, each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering), i.e. in each of the first and second subunits of the Fc domain, the leucine residue at position 234 is substituted with an alanine residue (L234A), the leucine residue at position 235 is substituted with an alanine residue (L235A), and the proline residue at position 329 is substituted with a glycine residue (P329G) (numbering according to the EU index of Kabat). In one such embodiment, the Fc domain is an IgG₁Fc domain, in particular human IgG₁An Fc domain. The combination of amino acid substitutions "P329G LALA" almost completely eliminated human IgG₁Fc gamma receptor (and complement) binding of Fc domains, as described in PCT publication No. WO 2012/130831, which is incorporated herein by reference in its entirety. WO 2012/130831 also describes methods of making such mutant Fc domains and methods of determining properties thereof, such as Fc receptor binding or effector function.

And IgG₁Antibody vs. IgG₄Antibodies exhibit reduced binding affinity to Fc receptors and reduced effector function. Thus, in some embodiments, the Fc domain of the antibody comprised in the immunoconjugate of the invention is an IgG₄Fc domain, in particular human IgG₄An Fc domain. In one embodiment, the IgG is₄The Fc domain comprises an amino acid substitution at position S228, in particular amino acid substitution S228P (numbering according to the Kabat EU index). To further reduce its binding affinity to Fc receptors and/or its effector function, in one embodiment, IgG₄The Fc domain comprises an amino acid substitution at position L235, in particular the amino acid substitution L235E (numbering according to the Kabat EU index). In another embodiment, the IgG is₄The Fc domain comprises an amino acid substitution at position P329, in particular the amino acid substitution P329G (according toEU index numbering of Kabat). In a particular embodiment, the IgG₄The Fc domain comprises amino acid substitutions at positions S228, L235 and P329, in particular amino acid substitutions S228P, L235E and P329G (numbering according to the EU index of Kabat). Such IgG₄Fc domain mutants and their Fc γ receptor binding properties are described in PCT publication No. WO 2012/130831, which is incorporated herein by reference in its entirety.

In a particular embodiment, the IgG is naturally associated with₁The Fc domain exhibiting reduced binding affinity to an Fc receptor and/or reduced effector function compared to an Fc domain is a human IgG comprising the amino acid substitutions L234A, L235A and optionally P329G₁An Fc domain, or a human IgG comprising the amino acid substitutions S228P, L235E and optionally P329G₄Fc domain (numbering according to EU index of Kabat).

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

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

The mutant Fc domain may be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide change can be verified, for example, by sequencing.

Binding to Fc receptors can be readily determined, for example, by ELISA or by Surface Plasmon Resonance (SPR) using standard instruments such as BIAcore instruments (GE Healthcare), and Fc receptors can be obtained, for example, by recombinant expression. Alternatively, cell lines known to express specific Fc receptors (such as human NK cells expressing Fc γ IIIa receptors) can be used to assess the binding affinity of the Fc domain or antibody comprising the Fc domain to the Fc receptor.

The effector function of an Fc domain, or an antibody comprising an Fc domain, can be measured by methods known in the art. Examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Pat. nos. 5,500,362; hellstrom et al, Proc Natl Acad Sci USA 83, 7059-; U.S. Pat. nos. 5,821,337; bruggemann et al, J Exp Med 166, 1351-. Alternatively, non-radioactive assay methods can be used (see, e.g., ACTI for flow cytometry)^TMNon-radioactive cytotoxicity assay (CellTechnology, inc. mountain View, CA); and Cytotox

Non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example in an animal model such as disclosed in Clynes et al, Proc Natl Acad Sci USA 95, 652-.

In some embodiments, Fc domain binding to complement components, particularly C1q, is reduced. Thus, in some embodiments, wherein the Fc domain is engineered to have reduced effector function, said reduced effector function comprises reduced CDC. A C1q binding assay may be performed to determine whether an Fc domain or an antibody comprising the Fc domain is capable of binding C1q and thus has CDC activity. See, e.g., WO 2006/029879 and WO 2005/100402 for C1q and C3C binding ELISA. To assess complement activation, CDC assays may be performed (see, e.g., Gazzano-Santoro et al, J Immunol Methods 202,163 (1996); Cragg et al, Blood 101, 1045-.

FcRn binding and in vivo clearance/half-life assays can also be performed using methods known in the art (see, e.g., Petkova, s.b. et al, Int' l.immunol.18(12): 1759-.

Particular aspects of the invention

In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to CD8,

wherein the IL-2 polypeptide mutant is a human IL-2 molecule comprising the amino acid substitutions F42A, Y45A and L72G (numbering relative to the human IL-2 sequence SEQ ID NO: 13); and is

Wherein the antibody comprises: (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 7; and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 8.

In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to CD8,

wherein the IL-2 polypeptide mutant is a human IL-2 molecule comprising the amino acid substitutions T3A, F42A, Y45A, L72G and C125A (corresponding to the amino acid sequence numbering for human IL-2 as set forth in SEQ ID NO: 13); and is

Wherein the antibody comprises: (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 7; and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 8.

In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to CD8,

wherein the IL-2 polypeptide mutant comprises an amino acid sequence shown as SEQ ID NO. 14; and is

Wherein the antibody comprises: (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 7; and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 8.

In accordance with the present inventionIn one embodiment of any of the above aspects, the antibody is an immunoglobulin of the IgG class comprising human IgG comprising a first subunit and a second subunit₁An Fc domain having a sequence that is complementary to the Fc domain,

wherein in said first subunit of said Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W); and in the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V), and optionally the threonine residue at position 366 is replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A) (numbering according to the Kabat EU index),

and wherein further each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering).

In this example, the IL-2 polypeptide mutant may be fused at its amino-terminal amino acid to the carboxy-terminal amino acid of the first subunit of the Fc domain via a linker peptide as set forth in SEQ ID NO. 15.

In one aspect, the invention provides an immunoconjugate comprising: a polypeptide comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO. 9, a polypeptide comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO. 10, and a polypeptide comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO. 11.

In a preferred embodiment, the present invention provides an immunoconjugate comprising a polypeptide comprising the amino acid sequence of SEQ ID NO 9, a polypeptide comprising the amino acid sequence of SEQ ID NO 10, and a polypeptide comprising the amino acid sequence of SEQ ID NO 11. In a more preferred embodiment, the present invention provides an immunoconjugate comprising two polypeptides comprising the amino acid sequence of SEQ ID NO 9, a polypeptide comprising the amino acid sequence of SEQ ID NO 10 and a polypeptide comprising the amino acid sequence of SEQ ID NO 11.

In one aspect, the invention provides an immunoconjugate comprising: a polypeptide comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO. 9, a polypeptide comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO. 10, and a polypeptide comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO. 12.

In a preferred embodiment, the present invention provides an immunoconjugate comprising a polypeptide comprising the amino acid sequence of SEQ ID NO 9, a polypeptide comprising the amino acid sequence of SEQ ID NO 10, and a polypeptide comprising the amino acid sequence of SEQ ID NO 12.

In another aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to CD8, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising the amino acid substitutions F42A, Y45A, and L72G (numbered relative to the human IL-2 sequence SEQ ID NO: 13); and wherein the antibody comprises: (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:7, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 29.

In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to CD8, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising the amino acid substitutions T3A, F42A, Y45A, L72G, and C125A (numbered relative to the human IL-2 sequence SEQ ID NO: 13); and wherein the antibody comprises: (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:7, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 29.

In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds CD8, wherein the mutant IL-2 polypeptide comprises the amino acid sequence of SEQ ID No. 14; and wherein the antibody comprises: (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:7, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 29.

In one embodiment according to any of the above aspects of the invention, the antibody is an IgG class immunoglobulin comprising a human IgG1 Fc domain comprising a first subunit and a second subunit,

wherein in said first subunit of said Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W); and in said second subunit of said Fc domain, the tyrosine residue at position 407 is replaced by a valine residue (Y407V), and optionally the threonine residue at position 366 is replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A) (numbering according to the Kabat EU index), and wherein further each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering).

In this example, the IL-2 polypeptide mutant may be fused at its amino-terminal amino acid to the carboxy-terminal amino acid of the first subunit of the Fc domain via a linker peptide as set forth in SEQ ID NO. 15.

In one aspect, the invention provides an immunoconjugate comprising: a polypeptide comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO. 30, a polypeptide comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO. 10, and a polypeptide comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO. 11.

In a preferred embodiment, the present invention provides an immunoconjugate comprising a polypeptide comprising the amino acid sequence of SEQ ID NO 30, a polypeptide comprising the amino acid sequence of SEQ ID NO 10, and a polypeptide comprising the amino acid sequence of SEQ ID NO 11. In a more preferred embodiment, the present invention provides an immunoconjugate comprising two polypeptides comprising the amino acid sequence of SEQ ID NO 30, a polypeptide comprising the amino acid sequence of SEQ ID NO 10 and a polypeptide comprising the amino acid sequence of SEQ ID NO 11.

