Modified interleukin-2 receptor beta-binding reduced agonist

Abstract

Modified IL2 polypeptides, IL2Rβ-binding reduced agonists, and fusion proteins thereof are provided herein. Also provided is a method of modulating an immune response by administering a modified IL2 polypeptide-binding reduced agonist or a fusion protein thereof.

Description

Background Art

[0001] Reference to Electronic Sequence Listing The content of the electronic sequence listing (300096_403WO_SEQUENCE_LISTING.xml, size: 266,559 bytes, creation date: May 8, 2023) is hereby incorporated by reference in its entirety into this specification.

[0002] Interleukin-2 (IL2) is a cytokine that regulates the proliferation and activation of lymphocytes. It has a length of 133 amino acids and its structure contains four antiparallel amphipathic C-helices. IL2 mediates its action by binding to the IL2 receptor (IL2R), which contains up to three distinct subunits. The association of all three subunits, namely the interleukin-2 receptor alpha chain (IL2Rα, or CD25), the interleukin-2 receptor beta chain (IL2Rβ, or CD122), and the interleukin-2 receptor gamma chain (IL2Rγ, or CD132), results in the trimeric IL2Rαβγ, which is the high-affinity receptor for IL2. The association of the IL2Rβ and IL2Rγ subunits results in the dimeric receptor IL2Rβγ, which is called the intermediate-affinity IL2R. The IL2Rα subunit forms a monomeric low-affinity IL2 receptor. The expression of IL2Rα is involved in the expansion of immunosuppressive regulatory T cells (Tregs), while the dimeric IL2Rβγ can cause the proliferation and death of cytolytic CD8 + T cells and NK cells in the absence of IL2Rα.

Summary of the Invention

[0003] The present disclosure provides a rationally designed modified IL2 polypeptide having an amino acid substitution in the IL2Rβ-binding region 2 that reduces binding to IL2Rβ as compared to wild-type IL2.

[0004] In one aspect, the present disclosure provides a modified interleukin-2 (IL2) polypeptide comprising a modified interleukin-2 receptor beta (IL2Rβ) binding region 2 motif, wherein the modified IL2Rβ binding region 2 motif is X1-X2-X3-X4-X-5-X6-X7-X8-X9-X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 (SEQ ID NO: 3), wherein X1 comprises a residue selected from R, S, L, N, F, K or T, X2 comprises a residue selected from A, F, S, L, R, T, I, H, P or N, X3 comprises a residue selected from K, R, T, S, I or P, X4 comprises a residue selected from G, D, R, A, Q, H, N, Y or E, X5 comprises a residue selected from I, P, T, S, K, F, V or L, X6 comprises a residue selected from I, R, V, M, T or L, X7 comprises a residue selected from A, R, M, I, S, N, G or S, X8 comprises a residue selected from E, N, H, T, K, Y, S, L, V, D or R, X9 comprises a residue selected from I, V, A, T, L, T or M, X 10 comprises a residue selected from N, G, V, Y, I, W, R, K, Q, A, D, S or D, X 11 comprises a residue selected from F, G, V, N, T, I, R, E or A, X 12 comprises a residue selected from I, S, R, V, P, G, T, L, M, F or Y, X 13 comprises a residue selected from V, I, F, D, P, H, A, V or L, X 14 comprises a residue selected from L, Q, R, E, P, K, H, W, F or V, X 15 comprises a residue selected from A, E, L, K, V, D, Y, R or Q, X 16 comprises a residue selected from L or I, the modified IL2Rβ binding region 2 motif does not include SEQ ID NO: 2, and the modified IL2 polypeptide binds to IL2Rβ with a reduced affinity compared to wild-type IL2.

[0005] In some embodiments, the present disclosure provides a modified IL2 polypeptide comprising a sequence having at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 46-102, 147-169, and 203-211. In some embodiments, the present disclosure provides a modified IL2 polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 46-102, 147-169, and 203-211 or comprising those sequences.

[0006] In some embodiments, the present disclosure provides a fusion polypeptide comprising a first polypeptide sequence and a second polypeptide sequence, wherein the first polypeptide sequence comprises a modified IL2 polypeptide provided herein. In some embodiments, the second polypeptide sequence of the fusion protein comprises an Fc domain, an antibody, an antigen-binding portion, a cytokine, a half-life extending molecule, a tag or marker polypeptide, a targeting domain, a transport molecule, an immunotoxin, NKG2D, a linker sequence, PEGylation, a chemically conjugated small molecule, a nucleic acid, or any combination thereof. In some embodiments, the second polypeptide sequence comprises an antibody heavy chain constant region. In some embodiments, the antibody heavy chain constant region is a human IgG heavy chain constant region. In some embodiments, the second polypeptide comprises an antigen-binding portion. For example, the antigen-binding portion can bind to PD-L1, PD-1, CTLA-4, TIM3, LAG3, B7-H2, B7-H3, CD4, CD8, or a cell marker.

[0007] In some embodiments, the present disclosure provides a monovalent modified IL2-Fc fusion polypeptide complex comprising (a) a first polypeptide comprising a fusion polypeptide as described herein and (b) a second polypeptide that forms a dimer with the first protein. In some embodiments, the second polypeptide comprises a heavy chain constant region.

[0008] In some embodiments, the present disclosure provides a protein complex comprising a first polypeptide that is a fusion polypeptide as described herein and a second polypeptide comprising an antigen-binding portion. In some embodiments, the antigen-binding portion can bind to PD-L1, PD-1, CTLA-4, TIM3, LAG3, B7-H2, B7-H3, CD4, CD8, or a cell marker.

[0009] In some embodiments, the present disclosure provides a bifunctional fusion protein comprising (a) a modified IL2 polypeptide comprising any one of the sequences of claims 1-2524 and (b) an antigen-binding portion. In some embodiments, the antigen-binding portion can bind to PD-L1, PD-1, CTLA-4, TIM3, LAG3, B7-H2, B7-H3, CD4, CD8, or a cell marker.

[0010] In some embodiments, the present disclosure provides an isolated polynucleotide encoding at least one polypeptide disclosed herein. In some embodiments, the present disclosure provides an expression vector comprising a polynucleotide encoding at least one polypeptide disclosed herein. In some embodiments, the present disclosure provides a modified cell comprising the isolated polynucleotide or expression vector disclosed herein.

[0011] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a modified IL2 polypeptide, a fusion polypeptide, a protein complex, a bifunctional fusion protein, a polynucleotide, a vector, or a modified cell as disclosed herein, and a pharmaceutically acceptable carrier.

[0012] In some embodiments, the present disclosure provides a method of modulating an immune response in a subject in need thereof. The method comprises administering an effective amount of a modified IL2 polypeptide, a fusion polypeptide, a protein complex, a bifunctional fusion protein, a polynucleotide, a vector, a modified cell, or a pharmaceutical composition as disclosed herein.

