IL-2 mutants and uses thereof

Abstract

The present disclosure relates to mutants of cytokine IL-2 having altered functional characteristics including increased expression titers and selective binding to different IL-2 receptor protein complexes, pharmaceutical compositions comprising these mutant IL-2 polypeptides and the use of these compositions as therapeutic agents, for example in the treatment of cancer and autoimmune disorders.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 385,610, filed on November 30, 2022, which is hereby incorporated by reference in its entirety for all purposes. Technical Field

[0003] The present disclosure relates to mutant IL-2 polypeptides, as well as fusions of these polypeptides with half-life extending proteins (such as Fc monomers or Fc dimers), serum albumin polypeptides and / or specific binding biomolecules (such as VHH polypeptides) and antibodies, pharmaceutical compositions comprising these polypeptides, and the use of these compositions as therapeutic agents, for example, in the treatment of cancer or autoimmune diseases.

[0004] Sequence Listing Reference

[0005] An official copy of the sequence listing is filed with this specification via the USPTO Patent Center as an XML file in WIPO standard ST.26 format, with the file name "17195-001PV1.xml," creation date November 30, 2022, and a size of 163,978 bytes. This sequence listing filed via the USPTO Patent Center is part of this specification and is incorporated herein by reference in its entirety. Background Art

[0006] Interleukin-2 (IL-2), also known as T cell growth factor (TCGF), is a multipotent cytokine produced primarily by activated T cells, particularly CD4+ T helper cells. IL-2 signaling is mediated by binding to three different receptor proteins: IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132). Immune cells express either dimer or trimeric complexes of the IL-2 receptor protein. The dimeric receptor (IL-2Rβγ) is expressed on cytotoxic CD8+ T cells and natural killer (NK) cells, while the trimeric receptor (IL-2Rαβγ) is primarily expressed on activated lymphocytes and CD4+CD25+FoxP3+ suppressive regulatory T cells (Treg). Treg cells express high levels of IL-2Rα (CD25), and Treg proliferation is stimulated by IL-2. However, resting effector T cells and NK cells do not have CD25 on their cell surface and are relatively insensitive to IL-2.

[0007] IL-2 binds to three different receptor proteins in distinct ways. IL-2 has a high affinity for the trimeric receptor, with a K D The affinity of the dimeric receptor is about 10 pM, with a moderate affinity, K Dis approximately 1 nM and has low affinity for monomeric IL-2Rα receptor, K D The IL-2 signaling activity mediated by different receptor complexes also varies significantly. Generally, it has been found that IL-2Rβ and IL-2Rγ are critical for IL-2 signaling, while IL-2Rα (CD25) is not essential.

[0008] The combination of IL-2 and IL-2 receptor proteins expressed on different cells mediates different immune responses. IL-2 can stimulate immune responses, such as: T cell proliferation and differentiation, cytotoxic T lymphocytes (CTL) production, B cell proliferation and differentiation, immunoglobulin synthesis, and the production, proliferation and activation of natural killer (NK) cells. IL-2 has been approved as an immunotherapeutic agent for the treatment of cancer and chronic viral infections. However, IL-2 can also promote the activation and proliferation of immunosuppressive CD4+CD25+Treg cells, thereby leading to immunosuppression (Fontenot et al., Nature Immunol.6,1142-51 (2005); D'Cruz and Klein, Nature Immunol.6,1152-59 (2005); Maloy and Powrie, Nature Immunol.6,1171-72 (2005)). In addition, IL-2 treatment is associated with vascular leak syndrome (VLS) and pulmonary edema in patients. Pulmonary edema is believed to result from direct binding of IL-2 to a trimeric receptor (IL-2Rαβγ) on lung endothelial cells (Krieg et al., Proc Nat Acad Sci USA 107, 11906-11 (2010)).

[0009] The engineering of IL-2 with mutations has been proposed to reduce these toxic side effects by changing the selectivity or preference of IL-2 for different IL-2 receptor subunits, thereby improving its therapeutic effect. For example, it has been proposed that IL-2 targeting cells expressing IL-2Rβ but not expressing IL-2Rα can induce the expansion of IL-2Rβ high cell populations, which improves the therapeutic effect of IL-2 therapy (Boyman et al., Science 311,1924-1927 (2006)). U.S. Patent Publication 2018 / 0142037A1 describes the introduction of mutations at IL-2 amino acid positions 42, 45 and 72, also to reduce the affinity of IL-2 for IL-2Rα receptors. Another mutant IL-2, called "IL-2H9," contains five mutations: L80F, R81D, L85V, I86V, and I92F, and exhibits enhanced binding to IL-2Rβ, leading to stimulation of CD25- cells (see Levin et al., Nature, vol. 484, pp. 529-533, DOI: 10.1038 / nature10975). The mutant IL-2 protein "IL-23x" has three mutations: R38D, K43E, and E61R, which result in very low binding affinity for IL-2Rα (see Rodrigo Vazquez-Lombardi et al., Nature Communications, 8: 15373, DOI: 10.1038 / ncomms15373). However, IL-23x still retains its preference for activating CD25+ cells, and the expression level of the mutant polypeptide is low, making it difficult to produce large-scale drugs.

[0010] Therefore, there remains a need for variant forms of IL-2 that exhibit improved functional properties for therapeutic use, such as in pharmaceutical compositions for the treatment of diseases such as infections, cancer, and autoimmune disorders. Summary of the Invention

[0011] The present disclosure generally relates to mutants of the cytokine IL-2, pharmaceutical compositions, and the use of these compositions as therapeutic agents, for example, in the treatment of cancer. This summary is intended to introduce the subject matter of the present disclosure and is not intended to encompass every embodiment, combination, or variation contemplated and described herein. Additional embodiments are contemplated and described by way of the detailed description, drawings, and claims.

[0012] In at least one embodiment, the present disclosure provides a mutant IL-2 polypeptide that specifically binds to an IL-2 receptor protein, wherein the polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 10 (wild-type IL-2) and has one or more amino acid differences relative to SEQ ID NO: 10 selected from the group consisting of:

[0013]

[0014]

[0015] In at least one embodiment, the polypeptide further comprises one or more amino acid differences relative to SEQ ID NO: 10 selected from the group consisting of K35N, F42A, Y45R, N88D, D109N, and Q126T.

[0016] In at least one embodiment of the mutant IL-2 polypeptides of the present disclosure, the polypeptide comprises a combination of amino acid differences relative to SEQ ID NO: 10 selected from the group consisting of:

[0017]

[0018]

[0019] In at least one embodiment of the mutant IL-2 polypeptides disclosed herein, the polypeptide comprises a combination of amino acid differences selected from the group consisting of:

[0020]

[0021]

[0022] In at least one embodiment of the mutant IL-2 polypeptides disclosed herein, the polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47 ,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85 , 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144 and 145.

[0023] In at least one embodiment of the mutant IL-2 polypeptides of the present disclosure, the polypeptide is characterized by one or more of the following properties relative to the C125S IL-2 polypeptide of SEQ ID NO: 10:

[0024] (i) increased titer when expressed in mammalian cell culture systems;

[0025] (ii) increased solubility;

[0026] (iii) reduced binding affinity to the IL-2Rα receptor; and / or

[0027] (iv) Reduced binding affinity to IL-2Rβ and IL-2Rγ receptors.

[0028] In at least one embodiment of the mutant IL-2 polypeptides disclosed herein, the polypeptide is fused to a monomeric or dimeric Fc polypeptide via a linker; optionally, the Fc polypeptide is a monomeric Fc polypeptide comprising the amino acid sequence of SEQ ID NO: 146, 147, or 148. In at least one embodiment, the linker consists of a polypeptide having a length of at least 1-50 amino acids. In at least one embodiment, the linker comprises a polypeptide having an amino acid sequence selected from the group consisting of (GGGGS)1 (SEQ ID NO: 149), (GGGGS)2 (SEQ ID NO: 150), (GGGGS)3 (SEQ ID NO: 151), (GGGGS)4 (SEQ ID NO: 152), (GRPGS)2 (SEQ ID NO: 153), (GRPGS)4 (SEQ ID NO: 154), and (GGGGS)3GGG (SEQ ID NO: 155). In at least one embodiment, the linker is conjugated to the C-terminus of the Fc polypeptide and the N-terminus of the mutant IL-2 polypeptide. In at least one embodiment, the linker is conjugated to the N-terminus of the Fc polypeptide and the C-terminus of the mutant IL-2 polypeptide.

[0029] In at least one embodiment, the present disclosure provides a polynucleotide encoding a mutant IL-2 polypeptide of the present disclosure. In at least one embodiment, the present disclosure provides an expression vector comprising a polynucleotide encoding a mutant IL-2 polypeptide of the present disclosure.

[0030] In at least one embodiment, the present disclosure further provides an isolated host cell comprising a polynucleotide encoding a mutant IL-2 polypeptide of the present disclosure or an expression vector comprising such a polynucleotide. In at least one embodiment, the host cell is a mammalian cell or a yeast cell; optionally, the mammalian cell is selected from Chinese hamster ovary (CHO) cells, myeloma cells (e.g., Y0, NS0, Sp2 / 0), monkey kidney cells (COS-7), human embryonic kidney cell line (293), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells, human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells, TR1 cells, Medical Research Council 5 (MRC 5) cells, and FS4 cells.

[0031] In at least one embodiment, the present disclosure also provides a method for producing a mutant IL-2 polypeptide of the present disclosure, the method comprising culturing a host cell comprising a polynucleotide or expression vector encoding the mutant IL-2 polypeptide of the present disclosure under conditions suitable for expressing the polypeptide.

[0032] In at least one embodiment, the present disclosure also provides a pharmaceutical composition comprising the mutant IL-2 polypeptide of the present disclosure and a pharmaceutically acceptable carrier.

[0033] In at least one embodiment, the present disclosure also provides a method for treating a disease (e.g., an IL-2-mediated disease) in a subject, the method comprising administering to the subject a therapeutically effective amount of a mutant IL-2 polypeptide of the present disclosure or administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a mutant IL-2 polypeptide of the present disclosure and a pharmaceutically acceptable carrier. In at least one embodiment of the method, the disease is cancer; optionally, the cancer is selected from colorectal cancer, pancreatic cancer, ovarian cancer, liver cancer, kidney cancer, breast cancer, lung cancer, esophageal cancer and gastric cancer, head and neck cancer, cervical cancer, prostate cancer, melanoma, bladder cancer, or oral cancer.