In one aspect, the invention provides an immunoconjugate comprising: a polypeptide comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO. 30, a polypeptide comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO. 10, and a polypeptide comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO. 12.

In a preferred embodiment, the present invention provides an immunoconjugate comprising a polypeptide comprising the amino acid sequence of SEQ ID NO 30, a polypeptide comprising the amino acid sequence of SEQ ID NO 10, and a polypeptide comprising the amino acid sequence of SEQ ID NO 12.

Polynucleotide

The invention also provides isolated polynucleotides encoding the immunoconjugates or fragments thereof as described herein. In some embodiments, the fragment is an antigen-binding fragment.

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

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

In one embodiment, the isolated polynucleotides of the invention encode the heavy chains of the antibodies (e.g., immunoglobulin heavy chains) and the IL-2 polypeptide mutants contained in the immunoconjugate. In another embodiment, the isolated polynucleotide of the invention encodes the light chain of the antibody comprised in the immunoconjugate.

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

Recombination method

Mutants of IL-2 polypeptides useful in the present invention may be prepared by deletion, substitution, insertion or modification by genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide change can be verified, for example, by sequencing. In this regard, the nucleotide sequence of native IL-2 has been described by Taniguchi et al (Nature 302,305-10(1983)), and nucleic acids encoding human IL-2 are available from public depositories such as the American Type Culture Collection (Rockville MD). The sequence of native human IL-2 is shown in SEQ ID NO 13. Substitutions or insertions may involve natural and unnatural amino acid residues. Amino acid modifications include well known chemical modification methods such as the addition of glycosylation sites or carbohydrate attachments, and the like.

The immunoconjugates of the invention can be obtained, for example, by solid-state peptide synthesis (e.g., Merrifield solid phase synthesis) or recombinant production. For recombinant production, one or more polynucleotides encoding the immunoconjugates (fragments), e.g., as described above, are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such polynucleotides can be readily isolated and sequenced using conventional methods. In one embodiment, a vector, preferably an expression vector, is provided comprising one or more of the polynucleotides of the present invention. Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequences for the immunoconjugates (fragments) and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination/genetic recombination. See, e.g., in Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. (1989); and techniques described in 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, virus, or may be a nucleic acid fragment. The expression vector includes an expression cassette into which a polynucleotide encoding an immunoconjugate (fragment), i.e., the coding region, is cloned in operable association with a promoter and/or other transcriptional or translational control elements. As used herein, a "coding region" is a portion of a nucleic acid that consists of codons that are translated into amino acids. Although the "stop codon" (TAG, TGA or TAA) is not translated into an amino acid, it (if present) can be considered part of the coding region, whereas 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. The two or more coding regions may be present in a single polynucleotide construct (e.g., on a single vector), or in separate polynucleotide constructs (e.g., on separate (different) vectors). In addition, any vector may contain a single coding region, or may contain two or more coding regions, e.g., a vector of the invention may encode one or more polypeptides that are separated into the final protein by proteolytic cleavage post-or post-translationally. In addition, the vectors, polynucleotides or nucleic acids of the invention may encode a heterologous coding region, which may or may not be fused to the polynucleotide encoding the immunoconjugate of the invention, or a variant or derivative thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as secretion signal peptides or heterologous functional domains. Operable association is when the coding region of a gene product (e.g., a polypeptide) is associated with one or more regulatory sequences in a manner such that expression of the gene product is under the influence or control of the regulatory sequences. Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated" if induction of promoter function results in transcription of mRNA encoding the desired gene product, and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression control sequences to direct expression of the gene product or with the ability of the gene template to be transcribed. Thus, if a promoter is capable of affecting transcription of the nucleic acid, the promoter region will be operably associated with the nucleic acid encoding the polypeptide. The promoter may be a cell-specific promoter that directs substantial transcription of DNA only in predetermined cells. In addition to promoters, other transcriptional control elements, such as enhancers, operators, repressors, and transcriptional termination signals, may be operably associated with a polynucleotide to direct cell-specific transcription. Suitable promoters and other transcriptional control regions are disclosed herein. Various transcriptional control regions are known to those skilled in the art. These transcriptional control regions include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (e.g., immediate early promoter-binding intron-a), simian virus 40 (e.g., early promoter), and retroviruses (such as, for example, rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes (such as actin, heat shock proteins, bovine growth hormone, and rabbit beta globin), as well as other sequences capable of controlling gene expression in eukaryotic cells. Other suitable transcriptional control regions include tissue-specific promoters and enhancers and inducible promoters (e.g., tetracycline-inducible promoters). Similarly, various translational control elements are known to those of ordinary skill in the art. These translation control elements include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly internal ribosome entry sites, or IRES, also known as CITE sequences). The expression cassette may also include other features, such as an origin of replication, and/or chromosomal integration elements, such as retroviral Long Terminal Repeats (LTRs), or adeno-associated virus (AAV) Inverted Terminal Repeats (ITRs).

The polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions encoding a secretion peptide or signal peptide which direct secretion of the polypeptide encoded by the polynucleotide of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein once the protein chain has been initiated to grow across the rough endoplasmic reticulum export. One of ordinary skill in the art will recognize 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 yield a secreted or "mature" form of the polypeptide. For example, human IL-2 is translated into a signal sequence of 20 amino acids at the N-terminus of the polypeptide, which is subsequently cleaved to yield mature human IL-2 of 133 amino acids. In certain embodiments, a native signal peptide (e.g., an IL-2 signal peptide or an immunoglobulin heavy or light chain signal peptide) is used, or a functional derivative of such a sequence that retains the ability to direct secretion of a polypeptide with which it is operably associated. Alternatively, a heterologous mammalian signal peptide or functional derivative thereof may be used. For example, the wild-type leader sequence may be substituted with the leader sequence of human Tissue Plasminogen Activator (TPA) or mouse β -glucuronidase.

DNA encoding short protein sequences (e.g., histidine tags) or to aid in labeling of the immunoconjugate, which may be used to facilitate subsequent purification, may be included within or at the end of the immunoconjugate (fragment) encoding polynucleotide.

In another embodiment, a host cell comprising one or more polynucleotides of the invention is provided. In certain embodiments, host cells comprising one or more vectors of the invention are provided. Multiple purposeThe nucleotide and the vector may incorporate, alone or in combination, any of the features described herein with respect to the polynucleotide and vector, respectively. In one such embodiment, the host cell comprises (e.g., has been transformed or transfected with) one or more vectors comprising one or more polynucleotides encoding the immunoconjugate of the invention. The term "host cell" as used herein refers to any kind of cellular system that can be engineered to produce an immunoconjugate of the invention, or a fragment thereof. Host cells suitable for replicating and supporting the expression of immunoconjugates are well known in the art. Such cells can be appropriately transfected or transduced with a particular expression vector, and large numbers of vector-containing cells can be grown for seeding large-scale fermentors to obtain sufficient quantities of the immunoconjugate for clinical use. Suitable host cells include prokaryotic microorganisms such as E.coli, or various eukaryotic cells such as Chinese hamster ovary Cells (CHO), insect cells, and the like. For example, the polypeptide may be produced in bacteria, particularly when glycosylation is not required. The polypeptide can be isolated from the bacterial cell paste after expression in a soluble fraction and can be further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding polypeptides, including fungi and yeast strains whose glycosylation pathways have been "humanized" resulting in the production of polypeptides having a partially or fully human glycosylation pattern. See Gerngross, Nat Biotech 22, 1409-. Suitable host cells for the expression (glycosylation) of polypeptides also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. A number of baculovirus strains have been identified which can be used with insect cells, particularly for transfecting Spodoptera frugiperda (Spodoptera frugiperda) cells. Plant cell cultures may also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIIES for antibody production in transgenic plants^TMA technique). Vertebral motionSomatic cells may also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney lines (293 or 293T cells, as described for example in Graham et al, J Gen Virol 36,59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells, as described for example in Mather, Biol Reprod 23, 243-. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including dhfr^-CHO cells (Urlaub et al, Proc Natl Acad Sci USA 77,4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63, and Sp 2/0. For a review of certain mammalian host cell lines suitable for protein production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol.248 (B.K.C.Lo eds., Humana Press, Totowa, NJ), pp.255-268 (2003). Host cells include cultured cells such as mammalian cultured cells, yeast cells, insect cells, bacterial cells and plant cells, to name a few, and also include cells contained in transgenic animals, transgenic plants or cultured plant or animal tissues. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, a Human Embryonic Kidney (HEK) cell, or a lymphocyte (e.g., Y0, NS0, Sp20 cell).