[0013] In some aspects, the present disclosure provides a method of treating a disease in a subject in need thereof. The method includes administering to the subject an effective amount of a modified IL2 polypeptide, a fusion polypeptide, a protein complex, a bifunctional fusion protein, a polynucleotide, a vector, a modified cell, or a pharmaceutical composition, as disclosed herein.

[0014] In some aspects, the present disclosure provides a cell culture medium comprising a modified IL2 polypeptide, a fusion polypeptide, a protein complex, a bifunctional fusion protein, a polynucleotide, a vector, or a cell, as disclosed herein. In some aspects, a method of culturing cells is disclosed herein. The method includes incubating the cells in a culture medium comprising a modified IL2 polypeptide, a fusion polypeptide, a protein complex, a bifunctional fusion protein, a polynucleotide, a vector, or a cell, as disclosed herein.

[0015] In some aspects, the present disclosure provides transgenic immune cells comprising a modified IL2 polypeptide, a fusion polypeptide, a protein complex, a bifunctional fusion protein, a polynucleotide, or a vector, as described herein. In some aspects, the immune cells are CD4+ T cells, CD8+ T cells, γδ T cells, NK cells, regulatory T cells, or any combination thereof. In some aspects, the immune cells further comprise a chimeric antigen receptor (CAR). In some aspects, the modified IL2 polypeptide, the fusion polypeptide, the protein complex, or the bifunctional fusion protein is secreted by the transgenic immune cells or expressed / localized on the surface of the cells.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0017] A rationally designed modified IL2 polypeptide having an amino acid substitution in the IL2Rβ binding region 2 that reduces binding to IL2Rβ compared to wild-type IL2 is presented herein. The modified IL2 polypeptide is an IL2Rβ binding-reducing agonist, which provides improved safety and persistent immunomodulation compared to wild-type IL2, and at the same time provides the advantage of stimulating NK cells and T effector cells. Thus, the modified IL2Rβ agonist is useful for regulating or activating the immune response, for example, for the treatment of cancer.

[0018] Definitions In this specification, any concentration range, percentage range, ratio range or integer range is understood to include any value or sub-range within the stated range, unless otherwise indicated. As used herein, the term "about" means ±20% of the indicated range or value, unless otherwise indicated.

[0019] Also, it should be noted that the term "or" is generally adopted in the sense of including "and / or" (i.e., meaning any one, both, or a combination thereof of the options), unless otherwise indicated in the content.

[0020] Also, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise.

[0021] Terms such as "comprising", "having", "including", and their variants are used synonymously and are to be construed as non-limiting.

[0022] As used herein, the term "combinations thereof" refers to all possible combinations of the items listed prior to that term. For example, "A, B, C, or combinations thereof" is intended to refer to any one of A, B, C, AB, AC, BC, or ABC. Similarly, as used herein, the term "combinations thereof" refers to all possible combinations of the items listed prior to that term. For example, "A, B, C, and combinations thereof" is intended to refer to all of A, B, C, AB, AC, BC, and ABC.

[0023] As used herein, the term "interleukin 2" or "IL2" refers to IL2 from any vertebrate source, including mammals such as humans or mice, unless otherwise indicated. This term encompasses precursor or unprocessed IL2, as well as any form of IL2 resulting from cellular processing. This term also encompasses naturally occurring variants of IL2, examples of which include splice variants or allelic variants. When used in reference to IL2, "wild-type" or "native" is intended to mean a mature IL2 molecule (e.g., SEQ ID NO: 1). As used herein, the term "modified IL2" or "modified IL2 polypeptide" encompasses IL2 having at least one residue different from native or wild-type IL2, including full-length IL2, truncated forms of IL2, and forms in which IL2 is linked or fused to another molecule such as another polypeptide. Various forms of modified IL2 are characterized by having at least one amino acid substitution that affects the interaction of IL2 with IL2Rβ. The modified IL2 referred to herein may be an IL2Rβ binding-reducing agonist. An IL2Rβ binding-reducing agonist has reduced binding to IL2Rβ compared to wild-type IL2 or IL2 having T3A and C125S substitutions relative to, for example, SEQ ID NO: 1 (e.g., SEQ ID NO: 171).

[0024] IL2Rβ binding region 1 and IL2Rβ binding region 2 are involved in the binding of IL2 to IL2Rβ. As used herein, "IL2Rβ binding region 1" refers to residues 11-23 of wild-type or native human IL2. As used herein, "IL2Rβ binding region 2" refers to residues 81-96 of wild-type or native human IL2. The amino acid sequence of IL2Rβ binding region 2 is shown in SEQ ID NO: 2.

[0025] IL2Rα binding region 1 and IL2Rα binding region 2 are involved in the binding of IL2 to IL2Rα. As used herein, "IL2Rα binding region 1" refers to residues 34-45 of wild-type or native human IL2.

[0026] As used herein, the terms "modified", "recombinant", or "non-natural" refer to a polypeptide / protein, nucleic acid molecule, vector, organism, microorganism, or cell that contains at least one genetic change or is modified by the introduction of an exogenous or heterologous nucleic acid molecule, and such change or modification is introduced by genetic engineering (i.e., human intervention). Genetic changes include, for example, modifications that introduce an expressible nucleic acid molecule encoding a functional RNA, protein, fusion protein, or enzyme, or the addition, deletion, substitution, or other functional disruption of the genetic material of a cell by another nucleic acid molecule. Additional modifications include, for example, non-coding regulatory regions whose expression of a polynucleotide, gene, or operon is changed by the modification.

[0027] As used herein, the term "substitution" or "residue substitution" refers to replacing a native or wild-type residue with a different residue. Similarly, "mutation" refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule as compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively. Mutations can result in several different types of changes in the sequence, including substitutions, insertions, or deletions of nucleotides and / or amino acids. In this specification, the same residue substitution may be indicated using various identifiers. For example, the substitution of threonine at position 3 with alanine can be indicated as T3A or 3A.

[0028] As used herein, "nucleic acid molecule" or "polynucleotide" or "poly nucleic acid" refers to a polymeric compound that includes covalently linked nucleotides and can be made from natural subunits (e.g., purine or pyrimidine bases) or non-natural subunits (e.g., morpholine rings). Purine bases include adenine, guanine, hypoxanthine, and xanthine, and pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid molecules include polyribonucleic acids (RNA) including mRNA, microRNA, siRNA, viral genomic RNA, and synthetic RNA, and polydeoxyribonucleic acids (DNA) including cDNA, genomic DNA, and synthetic DNA, and can be either single-stranded or double-stranded. In the case of single-stranded, the nucleic acid molecule can be either a coding strand or a non-coding (antisense) strand. A nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences encoding the same amino acid sequence. Also, some versions of the nucleotide sequence may include introns to the extent that the introns are removed by transcriptional or post-transcriptional mechanisms. In other words, due to the redundancy or degeneracy of the genetic code, or as a result of splicing, various nucleotide sequences may encode the same amino acid sequence.