[0034] In at least one embodiment of the method of treating cancer, the administered mutant IL-2 polypeptide exhibits a selectively decreased binding affinity to IL-2Rα relative to the IL-2Rα binding affinity of the C125 IL-2 polypeptide of SEQ ID NO: 10. In at least one embodiment of the method, the polypeptide comprises a combination of amino acid differences relative to SEQ ID NO: 10 selected from the group consisting of:

[0035] P65N, E67T; P65N, E67S; F42N、F44T K35N, P65N, E67T; K35N, P65N, E67S; K35N, Y45R, P65N, E67S; K35N, Y45R, P65N, E67T; K35N, F42N, F44T, D109N; K35N, F42N, F44S, D109N; F42A, R81N, P82A, R83T; F42A, R81N, P82A, R83S; Y45R, R81N, P82A, R83T; and Y45R, R81N, P82A, R83S.

[0036] In at least one embodiment of the method of treating a disease, the disease is an autoimmune disease; optionally, wherein the autoimmune disease is selected from Crohn's disease, ulcerative colitis, celiac disease, systemic lupus erythematosus, psoriatic arthritis, rheumatoid arthritis, Sjögren's syndrome, type 1 diabetes, atopic dermatitis, psoriasis, multiple sclerosis.

[0037] In at least one embodiment of the method of treating an autoimmune disease, the polypeptide exhibits selectively reduced IL-2Rβγ binding affinity relative to the IL-2Rβγ binding affinity of the C125 IL-2 polypeptide of SEQ ID NO: 10. In at least one embodiment of the method, the polypeptide comprises a combination of amino acid differences relative to SEQ ID NO: 10 selected from the group consisting of:

[0038]

[0039]

[0040] BRIEF DESCRIPTION OF THE DRAWINGS

[0041] A better understanding of the novel features and advantages of the present disclosure will be obtained by referring to the following detailed description and accompanying drawings (also referred to herein as "Figures") which illustrate exemplary embodiments in which the principles of the present disclosure are utilized, and in which:

[0042] Figure 1A 、 Figure 1B 、 Figure 1C 、 Figure 1D 、 Figure 1E 、 Figure 1F 、 Figure 1G 、 Figure 1H 、 Figure 1I and Figure 1J Depicted are the results of biolayer interferometry (BLI) measurements performed as described in Example 1 to measure binding of exemplary mutant IL-2 polypeptide Fc fusion constructs p132, p115, and p151 relative to a control p123 (C125S IL2) construct to IL-2Rα and IL-2Rβγ.

[0043] Figure 2A 、 Figure 2B 、 Figure 2C 、 Figure 2D 、 Figure 2E 、 Figure 2F 、 Figure 2G 、 Figure 2H 、 Figure 2I 、 Figure 2J 、 Figure 2K and Figure 2L Depicted are SEC profiles of exemplary mutant IL-2 polypeptide Fc fusion constructs p296, p307, p406, p297, p300, p308, p214, p310, p411, p298, and a p123 C125S IL2 fusion control, obtained as described in Example 1, as follows: Control p123 (C125S IL2) construct ( Figure 2A and Figure 2B )、p296( Figure 2C )、p307( Figure 2D )、p406( Figure 2E )、p297( Figure 2F )、p300( Figure 2G )、p308( Figure 2H )、p214( Figure 2I )、p310( Figure 2J )、p411( Figure 2K ) and p298( Figure 2L ).

[0044] Figure 3A 、 Figure 3B 、 Figure 3C 、 Figure 3D 、 Figure 3E、 Figure 3F 、 Figure 3G 、 Figure 3H 、 Figure 3I 、 Figure 3J 、 Figure 3K 、 Figure 3L 、 Figure 3M and Figure 3N Depicted are binding curves and EC for exemplary mutant IL-2 polypeptide Fc fusion constructs p124, p132, p167, p296, p300, p214, p307, p308, p310, p297, p298, and p411, as well as a p123 C125S IL2 fusion control, measured in the HEK Blue IL-2 reporter assay as described in Example 1. 50 The values ​​are as follows: p124, p132 and p167 ( Figure 3A 、 Figure 3B ); p296, p300 and p214( Figure 3C 、 Figure 3D ); p123, p307 and p214 ( Figure 3E 、 Figure 3F ); p123, p296 and p308 ( Figure 3G 、 Figure 3H ); p123, p310, and p214 ( Figure 3I 、 Figure 3J ); p296, p297, p298 and p214 ( Figure 3K 、 Figure 3L ); p411, p214, p123, p406 and p214 ( Figure 3M 、 Figure 3N ). DETAILED DESCRIPTION

[0045] The present disclosure provides mutant IL-2 polypeptides having mutations that alter the glycosylation of the polypeptide and affect various physicochemical and functional characteristics of IL-2, including recombinant expression titer, solubility, and binding affinity to the IL-2R chains IL-2Rα, IL-2Rβ, and IL-2Rγ in monomeric, dimer, and trimer forms. The altered binding characteristics of the mutant IL-2 polypeptides to different IL-2R chains allow for inhibition, reduction, and / or complete blocking of the function of the IL-2R receptor, particularly its function as a cell surface receptor in mediating immune regulation. Therefore, it is envisioned that any composition or formulation comprising the mutant IL-2 polypeptides of the present disclosure can be used as a therapeutic agent for the treatment of diseases mediated by the function of IL-2R or its cognate ligand IL-2, such as the treatment of cancer and autoimmune disorders. In addition, it is envisioned that the mutant IL-2 polypeptides of the present disclosure can be used as therapeutic agents in combination with other therapeutic agents (such as antibodies targeting immune checkpoint molecules).

[0046] Overview of terms and techniques

[0047] For purposes of the description herein and the appended claims, the singular forms "a," "an," and "an" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes more than one protein, and reference to "a compound" refers to more than one compound. It should also be noted that claims may be drafted to exclude any optional elements. Therefore, this description is intended to serve as a precondition for the use of such exclusive terminology as "only," "only," and the like in connection with the recitation of claim elements, or for the use of a "negative" limitation. The use of "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and are not intended to be limiting. It should also be understood that where the description of various embodiments uses the term "comprising," those skilled in the art will understand that, in some specific cases, the embodiments may alternatively be described using the language "consisting essentially of" or "consisting of."

[0048] In the case of providing the range of values, it should be understood that unless the context clearly indicates otherwise, each intermediate integer of the value between the upper and lower limits of the scope and each intermediate integer of the value and any other described or intermediate value in the described range are all encompassed within the present invention. These smaller ranges of upper and lower limits can be independently included in a smaller range, and are also encompassed within the present invention, except any definite limit beyond the described range. In the case where the scope includes one or two limits in these limits, excluding those included limits (i) any one or (ii) two scopes are also encompassed within the present invention. For example, "1 to 50" includes "2 to 25", "5 to 20", "25 to 50", "1 to 10" etc.

[0049] Generally, the nomenclature used herein and the techniques and procedures described herein include those well understood and commonly employed by those of ordinary skill in the art, such as described in, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 2012 (hereinafter “Sambrook”); and Current Protocols in Molecular Biology, F.M. Ausubel et al., eds. (Originally published in book form in 1987 by Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., with regular supplements until 2011, now available as CurrentProtocols inMolecularBiology,Vols.00-130 , (1987-2020) available online in journal form, published by Wiley & Sons, Inc. in the Wiley Online Library (hereinafter referred to as "Ausubel").

[0050] All publications, patents, patent applications, and other documents mentioned in this disclosure are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference herein for all purposes.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. It should be understood that the terms used herein are only used to describe specific embodiments and are not intended to be limiting. For the purpose of explaining this disclosure, the following terminology will be used, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0052] As used herein, "IL-2" or "IL-2 polypeptide" refers to the cytokine interleukin-2, and includes naturally occurring and recombinant forms of interleukin-2 polypeptides from humans, mice, rats, or non-human primates, as well as unprocessed forms (with signal peptides) and processed forms (without signal peptides). In addition, the term includes naturally occurring IL-2 variants, such as alleles and splice variants, isoforms, homologs, and species homologs, as well as recombinant (i.e., artificial) IL-2 variants or mutants, including mutant IL-2 polypeptides having 1-15 amino acid substitutions relative to the amino acid sequence of naturally occurring IL-2. For example, the term encompasses the recombinant human IL-2 amino acid sequence of UniProt P60568 having an amino acid substitution at position C125, such as C125S or C125A. The term is also intended to encompass IL-2 polypeptides covalently conjugated (or fused) to another polypeptide or protein. Exemplary IL-2 fusions of the present disclosure include mutant IL-2 polypeptides fused to other cytokines (eg, IL-15) or to half-life extending polypeptides (eg, monomeric Fc, dimeric Fc, or human serum albumin).

[0053] As used herein, "IL-2 receptor" or "IL-2R" refers to a heterotrimeric protein expressed on the surface of certain immune cells and endothelial cells, and also encompasses each of the polypeptide subunits IL-2Rα, IL-2Rβ, and IL-2Rγ (also known as cytokine receptor common subunit γ), such as IL-2Rβγ, in their monomeric and dimeric forms.

[0054] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an IL-2 polypeptide) and its binding partner (e.g., an IL-2 receptor). "Binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be expressed by the equilibrium dissociation constant (K D Affinity can be measured by common methods known in the art, including those described herein. "Specific binding" or "specific binding" means that the IL-2 polypeptide binds to its receptor with an affinity of no more than about 1 x 10 -7 The affinity value binding of M. Exemplary and exemplary embodiments for measuring binding affinity and / or specific binding are described elsewhere herein, including in the Examples.

[0055] As used herein, "host cell" refers to a cell that can be functionally modified with a recombinant nucleic acid and used to express a recombinant product (including a polypeptide and a compound produced by the activity of the polypeptide).

[0056] As used interchangeably herein, "nucleic acid" or "polynucleotide" refers to two or more nucleosides covalently linked together. Nucleic acid can be composed entirely of ribonucleosides (e.g., RNA), composed entirely of 2'-deoxyribonucleotides (e.g., DNA), or a mixture comprising ribonucleosides and 2'-deoxyribonucleosides. The nucleoside units of a nucleic acid can be linked together via phosphodiester bonds (e.g., as in naturally occurring nucleic acids), or the nucleic acid can comprise one or more non-natural bonds (e.g., phosphorothioate bonds). Nucleic acids or polynucleotides are intended to comprise single-stranded or double-stranded molecules, or molecules having both single-stranded and double-stranded regions. Nucleic acids or polynucleotides are intended to comprise molecules consisting of naturally occurring nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), or molecules comprising one or more modified and / or synthetic nucleobases (such as inosine, xanthine, hypoxanthine, etc.).