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

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

In the immunoconjugates of the invention, the IL-2 polypeptide mutants may be genetically fused to antibodies or may be chemically conjugated to antibodies. The genetic fusion of an IL-2 polypeptide to an antibody can be designed such that the IL-2 sequence is fused directly to the polypeptide or indirectly to the polypeptide through a linker sequence. The composition and length of the linker can be determined according to methods well known in the art, and the efficacy of the linker can be tested. Specific linker peptides are described herein. Additional sequences (e.g., endopeptidase recognition sequences) may also be included to incorporate cleavage sites to separate the fused individual components, if desired. Alternatively, IL-2 fusion proteins can be chemically synthesized using polypeptide synthesis methods well known in the art (e.g., Merrifield solid phase synthesis). The IL-2 polypeptide mutants can be chemically conjugated to other molecules (e.g., antibodies) using well-known chemical conjugation methods. Difunctional crosslinking agents, such as homofunctional and heterofunctional crosslinking agents well known in the art, may be used for this purpose. The type of cross-linking agent used depends on the nature of the molecule to which IL-2 is coupled and can be readily identified by the person skilled in the art. Alternatively or additionally, the IL-2 mutant and/or its intended conjugated molecule may be chemically derivatized such that both the IL-2 mutant and/or its intended conjugated molecule may be conjugated in separate reactions, as is also well known in the art.

The immunoconjugates of the invention comprise antibodies. Methods for producing Antibodies are well known in the art (see, e.g., Harlow and Lane, "Antibodies, a Laboratory Manual", Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be recombinantly produced (e.g., as described in U.S. patent No. 4,186,567), or can be obtained, for example, by screening combinatorial libraries comprising variable heavy and variable light chains (see, e.g., U.S. patent No. 5,969,108 to McCafferty). Immunoconjugates, antibodies and methods for their preparation are also described in detail, for example, in PCT publication nos. WO 2011/020783, WO2012/107417 and WO 2012/146628, each of which is incorporated herein by reference in its entirety.

Antibodies of any animal species may be used in the immunoconjugates of the invention. Non-limiting antibodies useful in the invention can be of murine, primate, or human origin. If the immunoconjugate is intended for human use, a chimeric form of the antibody may be used, wherein the constant region of the antibody is from a human. Antibodies in humanized or fully human form can also be prepared according to methods well known in the art (see, e.g., U.S. Pat. No. 5,565,332 to Winter). Humanization can be achieved by a variety of methods including, but not limited to, (a) grafting non-human (e.g., donor antibody) CDRs onto human (e.g., acceptor antibody) frameworks and constant regions with or without retaining critical framework residues (e.g., critical framework residues important for maintaining good antigen binding affinity or antibody function), (b) grafting only non-human specificity determining regions (SDRs or a-CDRs; residues critical for antibody-antigen interaction) onto human frameworks and constant regions, or (c) grafting entire non-human variable domains but "hiding" them with human-like regions by replacing surface residues. Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro and Fransson, front.biosci.13:1619-1633(2008), and further described, for example, in Riechmann et al, Nature 332:323-329 (1988); queen et al, Proc.nat' l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; kashmiri et al, Methods 36:25-34(2005) (describes Specificity Determining Region (SDR) grafting); padlan, mol.Immunol.28:489-498(1991) (described as "surface remodeling"); dall' Acqua et al, Methods 36:43-60(2005) (describing "FR shuffling"); and Osbourn et al, Methods 36:61-68(2005) and Klimka et al, Br.J. cancer,83:252-260(2000) (describing the "guided selection" method for FR shuffling). Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using the "best-fit" approach (see, e.g., Sims et al, J.Immunol.151:2296 (1993)); framework regions derived from consensus sequences of human antibodies having a particular subset of light or heavy chain variable regions (see, e.g., Carter et al, proc. natl. acad. sci. usa,89:4285 (1992); and Presta et al, j. immunol.,151:2623 (1993)); human mature (somatic mutation) framework region or human germline framework region (see, e.g., Almagro and Fransson, front. biosci.13:1619-1633 (2008)); and the framework regions derived from screening FR libraries (see, e.g., Baca et al, J.biol.chem.272:10678-10684(1997) and Rosok et al, J.biol.chem.271:22611-22618 (1996)).

Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr Opin Pharmacol 5,368-74(2001) and Lonberg, Curr Opin Immunol 20, 450-. Human antibodies can be made by: the immunogen is administered to a transgenic animal that has been modified to produce a fully human antibody or a fully antibody with human variable regions in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces an endogenous immunoglobulin locus, or is present extrachromosomally or randomly integrated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin loci have typically been inactivated. For an overview of the methods for obtaining human antibodies from transgenic animals, see Lonberg, nat. Biotech.23:1117-1125 (2005). See also, e.g., the description XENOMOUSE^TMU.S. Pat. nos. 6,075,181 and 6,150,584 to technology; description of the invention

U.S. patent numbers 5,770,429 for technology; description of K-M

U.S. Pat. No.7,041,870 to Art, and description

U.S. patent application publication No. US 2007/0061900 for technology). The human variable region from an intact antibody produced by such an animal may be further modified, for example byIn combination with different human constant regions.

Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cell lines have been described for the production of human monoclonal antibodies. (see, e.g., Kozbor J.Immunol.,133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp 51-63 (Marcel Dekker, Inc., New York,1987), and Boerner et al, J.Immunol.,147:86 (1991)), human antibodies produced via human B-cell hybridoma technology are also described in Li et al, Proc.Natl.Acad.Sci.USA,103:3557-3562 (2006). Additional methods include, for example, those described in U.S. Pat. No.7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268(2006) (describing human-human hybridomas). The human hybridoma technique (Trioma technique) is also described in Vollmers and Brandlens, Histology and Histopathology,20(3): 927-.

Human antibodies can also be produced by isolation from a library of human antibodies, as described herein.

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

In some phage display methods, the repertoire of VH and VL genes are individually cloned by Polymerase Chain Reaction (PCR) and randomly recombined in a phage library from which antigen-binding phage can then be screened, as described in Winter et al, Annual Review of Immunology 12:433-455 (1994). Phage typically display antibody fragments as single chain fv (scfv) fragments or Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, all natural components (e.g., all natural components from humans) can be cloned to provide a single source of antibodies to a wide range of non-self and self-antigens without any immunization, as described by Griffiths et al in EMBO Journal 12:725-734 (1993). Finally, natural libraries are also synthesized by: cloning unrearranged V gene segments from stem cells; and the use of PCR primers containing random sequences to encode highly variable CDR3 regions and complete in vitro rearrangement as described by Hoogenboom and Winter in the Journal of Molecular Biology 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. nos. 5,750,373; 7,985,840; 7,785,903, and 8,679,490, and U.S. patent publication nos. 2005/0079574, 2007/0117126, 2007/0237764, and 2007/0292936. Other examples of methods known in the art for screening combinatorial libraries of antibodies with one or more desired activities include ribosome and mRNA display, and methods of antibody display and selection on bacterial, mammalian, insect or yeast cells. Methods for yeast surface display are reviewed, for example, in Scholler et al, Methods in Molecular Biology 503:135-56(2012) and Cherf et al, Methods in Molecular Biology 1319:155-175(2015) and in Zhao et al, Methods in Molecular Biology 889:73-84 (2012). Methods for ribosome display are described, for example, in He et al, Nucleic Acids Research 25: 5132-.

Further chemical modifications of the immunoconjugates of the invention may be required. For example, the problems of immunogenicity and short half-life can be ameliorated by conjugation to a substantially linear polymer, such as polyethylene glycol (PEG) or polypropylene glycol (PPG) (see, e.g., WO 87/00056).

Immunoconjugates prepared as described herein can be purified by techniques known in the art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and will be apparent to those skilled in the art. For affinity chromatography purification, antibodies, ligands, receptors or antigens bound to the immunoconjugate may be used. For example, antibodies that specifically bind to mutants of IL-2 polypeptides may be used. For affinity chromatography purification of the immunoconjugates of the invention, matrices with protein a or protein G can be used. For example, immunoconjugates can be separated using sequential protein a or G affinity chromatography and size exclusion chromatography, substantially as described in the examples. The purity of the immunoconjugate can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high pressure liquid chromatography, and the like.

Compositions, formulations and routes of administration

In another aspect, the invention provides a pharmaceutical composition comprising an immunoconjugate as described herein, e.g., for use in any of the following methods of treatment. In one embodiment, the pharmaceutical composition comprises any of the immunoconjugates provided herein, and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises any of the immunoconjugates provided herein, and at least one additional therapeutic agent, e.g., as described below.