[0029] As used herein, "protein" or "polypeptide" refers to a polymer of amino acid residues. Proteins apply not only to natural amino acid polymers but also to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding natural amino acids, and to non-natural amino acid polymers. Variants of the proteins, peptides, and polypeptides of the present disclosure are also contemplated. In certain embodiments, variant proteins, peptides, and polypeptides include or consist of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to the amino acid sequence of a defined amino acid sequence or a reference amino acid sequence as described herein.

[0030] "Fusion polypeptide" or "fusion protein" refers to a polypeptide encoded by at least two different DNA sequences corresponding to genes or fragments thereof that are not naturally expressed from the same gene. An example of a fusion polypeptide is a modified IL2-Fc fusion polypeptide that includes the amino acid sequence of a modified IL2 polypeptide and the amino acid sequence of an Fc domain.

[0031] "Protein complex" or "multiprotein complex" refers to a group of two or more related polypeptide chains that interact to form a quaternary structure. The complex may form under energetically favorable circumstances. For example, a protein complex may form by ionic and / or hydrophobic interactions. A protein complex may include one or more disulfide bonds or two or more protein subunits linked by disulfide bonds. An antibody that includes at least one heavy chain and at least one light chain is an example of a protein complex. A protein complex can include one or more fusion proteins. For example, an IL2-Fc fusion polypeptide may form a protein complex with the heavy and light chains of an antibody.

[0032] "Bifunctional fusion protein" or "bispecificity" refers to a protein, fusion protein, and / or heterodimeric protein pair that contains one or more functional domains. Examples of functional domains include antigen-binding sites, antibody fragments (e.g., Fab, scFv, etc.), antibody heavy and light chains, and cytokines (e.g., IL-2, IL-15). A bifunctional fusion protein or bispecific protein can refer to an antibody that includes a fusion with a non-antibody polypeptide such as a cytokine. For example, a bifunctional protein can include the heavy and light chains of an antibody, and the heavy chain constant region is fused to a modified IL-2. Further, a bifunctional fusion protein can include the heavy and light chains of an antibody, and the heavy chain constant region can form a heterodimer with a polypeptide or protein that does not contain an antigen-binding site. For example, a bifunctional fusion protein can include a heavy chain, a light chain, and a modified IL-2 fusion protein, and this modified IL-2 fusion protein includes an antibody Fc domain that can form a heterodimer with the Fc domain of an antibody heavy chain.

[0033] As used herein, "percent sequence identity" refers to the relationship determined by comparing two or more sequences. Preferred methods for determining sequence identity are designed to give the best match between the sequences being compared. For example, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment). Additionally, non-homologous sequences may be disregarded for comparison purposes. The percent sequence identity referred to herein is calculated over the length of the reference sequence, unless otherwise indicated. Methods for determining sequence identity and sequence similarity can be found in publicly available computer programs. Calculation of sequence alignment and percent identity may be performed using the BLAST program (e.g., BLAST 2.0, BLASTP, BLASTN, or BLASTX). The mathematical algorithms used in the BLAST program are described in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997. In the context of this disclosure, it will be understood that when sequence analysis software is used for an analysis, the results of the analysis are based on the "default values" of the program being referenced. "Default values" mean any set of values or parameters that are initially read in with the software when it is first initialized.

[0034] The term "isolated" means that a substance has been removed from its original environment (e.g., its natural environment if it is naturally occurring). For example, a naturally-occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide is isolated if it is separated from some or all of the substances that coexist in the natural system. Such nucleic acids can be part of a vector, and / or such nucleic acids or polypeptides can be part of a composition (e.g., a cell lysate), and such a vector or composition is still isolated in that it is not part of the natural environment of the nucleic acid or polypeptide.

[0035] "Affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., a receptor and a ligand). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (K D ). This is the ratio of the dissociation rate constant and the association rate constant (k off and k on ), respectively. Thus, equivalent affinities may include different rate constants as long as the ratio of the rate constants remains the same. Affinity can be measured by methods known to those skilled in the art, including the methods described herein.

[0036] "Immunoglobulin" refers to a protein having the structure of a naturally occurring antibody. As an example, an immunoglobulin of the IgG class is a heterotetrameric glycoprotein in which two light chains and two heavy chains are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called the heavy chain constant regions. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called the variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain, also called the light chain constant region. The heavy chain of an immunoglobulin may be assigned to one of five classes called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), and a portion of it may be further divided into subclasses, examples of which include γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chain of an immunoglobulin may be assigned to one of two types called kappa (κ) and lambda (λ) based on the sequence of the constant domain. An immunoglobulin contains two Fab molecules and one Fc domain linked via an immunoglobulin hinge region.

[0037] As used herein, the term "Fc domain" or "Fc region" refers to a polypeptide derived from the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes polypeptides having a native sequence Fc region or variants thereof. The boundaries of the Fc region of the IgG heavy chain may vary slightly, but the human IgG heavy chain Fc region is typically defined as extending from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Examples of Fc regions are disclosed in U.S. Patent No. 7,317,091, U.S. Patent No. 8,735,545, U.S. Patent No. 7,371,826, U.S. Patent No. 7,670,600, and US9,803,023, all of which are incorporated herein by reference in their entirety.

[0038] As used herein, the term "antibody" encompasses various antibody structures so long as they exhibit the desired antigen-binding activity, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, bifunctional antibodies), antibody fusion proteins, antibodies that form heterodimers in modified proteins, and antibody fragments.

[0039] "Antibody fragment" refers to a polypeptide or protein other than an intact antibody that includes a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab′, Fab′-SH, F(ab′)2, diabody, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0040] The "antigen-binding portion" or "antigen-binding site" is used interchangeably herein and refers to the site (i.e., amino acid residues) of an antigen-binding molecule (e.g., an antibody) that provides interaction with an antigen epitope. The antigen-binding portion may include one or more antibody variable domains (also referred to as antibody variable regions). In a human antibody, the antigen-binding site is formed by the amino acid residues of the N-terminal variable ("V") regions of the heavy chain ("H") and the light chain ("L"). The "hypervariable regions" are three highly diverse regions within the V regions of the heavy and light chains and are inserted between relatively conserved adjacent regions, the "framework regions" ("FR"). The term "FR" refers to the amino acid sequences that naturally exist between and in the vicinity of the hypervariable regions of an immunoglobulin. In a human antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen. Each of the three hypervariable regions of the heavy chain ("H") and the light chain ("L") is called a "complementary determining region" or "CDR". The antigen-binding site can be present in an intact antibody, in an antigen-binding fragment of an antibody that retains the antigen-binding surface, or in a recombinant polypeptide such as an scFv, connecting the heavy chain variable domain to the light chain variable domain in a single polypeptide using a peptide linker. The antigen-binding site can include the variable region of the light chain (VL) of the antibody and the variable region of the heavy chain (VH) of the antibody. Examples of antigen-binding portions include immunoglobulins, Fab molecules, scFv, bispecific antibodies, diabodies, bispecific T cell engagers, and nanobodies. Specific examples of antigen-binding portions include nivolumab, pembrolizumab, pidilizumab, atezolizumab, ipilimumab, tremelimumab, rituximab, ofatumumab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, tositumomab, ublituximab, and bevacizumab.