[0057] "Protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modifications (e.g., glycosylation, phosphorylation, lipidation, myristylation, ubiquitination, etc.). As used herein, a "protein" or "polypeptide" or "peptide" polymer may comprise D-amino acids and L-amino acids, as well as mixtures of D-amino acids and L-amino acids.

[0058] As used herein, "naturally occurring" or "wild type" refers to a form found in nature. For example, a naturally occurring nucleic acid sequence is a sequence found in an organism that can be isolated from a source in nature and has not been intentionally modified by human manipulation.

[0059] "Recombinant," "engineered," or "non-naturally occurring," when used herein with respect to, for example, cells, nucleic acids, or polypeptides, refers to a material, or a material corresponding to the natural or native form of the material, that has been modified in a manner not otherwise found in nature, or is identical thereto, but is produced or derived from synthetic materials and / or manipulations using recombinant techniques. Non-limiting examples include, inter alia, recombinant cells that express genes not present in the natural (non-recombinant) form of the cell or that express natural genes that are otherwise expressed at different levels.

[0060] As used herein, "nucleic acid derived from" refers to a nucleic acid having a sequence that is at least substantially identical to a sequence naturally occurring in an organism. For example, a cDNA molecule prepared by reverse transcribing mRNA isolated from an organism or a synthetically prepared nucleic acid molecule having a sequence that is at least substantially identical to a nucleic acid sequence present in the organism or a sequence that hybridizes therewith.

[0061] "Coding sequence" refers to the portion of a nucleic acid (eg, a gene) that encodes the amino acid sequence of a protein.

[0062] As used herein, "heterologous nucleic acid" refers to any polynucleotide that is introduced into a host cell by laboratory techniques and includes polynucleotides that are removed from a host cell, subjected to laboratory manipulation, and then reintroduced into the host cell.

[0063] "Codon-optimized" refers to changing the codons of a polynucleotide encoding a protein to codons that are preferentially used in a particular organism so that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate, in that most amino acids are represented by several codons (referred to as "synonymous" codons), it is well known that the codon usage of a particular organism is non-random and biased towards specific codon triplets. This codon usage bias may be higher for a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and aggregated protein coding regions of an organism's genome. In some embodiments, the polynucleotide encoding an imine reductase may be codon-optimized for optimal production from a host organism selected for expression.

[0064] "Preferred, optimal, high codon usage bias codons" refer to codons that are used more frequently in protein coding regions than other codons encoding the same amino acid. Preferred codons can be determined based on codon usage in a single gene, a group of genes of common function or origin, highly expressed genes, codon frequency in aggregate protein coding regions of an entire organism, codon frequency in aggregate protein coding regions of related organisms, or a combination thereof. Codons whose frequency increases with gene expression level are generally the optimal codons for expression. A variety of methods are known for determining codon frequency (e.g., codon usage, relative synonymous codon usage) and codon bias in a particular organism, including multivariate analysis, such as using cluster analysis or correspondence analysis, and the effective number of codons used in a gene (see GCG Codon Preference, Genetics Computer Group Wisconsin Package; Codon W, John Peden, University of Nottingham; McInerney, JO, 1998, Bioinformatics 14:372-73; Stenico et al., 1994, Nucleic Acids Res. 222 437-46; Wright, F., 1990, Gene 87:23-29). Codon usage tables are available for a growing list of organisms (see, e.g., Wada et al., 1992, Nucleic Acids Res. 20:2111-2118; Nakamura et al., 2000, Nucl. Acids Res. 28:292; Duret et al., supra; Henaut and Danchin, "Escherichia coli and Salmonella," 1996, Neidhardt et al., eds., ASM Press, Washington DC, pp. 2047-2066). The data source for obtaining codon usage can rely on any available nucleotide sequence capable of encoding a protein.These data sets include nucleic acid sequences that are actually known to encode expressed proteins (e.g., complete protein coding sequences - CDS), expressed sequence tags (ESTs), or predicted coding regions of genomic sequences (see, e.g., Mount, D., Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001; Uberbacher, EC, 1996, Methods Enzymol. 266:259-281; ​​Tiwari et al., 1997, Comput. Appl. Biosci. 13:263-270).

[0065] As used herein, "control sequences" refers to all sequences that are necessary or advantageous for the expression of polynucleotides and / or polypeptides as used in the present disclosure. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader sequence, a promoter, a polyadenylation sequence, a propeptide sequence, a signal peptide sequence, and a transcription terminator. At a minimum, a control sequence typically comprises a promoter, and transcriptional and translational termination signals. The control sequence may be provided with a linker to introduce specific restriction sites that facilitate ligation of the control sequence to the coding region of the nucleic acid sequence encoding the polypeptide.

[0066] As used herein, "operably linked" refers to a configuration in which a control sequence is appropriately placed (e.g., in a functional relationship) at a position relative to a polynucleotide sequence or polypeptide sequence of interest such that the control sequence directs or regulates the expression of the sequence of interest.

[0067] "Promoter sequence" refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest (such as a coding sequence). The promoter sequence contains transcriptional control sequences that mediate expression of the polynucleotide of interest. The promoter can be any nucleic acid sequence that shows transcriptional activity in the selected host cell, including mutant, truncated, and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0068] "Percentage of sequence identity," "percent sequence identity," "percent homology," or "percent homology" are used interchangeably herein and refer to a value that quantifies the comparison of a polynucleotide or polypeptide sequence and is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (or gaps) compared to the reference sequence for optimal alignment of the two sequences. The percentage value can be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in the two sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Alternatively, the percentage can be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in the two sequences or at which a nucleic acid base or amino acid residue is aligned with a gap to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. It will be appreciated by those skilled in the art that there are many established algorithms that can be used to align two sequences. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin software package), or by visual inspection (see generally, Current Protocols in Molecular Biology, FM Ausubel et al., eds., Current Protocols, a joint venture of Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1995 supplement) (Ausubel)). Examples of algorithms suitable for determining percent sequence identity and percent sequence similarity are BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1990, J. Mol. Biol. 215:403-410 and Altschul et al., 1977, Nucleic Acids Res. 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website.The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy some positive-valued threshold score T when aligned with a word of the same length in the database sequence. T is called the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence as far as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. Extension of the word hits in each direction is stopped when: the cumulative alignment score drops by the amount X from its maximum achieved value; the cumulative score approaches zero or lower due to the accumulation of one or more negatively scored residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:10915). Sequence alignments and exemplary determinations of percent sequence identity can be performed using the BESTFIT or GAP programs in the GCG Wisconsin software package (Accelrys, Madison Wis.) using the default parameters provided.

[0069] A "reference sequence" refers to a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length nucleic acid or polypeptide sequence. The length of a reference sequence is typically at least 20 nucleotide or amino acid residue units, but may also be the full length of a nucleic acid or polypeptide. Because two polynucleotides or polypeptides may each (1) contain a sequence that is similar between the two sequences (i.e., a portion of the complete sequence), and (2) may also contain a sequence that is different between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing the sequences of the two polynucleotides or polypeptides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least about 20 consecutive nucleotide positions or amino acid residues in which a sequence may be compared to a reference sequence of at least 20 consecutive nucleotides or amino acids, and wherein the portion of the sequence in the comparison window may contain 20% or less additions or deletions (or gaps) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences.

[0070] "Substantial identity" or "substantially identical" refers to a polynucleotide or polypeptide sequence that has at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity compared to a reference sequence over a comparison window of at least 20 nucleotide or amino acid residue positions, typically over a window of at least 30-50 positions, wherein the percent sequence identity is calculated by comparing the reference sequence to a sequence comprising deletions or additions that total 20% or less of the reference sequence over the comparison window.

[0071] When used in the context of numbering a given amino acid or polynucleotide sequence, "corresponding to," "reference," or "relative to," refers to the numbering of the residues of a given reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue numbers or residue positions of a given polymer are designated relative to the reference sequence, rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence (such as the amino acid sequence of an engineered imine reductase) can be aligned to a reference sequence by introducing gaps to optimize residue matching between the two sequences. In these cases, the numbering of the residues in a given amino acid or polynucleotide sequence is relative to the reference sequence to which it is aligned, despite the presence of the gaps.

[0072] As used herein, "isolated" with respect to a molecule means that the molecule (e.g., a cannabinoid, a polynucleotide, a polypeptide) is substantially separated from other compounds with which it naturally accompanies it (e.g., proteins, lipids, and polynucleotides). The term includes nucleic acids that have been removed or purified from their naturally occurring environment or expression system (e.g., a host cell or in vitro synthesis).

[0073] "Substantially pure" refers to a composition in which the desired molecule is the predominant species present (i.e., it is more abundant on a molar or weight basis than any other individual macromolecular species in the composition), and is generally a substantially purified composition when the species of interest constitutes at least about 50% of the macromolecular species present on a molar or weight % basis.

[0074] An "immunoconjugate" refers to an antibody conjugated to one or more heterologous molecules, including but not limited to cytokines such as IL-2.

[0075] "Treatment," "treat," or "treating" refers to clinical intervention that attempts to alter the natural course of a condition in the individual being treated, and may be used prophylactically or during clinical pathology. Desirable therapeutic outcomes may include, but are not limited to, preventing the occurrence or recurrence of a condition, alleviating symptoms, reducing any direct or indirect pathological consequences of a condition, preventing metastasis, reducing the rate of progression, ameliorating or relieving the disease state, and alleviating or improving prognosis. For example, treatment may comprise administering to a subject a therapeutically effective amount of a pharmaceutical formulation comprising an IL-2 mutant polypeptide to delay the development or slow the progression of a disease or condition mediated by IL-2R or in which IL-2R may play a role in the pathogenesis and / or progression.

[0076] A "pharmaceutical formulation" refers to a formulation that is in a form that permits the biological activity of the active ingredient to be effective and that does not contain additional components that are toxic to the subject to which the formulation is administered. A pharmaceutical formulation may contain one or more active agents. For example, a pharmaceutical formulation may contain a mutant IL-2 polypeptide as the sole active agent of the formulation, or may contain a mutant IL-2 polypeptide and one or more additional active agents, such as an immune checkpoint inhibitor.