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

The pharmaceutical compositions of the invention comprise a therapeutically effective amount of the immunoconjugate dissolved or dispersed in a pharmaceutically acceptable carrier. The term "pharmaceutically or pharmacologically acceptable" means that the molecular entities and compositions are generally non-toxic to recipients at the dosages and concentrations employed, i.e., do not produce adverse, allergic, or other untoward reactions when administered to an animal (e.g., a human) as appropriate. In accordance with the present disclosure, the preparation of Pharmaceutical compositions containing immunoconjugates and optionally additional active ingredients will be known to those skilled in the art, as exemplified by Remington's Pharmaceutical Sciences, 18 th edition, Mack Printing Company,1990, which is incorporated herein by reference. Further, for animal (e.g., human) administration, it is understood that the preparation should meet sterility, thermogenicity, general safety and purity standards as required by FDA office of biological standards or corresponding authorities in other countries/regions. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, the like, and combinations thereof, as would be known to one of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18 th edition, Mack Printing Company,1990, 1289-1329, which is incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, use of the carrier in the therapeutic or pharmaceutical compositions is contemplated.

The immunoconjugates (and any additional therapeutic agents) of the invention can be administered by any suitable means, including parenterally, intrapulmonary and intranasally, and if desired for topical, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Dosing may be by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is transient or chronic.

Parenteral compositions include those compositions designed for administration by injection (e.g., subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal, or intraperitoneal injection). For injection, the 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 formulating agents (formulations), such as suspending, stabilizing and/or dispersing agents. Alternatively, the immunoconjugate may be in powder form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to use. Sterile injectable solutions are prepared by incorporating the immunoconjugate of the invention in the required amount in the appropriate solvent with various other ingredients enumerated below, as required. For example, sterility can be readily achieved by filtration through sterile filtration membranes. Generally, 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 drying or lyophilization techniques that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium. The liquid medium should be suitably buffered if necessary, and sufficient saline or glucose should first be used to make the liquid diluent isotonic prior to injection. The composition must be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept to a minimum at a safe level, for example below 0.5ng/mg protein. Suitable pharmaceutically acceptable carriers include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexa-hydrocarbyl quaternary ammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc protein complexes); and/or a non-ionic surfactant, such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, and the like. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil; or synthetic fatty acid esters such as ethyl oleate or triglycerides; or liposomes.

The active ingredient may be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively); in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules); or in a coarse emulsion. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18 th edition, Mack Printing Company, 1990). Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g., films, or microcapsules. In certain embodiments, prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate, gelatin or combinations thereof.

In addition to the previously described compositions, the immunoconjugates can also be formulated as depot preparations. Such long acting formulations may be administered by implantation (e.g. subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the immunoconjugate may be formulated with a suitable polymeric or hydrophobic material (e.g., as an emulsion in an acceptable oil) or with an ion exchange resin, or as a sparingly soluble derivative, e.g., as a sparingly soluble salt.

Pharmaceutical compositions comprising the immunoconjugates of the invention can be prepared by conventional means of mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilizing. The pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the proteins into preparations which can be used pharmaceutically. The appropriate formulation depends on the route of administration chosen.

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

Therapeutic methods and compositions

Any of the immunoconjugates provided herein can be used in methods of treatment. The immunoconjugates of the invention are useful as immunotherapeutics, e.g., for the treatment of cancer.

For use in a method of treatment, the immunoconjugates of the invention will be formulated, dosed and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to the practitioner.

The immunoconjugates of the invention are particularly useful in the treatment of disease states in which stimulation of the host immune system is beneficial, particularly diseases in which an enhanced cellular immune response is desired. These disease states may include those in which the host immune response is inadequate or absent. Disease states in which the immunoconjugates of the invention can be administered include, for example, tumors or infections in which cellular immune response is a key mechanism for specific immunity. The immunoconjugates of the invention can be administered as such or in any suitable pharmaceutical composition.

In one aspect, the immunoconjugates of the invention are provided for use as a medicament. In other aspects, the immunoconjugates of the invention are provided for use in treating a disease. In certain embodiments, immunoconjugates of the invention are provided for use in methods of treatment. In one embodiment, the invention provides an immunoconjugate as described herein for use in treating a disease in an individual in need thereof. In certain embodiments, the invention provides an immunoconjugate for use in a method of treating an individual having a disease, the method comprising administering to the individual a therapeutically effective amount of the immunoconjugate. In certain embodiments, the disease to be treated is a proliferative disease. In a particular embodiment, the disease is cancer. In certain embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., if the disease to be treated is cancer, an anti-cancer agent. In other embodiments, the invention provides immunoconjugates for stimulating the immune system. In certain embodiments, the present invention provides an immunoconjugate for use in a method of stimulating the immune system of an individual, the method comprising administering to the individual an effective amount of the immunoconjugate to stimulate the immune system. An "individual" according to any of the above embodiments is a mammal, preferably a human. The "stimulation of the immune system" according to any of the above embodiments may comprise any one or more of the following: general enhancement of immune function, enhancement of T cell function, enhancement of B cell function, restoration of lymphocyte function, increase in IL-2 receptor expression, enhancement of T cell reactivity, enhancement of natural killer cell activity or Lymphokine Activated Killer (LAK) cell activity, and the like.

In another aspect, the invention provides the use of an immunoconjugate of the invention in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treating a disease in an individual in need thereof. In one embodiment, the medicament is for use in a method of treating a disease, the method comprising administering to an individual having the disease a therapeutically effective amount of the medicament. In certain embodiments, the disease to be treated is a proliferative disease. In a particular embodiment, the disease is cancer. In one embodiment, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., if the disease to be treated is cancer, an anti-cancer agent. In another embodiment, the medicament is for stimulating the immune system. In another embodiment, the medicament is for use in a method of stimulating the immune system of an individual, the method comprising administering to the individual an effective amount of the medicament to stimulate the immune system. An "individual" according to any of the above embodiments may be a mammal, preferably a human. The "stimulation of the immune system" according to any of the above embodiments may comprise any one or more of the following: general enhancement of immune function, enhancement of T cell function, enhancement of B cell function, restoration of lymphocyte function, increase in IL-2 receptor expression, enhancement of T cell reactivity, enhancement of natural killer cell activity or Lymphokine Activated Killer (LAK) cell activity, and the like.

In another aspect, the invention provides a method of treating a disease in an individual. In one embodiment, the method comprises administering to an individual having such a disease a therapeutically effective amount of an immunoconjugate of the invention. In one embodiment, the individual is administered a composition comprising an immunoconjugate of the invention in a pharmaceutically acceptable form. In certain embodiments, the disease to be treated is a proliferative disease. In a particular embodiment, the disease is cancer. In certain embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., if the disease to be treated is cancer, an anti-cancer agent. In another aspect, the invention provides a method of stimulating the immune system of an individual, the method comprising administering to the individual an effective amount of an immunoconjugate to stimulate the immune system. An "individual" according to any of the above embodiments may be a mammal, preferably a human. The "stimulation of the immune system" according to any of the above embodiments may comprise any one or more of the following: general enhancement of immune function, enhancement of T cell function, enhancement of B cell function, restoration of lymphocyte function, increase in IL-2 receptor expression, enhancement of T cell reactivity, enhancement of natural killer cell activity or Lymphokine Activated Killer (LAK) cell activity, and the like.

In certain embodiments, the disease to be treated is a proliferative disease, in particular cancer. Non-limiting examples of cancer include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell cancer, bone cancer, and renal cancer. Other cell proliferation disorders that can be treated using the immunoconjugates of the invention include, but are not limited to, tumors located in: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testis, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral nervous system), lymphatic system, pelvis, skin, soft tissue, spleen, chest, and urogenital system. Also included are precancerous conditions or lesions and metastases. In certain embodiments, the cancer is selected from the group consisting of: kidney, skin, lung, colorectal, breast, brain, head and neck, prostate and bladder cancer. The skilled artisan will readily recognize that in many cases, immunoconjugates may not provide a cure, but may only provide partial benefit. In some embodiments, physiological changes with some benefit are also considered therapeutically beneficial. Thus, in some embodiments, the amount of 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 particularly a human.

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

For the prevention or treatment of disease, the appropriate dosage of the immunoconjugate of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend upon the type of disease to be treated, the route of administration, the body weight of the patient, the type of molecule (e.g., with or without an Fc domain), the severity and course of the disease, whether the immunoconjugate is administered for prophylactic or therapeutic purposes, previous or concurrent therapeutic intervention, the patient's clinical history and response to the immunoconjugate, and the judgment of the attending physician. In any case, the practitioner responsible for administration will determine the concentration and appropriate dosage of the active ingredient in the composition for the individual subject. Various dosing schedules are contemplated herein, including but not limited to single or multiple administrations at various time points, bolus administrations, and pulsed infusions.