[0041] The numbering of CDRs and framework regions may be performed according to any known method or scheme, examples of which include Kabat, Chothia, EU, IMGT, and AHo numbering schemes (e.g., see Kabat et al., “Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5 th ed.; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); Lefranc et al., Dev. Comp. Immunol. 27:55, 2003; Honegger and Pluckthun, J. Mol. Bio. 309:657-670 (2001)). For different molecules to be compared, the positions of equivalent residues can be annotated using the Antigen receptor Numbering and Receptor Classification (ANARCI) software tool (2016, Bioinformatics 15:298-300). The CDRs of the antigen-binding site can be determined according to known methods, examples of which include Kabat, Chothia, EU, IMGT, AHo as described above. The CDRs determined under these definitions usually contain overlapping amino acid residues, or subsets of amino acid residues, when compared to each other. The heavy-chain CDRs and light-chain CDRs of an antibody can be defined using various numbering conventions. For example, in certain embodiments, the heavy-chain CDRs are defined according to Chothia (as above), and the light-chain CDRs are defined according to Kabat (as above). CDRH1, CDRH2, and CDRH3 denote the heavy-chain CDRs, and CDRL1, CDRL2, and CDRL3 denote the light-chain CDRs.

[0042] A “Fab molecule” or “antigen-binding fragment” is an antigen-binding fragment of an antibody that includes the variable and constant domains of the light chain, as well as the variable domain and CH1 domain of the heavy chain.

[0043] "Single-chain variable domain" or "scFv" refers to an antigen-binding moiety that includes variable domains of a heavy chain and a light chain linked by a linker peptide.

[0044] "Bispecific antibody" refers to an artificial antibody that has two different antigen-binding sites. A bispecific antibody can refer to a complete immunoglobulin protein that has two different antigen-binding sites, or can refer to other molecules that have two antigen-binding moieties, such as a fusion protein that includes two Fabs or two scFvs.

[0045] "Diabody" refers to a class of antigen-binding molecules that are bivalent and bispecific. The fragment includes a heavy chain variable domain (VH) that is bound to a light chain variable domain (VL) on the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains pair with complementary domains on another chain to form two antigen-binding sites.

[0046] "Bispecific T cell engager" refers to a class of bispecific antibodies that have a first antigen-binding moiety that binds to a T cell (e.g., by binding to CD3) and a second antigen-binding moiety that binds to a different antigen (e.g., a tumor antigen).

[0047] "VHH antibody", "nanobody" or "single-domain antibody" refers to an antigen-binding moiety that consists of a single monomeric variable antibody domain.

[0048] "Transferrin" is an iron transport protein that can be used in a fusion protein to extend the half-life. Human transferrin has a half-life of 12 days in serum.

[0049] As used herein, "cytokine" refers to a class of small (less than 25 kDa) proteins involved in cell signaling and immune regulation. Cytokines include, for example, IL2, interleukin 10 (IL-10), interleukin 1 (IL-1), interleukin 17 (IL-17), interleukin 18 (IL-18), interferon α, interferon β, interferon γ, TGF-β1, TGF-β2, and TGF-β3, chemokine (C-C motif) ligand 2 (CCL2), and chemokine (C-C motif) ligand 19 (CCL19).

[0050] As used herein, "half-life extending molecule" refers to a molecule that, when bound (e.g., covalently) to a second molecule, extends the half-life of the second molecule. Examples of half-life extending molecules include the Fc domain, human serum albumin (HSA), HSA-binding molecules, polyethylene glycol (PEG), and polypropylene glycol (PPG).

[0051] "Human serum albumin" or "HSA" refers to serum albumin found in human blood. A commonly used form of HSA has a molecular mass of 66.5 kDa and a half-life of about 20 days. Examples of HSA molecules are disclosed in U.S. Patent No. 8,143,026 and U.S. Patent No. 7,189,690 (which are incorporated herein by reference in their entirety).

[0052] "HSA-binding molecule" refers to a molecule that specifically binds to human serum albumin (HSA), such as an antigen-binding portion having an HSA-binding domain.

[0053] "Polyethylene glycol" or "PEG", also called polyethylene oxide or polyoxyethylene, is a polyether polymer that can be used to extend the half-life.

[0054] "Polypropylene glycol" or "PPG", also called polypropylene oxide, is a polymer of propylene glycol that can be used to extend the half-life.

[0055] The "subject" according to any of the above embodiments is a mammal. Mammals include, but are 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). Preferably, the subject is a human.

[0056] "Modulating an immune response" may include one or more of a general increase, an increase in T effector cell response (e.g., cytotoxicity against tumor cells and virus-infected cells), an increase in B cell activation, a recovery of lymphocyte activation and proliferation, an increase in the expression of the IL2 receptor, an increase in T cell responsiveness, an increase in natural killer cell activity or lymphokine-activated killer (LAK) cell activity, and a decrease in regulatory T cell response against other T cells.

[0057] "Regulatory T cell" or "Treg cell" refers to a special type of CD4+ T cell that can function to suppress the response of other T cells. Treg cells express the α subunit of the IL2 receptor (CD25) and the transcription factor forkhead box P3 (FOXP3) (Sakaguchi, Annu Rev Immunol 22, 531-62 (2004)) and are involved in the induction and maintenance of peripheral self-tolerance to antigens, including those expressed by tumors. Treg cells require IL2 for improvement and induction of function as well as suppression characteristics.

[0058] "T effector cell" refers to a population of T cells that respond to stimuli such as IL2. T effector cells include CD8+ cytotoxic T cells and CD4+ helper T cells. As used herein, T effector cells do not include regulatory T cells.

[0059] "Natural killer cell" or "NK cell" is a component of the innate immune system and is a cytotoxic lymphocyte that plays a major role in the rejection of tumors and virus-infected cells.

[0060] A "chimeric antigen receptor" or "CAR" is a modified antigen-binding receptor that, when expressed in certain types of immune cells, activates the immune cells upon antigen binding. A CAR typically includes an extracellular domain that contains an antigen-binding portion (e.g., scFv), a transmembrane domain, and an intracellular immune signaling domain (e.g., including signaling domains from CD3ζ, 4-1BB, and / or CD28). A CAR may be expressed, for example, by T cells or NK cells and may include an antigen-binding portion that targets a cancer antigen such as CD19 or ROR1.

[0061] "Treatment", "treating", or "alleviating" refers to the medical management of a subject (e.g., a patient) with a medical condition, disease, or disorder, which may be a therapeutic, prophylactic / preventive, or a combination thereof treatment.