[0077] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation other than the active ingredient that is non-toxic to the subject to which it is administered. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0078] "Therapeutically effective amount" refers to the amount of an active ingredient or agent (e.g., a pharmaceutical formulation) that achieves a desired treatment or prevention result (e.g., treating or preventing a disease, disorder, or condition in a subject). In the case of an IL-2-mediated disease or condition, a therapeutically effective amount of a therapeutic agent is an amount that reduces, prevents, suppresses, and / or alleviates one or more symptoms associated with the disease, disorder, or condition to a certain extent. For cancer therapy, in vivo efficacy can be measured, for example, by assessing the growth of primary tumors, the occurrence and / or growth of secondary tumors, the occurrence and / or number of metastases, the duration, severity, and / or recurrence of symptoms, response rate (RR), response duration, and / or quality of life.

[0079] "Individual" or "subject" refers to mammals, including but not limited to domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0080] IL-2 receptor

[0081] IL-2 signal transduction is mediated by binding to three different IL-2 receptor protein subunits: IL-2Rα (CD25), IL-2Rβ (CD122) and IL-2Rγ (CD132). Immune cells express dimer or trimer IL-2 receptors. Dimeric receptors are expressed on cytotoxic CD8+ T cells and natural killer cells (NK), while trimeric receptors are mainly expressed on activated lymphocytes and CD4+CD25+FoxP3+ inhibitory regulatory T cells (Treg) (see Byman et al., J.Nat.Rev.Immunol.12,180-190 (2012)). Resting effector T cells and NK cells do not have CD25 on the cell surface and are therefore relatively insensitive to IL-2. However, Treg cells express high levels of CD25, so Treg proliferation is stimulated by IL-2.

[0082] The trimeric receptor IL-2Rαβγ, which is formed by the combination of IL-2Rα, IL-2Rβ and IL-2Rγ, is K D It is a high affinity receptor for IL-2 of about 10 pM. The dimeric receptor (IL-2Rβγ) is K D It is a medium affinity receptor of about 1 nM. The monomeric IL-2Rα receptor is a low affinity IL-2 receptor. The IL-2 signaling activity mediated by the receptor and its complex is also significantly different. Generally, it has been found that IL-2Rβ and IL-2Rγ are critical for IL-2 signaling, while IL-2Rα (CD25) is not essential for signaling, but the presence of IL-2Rα enables high-affinity IL-2 to bind to the receptor complex (see, for example, Krieg et al., Proc Natl Acad Sci 107, 11906-11 (2010)).

[0083] The amino acid sequence of the IL-2R alpha subunit can be found in UniProt P01589 and is set forth herein as SEQ ID NO: 2 (the polynucleotide sequence encoding SEQ ID NO: 2 is included in the sequence listing as SEQ ID NO: 1). The amino acid sequence of the IL-2R beta subunit can be found in UniProt P14784 and is set forth herein as SEQ ID NO: 4 (the polynucleotide sequence encoding SEQ ID NO: 4 is included in the sequence listing as SEQ ID NO: 3). The amino acid sequence of the IL-2R gamma subunit can be found in UniProt 31785 and is set forth herein as SEQ ID NO: 6 (the polynucleotide sequence encoding SEQ ID NO: 6 is included in the sequence listing as SEQ ID NO: 5). Table 1 below provides a summary of the amino acid sequences of various IL-2R polypeptides disclosed herein and their sequence identifiers. The sequences are also included in the accompanying sequence listing.

[0084] Table 1: IL-2R protein subunits

[0085]

[0086]

[0087] Mutant IL-2 polypeptide

[0088] The present disclosure provides IL-2 polypeptides having mutations (relative to wild-type or C125S IL-2) that alter glycosylation, thereby affecting the functional properties of the polypeptide, including increased expression titer, increased solubility, and / or altered binding affinity for the IL-2 receptor complex. The altered glycosylation and functional properties resulting from the mutations (also referred to as amino acid substitutions or differences) in the amino acid sequence relative to the parent IL-2 polypeptide result in IL-2 polypeptides having improved properties for use in pharmaceutical compositions for the treatment of IL-2-mediated diseases such as cancer and autoimmune diseases.

[0089] Naturally occurring human IL-2 is a 153 amino acid polypeptide sequence (Uniprot: P60568; disclosed herein as SEQ ID NO: 8; the corresponding polynucleotide sequence encoding SEQ ID NO: 8 is included in the sequence listing as SEQ ID NO: 7) that includes a 20 amino acid N-terminal signal peptide:

[0090] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQ MILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT(SEQ ID NO:8)

[0091] The structure of the IL-2 polypeptide comprises four antiparallel and amphipathic α-helices that form a quaternary structure that is essential for its function (see, e.g., Smith, Science 240, 1169-76 (1988); Bazan, Science 257, 410-413 (1992)). The 153 amino acid precursor IL-2 of SEQ ID NO: 8 is processed to remove the signal peptide, resulting in the 133 amino acid mature secreted IL-2 polypeptide of SEQ ID NO: 9 shown below:

[0092] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT(SEQ ID NO:9)

[0093] The mature IL-2 polypeptide of SEQ ID NO: 9 has been engineered for human pharmaceutical use by removing the cysteine ​​residue at position C125, thereby reducing aggregation of the polypeptide. The C125S IL-2 mutant polypeptide (disclosed herein as SEQ ID NO: 10) is the active ingredient in aldesleukin, a drug approved for human cancer treatment.

[0094] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT(SEQ ID NO:10)

[0095] Exemplary mutant IL-2 polypeptides with improved properties disclosed herein are derived from the parent C125S IL-2 polypeptide of SEQ ID NO: 10. As described elsewhere herein, the mutant IL-2 polypeptides have been engineered with amino acid substitutions relative to the C125S IL-2 of SEQ ID NO: 10 that provide new N-glycosylation sites on the expressed mutant IL-2 polypeptide. The engineering of N-glycosylation motifs in proteins is well known in the art. Typically, the three amino acid sequence motifs asparagine-X-serine (NXS) and asparagine-X-threonine (NXT), where X can be any amino acid except proline, provide potential N-glycosylation sites on a polypeptide. Depending on the sequence of the polypeptide, N-glycosylation sites can be introduced into the polypeptide by engineering amino acid substitutions at one, two, or three positions in the amino acid sequence.

[0096] Table 2 below provides a summary of the sequences of exemplary mutant IL-2 polypeptides of the present disclosure that have been engineered with one or more N-glycosylation sites that are not present in the naturally occurring mature IL-2 polypeptide of SEQ ID NO: 9 or the C125S IL-2 polypeptide of SEQ ID NO: 10. The complete amino acid sequences and polynucleotide sequences encoding the polypeptides are provided in the accompanying sequence listing.

[0097] Table 2: Exemplary mutant IL-2 polypeptides

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111] Altered expression titer and solubility of mutant IL-2 polypeptides

[0112] As described elsewhere herein, including in the Examples, the mutant IL-2 polypeptides of the present disclosure engineered with N-glycosylation sites exhibit surprising and advantageous technical effects relative to the parent C125S IL-2 polypeptide of SEQ ID NO: 10. The technical effects exhibited include increased IL-2 polypeptide expression titers in mammalian cell culture during production, increased solubility, and altered binding affinity to trimeric and dimeric IL-2R complexes formed by the IL-2Rα subunit of SEQ ID NO: 2, the IL-2Rβ subunit of SEQ ID NO: 4, and the IL-2Rγ subunit of SEQ ID NO: 6. For example, certain glycosylation mutations disclosed herein reduce the affinity of the mutant IL-2 for binding to the IL-2Rα subunit, but do not reduce the affinity for binding to dimeric IL-2Rβγ.

[0113] It has been observed that engineered N-glycosylation mutations in recombinant genes encoding polypeptides can result in increased titers of polypeptides expressed in mammalian cell culture systems. Additionally, it has been observed that engineered N-glycosylation mutations in recombinant genes encoding polypeptides can result in reduced aggregation and increased solubility of the resulting polypeptides, as indicated by an increase in the amount of monomeric form of the polypeptide. Among the mutant IL-2 polypeptides disclosed herein, it has been found that certain N-glycosylation mutations, alone or in combination, result in IL-2 polypeptides exhibiting increased expression titers and increased solubility (measured as % monomeric polypeptide) in mammalian cell culture systems. Table 3 below provides a collection of exemplary amino acid differences of the present disclosure that exhibit these functional improvements of increased expression titer and increased solubility.

[0114] Table 3

[0115]

[0116]

[0117]

[0118]

[0119] Altered IL-2R binding affinity of mutant IL-2 polypeptides

[0120] It has also been observed that engineered N-glycosylation mutations in recombinant genes encoding IL-2 polypeptides can result in mutant IL-2 polypeptides that exhibit altered binding affinity for the different IL-2R receptor subunits IL-2Rα, IL-2Rβ, and IL-2Rγ in the form of monomers, dimers, and trimeric complexes. As described elsewhere herein, in at least one embodiment, the mutant IL-2 polypeptide exhibits reduced affinity for IL-2Rα, while exhibiting little or no loss of binding affinity for IL-2β, IL-2γ, or IL-2βγ dimer complexes. It is believed that such mutant IL-2 polypeptides that exhibit reduced binding affinity for IL-2Rα may provide improved therapeutic compounds for the treatment of cancer, for example, due to reduced or no stimulation of immunosuppressive CD25+ cells. Additionally, it is believed that mutant IL-2 polypeptides that exhibit reduced binding affinity for IL-2Rβγ with little or no reduced binding affinity for IL-2Rα may provide improved therapeutic compounds for treating autoimmune disorders, for example due to preferential stimulation of immunosuppressive CD25+ cells. An exemplary set of amino acid differences of the present disclosure that exhibit selectively reduced binding affinity for IL-2Rα and / or IL-2Rβγ is provided in Table 4 below.