The immunoconjugate is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μ g/kg to 15mg/kg (e.g., 0.1mg/kg-10mg/kg) of the immunoconjugate may be an initial candidate dose for administration to a patient, whether, for example, by one or more separate administrations, or by continuous infusion. Depending on the factors mentioned above, a typical daily dose may range from about 1. mu.g/kg to 100mg/kg or more. For repeated administrations over several days or longer, depending on the condition, the treatment will generally continue until the desired suppression of disease symptoms occurs. An exemplary dose of the immunoconjugate should be in the range of about 0.005mg/kg to about 10 mg/kg. In other non-limiting examples, the dose can further comprise about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derived therefrom. In non-limiting examples of ranges derivable from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 micrograms/kg/body weight to about 500 milligrams/kg/body weight, and the like, may be administered based on the above-mentioned values. Thus, one or more dose(s) of about 0.5mg/kg, 2.0mg/kg, 5.0mg/kg, or 10mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, such as weekly or every three weeks (e.g., such that a patient receives about two to about twenty doses, or, for example, about six doses of the immunoconjugate). An initial higher loading dose may be administered followed by one or more lower doses. However, other dosage regimens may be useful. The progress of the therapy can be readily monitored by conventional techniques and assays.

The immunoconjugates of the invention will generally be used in an amount effective to achieve the intended purpose. For use in treating or preventing a disorder, the immunoconjugate of the invention or pharmaceutical composition thereof is administered or applied in a therapeutically effective amount. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, particularly in light of the detailed disclosure provided herein.

For systemic administration, a therapeutically effective dose may be initially estimated from in vitro assays, such as cell culture assays. Doses can then be formulated in animal models to achieve IC including as determined in cell culture₅₀Circulating concentration range. Such information can be used to more accurately determine useful doses for humans.

Initial dosages can also be estimated from in vivo data (e.g., animal models) using techniques well known in the art. Administration to humans can be readily optimized by one of ordinary skill in the art based on animal data.

The amount and spacing of the doses can be adjusted individually to provide plasma levels of the immunoconjugate sufficient to maintain the therapeutic effect. The usual patient dose for administration by injection is in the range of about 0.1 to 50 mg/kg/day, usually about 0.5 to 1 mg/kg/day. Therapeutically effective plasma levels can be achieved by administering multiple doses per day. Levels in plasma can be measured, for example, by HPLC.

In the case of topical administration or selective uptake, the effective local concentration of the immunoconjugate may not be related to the plasma concentration. One skilled in the art will be able to optimize therapeutically effective local dosages without undue experimentation.

A therapeutically effective dose of the immunoconjugate described herein will generally provide therapeutic benefit without causing substantial toxicity. Toxicity and therapeutic efficacy of the immunoconjugates can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Cell culture assays and animal studies can be used to determine LD₅₀(dose of 50% of lethal population) and ED₅₀(a therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD₅₀/ED₅₀. Immunoconjugates exhibiting a large therapeutic index are preferred. In one embodiment, the immunoconjugate according to the invention exhibits a high therapeutic index. Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages suitable for use in humans. The dosage is preferably selected to include ED with little or no toxicity₅₀In the circulating concentration range of (c). The dosage may vary within this range depending upon a variety of factors, such as the dosage form employed, the route of administration utilized, the condition of the subject, and the like. The exact formulation, route of administration and dosage may be selected by the individual physician in accordance with the condition of the patient. (see, e.g., Fingl et al, 1975, in: The pharmaceutical Basis of Therapeutics, Chapter 1, page 1, The entire contents of which are incorporated herein by reference).

The attending physician of a patient treated with an immunoconjugate of the invention will know how and when to terminate, discontinue or regulate administration due to toxicity, organ dysfunction, and the like. Conversely, if the clinical response is not sufficient (toxicity excluded), the attending physician will also know to adjust the treatment to higher levels. The size of the dose administered in the management of the target disorder will vary with the severity of the condition to be treated, the route of administration, and the like. For example, the severity of a condition can be assessed, in part, by standard prognostic assessment methods. In addition, the dose and possibly the frequency of dosing will also vary according to the age, weight and response of the individual patient.

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

Other Agents and treatments

The immunoconjugates according to the invention may be administered in combination with one or more other agents in therapy. For example, the 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 or disease in an individual in need of such treatment. Such additional therapeutic agents may comprise any active ingredient suitable for the particular indication being treated, preferably active ingredients having complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is an immunomodulator, cytostatic, cell adhesion inhibitor, cytotoxic agent, apoptosis activator, or an agent that increases the sensitivity of a cell to an apoptosis-inducing agent. In a particular embodiment, the additional therapeutic agent is an anti-cancer agent, such as a microtubule disrupting agent, an anti-metabolite, a topoisomerase inhibitor, a DNA intercalating agent, an alkylating agent, a hormonal therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an anti-angiogenic agent.

Such other agents are suitably present in combination in an amount effective for the intended purpose. The effective amount of such other agents depends on the amount of immunoconjugate used, the type of disorder or treatment, and other factors discussed above. The immunoconjugate is typically used at the same dosage and route of administration as described herein, or at about 1% to 99% of the dosage described herein, or at any dosage and any route empirically/clinically determined to be appropriate.

Such combination therapies described above include combined administration (where two or more therapeutic agents are included in the same or different compositions) and separate administration, in which case administration of the immunoconjugate of the invention may occur prior to, concurrently with, and/or after administration of additional therapeutic agents and/or adjuvants. The immunoconjugates of the invention can also be used in combination with radiation therapy.

Article of manufacture

In another aspect of the invention, an article of manufacture is provided that contains materials useful for the treatment, prevention and/or diagnosis of the above-mentioned conditions. The article of manufacture comprises a container and a label or package insert (package insert) on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, Intravenous (IV) solution bags, and the like. The container may be formed from a variety of materials such as glass or plastic. The container contains a composition that is effective, by itself or in combination with another composition, for treating, preventing and/or diagnosing a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an immunoconjugate of the invention. The label or package insert indicates that the composition is for use in treating the selected condition. In addition, the article of manufacture can comprise (a) a first container comprising a composition, wherein the composition comprises an immunoconjugate of the invention; and (b) a second container containing a composition, wherein the composition comprises an additional cytotoxic or other therapeutic agent. The article of manufacture of this embodiment of the invention may further comprise a package insert indicating that the composition is useful for treating a particular condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. The article of manufacture may also include other materials as desired from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

Amino acid sequence

Examples of the invention

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

Example 1

Example 1.1 Generation of anti-CD 8-IL2v TA and anti-CD 8-IL2v OA conjugates targeting human CD8

The abbreviation "TA" means "double arm" herein. The abbreviation "OA" refers herein to "one-armed". The terms "anti-CD 8-IL2v TA" and "CD 8-IL2v TA" are used interchangeably herein. The terms "anti-CD 8-IL2v OA" and "CD 8-IL2v OA" are used interchangeably herein.

Expression of all genes is controlled by the human CMV promoter-intron a-5' UTR cassette. The BGH polyadenylation signal is located downstream of the gene. For use in HEK293 EBNA cells, the vector contains an oriP element for stable episomal maintenance of the plasmid.

Example 1.2 production of IgG-like proteins in HEK293 EBNA cells

IgG-IL2 molecules were produced by transient transfection of HEK293 EBNA cells. For anti-CD 8-IL2v TA, cells were transfected with the corresponding expression vectors in the following ratios: 1:1:2 ("vector heavy chain (VH-CH1-CH2-CH 3)" vector heavy chain (VH-CH1-CH2-CH3-IL2v) ": vector light chain (VL-CL)"). For anti-CD 8-IL2v OACells were transfected with the corresponding expression vectors in the following ratios: 1:1:1 ratio ("vector heavy chain (VH-CH1-CH2-CH 3)" vector heavy chain (CH2-CH3-IL2v) "to" vector light chain (VL-CL) "). The cells were centrifuged and then the pre-warmed CD CHO medium (Thermo Fisher, Cat. No. 10743029) was used in place of the original medium. The expression vectors were mixed in CD CHO medium, PEI (polyethyleneimine, Polysciences, Inc, cat # 23966-1) was added, the solution was vortexed, and incubated at room temperature for 10 minutes. Then, the cells (2Mio/ml) were mixed with the carrier/PEI solution, transferred to flasks, and placed in a shaking incubator at 5% CO₂Was incubated at 37 ℃ for 3 hours under the atmosphere of (2). After incubation, Excell medium (W.Zhou and A.Kantardjieff, Mammalian Cell Cultures for Biologics management, DOI: 10.1007/978-3-642-54050-9; 2014) containing supplements (80% of the total volume) was added. 1 day after transfection, supplement (feed, 12% of total volume) was added. After 7 days, the cell supernatant was harvested by centrifugation and subsequent filtration (0.2 μm filter), and the protein was purified from the harvested supernatant using standard methods as shown below.