[0062] "Effective amount" or "therapeutically effective amount" can refer to the amount of a therapeutic agent (e.g., a modified IL2 polypeptide or a modified IL2 fusion polypeptide described herein) that provides a desired physiological change such as an anti-cancer effect. The desired physiological change may be, for example, a decrease in the symptoms of a disease, or a decrease in the severity of a disease, or a reduction in the progression of a disease. With respect to cancer, the desired physiological change may include, for example, tumor regression, a decrease in the rate of tumor progression, a decrease in the level of a cancer biomarker, a decrease in cancer-related symptoms, prevention or delay of metastasis, or a clinical remission.

[0063] As used herein, the term "inhibiting" refers to a decrease in a particular activity (e.g., immunosuppression or tumor growth). Unless otherwise specified, an activity is considered inhibited if it is decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% as measured by the methods disclosed herein or methods known in the art.

[0064] "Checkpoint inhibitor" refers to an agent that reduces the activity of immune checkpoint proteins. A checkpoint inhibitor can be an antigen-binding portion that binds to an immune checkpoint protein and reduces its activity. Immune checkpoint proteins include, for example, programmed cell death protein 1 (PD-1 or CD279), programmed death ligand 1 (PD-L1 or CD274), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4 or CD152), T cell immunoglobulin mucin 3 (TIM3), lymphocyte activation 3 (LAG3 or CD223), B7-H2 (ICOSL or CD275), and B7-H3 (CD276). Examples of checkpoint inhibitors include ipilimumab (anti-CTLA-4 antibody), nivolumab (anti-PD-1 antibody), and pembrolizumab (anti-PD-1 antibody).

[0065] "Tumor-infiltrating lymphocytes" or "TIL" refers to lymphocytes that are isolated from tumor tissue, manipulated in vitro (e.g., stimulated using cytokines such as interleukin 2), and then injected back into the patient, whereupon the activated TIL return to the tumor site and induce tumor regression.

[0066] "Tumor microenvironment inhibitor" refers to an agent that suppresses one or more conditions or cell types present in the local environment surrounding a tumor that promote tumor growth. For example, bevacizumab can inhibit the tumor microenvironment by reducing angiogenesis in the tumor microenvironment.

[0067] As used herein, PD-L1 (also known as "programmed cell death ligand 1" or CD274 in humans) refers to the protein of UniProt accession number Q0GN75 (human) and related isoforms and orthologs. As used herein, PD-1 (also known as "programmed cell death protein 1", "PDCD1" or CD279) refers to the protein of UniProt accession number Q15116 (human) and related isoforms and orthologs.

[0068] The recombinant DNA, molecular cloning, and gene expression techniques used in the present disclosure are known in the art and are described in references such as ambrook et al., Molecular Cloning: A Laboratory Manual, 3 rd Ed., Cold Spring Harbor Laboratory, New York, 2001, and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD, 1999.

[0069] Modified interleukin-2 polypeptide As described above, the IL2 polypeptide of the present disclosure includes an IL2Rβ-binding reduced agonist having a modified IL2 receptor beta (IL2Rβ) binding region 2. In some embodiments, the IL2Rβ binding region 2 is X1-X2-X3-X4-X-5-X6-X7-X8-X9-X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 (SEQ ID NO: 3), wherein X1 includes a residue selected from R, S, L, N, F, K, or T, X2 includes a residue selected from A, F, S, L, R, T, I, H, P, or N, X3 includes a residue selected from K, R, T, S, I, or P, X4 includes a residue selected from G, D, R, A, Q, H, N, Y, or E, X5 includes a residue selected from I, P, T, S, K, F, V, or L, X6 includes a residue selected from I, R, V, M, T, or L, X7 includes a residue selected from A, R, M, I, S, N, G, or S, X8 includes a residue selected from E, N, H, T, K, Y, S, L, V, D, or R, X9 includes a residue selected from I, V, A, T, L, T, or M, X 10 includes a residue selected from N, G, V, Y, I, W, R, K, Q, A, D, S, or D, X 11 includes a residue selected from F, G, V, N, T, I, R, E, or A, X12 comprises a residue selected from I, S, R, V, P, G, T, L, M, F or Y, and X 13 comprises a residue selected from V, I, F, D, P, H, A, V or L, and X 14 comprises a residue selected from L, Q, R, E, P, K, H, W, F or V, and X 15 comprises a residue selected from A, E, L, K, V, D, Y, R or Q, and X 16 comprises a residue selected from L or I.

[0070] For example, in some embodiments, the modified IL2 polypeptide has the amino acid sequence: APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPLLTDMLTRKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHL-X1-X2-X3-X4-X-5-X6-X7-X8-X9-X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -KGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 45), wherein X1 comprises a residue selected from R, S, L, N, F, K or T, X2 comprises a residue selected from A, F, S, L, R, T, I, H, P or N, X3 comprises a residue selected from K, R, T, S, I or P, X4 comprises a residue selected from G, D, R, A, Q, H, N, Y or E, X5 comprises a residue selected from I, P, T, S, K, F, V or L, X6 comprises a residue selected from I, R, V, M, T or L, X7 comprises a residue selected from A, R, M, I, S, N, G or S, X8 comprises a residue selected from E, N, H, T, K, Y, S, L, V, D or R, X9 comprises a residue selected from I, V, A, T, L, T or M, and X 10 comprises a residue selected from N, G, V, Y, I, W, R, K, Q, A, D, S or D, and X 11 comprises a residue selected from F, G, V, N, T, I, R, E or A, and X 12contains a residue selected from I, S, R, V, P, G, T, L, M, F or Y, and X 13 contains a residue selected from V, I, F, D, P, H, A, V or L, and X 14 contains a residue selected from L, Q, R, E, P, K, H, W, F or V, and X 15 contains a residue selected from A, E, L, K, V, D, Y, R or Q, and X 16 contains a residue selected from L or I.

[0071] The modified IL2 polypeptide disclosed herein does not contain the wild-type IL2Rβ binding region 2 motif, i.e., SEQ ID NO: 2. The modified IL2 polypeptide binds to IL2Rβ with decreased affinity compared to wild-type IL2 (SEQ ID NO: 1) or IL2 having T3A and C125S substitutions relative to SEQ ID NO: 1 (e.g., SEQ ID NO: 171).