[0121] Table 4

[0122]

[0123]

[0124]

[0125]

[0126] Mutant IL-2 polypeptide fusion

[0127] As described elsewhere herein, it is contemplated that the mutant IL-2 polypeptides of the present disclosure may be conjugated (or fused) to other polypeptides or proteins, including but not limited to immunoglobulin molecules, antibody fragments, and / or other cytokines (e.g., IL-15). Such fusions may result in improved properties of the IL-2 polypeptide. For example, the mutant IL-2 polypeptides of the present disclosure (e.g., the polypeptides of Table 2) may be fused to immunoglobulin Fc region polypeptides. Conjugation to Fc region polypeptides may result in improved pharmacokinetic properties of the entire fusion molecule, such as half-life, thereby allowing for better pharmaceutical compositions for therapeutic use. In at least one embodiment, the present disclosure provides mutant IL-2 polypeptides fused to a polypeptide linker of an Fc polypeptide, such as the wild-type monomeric human IgG1 Fc lower hinge region polypeptide of SEQ ID NO: 146 as shown below:

[0128] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO:146).

[0129] Certain variants of the wild-type human IgG1 Fc polypeptide of SEQ ID NO: 146 can be used for fusion with mutant IL-2 polypeptides, including the "KK" variant of SEQ ID NO: 147 and the "DSDL" variant of SEQ ID NO: 148, both of which are shown below:

[0130] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSPGK(SEQID NO:147)

[0131] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLSSDLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSPGK(SEQID NO:148).

[0132] Although only three human IgG1 Fc lower hinge region polypeptides are described above, it is contemplated that the mutant IL-2 polypeptide fusions disclosed herein can be prepared using other Fc region fragments and variants known to provide improved properties when conjugated to polypeptides such as cytokines for therapeutic use. For example, an Fc polypeptide variant with effector function removed, such as an Fc region having the amino acid substitution L234A / L235A ("LALA") (Woodle, E. Steve et al., Transplantation, 68(5):608-616 (1999)) can be used. Other effector-free Fc region mutations are well known in the art, such as L234A / L235A / P329G ("LALAPG") (see, e.g., Schlothauer, T. et al., "Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions", Protein Eng. Des. Sel., 29(10):457–466 (2016)), or when the Fc is of the isotype IgG2 or IgG4, the amino acid substitutions S228P and / or L235E.

[0133] In at least one embodiment, it is envisioned that the mutant IL-2 polypeptides of the present disclosure can be conjugated to other polypeptides or proteins (e.g., Fc polypeptides) via linkers. Any of a variety of synthetic chain molecules known in the art that can be used as linkers between biomolecules can be used to fuse the mutant IL-2 polypeptide with other polypeptides. In at least one embodiment, a polypeptide linker can be used. Such polypeptide linkers comprise an amino acid chain, wherein each end of the chain is covalently linked to one of two different polypeptides, thereby acting as a conjugate or fusion with them. Typically, such polypeptide linkers comprise chains of 5 to 30 amino acids. A variety of polypeptide linkers are known in the art and can be used in the mutant IL-2 polypeptide fusions of the present disclosure. Exemplary polypeptide linkers that can be used in the IL-2 fusions of the present disclosure include, but are not limited to: (GGGGS)1 (SEQ ID NO: 149), (GGGGS)2 (SEQ ID NO: 150), (GGGGS)3 (SEQ ID NO: 151), (GGGGS)4 (SEQ ID NO: 152), (GRPGS)2 (SEQ ID NO: 153), (GRPGS)4 (SEQ ID NO: 154), (GGGGS)3GGG (SEQ ID NO: 155). Additional polypeptides known in the art that can be used include: (GGGGS) n (n is 1-10), (SSSSG) n (n is 1-10), (GGGG)(SGGGG) n (n is 1-10), (EAAAK) n (n is 1-10), (XP) n (n is 1-10) and ENLYFQ(-G / S).

[0134] The present disclosure exemplifies fusions of mutant IL-10 polypeptides (e.g., SEQ ID NOs: 11-145) fused to monomeric Fc polypeptides (e.g., SEQ ID NOs: 146, 147, or 148), wherein the monomeric Fc polypeptide is conjugated from its C-terminus to the N-terminus of the mutant IL-10 polypeptide via a polypeptide linker (e.g., SEQ ID NOs: 149-155). However, one of ordinary skill in the art can envision and will appreciate that other orientations of the mutant IL-2 polypeptide fusions of the present disclosure can be used. For example, in at least one embodiment, a mutant IL-2 polypeptide of the present disclosure can be conjugated to another polypeptide or protein (such as a monomeric Fc polypeptide) via its N-terminus or its C-terminus, optionally via a linker (e.g., a polypeptide linker).

[0135] In addition to mutant IL-2 polypeptides fused to monomeric Fc polypeptides, the present disclosure also contemplates mutant IL-2 fused to immunoglobulin molecules with specific antigen binding capabilities, such as antibodies or antibody fragments (e.g., Fab molecules, scFv or VHH). In at least one embodiment, the fused antibody or antibody fragment provides a specific antigen binding affinity that targets the fusion to cancer cells or other cells in the tumor environment. In at least one embodiment, the specific antigen binding affinity targets the cytokine molecule CD8, and the antibody is a VHH antibody. Exemplary anti-CD8 VHH antibodies are disclosed in, for example, U.S. Provisional Patent Application No. 63 / 477,529, filed on December 28, 2022, which is hereby incorporated by reference herein. In at least one embodiment, the specific antigen binding affinity targets the immune checkpoint inhibitor molecule PD-1, and the antibody is a VHH antibody. Exemplary anti-PD-1 VHH antibodies are disclosed in, for example, U.S. Provisional Patent Application No. 63 / 488,176, filed on March 3, 2023, which is hereby incorporated by reference herein.

[0136] Recombinant methods and compositions

[0137] The mutant IL-2 polypeptides disclosed herein can be prepared using recombinant methods and materials well known in the art of polypeptide and protein preparation. In some embodiments, the present disclosure provides an isolated nucleic acid encoding a mutant IL-2 polypeptide. The nucleic acid may encode an amino acid sequence comprising only an IL-2 polypeptide or a fusion thereof with another polypeptide (such as a monomeric Fc polypeptide). In some embodiments, one or more vectors (e.g., expression vectors) are provided, comprising a nucleic acid sequence encoding a mutant IL-2 polypeptide disclosed herein. In some embodiments, a host cell is provided, comprising a nucleic acid sequence encoding a mutant IL-2 polypeptide disclosed herein. In one embodiment, the host cell has been transformed with a vector comprising a nucleic acid encoding an amino acid sequence comprising a mutant IL-2 polypeptide. In some embodiments, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20).

[0138] In at least one embodiment, a method for preparing a mutant IL-2 polypeptide is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the polypeptide as provided above under conditions suitable for expression of the polypeptide, and optionally recovering the polypeptide from the host cell (or host cell culture medium). Briefly, recombinant preparation of the mutant IL-2 polypeptide is performed by synthesizing or isolating a nucleic acid encoding the mutant IL-2 polypeptide (e.g., as described herein) and inserting the nucleic acid into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids are readily isolated and sequenced using conventional procedures well known in the art (e.g., by using oligonucleotide probes capable of specifically binding to a gene encoding IL-2). Suitable host cells and culture methods for cloning or expressing vectors encoding IL-2 polypeptides are well known in the art and include prokaryotic or eukaryotic cells. Typically, after expression, the mutant IL-2 polypeptide can be isolated from the cell paste in a soluble fraction and further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for the vector.

[0139] Examples of suitable mammalian host cell lines that can be used to prepare the mutant IL-2 polypeptides of the present disclosure include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (see, e.g., Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); myeloma cell lines, such as Y0, NS0, and Sp2 / 0; monkey kidney CV1 cell line transformed by SV40 (COS-7); human embryonic kidney cell line (293 or 293 cells, such as, e.g., Graham et al., J. Gen. Biol., 1996; and human embryonic kidney cell line (293 or 293 cells, such as, e.g., Graham et al., J. Gen. Biol., 1996). Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, for example, in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; Buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TR1 cells (see, for example, Mather et al., Annals N Y. Acad. Sci. 383:44-68 (1982) and US 6,235,498); Medical Research Council 5 (MRC 5) cells (such as those available from ATCC, also known as CCL-171); and foreskin 4 (FS-4) cells (see, e.g., Vilcek et al., Ann. NY Acad. Sci. 284:703-710 (1977); Gardner & Vilcek. J. Gen. Virol. 44:161-168 (1979); and Pang et al., Proc. Natl. Acad. Sci. USA 77:5341-5345 (1980)).

[0140] Pharmaceutical compositions and preparations of mutant IL-2 polypeptides

[0141] The present disclosure also provides pharmaceutical compositions and pharmaceutical formulations comprising mutant IL-2 polypeptides. In some embodiments, the present disclosure provides a pharmaceutical formulation comprising a mutant IL-2 polypeptide as described herein and a pharmaceutically acceptable carrier. In some embodiments, the mutant IL-2 polypeptide is the sole active agent in the pharmaceutical composition. Such pharmaceutical formulations can be prepared by mixing a mutant IL-2 polypeptide having a desired purity with one or more pharmaceutically acceptable carriers. Typically, such mutant IL-2 polypeptide formulations can be prepared as aqueous solutions or lyophilized formulations.

[0142] Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed. A variety of such pharmaceutically acceptable carriers are well known in the art (see, for example, Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). Exemplary pharmaceutically acceptable carriers that can be used in the formulations of the present disclosure may include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl paraben 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., Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG).

[0143] Pharmaceutically acceptable carriers useful in the formulations of the present disclosure may also include interstitial drug dispersion agents, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP) (see, e.g., U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968), such as human soluble PH-20 hyaluronidase glycoprotein (e.g., rHuPH20 or Baxter International, Inc.).

[0144] It is also contemplated that the formulations disclosed herein may contain active ingredients other than mutant IL-2 polypeptides, as necessary for the particular indication being treated for the subject to which the formulation is administered. Preferably, any additional active ingredient has an activity that is complementary to the activity of IL-2, and the activities do not adversely affect each other.

[0145] As disclosed elsewhere herein, including in the Examples, it has been demonstrated that the mutant IL-2 polypeptides of the present disclosure can be used as fusions with Fc polypeptides to provide improved therapeutic effects in the treatment of autoimmune disorders and / or cancer.