Example 1.3 purification of IgG-like proteins

The protein was purified from the filtered cell culture supernatant according to standard protocols. Briefly, Protein A affinity chromatography (equilibration buffer: 20mM sodium citrate, 20mM sodium phosphate, pH 7.5; elution buffer: 20mM sodium citrate, 100mM NaCl, 100mM glycine pH 3.0) was used. Elution was achieved at pH 3.0, followed by immediate neutralization of the pH of the sample. By centrifugation (Millipore)

ULTRA-15(Art. Nr.: UFC903096) concentrates the protein and then separates aggregated from monomeric protein using size exclusion chromatography in 20mM histidine, 140mM sodium chloride (pH 6.0).

Example 1.4 analysis of IgG-like proteins

The concentration of the purified Protein was determined by measuring the absorbance at 280nm using the method based on the amino acid sequence according to Pace et al (Protein Science,1995,4,2411-1423)The resulting mass extinction coefficient is listed. The purity and molecular weight of the proteins were analyzed by CE-SDS in the presence and absence of reducing agents using LabChipGXII (Perkin Elmer). Determination of the aggregate content by HPLC chromatography at 25 ℃ is carried out as follows: using an analytical size exclusion column (TSKgel G3000 SW XL or UP-SW3000), in running buffer (25 mM K, respectively)₂HPO₄125mM NaCl, 200mM L-arginine monohydrochloride (pH 6.7) or 200mM KH₂PO₄250mM KCl (pH 6.2)).

Both IgG-IL2v constructs were purified with comparable and good quality, with monomer content higher than 98% as determined by size exclusion chromatography. The percentage of main peaks (determined by CE-SDS) was higher than 92%. The yield after purification (amount of purification in mg divided by volume of supernatant produced in L) was 2.1mg/L for anti-CD 8-IL2v TA and 7.8mg/L for anti-CD 8-IL2v OA.

anti-CD 8-IL2v TA consists of two polypeptides having the amino acid sequence of SEQ ID NO:9, one polypeptide having the amino acid sequence of SEQ ID NO:10 and one polypeptide having the amino acid sequence of SEQ ID NO:11 (form: A2 HK). anti-CD 8-IL2v OA consists of one polypeptide having the amino acid sequence of SEQ ID NO 9, one polypeptide having the amino acid sequence of SEQ ID NO 10 and one polypeptide having the amino acid sequence of SEQ ID NO 12 (form: AHK).

anti-CD 8(OKT8.v11) -IL2v TA consists of two polypeptides having the amino acid sequence of SEQ ID NO:30, one polypeptide having the amino acid sequence of SEQ ID NO:10 and one polypeptide having the amino acid sequence of SEQ ID NO:11 (form: A2 HK). The terms "anti-CD 8(okt8.v11) -IL2v TA", "anti-CD 8-IL2v okt8.v11 TA" and "CD 8-IL2v okt8.v11 TA" are used interchangeably herein. anti-CD 8(OKT8.v11) -IL2v OA consists of one polypeptide having the amino acid sequence of SEQ ID NO:30, one polypeptide having the amino acid sequence of SEQ ID NO:10 and one polypeptide having the amino acid sequence of SEQ ID NO:12 (form: AHK). The terms "anti-CD 8(okt8.v11) -IL2v OA", "anti-CD 8-IL2v okt8.v11 OA" and "CD 8-IL2v okt8.v11 OA" are used interchangeably herein.

The conjugates prepared in this example were further used in the following examples.

Example 2

Binding of anti-CD 8-IL2v TA and anti-CD 8-IL2v OA to CD 8T cells

Freshly isolated PBMCs from healthy donors were counted and transferred to 96-well round bottom plates (100,000 cells per well). Cells were washed with FACS buffer (PBS, 2% FBS, 5mM EDTA, 0.025% NaN3) and stained with 30 μ Ι of the indicated molecule in FACS buffer for 30 minutes at 4 ℃. The cells were washed twice with FACS buffer to remove unbound molecules. Mu.l of diluted FITC anti-human Fc specific secondary antibody (1:50 dilution, 109-. After incubation at 4 ℃ for 30min, unbound antibody was removed by washing twice with FACS buffer. Finally, cells were resuspended in FACS buffer and CD3 was gated using BD Fortessa⁺CD8⁺Cells (CD 8T cells) were measured.

The ability of anti-CD 8-IL2v TA and anti-CD 8-IL2v OA molecules to bind to CD 8T cells within PBMCs was evaluated in comparison to FAP-IL2v (e.g., as disclosed in WO2012107417a1, which is incorporated herein by reference; FAP-IL2v comprises polypeptides according to SEQ ID NOs: 25, 26 and 27). As shown in FIG. 2, anti-CD 8-IL2v TA showed very strong binding to CD 8T cells, and anti-CD 8-IL2v OA bound weakly to CD 8T cells, but still much stronger than FAP-IL2 v. This is consistent with the fact that anti-CD 8-IL2v OA can only bind monovalently to CD8, whereas anti-CD 8-IL2v TA can bind bivalently to CD8, resulting in a higher affinity of the latter for CD 8. IL2v contributed little to binding to CD 8T cells, as seen with FAP-IL2v, because IL2R β/γ expression was lower in resting CD 8T cells compared to CD8 expression (fig. 2).

Example 3

STAT5 phosphorylation of immune cells following treatment with anti-CD 8-IL2v TA and anti-CD 8-IL2v OA freshly isolated PBMC from healthy donors were seeded in warm medium (RPMI1640, 10% FCS,2mM glutamine) in 96-well round bottom plates (200,000 cells/well). Will boardCentrifuge at 300g for 10min and remove supernatant. Cells were resuspended in 100. mu.l of medium containing IL2v conjugate and stimulated at 37 ℃ for 20 min. To maintain the phosphorylation state, cells were fixed with an equal amount of pre-warmed Cytofix buffer (554655, BD Bioscience) for 10min at 37 ℃ immediately after stimulation. The plate was then centrifuged at 300g for 10min and the supernatant removed. To allow intracellular staining, cells were permeabilized in 200. mu.l Phosflow Perm buffer III (558050, BD Bioscience) for 30min at 4 ℃. The cells were then washed twice with 150 μ l cold FACS buffer and separated into two 96-well round bottom plates and stained with 20 μ l each of antibody mix I or II in the refrigerator for 60 min. Antibody cocktail I was used to stain pSTAT5 in CD 4T cells and regulatory T cells, and antibody cocktail II was used to stain pSTAT5 in CD 8T cells and NK cells. The cells were then washed twice with FACS buffer and resuspended in 200 μ l FACS buffer containing 2% PFA per well. Gating of CD 8T cells using BD Fortessa flow cytometer (CD 3)⁺CD8⁺) NK cells (CD 3)^-CD56⁺CD 4T cell (CD 4)⁺) And Tregs (CD 4)⁺CD25⁺FoxP3⁺) An analysis is performed.

TABLE 1 FACS antibody cocktail I (CD 4T cells and regulatory T cells)

TABLE 2 FACS antibody cocktail II (CD 8T cells and NK cells)

The functional activity of anti-CD 8-IL2v TA and anti-CD 8-IL2v OA to induce STAT5 phosphorylation was compared to the functional activity of FAP-IL2v on different subpopulations of immune cells within PBMCs. STAT5 phosphorylation serves as a marker for activation of the IL2 receptor (IL 2R). As shown in figure 3, all three test molecules have the same activity in inducing STAT5 phosphorylation on CD 4T cells and regulatory T cells (tregs). On CD 8T cells, anti-CD 8-IL2v TA and anti-CD 8-IL2v OA had higher potency in inducing STAT5 phosphorylation, while anti-CD 8-IL2v TA was slightly more potent than anti-CD 8-IL2v OA. On NK cells, anti-CD 8-IL2v TA and anti-CD 8-IL2v OA were slightly more potent than FAP-IL2v, probably because a fraction of NK cells were positive for CD 8. These data indicate that targeting IL2v to CD 8T cells can strongly enhance activation of IL2R via IL2v, and this effect is only mediated in cis, as IL2R activation on CD8 negative T cells is not increased (fig. 3).