[0072] In some embodiments, the modified IL2 polypeptide is RFKALIIEINFIVQLL (SEQ ID NO: 4), RSRQLISNMNGIILKL (SEQ ID NO: 5), RLTHLRNVIGVILVQL (SEQ ID NO: 6), LSLREPIGNIVTSVRE (SEQ ID NO: 7), NRTDLVGDVNATIKAL (SEQ ID NO: 8), RNKGILGDISNIVLAL (SEQ ID NO: 9), RSREVVSRIDAIILEL (SEQ ID NO: 10), RPRGLISDISNIVLAL (SEQ ID NO: 11), RPRGLIGNISNIVLAL (SEQ ID NO: 12), RPRGLIGDINNIVLAL (SEQ ID NO: 13), RPKGLISNISNIVLAL (SEQ ID NO: 14), RPKGLISDINNIVLAL (SEQ ID NO: 15), RPKGLIGNINNIVLAL (SEQ ID NO: 16), RNRGLISNISNIVLAL (SEQ ID NO: 17), RNRGLISDINNIVLAL (SEQ ID NO: 18), RNRGLIGNINNIVLAL (SEQ ID NO: 19), RNKGLISNINNIVLAL (SEQ ID NO: 20), RPRGLIGDISNIVLAL (SEQ ID NO: 21), RPKGLISDISNIVLAL (SEQ ID NO: 22), RPKGLIGNISNIVLAL (SEQ ID NO: 23), RPKGLIGDINNIVLAL (SEQ ID NO: 24), RNRGLISDISNIVLAL (SEQ ID NO: 25), RNRGLIGNISNIVLAL (SEQ ID NO: 26), RNRGLIGDINNIVLAL (SEQ ID NO: 27), RNKGLISNISNIVLAL (SEQ ID NO: 28), RNKGLISDINNIVLAL (SEQ ID NO: 29), RNKGLIGNINNIVLAL (SEQ ID NO: 30), RPRDLISDISNIVLAL (SEQ ID NO: 31), RPRGLISDINNIVLAL (SEQ ID NO: 32), RPRGLISDISVIVLAL (SEQ ID NO: 33), RPRGLISDISNIVLEL (SEQ ID NO: 34), RPRDLISDINNIVLAL (SEQ ID NO: 35), RPRDLISDISVIVLAL (SEQ ID NO: 36), RPRDLISDISNIVLEL (SEQ ID NO: 37), RPRGLISDINVIVLAL (SEQ ID NO: 38), RPRGLISDINNIVLEL (SEQ ID NO: 39), RPRGLISDISVIVLEL (SEQ ID NO: 40), RPKDLISNISNIVLAL (SEQ ID NO: 41), RPKGLISNINNIVLAL (SEQ ID NO: 42),It contains an IL2Rβ binding region 2 selected from RPKGLISNISVIVLAL (SEQ ID NO: 43), RPKGLISNISNIVLEL (SEQ ID NO: 44), RPKGLISNISVIVLEL (SEQ ID NO: 194), RPRGLISNISVIVLEL (SEQ ID NO: 195), RPRDLISNISNIVLEL (SEQ ID NO: 196), RPKGLISNINNIVLEL (SEQ ID NO: 197), RPKGLISDINNIVLEL (SEQ ID NO: 198), RPRDLISRIDAIVLEL (SEQ ID NO: 199), RNRGLIGNINNIVLEL (SEQ ID NO: 200), RPKGLISEINNIVLEL (SEQ ID NO: 201), and RPKGLISRINNIVLEL (SEQ ID NO: 202).

[0073] In some embodiments, the modified IL2 polypeptide contains an IL2Rβ binding region 2 selected from RFKALIIEINFIVQLL (SEQ ID NO: 4), RSRQLISNMNGIILKL (SEQ ID NO: 5), RLTHLRNVIGVILVQL (SEQ ID NO: 6), RNKGILGDISNIVLAL (SEQ ID NO: 9), RSREVVSRIDAIILEL (SEQ ID NO: 10), RPRGLISDISNIVLAL (SEQ ID NO: 11), RPRGLIGDINNIVLAL (SEQ ID NO: 13), RPKGLISNISNIVLAL (SEQ ID NO: 14), RPRGLIGDISNIVLAL (SEQ ID NO: 21), RPKGLISDISNIVLAL (SEQ ID NO: 22), RPKGLIGDINNIVLAL (SEQ ID NO: 24), RNKGLISNISNIVLAL (SEQ ID NO: 28), RNKGLISDINNIVLAL (SEQ ID NO: 29), RPKGLISNISVIVLEL (SEQ ID NO: 194), RPRGLISNISVIVLEL (SEQ ID NO: 195), RPRDLISNISNIVLEL (SEQ ID NO: 196), RPKGLISNINNIVLEL (SEQ ID NO: 197), RPKGLISDINNIVLEL (SEQ ID NO: 198), RPRDLISRIDAIVLEL (SEQ ID NO: 199), RNRGLIGNINNIVLEL (SEQ ID NO: 200), RPKGLISEINNIVLEL (SEQ ID NO: 201), and RPKGLISRINNIVLEL (SEQ ID NO: 202).

[0074] In some embodiments, the modified IL2 polypeptide has a reduced affinity for IL2Rβ compared to wild-type IL2 or IL2 having the sequence of SEQ ID NO: 171. In certain embodiments, the binding of the modified IL2 polypeptide to IL2Rβ is at least 10-fold less, at least 15-fold less, at least 20-fold less, at least 25-fold less, or at least 30-fold less than that of wild-type IL2 or IL2 having the sequence of SEQ ID NO: 171, with a K D value. In some embodiments, the modified IL2 polypeptide binds to IL2Rβ with a K D that is at least 30-fold lower than that of wild-type IL2 or IL2 having the sequence of SEQ ID NO: 171. In some embodiments, the modified IL2 polypeptide has a reduced affinity for IL2Rβ compared to wild-type IL2 or IL2 having the sequence of SEQ ID NO: 171, with the affinity being reduced by at least one-tenth, at least one-fifteenth, at least one-twentieth, at least one-twenty-fifth, or at least one-thirtieth. Assays used to detect IL2 binding may include ELISA or surface plasmon resonance (SPR) detection.

[0075] In some embodiments, the modified IL2 polypeptide has a reduced affinity for IL2 receptor α (IL2Rα) compared to wild-type IL2 or IL2 having the sequence of SEQ ID NO: 171. In certain embodiments, the modified IL2 polypeptide has an affinity for IL2Rα that is at least 5% reduced, at least 10% reduced, at least 15% reduced, or at least 20% reduced compared to wild-type IL2 or IL2 having the sequence of SEQ ID NO: 171. In some embodiments, the modified IL2 polypeptide has no detectable binding to IL2Rα compared to wild-type IL2 or IL2 having the sequence of SEQ ID NO: 171. Assays used to detect IL2 binding may include ELISA or SPR detection.

[0076] In some embodiments, the modified IL2 polypeptide has a similar affinity for IL2Rα compared to wild-type IL2. In certain embodiments, the modified IL2 polypeptide has an affinity for IL2Rα that is different from the affinity of wild-type IL2 for IL2Rα by no more than ±20%, ±15%, ±10%, or ±5%.

[0077] Some embodiments of the present disclosure provide a modified IL2 polypeptide comprising a modified IL2Rα binding region 1. The modified IL2Rα binding region 1 can include substitutions selected from a substitution at position K35, a substitution at position R38, a substitution at position F42, a substitution at position Y45, or combinations thereof. In some embodiments, the modified IL2 polypeptide binds to IL2Rα with a binding reaction rate that is reduced by at least one-half compared to wild-type IL2.

[0078] In some embodiments, the modified IL2 polypeptide may include a substitution at position K35. In some embodiments, the substitution at position K35 includes a non-basic residue. In some embodiments, the substitution at position K35 includes an uncharged residue or an acidic residue. In some embodiments, the substitution at position K35 is selected from K35G, K35L, K35S, K35V, K35D, K35E, and K35C.