[0146] As described elsewhere herein, in some embodiments, the present disclosure provides pharmaceutical compositions or formulations for use in therapeutic methods comprising a mutant IL-2 polypeptide conjugated to another polypeptide or protein, such as a half-life-extending Fc polypeptide. In some embodiments, the pharmaceutical composition or formulation may comprise a mutant IL-2 polypeptide covalently fused to an Fc polypeptide via a linker, such as a polypeptide linker of the amino acid sequence of SEQ ID NOs: 149-155. The Examples demonstrate such mutant IL-2 polypeptide fusions with the monomeric Fc polypeptide of SEQ ID NOs: 146, 147, or 148 and their use in pharmaceutical compositions for reducing the treatment of cancer or autoimmune disorders as described elsewhere herein.

[0147] In some embodiments, a pharmaceutical composition may comprise a mutant IL-2 polypeptide of the present invention and an additional active agent for treating cancer, such as an immune checkpoint inhibitor. Checkpoint inhibitors that can be used in such embodiments include, but are not limited to, antibodies specific for an antigen that is an immune checkpoint molecule such as PD1, LAG3, CTLA-4, A2AR, TIM-3, BTLA, CD276, CD328, VTCN1, KIR, NOX2, VISTA, OX40, CD27, CD28, CD40, CD122, CD137, GITR, or ICOS.

[0148] Typically, the active ingredient of the pharmaceutical composition can be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980).

[0149] In some embodiments, the formulation of the mutant IL-2 polypeptide can be a sustained-release formulation of the polypeptide and / or other active ingredients. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the mutant IL-2 polypeptide, which matrices are in the form of shaped articles, such as films or microcapsules.

[0150] Typically, the formulations of the present disclosure to be administered to a subject are sterile. Sterile formulations can be readily prepared using well-known techniques, such as filtration through sterile filtration membranes.

[0151] Uses and treatments

[0152] It is contemplated that any composition or formulation comprising a mutant IL-2 polypeptide disclosed herein can be used in any method or use, such as a therapeutic method, that exploits the ability of the polypeptide to specifically bind to an IL-2 receptor protein. When IL-2 is expressed on different cells, its binding to the IL-2 receptor protein mediates different immune responses. IL-2 binding can stimulate immune responses, such as T cell proliferation and differentiation, cytotoxic T lymphocyte (CTL) production, B cell proliferation and differentiation, immunoglobulin synthesis and production, and NK cell proliferation and activation. IL-2 polypeptides such as C125S IL-2 (Proleukin) have been approved as immunotherapeutic agents for the treatment of cancer and chronic viral infections. However, IL-2 polypeptides can also promote the activation and proliferation of immunosuppressive CD4+CD25+ Treg cells, leading to immunosuppression. Therefore, there is a range of diseases, disorders, and conditions that can be potentially treated by altering the immunomodulatory and / or immune signaling activity of IL-2 binding to the IL-2 receptor protein, particularly the effect of IL-2 on tumor progression. Diseases, disorders, and conditions include, but are not limited to, cancer, including, but not limited to, colon cancer, pancreatic cancer, ovarian cancer, liver cancer, kidney cancer, breast cancer, lung cancer, stomach cancer, head and neck cancer, or oral cancer. It is contemplated that any composition or formulation comprising a mutant IL-2 polypeptide of the present disclosure (including a mutant IL-2 polypeptide fusion with a monomeric Fc polypeptide) can be used in a method or use for treating any of the above-listed cancers. Thus, in at least one embodiment, the present disclosure provides a method for treating cancer in a subject, wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of a mutant IL-2 polypeptide of the present disclosure or administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a mutant IL-2 polypeptide of the present disclosure and a pharmaceutically acceptable carrier.

[0153] As disclosed herein, including in the following examples, the mutant IL-2 polypeptides of the present disclosure have the ability to differentially and specifically bind to IL-2 receptor proteins, thereby differentially altering immune signaling pathways mediated by the binding of IL-2 to IL-2 receptor proteins expressed on different cells. Thus, in some embodiments, the present disclosure provides a method for treating an IL-2-mediated disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of a mutant IL-2 polypeptide of the present disclosure or administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a mutant IL-2 polypeptide of the present disclosure and a pharmaceutically acceptable carrier. Similarly, in some embodiments, the present disclosure provides a method for treating a disease mediated by the binding of IL-2 to an IL-2 receptor protein expressed on cells of a subject, the method comprising administering to the subject a therapeutically effective amount of a mutant IL-2 polypeptide of the present disclosure or administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a mutant IL-2 polypeptide of the present disclosure and a pharmaceutically acceptable carrier.

[0154] Administration of a mutant IL-2 polypeptide, composition, or pharmaceutical formulation according to the treatment method provides an antibody-induced therapeutic effect that protects the subject from the effects of an IL-2-mediated disease and / or slows the progression of an IL-2-mediated disease in the subject. In some embodiments, the treatment method may further comprise administering one or more additional therapeutic agents or treatments known to those skilled in the art to prevent and / or treat an IL-2-mediated disease or condition. Such methods comprising administering one or more additional agents may encompass both combined administration (wherein two or more therapeutic agents are contained in the same or separate formulations) and separate administration, in which case administration of the mutant IL-2 polypeptide composition or formulation may be prior to, concurrently with, and / or subsequent to administration of the additional therapeutic agent.

[0155] In some embodiments of the treatment methods of the present disclosure, a mutant IL-2 polypeptide or a pharmaceutical formulation comprising a mutant IL-2 polypeptide is administered to a subject by any mode of administration that delivers the agent systemically or to a desired target tissue. Systemic administration generally refers to any mode of administration of an antibody to a site in a subject other than directly to the desired target site, tissue, or organ, such that the antibody or formulation thereof enters the subject's circulatory system and thereby undergoes metabolism and other similar processes. Thus, modes of administration that can be used in the treatment methods of the present disclosure may include, but are not limited to, injection, infusion, instillation, and inhalation. Administration by injection may include intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardial, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion.

[0156] In at least one embodiment, the pharmaceutical formulation of the mutant IL-2 polypeptide is formulated such that the IL-2 is protected from inactivation in the intestine. Thus, the method of treatment may comprise oral administration of the formulation.

[0157] In at least one embodiment, the use of a composition or formulation comprising a mutant IL-2 polypeptide of the present disclosure as a medicament is also provided. Furthermore, in some embodiments, the present disclosure further provides the use of a composition or formulation comprising a mutant IL-2 polypeptide in the manufacture or preparation of a medicament, particularly a medicament for treating an IL-2-mediated disease. In another embodiment, the medicament is used in a method of treating a disease, the method comprising administering an effective amount of the medicament to a subject suffering from the disease. In certain embodiments, the medicament further comprises an effective amount of at least one additional therapeutic agent or treatment.

[0158] As disclosed elsewhere herein, it is also contemplated that additional therapeutic agents or treatments may be used in conjunction with the mutant IL-2 polypeptides of the present disclosure for such drugs. In general, it is contemplated that the mutant IL-2 polypeptides of the present disclosure may be used together with any therapeutic agent or treatment (such as a therapeutic antibody) that specifically targets cell surface receptors on immune cells, tumor cells, or myeloid cells. In at least one embodiment, additional therapeutic agents may include, but are not limited to, therapeutic antibodies that specifically bind to immune checkpoint molecules, such as PD1, PD-L1, LAG3, CTLA-4, A2AR, TIM-3, BTLA, CD276, CD328, VTCN1, IDO, KIR, NOX2, VISTA, OX40, CD27, CD28, CD40, CD122, CD137, GITR, ICOS.

[0159] In another embodiment, the medicament is used to treat an IL-2-mediated disease, such as cancer, in a subject, comprising administering to the subject an effective amount of the medicament to treat, inhibit, or prevent the IL-2-mediated disease. The appropriate dosage of the mutant IL-2 polypeptides contained in the compositions and formulations of the present disclosure (when used alone or in combination with one or more other additional therapeutic agents) will depend on the specific disease or condition being treated, the severity and course of the disease, whether the dose is administered for preventive or therapeutic purposes, the therapy previously administered to the patient, the patient's medical history and response to the mutant IL-2 polypeptide composition, and the discretion of the attending physician. It is contemplated that the mutant IL-2 polypeptides contained in the compositions and formulations described herein can be appropriately administered to the patient at one time or over a series of treatments. Various dosing regimens are contemplated herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulse infusions.

[0160] Depending on the type and severity of the disease, about 1 μg / kg to 30 mg / kg of the mutant IL-2 polypeptide in the formulations of the present disclosure is an initial candidate dose for administration to a human subject, whether, for example, by one or more separate administrations or by continuous infusion. Typically, the dosage of the antibody administered will be in the range of about 0.05 mg / kg to about 10 mg / kg. In some embodiments, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the patient.

[0161] Dosage administration may be continued for several days or longer, depending on the condition of the subject, for example, until the IL-2 mediated disease is adequately treated, as determined by methods known in the art. In some embodiments, an initial higher loading dose may be administered, followed by one or more lower doses. However, other dosing regimens may be useful. The progression of the therapeutic effect of dosing can be monitored by conventional techniques and assays. Thus, in some embodiments of the methods of the present disclosure, administration of the mutant IL-2 polypeptide comprises a daily dose of about 0.05 mg / kg to about 50 mg / kg, at least about 0.5 mg / kg to about 30 mg / kg, or at least about 1 mg / kg to about 25 mg / kg. In some embodiments, the dose of the mutant IL-2 polypeptide comprises a daily dose of at least about 0.1 mg / kg, at least about 1 mg / kg, at least about 5 mg / kg, at least about 10 mg / kg, at least about 20 mg / kg, at least about 30 mg / kg, at least about 40 mg / kg, or at least about 50 mg / kg.

[0162] Example

[0163] Various features and embodiments of the present disclosure are illustrated in the following representative examples, which are intended to be illustrative and non-limiting. Those skilled in the art will readily appreciate that the specific examples are merely illustrative of the present invention, as more fully described in the claims that follow. Each embodiment and feature described in this application should be understood to be interchangeable and combinable with each embodiment contained therein.

[0164] Example 1: Preparation of IL-2 glycosylation mutant polypeptides

[0165] This example illustrates the preparation and screening of mutant IL-2 polypeptides with amino acid changes that alter glycosylation and thereby alter other functional properties, including expression titer, solubility, and binding affinity to IL-2Rα and IL-2Rβγ.

[0166] Materials and methods

[0167] A. Preparation of Mutant IL-2 Gene : Gene fragments encoding mutant IL-2 polypeptides were reverse transcribed and then synthesized into gene fragments (gblock or eblock) by Integrated DNA Technologies, Inc. These gene fragments were then cloned into expression vectors as described below.