Example 4

Proliferation of immune cells following treatment with CD8-IL2v and CD8-IL2v OA

Freshly isolated PBMCs from healthy donors were labeled with CFSE (5(6) -carboxyfluorescein diacetate N-succinimidyl ester, 21888, Sigma-Aldrich). Briefly, 3000 ten thousand PBMCs were washed once with PBS. In parallel, CSFE stock solutions (2mM in DMSO) were diluted 1:20 in PBS. The PBMCs were resuspended in 30ml of pre-warmed PBS, 30. mu.l of CFSE solution was added and the cells were immediately mixed. For optimal labeling, cells were incubated at 37 ℃ for 15 minutes. 10ml of pre-warmed medium (RPMI1640, 10% FCS, 1% glutamine) was then added to stop the labeling reaction. Cells were spun down (centrifuged) at 400g for 10min, then resuspended in 20ml fresh medium and incubated at 37 ℃ for a further 30 min. Finally, the cells were washed once with medium and resuspended in fresh medium at 100 ten thousand cells per ml. Labeled PBMCs were seeded in 96-well round bottom plates (100' 000 cells per well) and treated with the indicated molecules for 5 days. After incubation, cells were washed once with FACS buffer and stained with 20 μ l of a mixture of CD3 APC-Cy7(557834, BD Bioscience), CD8 APC (clones SK1, 344722, BioLegend), CD4 PE (300508, BioLegend) and CD56 BV421(318328, BioLegend) in FACS buffer for 30 minutes at 4 ℃. The PBMCs were then washed twice with FACS buffer, then they were fixed with 1% PFA in FACS buffer and fluorescence was measured with BD Fortessa. By measuring CD 8T cells (CD 3)⁺CD8⁺) CD 4T cell (CD 3)⁺CD4⁺) And NK cells (CD 3)^-CD56⁺) CFSE dilution of (a) to determine proliferation.

The ability of CD8-IL2v and CD8-IL2v OA to induce proliferation of CD 8T cells, CD 4T cells and NK cells was tested in comparison to FAP-IL2 v. As shown in FIG. 4, CD8-IL2v TA and CD8-IL2v OA have activity of inducing proliferation of CD 4T cells and NK cells similar to that of FAP-IL2 v. In contrast, on CD 8T cells, these two molecules were more potent than FAP-IL2v in inducing proliferation because these molecules directly target these cells via CD 8. The potency of CD8-IL2v TA was about 1300-fold higher, while the potency of CD8-IL2v OA was about 200-fold higher. This is consistent with the differences in binding and STAT5 phosphorylation observed on CD 8T cells.

Example 4

STAT5 phosphorylation of immune cells following treatment with CD8-IL2v TA and CD8-IL2v OKT8.v11 TA

Freshly isolated PBMCs from healthy donors were seeded in warm medium (RPMI1640, 10% FCS,2mM glutamine) in 96-well round bottom plates (200,000 cells/well). Plates were centrifuged at 300g for 10min and supernatants were removed. The cells were resuspended in 100. mu.l of medium containing IL2v molecule and stimulated at 37 ℃ for 20 min. To maintain the phosphorylation state, cells were fixed with an equal amount of pre-warmed Cytofix buffer (554655, BD Bioscience) for 10min at 37 ℃ immediately after stimulation. The plate was then centrifuged at 300g for 10min and the supernatant removed. To allow intracellular staining, cells were permeabilized in 200. mu.l Phosflow Perm buffer III (558050, BD Bioscience) for 30min at 4 ℃. The cells were then washed twice with 150 μ l cold FACS buffer and separated into two 96-well round bottom plates and stained with 20 μ l each of antibody mix I or II in the refrigerator for 60 min. Antibody cocktail I was used to stain pSTAT5 in CD 4T cells and regulatory T cells, and antibody cocktail II was used to stain pSTAT5 in CD 8T cells and NK cells. The cells were then washed twice with FACS buffer and resuspended in 200 μ l FACS buffer containing 2% PFA per well. Gating of CD 8T cells using BD Fortessa flow cytometer (CD 3)⁺CD8⁺) NK cells (CD 3)^-CD56⁺CD 4T cell (CD 4)⁺) And Tregs (CD 4)⁺CD25⁺FoxP3⁺) An analysis is performed.

TABLE 3 FACS antibody cocktail I (CD 4T cells and regulatory T cells)

TABLE 4 FACS antibody cocktail II (CD 8T cells and NK cells)

The functional activity of CD8-IL2v TA and CD8-IL2v okt8.v11 TA to induce STAT5 phosphorylation was compared to the functional activity of FAP-IL2v on different subpopulations of immune cells within PBMCs. STAT5 phosphorylation serves as a marker for activation of the IL2 receptor (IL 2R). On CD 4T cells and regulatory T cells (tregs), all three test molecules showed the same activity in inducing STAT5 phosphorylation. On CD 8T cells, CD8-IL2v TA and CD8-IL2v okt8.v11 TA showed higher potency in inducing STAT5 phosphorylation, but there was no difference in activation between CD8-IL2v TA and CD8-IL2v okt8.v11 TA. On NK cells, CD8-IL2v TA and CD8-IL2v OKT8.v11 TA were slightly more potent than FAP-IL2v, probably because some of the NK cells were positive for CD 8. These data indicate that targeting IL2v to CD 8T cells can strongly enhance activation of IL2R via IL2v, and this effect is only mediated in cis, as IL2R activation on CD8 negative T cells is not increased (fig. 5).

***

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the same is not to be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Sequence listing

<110> Haofmai Roche Ltd

<120> immunoconjugates

<130> P35369

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<170> PatentIn version 3.5

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Asp Thr Tyr Ile His

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Arg Ile Asp Pro Ala Asn Asp Asn Thr Leu Tyr Ala Ser Lys Phe Gln

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Gly

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Gly Tyr Gly Tyr Tyr Val Phe Asp His

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Arg Thr Ser Arg Ser Ile Ser Gln Tyr Leu Ala

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Ser Gly Ser Thr Leu Gln Ser

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Gln Gln His Asn Glu Asn Pro Leu Thr

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Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

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Gly Arg Ile Asp Pro Ala Asn Asp Asn Thr Leu Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

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Gly Arg Gly Tyr Gly Tyr Tyr Val Phe Asp His Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 8

<211> 107

<212> PRT

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<400> 8

Asp Val Gln Ile Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Arg Ser Ile Ser Gln Tyr

20 25 30

Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile

35 40 45

Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu Asn Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 9

<211> 214

<212> PRT

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<400> 9

Asp Val Gln Ile Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Arg Ser Ile Ser Gln Tyr

20 25 30

Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile

35 40 45

Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu Asn Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 10

<211> 448

<212> PRT

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<400> 10

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Ala Asn Asp Asn Thr Leu Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Gly Arg Gly Tyr Gly Tyr Tyr Val Phe Asp His Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys

355 360 365

Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 11

<211> 595

<212> PRT

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Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Ala Asn Asp Asn Thr Leu Tyr Ala Ser Lys Phe

50 55 60

Gln Gly Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Gly Arg Gly Tyr Gly Tyr Tyr Val Phe Asp His Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Pro

450 455 460

Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu

465 470 475 480

Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro

485 490 495

Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Ala Met Pro Lys Lys Ala

500 505 510

Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu

515 520 525

Glu Glu Val Leu Asn Gly Ala Gln Ser Lys Asn Phe His Leu Arg Pro

530 535 540

Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly

545 550 555 560

Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile

565 570 575

Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile Ile Ser

580 585 590

Thr Leu Thr

595

<210> 12

<211> 374

<212> PRT

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Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu

245 250 255

His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr

260 265 270

Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Ala Met Pro

275 280 285

Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu

290 295 300

Lys Pro Leu Glu Glu Val Leu Asn Gly Ala Gln Ser Lys Asn Phe His

305 310 315 320

Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu

325 330 335

Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr

340 345 350

Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser

355 360 365

Ile Ile Ser Thr Leu Thr

370

<210> 13

<211> 133

<212> PRT

<213> Intelligent people

<400> 13

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 14

<211> 133

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<400> 14

Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Ala Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Gly Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 15

<211> 15

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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 16

<211> 133

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<400> 16

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 17

<211> 133

<212> PRT

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<220>

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<400> 17

Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Ala Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Gly Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 18

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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 19

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<400> 19

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser

20

<210> 20

<211> 133

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<400> 20

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 21

<211> 225

<212> PRT

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<220>

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<400> 21

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro

225

<210> 22

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 22

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 23

<211> 105

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 23

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

1 5 10 15

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

20 25 30

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

35 40 45

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

50 55 60

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

65 70 75 80

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

85 90 95

Lys Thr Val Ala Pro Thr Glu Cys Ser

100 105

<210> 24

<211> 328

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 24

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro

325

<210> 25

<211> 604

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 25

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ile Gly Ser Gly Ala Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Trp Phe Gly Gly Phe Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu

115 120 125

Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys

130 135 140

Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser

145 150 155 160

Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp

180 185 190

Pro Ser Gln Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr

195 200 205

Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys

210 215 220

Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys

225 230 235 240

Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val

245 250 255

Val Val Ala Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe

260 265 270

Val Asp Asp Val Glu Val His Thr Ala Gln Thr Lys Pro Arg Glu Glu

275 280 285

Gln Ile Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His

290 295 300

Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala

305 310 315 320

Ala Phe Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg

325 330 335

Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met

340 345 350

Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asn Phe Phe Pro

355 360 365

Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn

370 375 380

Tyr Asp Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val

385 390 395 400

Tyr Ser Asp Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr

405 410 415

Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu

420 425 430

Lys Ser Leu Ser His Ser Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly

435 440 445

Gly Ser Gly Gly Gly Gly Ser Ala Pro Ala Ser Ser Ser Thr Ser Ser

450 455 460

Ser Thr Ala Glu Ala Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln

465 470 475 480

Gln His Leu Glu Gln Leu Leu Met Asp Leu Gln Glu Leu Leu Ser Arg

485 490 495

Met Glu Asn Tyr Arg Asn Leu Lys Leu Pro Arg Met Leu Thr Ala Lys

500 505 510

Phe Ala Leu Pro Lys Gln Ala Thr Glu Leu Lys Asp Leu Gln Cys Leu

515 520 525

Glu Asp Glu Leu Gly Pro Leu Arg His Val Leu Asp Gly Thr Gln Ser

530 535 540

Lys Ser Phe Gln Leu Glu Asp Ala Glu Asn Phe Ile Ser Asn Ile Arg

545 550 555 560

Val Thr Val Val Lys Leu Lys Gly Ser Asp Asn Thr Phe Glu Cys Gln

565 570 575

Phe Asp Asp Glu Ser Ala Thr Val Val Asp Phe Leu Arg Arg Trp Ile

580 585 590

Ala Phe Ala Gln Ser Ile Ile Ser Thr Ser Pro Gln

595 600

<210> 26

<211> 441

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 26

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ile Gly Ser Gly Ala Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Trp Phe Gly Gly Phe Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu

115 120 125

Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys

130 135 140

Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser

145 150 155 160

Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp

180 185 190

Pro Ser Gln Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr

195 200 205

Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys

210 215 220

Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys

225 230 235 240

Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val

245 250 255

Val Val Ala Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe

260 265 270

Val Asp Asp Val Glu Val His Thr Ala Gln Thr Lys Pro Arg Glu Glu

275 280 285

Gln Ile Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His

290 295 300

Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala

305 310 315 320

Ala Phe Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg

325 330 335

Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Lys Gln Met

340 345 350

Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asn Phe Phe Pro

355 360 365

Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn

370 375 380

Tyr Lys Asn Thr Gln Pro Ile Met Lys Thr Asp Gly Ser Tyr Phe Val

385 390 395 400

Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr

405 410 415

Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu

420 425 430

Lys Ser Leu Ser His Ser Pro Gly Lys

435 440

<210> 27

<211> 215

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 27

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Thr Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Asn Val Gly Ser Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Ile Met Leu Pro

85 90 95

Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Ala Asp Ala

100 105 110

Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser

115 120 125

Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp

130 135 140

Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val

145 150 155 160

Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met

165 170 175

Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser

180 185 190

Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys

195 200 205

Ser Phe Asn Arg Asn Glu Cys

210 215

<210> 28

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 28

Gln Gln Val Asn Glu Phe Pro Pro Thr

1 5

<210> 29

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 29

Asp Val Gln Ile Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Arg Ser Ile Ser Gln Tyr

20 25 30

Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile

35 40 45

Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Asn Glu Phe Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 30

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 30

Asp Val Gln Ile Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Arg Ser Ile Ser Gln Tyr

20 25 30

Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile

35 40 45

Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Asn Glu Phe Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

Claims (38)

1. An immunoconjugate comprising a mutant interleukin-2 (IL-2) polypeptide and an antibody that binds to CD8, wherein the IL-2 polypeptide is a mutant of IL-2 polypeptide comprising the amino acid substitutions F42A, Y45A, and L72G (numbered relative to the human IL-2 sequence of SEQ ID NO: 13).

2. The immunoconjugate according to claim 1,

wherein the IL-2 polypeptide mutant is a human IL-2 molecule comprising the amino acid substitutions F42A, Y45A and L72G (numbering relative to the human IL-2 sequence SEQ ID NO: 13); and is

Wherein the antibody comprises: (a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HCDR)1 comprising the amino acid sequence of SEQ ID NO:1, HCDR2 comprising the amino acid sequence of SEQ ID NO:2, HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; and (b) a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR)1 comprising the amino acid sequence of SEQ ID NO:4, LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and LCDR3 comprising the amino acid sequence of SEQ ID NO:6 or SEQ ID NO: 28.

3. The immunoconjugate according to claim 1 or 2, wherein the IL-2 polypeptide is an IL-2 polypeptide mutant,

wherein the IL-2 polypeptide mutant is a human IL-2 molecule comprising the amino acid substitutions F42A, Y45A and L72G (numbering relative to the human IL-2 sequence SEQ ID NO: 13); and is

Wherein the antibody comprises: (a) a heavy chain variable region (VH) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 7; and (b) a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:8 or SEQ ID NO: 29.

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

5. The immunoconjugate of any one of claims 1 to 4, wherein the mutant IL-2 polypeptide comprises the sequence of SEQ ID NO 14.

6. The immunoconjugate according to any one of claims 1 to 5, wherein the immunoconjugate comprises no more than one mutant of an IL-2 polypeptide.

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

8. The immunoconjugate according to claim 7, wherein the Fc domain is an IgG class Fc domain, in particular an IgG₁A subclass Fc domain.

9. The immunoconjugate according to claim 7 or 8, wherein the Fc domain is a human Fc domain.

10. The immunoconjugate according to any one of claims 1 to 9, wherein the antibody is an immunoglobulin of the IgG class, in particular IgG₁Subclass immunoglobulin.

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

12. The immunoconjugate according to any one of claims 7 to 11, wherein in the CH3 domain of the first subunit of the Fc domain, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby creating a protuberance within the CH3 domain of the first subunit that is positionable in a cavity within the CH3 domain of the second subunit; and in the CH3 domain of the second subunit of the Fc domain, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.

13. The immunoconjugate according to any one of claims 9 to 12, wherein in the first subunit of the Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W); and in the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V), and optionally the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the Kabat EU index).

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

15. The immunoconjugate according to any one of claims 9 to 14, wherein the mutant IL-2 polypeptide is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the subunits of the Fc domain, in particular to the carboxy-terminal amino acid of the first subunit of the Fc domain, optionally via a linking peptide.

16. The immunoconjugate according to claim 15, wherein the linking peptide has the amino acid sequence of SEQ ID No. 15.

17. The immunoconjugate according to any one of claims 9 to 15, wherein said Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors, particularly fey receptors, and/or reduce effector function, particularly antibody-dependent cell-mediated cytotoxicity (ADCC).

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

19. The immunoconjugate of any one of claims 9 to 18, wherein each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbering).

20. The immunoconjugate according to any one of claims 1 to 19, comprising: a polypeptide comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO 9 or 30, a polypeptide comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO 10, and a polypeptide comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO 11 or 12.

21. The immunoconjugate according to any one of claims 1 to 20, comprising a polypeptide comprising the amino acid sequence of SEQ ID No. 9 or SEQ ID No. 30, a polypeptide comprising the amino acid sequence of SEQ ID No. 10, and a polypeptide comprising the amino acid sequence of SEQ ID No. 11.

22. The immunoconjugate according to any one of claims 1 to 20, comprising a polypeptide comprising the amino acid sequence of SEQ ID No. 9 or SEQ ID No. 30, a polypeptide comprising the amino acid sequence of SEQ ID No. 10, and a polypeptide comprising the amino acid sequence of SEQ ID No. 12.

23. The immunoconjugate of any one of claims 1 to 22, consisting essentially of an IL-2 polypeptide mutant and an IgG joined by a linking sequence₁Immunoglobulin molecule composition.

24. One or more isolated polynucleotides encoding the immunoconjugate of any one of claims 1 to 23.

25. One or more vectors, in particular expression vectors, comprising a polynucleotide according to claim 24.

26. A host cell comprising the polynucleotide of claim 24 or the vector of claim 25.

27. A method of producing an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to CD8, the method comprising (a) culturing the host cell of claim 25 under conditions suitable for expression of the immunoconjugate, and optionally (b) recovering the immunoconjugate.

28. An immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to CD8, produced by the method of claim 27.

29. A pharmaceutical composition comprising the immunoconjugate of any one of claims 1 to 23 or claim 28 and a pharmaceutically acceptable carrier.

30. The immunoconjugate according to any one of claims 1 to 23 or claim 28 for use as a medicament.

31. The immunoconjugate according to any one of claims 1 to 23 or claim 28 for use in treating a disease.

32. The immunoconjugate for use in treating a disease according to claim 31, wherein the disease is cancer.

33. Use of an immunoconjugate according to any one of claims 1 to 23 or claim 28 in the manufacture of a medicament for the treatment of a disease.

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

35. A method of treating a disease in an individual comprising administering to the individual a therapeutically effective amount of a composition comprising the immunoconjugate of any one of claims 1 to 23 or claim 28 in a pharmaceutically acceptable form.

36. The method of claim 35, wherein the disease is cancer.

37. A method of stimulating the immune system of an individual comprising administering to the individual an effective amount of a composition comprising the immunoconjugate of any one of claims 1 to 23 or claim 28 in a pharmaceutically acceptable form.

38. The invention as hereinbefore described.

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