[0079] In some embodiments, the modified IL2 polypeptide includes a substitution at position R38. In some embodiments, the substitution at position R38 includes a non-basic residue. In some embodiments, the substitution at position R38 includes an uncharged residue or an acidic residue. In some embodiments, the substitution at position R38 is selected from R38V, R38D, R38E, R38S, R38I, R38A, R38Y, R38G, R38C, and R38N.

[0080] In some embodiments, the modified IL2 polypeptide may include a substitution at position F42. In some embodiments, the substitution at position F42 includes an uncharged residue. In some embodiments, the substitution at position F42 includes a basic residue. In some embodiments, the substitution at position F42 is selected from F42A, F42R, F42G, F42I, F42L, F42P, and F42H.

[0081] In some embodiments, the modified IL2 polypeptide may include a substitution at position Y45. In some embodiments, the substitution at position Y45 includes an uncharged residue. In some embodiments, the substitution at position Y45 includes an uncharged polar residue or an uncharged nonpolar residue. In some embodiments, the Y45 substitution is Y45S, Y45P, Y45A, Y45V, Y45C, Y45T, and Y45F.

[0082] In some embodiments, the modified IL2 polypeptide may include a substitution at position K35 and a substitution at position R38. In some embodiments, the modified IL2 polypeptide includes the K35G substitution and the R3E8 substitution.

[0083] In some embodiments, the modified IL2 polypeptide may include a substitution at position K35 and a substitution at position F42. In some embodiments, the modified IL2 polypeptide includes the K35S substitution and the F42G substitution.

[0084] In some embodiments, the modified IL2 polypeptide may include a substitution at position K35, a substitution at position R38, and a substitution at position F42. In some embodiments, the modified IL2 polypeptide includes the K35L substitution, the R38D substitution, and the F42R substitution.

[0085] In some embodiments, the modified IL2 polypeptide may include a substitution at position R38 and a substitution at position Y45S. In some embodiments, the modified IL2 polypeptide includes the R38D substitution and the Y45S substitution. In some embodiments, the modified IL2 polypeptide includes the R38V substitution and the Y45S substitution.

[0086] In some embodiments, the modified IL2 polypeptide binds to IL2Rα with a binding reaction rate that is at least one-tenth less than that of wild-type IL2.

[0087] In some embodiments, the modified IL2 is further modified to include a T3A substitution. The T3A substitution may be made with respect to the wild-type sequence. Additionally, the T3A substitution may be made with respect to the modified IL2 sequences disclosed herein. In some embodiments, the modified IL2 is further modified to include a C125S substitution. The C125S substitution may be made with respect to the wild-type sequence. Additionally, the C125S substitution may be made with respect to the modified IL2 sequences disclosed herein. Optionally, the modified IL2 sequence further includes both the T3A and C125S modifications.

[0088] In some embodiments, the modified IL2 polypeptide shares at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with the residues outside of IL2Rβ binding region 2 of SEQ ID NO: 1 (i.e., residues 1-80 and 97-133) and binds to IL2Rβ. In some embodiments, the modified IL2 polypeptide shares at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with the residues outside of IL2Rβ binding region 2 of SEQ ID NO: 171 (i.e., residues 1-80 and 97-133) and binds to IL2Rβ.

[0089] In some embodiments, the modified IL2 polypeptide has a sequence corresponding to any one of SEQ ID NOs: 46-102, 147-169, and / or 203-211. In certain embodiments, the modified IL2 polypeptide has at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with the residues outside of IL2Rβ binding region 2 of any one of SEQ ID NOs: 46-102, 147-169, and / or 203-211 (i.e., residues 1-80 and 97-133) and binds to IL2Rβ.

[0090] In some embodiments, the modified IL2 polypeptide is selected from any one of SEQ ID NOs: 46-102, 147-169, and / or 203-211 and optionally includes (or excludes) a C-terminal histidine tag. In some embodiments, the C-terminal histidine tag is replaced with a linker such as a Gly-Ser linker.

[0091] Modified IL2 fusion polypeptide Some embodiments of the present disclosure provide modified IL2 fusion polypeptides. The modified IL2 fusion polypeptide may include a modified IL2 polypeptide (see, e.g., SEQ ID NOs: 3-102, 147-169, 203-211, and Tables 2, 5, and 7 as described herein) and at least one additional molecule covalently bound to the modified IL2 polypeptide via a peptide bond or other chemical bond. In certain embodiments, the modified IL2 fusion polypeptide includes a modified IL2 polypeptide that shares at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with the residues outside of IL2Rβ binding region 2 of any one of SEQ ID NOs: 46-102, 147-169, and / or 203-211 (i.e., residues 1-80 and 97-133) and binds to IL2Rβ. In some embodiments, the modified IL2 fusion polypeptide includes a first polypeptide sequence and a second polypeptide sequence, and the first polypeptide sequence includes a modified IL2 polypeptide as described herein. In some embodiments, the second polypeptide sequence of the modified IL2 fusion polypeptide includes a polypeptide having a length of 5 to 500 amino acids, 5 to 400 amino acids, 5 to 300 amino acids, 5 to 250 amino acids, 5 to 200 amino acids, 50 to 500 amino acids, 50 to 400 amino acids, 50 to 300 amino acids, 50 to 250 amino acids, or 50 to 200 amino acids. In some embodiments, the second polypeptide sequence of the modified IL2 fusion polypeptide includes a polypeptide having a length of at least 5, at least 20, at least 40, at least 60, at least 80, 100, at least 150, at least 200, at least 250, or at least 300 amino acids.Non-limiting examples of polypeptides that make up the second polypeptide sequence of the fusion protein can include an Fc domain, an antibody, an antigen-binding portion, a cytokine, a half-life extending molecule, a tag or marker polypeptide, a targeting domain, a transport molecule, an immunotoxin (e.g., diphtheria toxin), NKG2D, a linker sequence, a chemically conjugated small molecule, a nucleic acid, PEGylation, or any combination thereof. In some embodiments, the second polypeptide sequence comprises a ligand or a scaffold protein.