[0168] B. Preparation of mutant IL-2 expression constructs and production of mutant polypeptides:The expression vector pcDNA3.1(+) (Thermo Fisher Scientific) was digested with PstI and XbaI restriction enzymes according to the protocol provided by New England Biolabs, Inc., and purified by agarose gel and dissolved in TE buffer (10mM Tris, pH 8.0 and 1mM EDTA). The synthetic DNA fragment encoding the mutant IL-2 polypeptide was dissolved in water. Equal volumes of vector and insert were added to 2x Gibson assembly master mix (New England Biolabs) at a molar ratio of 1:3 and incubated at 50°C for 1-2 hours. Subsequently, the ligated DNA was transformed into DH5α Escherichia coli (E. coli) competent cells (New England Biolabs). The resulting plasmid DNA was sequenced according to standard molecular biology techniques. PureLink DNA from Thermo Fisher Scientific was used. TM Correct plasmid DNA was prepared using the Hipure Maxiprep Kit (Cat. No. K210006) to recover sterile, salt-free, and supercoiled DNA.

[0169] result Table 2 (above) lists the amino acid sequences of 144 exemplary mutant IL-2 polypeptides prepared as described above and their sets of specific mutations (or amino acid differences) relative to the parent C125S IL-2 polypeptide of SEQ ID NO: 10.

[0170] C. Expression of IL-2 fusion protein in Chinese hamster ovary (CHO) cells :

[0171] ExpiCHO cells (Thermo Fisher Scientific) were maintained in 60 mL of ExpiCHO expression medium in Erlenmeyer flasks at 37° C., 5% CO 2 and 130 rpm. TM Transfection was performed according to the instructions provided by the ELISA expression system kit (ThermoFisher - catalog number A29133). Briefly, when the cells reached 6×10 6 When the viable cell density (VCD) is 10 cells / mL and the doubling time is 18-20h, 1 μg plasmid DNA / 10 6The cells and ExpiFectamine CHO were mixed by repeated inversion, diluted with cold OptiPRO serum-free medium, and finally complexed with diluted plasmid DNA at room temperature. After 5 minutes, the mixture was added dropwise to the cell culture at room temperature. After transfection, cells were cultured at 200 RPM for 24-well plates or 130 RPM for 25 mL or large flasks. 7-12 days after transfection, the culture medium was centrifuged at 25°C and 500 × g for 5 minutes to pellet the cells, followed by centrifugation at 4°C and 4500 × g for 30 minutes. The clarified supernatant was filtered through 0.45 μm filtration, and the fusion protein was purified by protein A affinity column chromatography.

[0172] D. Determination of relative expression titer levels: The expression of protein titer levels was measured on an Agilent 1100 HPLC system (Santa Clara, California) coupled to a POEOS™ A20 μm column (Catalog Number: 2100100, ThermoFisher Scientific). The equilibrium buffer was 1X PBS (buffer A) and the elution buffer was 0.1% HCl (buffer B) containing 150 mM NaCl. In a typical HPLC run, the column was washed with 6 column volumes (CV) of 1X PBS after sample injection. The bound protein was eluted with a stepwise elution of 10 CV elution buffer and the UV signal at a wavelength of 280 nm was recorded. Prior to sample measurement, a standard curve was generated by injecting a known amount of human IgG reference. For sample titer measurement, 40 μl of liquid culture medium was injected, and the titer was calculated by the area of ​​the elution peak and calculated using a standard curve of human IgG.

[0173] result Table 5 below summarizes the expression titer levels measured for different mutant IL-2 polypeptide Fc fusion constructs. These results demonstrate that genes encoding mutant IL-2 polypeptides engineered with the following set of glycosylation mutations (see also the relative values ​​listed in Table 3) provide significantly increased expression titers relative to C125S IL-2 in mammalian expression systems.

[0174] Table 5

[0175]

[0176] E. Determination of IL-2Rα and IL-2Rβγ binding affinity:

[0177] The association and dissociation constants of the binding kinetics of mutant IL-2 polypeptides to their receptors were measured by biolayer interferometry (BLI) using a Gator Prime instrument (Gator Bio, Inc.). Binding kinetics were measured at 30°C and analyzed using GatorOne analysis software. Biotinylated IL-2Rα protein (Acro Biosystems, Inc.; Catalog No. ILA-H82E6) or biotinylated IL-2Rβγ heterodimer protein (Acro Biosystems, Inc.; Catalog No. ILG-H82F3) was captured using a streptavidin (SA) sensor probe. Samples were prepared using purified protein (single peak confirmed by SEC) in Tris buffer. The streptavidin sensor was set up as follows: baseline: 1x KB buffer; loading: biotinylated IL-2Rα or IL-2Rβγ obtained from Acro Biosystems, Inc.; association: purified protein; dissociation: 1x KB buffer. The sensor probe was immersed in serial dilutions of the parental C125S IL-2 polypeptide or mutant IL-2 polypeptide. Wells with buffer only were set as reference wells for background subtraction during data processing.For each cytokine-receptor binding, the data were fit with a 1:1 Langmuir model for association and dissociation using Rmax coupled to a global fit.

[0178] result :like Figure 1A 、 Figure 1B 、 Figure 1C 、 Figure 1G and Figure 1H As shown in the BLI results plotted in FIG, an exemplary mutant IL-2 polypeptide Fc fusion construct p132 (K35N, Y45R, C125S) ( Figure 1B ), p115(K35N, F42A, C125S)( Figure 1C ) and p151(F42A, Y45A, L72G, C125S)( Figure 1H ) relative to the control p123(C125S IL2) construct ( Figure 1A and Figure 1G ) showed reduced binding to IL-2Rα. For comparison, the results of BLI-measured binding to IL-2Rβγ are also shown below. Figure 1D 、 Figure 1E 、 Figure 1F 、 Figure 1I and Figure 1J Middle: Control p132(C125 IL2) construct ( Figure 1D and Figure 1I ) relative to the Fc fusion construct p132(K35N, Y45R, C125S) ( Figure 1E), p115(K35N, F42A, C125S)( Figure 1F ) and p151(F42A, Y45A, L72G, C125S)( Figure 1J ).

[0179] Table 4 (above) also summarizes exemplary results showing that many mutant IL-2 polypeptides had significantly reduced IL-2Rα subunit binding affinities (indicated by "+++") relative to the affinity observed for the parent C125S IL-2 polypeptide. Furthermore, the results in Table 4 show that many of these same mutant IL-2 polypeptides exhibited little or no reduction in IL-2Rβγ binding affinity relative to the parent C125S IL-2 polypeptide. This differential reduction in IL-2Rα binding suggests that mutant IL-2 polypeptides with this combination of amino acid differences will exhibit higher differential binding and stimulation of cells expressing IL-2Rβγ, such as cytotoxic CD8+ T cells and natural killer (NK) cells, and therefore will be more suitable for treating cancer.

[0180] Table 4 also lists the results showing that IL-2Rα binding was unchanged, but IL-2Rβγ binding affinity was significantly reduced for many mutant IL-2 polypeptides. This differential reduction in IL-2Rβγ binding suggests that mutant IL-2 polypeptides with this combination of amino acid differences will exhibit higher differential binding and stimulation of cells expressing IL-2Rαβγ (such as activated lymphocytes and CD4+CD25+FoxP3+ suppressive regulatory T cells (Tregs)), and therefore will be immunosuppressive and more suitable for treating autoimmune disorders.

[0181] F. Preparation of Monomeric Fc Conjugates of IL-2 Mutants :

[0182] The gene fragments encoding monomeric Fc and mutant IL-2 polypeptides were reverse transcribed and then synthesized into gene fragments (gblock or eblock) by Integrated DNA Technologies, Inc. These fragments were then cloned into expression vectors as described below. The expression vector pcDNA3.1(+) was digested with PstI and XbaI restriction enzymes (New England Biolabs, Inc.) and purified by agarose gel and dissolved in TE buffer (10mM Tris, pH 8.0 and 1mM EDTA). The synthesized DNA fragments encoding monomeric Fc and mutant IL-2 polypeptides were dissolved in water. Equal volumes of vector and insert were added to 2x Gibson assembly master mix (New England Biolabs) at a molar ratio of 1:3 and incubated at 50°C for 1-2 hours. Subsequently, the ligated DNA was transformed into Escherichia coli competent cells (New England Biolabs). The resulting plasmid DNA was sequenced according to standard molecular biology techniques. PureLink DNA from Thermo Fisher Scientific was used. TM Correct plasmid DNA was prepared using the Hipure Maxiprep Kit (Cat. No. K210006) to recover sterile, salt-free, and supercoiled DNA.

[0183] G. Expression of mutant IL-2 polypeptide fusions with monomeric Fc in Chinese hamster ovary (CHO) cells: ExpiCHO cells (Thermo Fisher Scientific) were maintained in 60 mL of ExpiCHO expression medium using Erlenmeyer flasks at 37°C, 5% CO2, and 130 rpm. TM Transfection was performed according to the instructions provided by the ELISA expression system kit (Thermo Fisher Scientific - catalog number A29133). Briefly, when the cells reached 6×10 6 When the viable cell density (VCD) is 10 cells / mL and the doubling time is 18-20h, 1 μg plasmid DNA / 10 6The cells and ExpiFectamine CHO were mixed by repeated inversion, diluted with cold OptiPRO serum-free medium, and finally complexed with diluted plasmid DNA at room temperature. After 5 minutes, the mixture was added dropwise to the cell culture at room temperature. After transfection, cells were cultured at 200 RPM for 24-well plates or 130 RPM for 25 mL or large flasks. 7-12 days after transfection, the culture medium was centrifuged at 25°C and 500 × g for 5 minutes to pellet the cells, followed by centrifugation at 4°C and 4500 × g for 30 minutes. The clarified supernatant was filtered through 0.45 μm filtration, and the fusion protein was purified by protein A affinity column chromatography.

[0184] H. Relative Solubility Determination of Mutant IL-2 Polypeptide Fusions to Monomeric Fc: Protein relative solubility (turbidity) was determined using a Costar half-area 96-well assay plate (Corning) on ​​an Infinite M Plex multimode microplate reader (Tecan Systems, Inc.). 0.1 mL of a 1 mg / mL protein stock solution was added to the plate wells and the solution was adjusted to the desired pH using 1 M Tris. The adsorption of the solution at a wavelength of 340 nm (OD) was measured. 340 ). The first OD value measured immediately after pH adjustment 340 nm was set to T = 0. Time course OD was measured at 15 min, 30 min, 1 h and 2 h 340 .

[0185] result: The relative solubility of different mutant IL-2 polypeptides, expressed as % of monomer measured, is listed in Table 3. These results indicate that mutant IL-2 polypeptides engineered with certain sets of glycosylation mutations listed in Table 3 also provide significantly increased solubility relative to C125S IL-2 when fused to a monomeric Fc.

[0186] J. Size Exclusion Chromatography Size exclusion chromatography (SEC-HPLC) analysis was performed on an Agilent 1100 HPLC system (Santa Clara, California) using 1X PBS as running buffer. Samples were injected into a pre-filled Superose 12-300 column (Cytiva) balanced with 1X PBS buffer. The flow rate was 0.65 mL / min, and the total run time was 40 min.

[0187] result: Figures 2A to 2L The following control p123(C125S IL2) constructs are shown ( Figure 2A and Figure 2B) and exemplary SEC-HPLC profiles of exemplary mutant IL-2 polypeptide Fc fusions: p296 ( Figure 2C )、p307( Figure 2D )、p406( Figure 2E )、p297( Figure 2F )、p300( Figure 2G )、p308( Figure 2H )、p214( Figure 2I )、p310( Figure 2J )、p411( Figure 2K ) and p298( Figure 2L ).

[0188] K. IL-2 reporter gene assay using HEK Blue cells: IL-2 activity was measured using HEKBlue IL-2 cells. In this cell system, HEK293 cells stably express IL-2 receptor subunits (IL2Rα is CD25, IL2Rβγ is CD122 / CD132, and STAT5 inducible SEAP reporter gene). During the assay, a cell suspension with activity higher than 90% was prepared. The test article at the indicated concentration in each experiment was prepared by serial dilution in DMEM+10% heat-inactivated FBS, added to the cell suspension in a flat-bottomed 96-well plate at 50K / well, and incubated at 37°C for 20-24 hours. The next day, QuantI-Blue solution was prepared according to the instructions from Invivogen. 20 μl of induced HEK Blue cell supernatant per well was added to a flat-bottomed 96-well plate, and then 100 μl of resuspended QUANTI-Blue solution was added to each well, and the plate was incubated at 37°C for 1-3 hours. The absorbance at 630 nm was read using a spectrophotometer (OD 630 ), determine SEAP levels.

[0189] result: Figures 3A to 3N Shown are exemplary CD25+ and CD25- binding curves and EC values ​​measured in a HEK Blue IL-2 reporter assay for a p123 C125S IL2 fusion control and exemplary mutant IL-2 polypeptide Fc fusions. 50 The values ​​are as follows: p124, p132 and p167 ( Figure 3A 、 Figure 3B ); p296, p300 and p214( Figure 3C 、 Figure 3D ); p123, p307 and p214 ( Figure 3E 、 Figure 3F ); p123, p296 and p308 ( Figure 3G 、 Figure 3H ); p123, p310, and p214 ( Figure 3I、 Figure 3J ); p296, p297, p298 and p214 ( Figure 3K 、 Figure 3L ); p411, p214, p123, p406 and p214 ( Figure 3M 、 Figure 3N ).

[0190] Although the foregoing disclosure of the present invention has been described in considerable detail by way of example and illustration for purposes of clarity and understanding, the present disclosure, including the examples, descriptions, and embodiments described herein, is for illustrative purposes, is intended to be exemplary, and should not be construed as limiting the present disclosure. It will be clear to those skilled in the art that various modifications or changes may be made to the examples, descriptions, and embodiments described herein, and that such modifications or changes are included within the spirit and scope of the present disclosure and the appended claims. In addition, those skilled in the art will recognize that there are many methods and procedures equivalent to the methods and procedures described herein. All such equivalents should be understood to be within the scope of the present disclosure and to be covered by the appended claims.

[0191] Further embodiments of the invention are set forth in the following claims.

[0192] The disclosures of all publications, patent applications, patents, or other documents mentioned herein are expressly incorporated by reference in their entirety for all purposes, to the same extent as if each such individual publication, patent, patent application, or other document were individually and specifically indicated to be incorporated by reference in its entirety for all purposes and were set forth in its entirety herein. In the event of a conflict, the present specification, including designated terminology, will control.

Claims

1. A polypeptide that specifically binds to an IL-2 receptor, wherein the polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 10 (C125 IL-2) and has one or more amino acid differences relative to SEQ ID NO: 10 selected from the group consisting of:

2. The polypeptide of claim 1, wherein the polypeptide further comprises one or more amino acid differences selected from the group consisting of K35N, F42A, Y45R, N88D, D109N, and Q126T.

3. The polypeptide of claim 2, wherein the polypeptide comprises a combination of amino acid differences selected from the group consisting of:

4. The polypeptide of claim 1 , wherein the polypeptide comprises a combination of amino acid differences selected from the group consisting of:

5. The polypeptide of claim 1 , wherein the polypeptide comprises a sequence selected from the group consisting of: SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 , 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144 and 145.

6. The polypeptide of claim 1, wherein the polypeptide is fused to a monomeric or dimeric Fc polypeptide; optionally, wherein the Fc polypeptide is a monomeric Fc polypeptide.

7. The polypeptide of claim 6, wherein the monomeric or dimeric Fc polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 146, 147, and 148.

8. The polypeptide of claim 6, wherein the polypeptide is fused via a linker; optionally, wherein the linker is a polypeptide comprising an amino acid sequence selected from the group consisting of: (GGGGS)1 (SEQ ID NO: 149), (GGGGS)2 (SEQ ID NO: 150), (GGGGS)3 (SEQ ID NO: 151), (GGGGS)4 (SEQ ID NO: 152), (GRPGS)2 (SEQ ID NO: 153), (GRPGS)4 (SEQ ID NO: 154), and (GGGGS)3GGG (SEQ ID NO: 155).

9. The polypeptide of claim 6, wherein the polypeptide is fused from the C-terminus of the monomeric or dimeric Fc polypeptide and the N-terminus of the mutant IL-2 polypeptide via a linker.

10. The polypeptide of claim 1, wherein the polypeptide is characterized by having one or more of the following properties relative to the IL-2 polypeptide of SEQ ID NO: 10: (i) increased titer when expressed in mammalian cell culture systems; (ii) increased solubility; (iii) reduced binding affinity to the IL-2Rα receptor subunit of SEQ ID NO: 2; and / or (iv) Reduced binding affinity to heterodimeric IL-2Rβγ receptor protein. A polynucleotide encoding the polypeptide of claim 1 . An expression vector comprising the polynucleotide according to claim 11 .

13. An isolated host cell comprising the polynucleotide of claim 11; optionally, wherein, The host cell is a mammalian cell or a yeast cell.

14. The isolated host cell of claim 13, wherein the host cell is a mammalian cell selected from the group consisting of Chinese hamster ovary (CHO) cells, myeloma cells (e.g., Y0, NS0, Sp2 / 0), monkey kidney cells (COS-7), human embryonic kidney cell line (293), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells, human lung cells (W138), human liver cells (HepG2), mouse mammary tumor cells, TR1 cells, Medical Research Council 5 (MRC 5) cells, and FS4 cells.

15. A method for preparing a mutant IL-2 polypeptide, the method comprising culturing the host cell according to any one of claims 13 under conditions suitable for expression of the polypeptide.

16. A pharmaceutical composition comprising the polypeptide according to claim 1 and a pharmaceutically acceptable carrier.

17. A method for treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of the mutant IL-2 polypeptide of claim 1, or administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 16.

18. The method of claim 17, wherein the disease is cancer; optionally, wherein the cancer is selected from colorectal cancer, pancreatic cancer, ovarian cancer, liver cancer, kidney cancer, breast cancer, lung cancer, esophageal cancer and gastric cancer, head and neck cancer, cervical cancer, prostate cancer, melanoma, bladder cancer, oral cancer or a hematological malignancy.

19. The method of claim 17, wherein the polypeptide exhibits a selectively decreased binding affinity to IL-2Rα relative to the IL-2Rα binding affinity of the C125 IL-2 polypeptide of SEQ ID NO:

10.

20. The method of claim 19, wherein the polypeptide comprises a combination of amino acid differences relative to SEQ ID NO: 10 selected from the group consisting of:

21. The method of claim 17, wherein the IL-2 mediated disease is an autoimmune disease; optionally, wherein the autoimmune disease is selected from Crohn's disease, ulcerative colitis, celiac disease, systemic lupus erythematosus, psoriatic arthritis, rheumatoid arthritis, Sjögren's syndrome, type 1 diabetes, atopic dermatitis, psoriasis, and multiple sclerosis.

22. The method of claim 21, wherein the polypeptide exhibits selectively decreased IL-2Rβγ binding affinity relative to the IL-2Rβγ binding affinity of the C125 IL-2 polypeptide of SEQ ID NO:

10.

23. The method of claim 22, wherein the polypeptide comprises a combination of amino acid differences relative to SEQ ID NO: 10 selected from the group consisting of:

24. The method of claim 17, wherein the polypeptide comprises a sequence selected from the group consisting of: SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 , 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144 and 145.

25. The method of claim 17, wherein the polypeptide is fused to a monomeric or dimeric Fc polypeptide; optionally, wherein the Fc polypeptide is a monomeric Fc polypeptide.

26. The method of claim 25, wherein the monomeric or dimeric Fc polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 146, 147, and 148.

27. The method of claim 25, wherein the polypeptide is fused via a linker; optionally, wherein the linker is a polypeptide comprising an amino acid sequence selected from the group consisting of: (GGGGS)1 (SEQ ID NO: 149), (GGGGS)2 (SEQ ID NO: 150), (GGGGS)3 (SEQ ID NO: 151), (GGGGS)4 (SEQ ID NO: 152), (GRPGS)2 (SEQ ID NO: 153), (GRPGS)4 (SEQ ID NO: 154), and (GGGGS)3GGG (SEQ ID NO: 155).

28. The method of claim 25, wherein the polypeptide is fused from the C-terminus of the monomeric or dimeric Fc polypeptide and the N-terminus of the mutant IL-2 polypeptide via a linker.

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