[0092] In certain embodiments, the modified IL2 fusion polypeptide comprises a modified IL2 polypeptide as described herein and an IgG heavy chain constant region (e.g., Fc domain). The IL2 fusion polypeptide comprising an IgG heavy chain constant region may be referred to herein as an IL2-Fc fusion protein or an IL2-Fc fusion polypeptide. In some embodiments, the IgG heavy chain constant region is derived from an IgG antibody. Human IgG antibodies have several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. In certain embodiments, the IgG heavy chain constant region is derived from a human IgG1 antibody or an IgG4 antibody. In some embodiments, the IgG heavy chain constant region has one or more substitutions that reduce the effector function of the IgG heavy chain constant region. In certain embodiments, the IgG heavy chain constant region comprises one or more mutations selected from L234A, L235A, P329G, Y349C, S354C, T366S, T366W, L368A, F405K, K409A, and Y407V numbered according to the EU numbering system as compared to human IgG. In some embodiments, the IgG heavy chain constant region comprises the sequence of SEQ ID NO: 137 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 137. In some embodiments, the Fc domain comprises the LALAPG mutation. The LALAPG mutation refers to the changes of L234A, L235A, and P329G in the CH2-CH3 region of the IgG heavy chain constant region (e.g., IgG1) (see, e.g., Schlothauer et al., Protein Engineering, Design & Selection. 29(10):457-466, 2016; Lo et al., J. Biol. Chem. 292(9):3900-3908, 2017, incorporated herein by reference). In some embodiments, the antibody IgG heavy chain constant region comprises a knob mutation or a hole mutation.The knob-into-hole mutation is a modification to the IgG heavy chain constant region that enables heterodimerization of the IgG heavy chain constant region, which contains the knob mutation and the hole mutation, respectively. The knob mutation or the hole mutation enables preferential heterodimer formation in vitro with a low level of homodimer contaminant. The knob-into-hole mutation is disclosed in Merchant et al., Nat. Biotechnol. 16:677-681, 1998; and Wei et al., Oncotarget. 8(31):51037-51049, 2017, which are incorporated herein by reference. Examples of the knob mutation include the S354C, T366W, and K409A mutations in the IgG heavy chain constant region. Examples of the hole mutation include the Y349C, T366S, L368A, F405K, and Y407V mutations in the IgG heavy chain constant region. In some embodiments, the IgG heavy chain constant region includes the LALAPG mutation and the hole mutation or the knob mutation. For example, in some embodiments, the IgG heavy chain constant region can include the L234A, L235A, P329G, S354C, T366W, and K409A mutations. In some embodiments, the IgG heavy chain constant region can include the L234A, L235A, P329G, Y349C, T366S, L368A, F405K, and Y407V mutations. In certain embodiments, the IgG heavy chain constant region includes the amino acid sequence of SEQ ID NO: 123 or 138. In certain embodiments, the protein complex can be formed by a first polypeptide comprising a first antibody IgG heavy chain constant region containing one or more mutations selected from S354C, T366W, and K409A with respect to SEQ ID NO: 137, and a second polypeptide comprising a second IgG heavy chain constant region containing one or more mutations selected from Y349C, T366S, L368A, F405K, and Y407V with respect to SEQ ID NO: 137. However, the numbering follows the EU numbering system.In certain embodiments, the protein complex comprises a first antibody IgG heavy chain constant region comprising one or more mutations selected from L234A, L235A, P329G, S354C, T366W, and K409A, and a second antibody IgG heavy chain constant region comprising one or more mutations selected from L234A, L235A, P329G, Y349C, T366S, L368A, F405K, and Y407V, relative to SEQ ID NO: 137. However, the numbering follows the EU numbering system. In some embodiments, the IgG heavy chain constant region comprises a modification of at least one amino acid residue for increasing the serum half-life. Representative modifications to the IgG heavy chain constant region are described in US Patent No. 7,317,091, US Patent No. 8,735,545, US Patent No. 7,371,826, US Patent No. 7,670,600, and US Patent No. 9,803,023.

[0093] In some embodiments, the modified IL2 fusion polypeptide comprises a modified IL2 polypeptide linked to a fusion partner such as an IgG heavy chain constant region. Various linkers are known in the art and may be used to covalently attach the modified IL2 described herein to a fusion partner such as the Fc region. As used herein, "linker" and "linker sequence" mean a molecule or group of molecules (e.g., monomer or polymer) that connects two molecules and often serves to position the two molecules in a preferred configuration. The linker may contain amino acid residues that provide flexibility. Thus, the linker peptide may mainly comprise the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide must have a length sufficient to link the two molecules so that they adopt a suitable conformation relative to each other to retain the desired activity. A length suitable for this purpose comprises at least one and at most 30 amino acid residues. Preferably, the linker is about 1 to 30 amino acids in length, and linkers of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 amino acids in length are preferred. Useful linkers include glycine-serine polymers (e.g., (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. In some embodiments, the fusion polypeptide is a bivalent fusion polypeptide. The bivalent fusion polypeptide may refer to a molecular complex comprising two copies of the modified IL2 polypeptide, which may be of the same or different sequences. The molecular complex may be non-covalently bound. For example, the bivalent fusion polypeptide may comprise two Fc regions linked by one or more disulfide bridges or knob-into-hole chemistry.

[0094] In some embodiments, the fusion polypeptide is a monovalent fusion polypeptide, for example, a monovalent modified IL2-Fc fusion polypeptide. A monovalent fusion polypeptide refers to a fusion polypeptide having one copy of a modified IL2 polypeptide. In some embodiments, the fusion polypeptides disclosed herein have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NOs: 124-128 and / or 190-193.

[0095] In some embodiments, the monovalent modified IL2-Fc fusion polypeptide forms a complex with at least one additional protein. In some embodiments, the monovalent modified IL2-Fc fusion polypeptide complex comprises a first protein comprising the fusion polypeptide as described herein and a second protein that dimerizes with the first protein. For example, the second protein can be an IgG heavy chain constant region (Fc domain). The second protein can be an IgG heavy chain constant region having the sequence of SEQ ID NO: 123. In certain embodiments, the monovalent modified IL2-Fc fusion polypeptide complex comprises a first polypeptide sequence selected from SEQ ID NOs: 124-128 and / or 190-193 and a second polypeptide having the sequence of SEQ ID NO: 123. For example, the monovalent modified IL2-Fc fusion polypeptide complex can comprise SEQ ID NO: 124 and 123, SEQ ID NO: 125 and 123, SEQ ID NO: 126 and 123, SEQ ID NO: 127 and 123, SEQ ID NO: 128 and 123, SEQ ID NO: 190 and 123, SEQ ID NO: 191 and 123, SEQ ID NO: 192 and 123, and / or SEQ ID NO: 193 and 123.

[0096] In some embodiments, the fusion polypeptides disclosed herein include an antigen-binding portion. In some embodiments, the antigen-binding portion includes an immunoglobulin, a Fab molecule, a scFv, a bispecific T cell engager, a diabody, a single-domain antibody, or a VHH antibody (nanobody). The antigen-binding portion may bind, for example, PD-L1, PD-1, CTLA-4, TIM3, LAG3, B7-H2, B7-H3, carcinoembryonic antigen (CEA), GD2, CD20, CD4, CD8, or a cell marker. Examples of PD-L1 antigen-binding portions include atezolizumab, avelumab, and durvalumab. Examples of PD-1 antigen-binding portions include nivolumab and pembrolizumab. An example of a CEA antigen portion is CH1A1A-2F. An example of a GD-2 antigen-binding portion is dinutuximab, and an example of a CD20 antigen-binding portion is rituximab.

[0097] In some embodiments, a protein complex is disclosed herein that includes a first polypeptide that is a fusion polypeptide as disclosed herein and a second polypeptide or protein complex that includes an antigen-binding portion. In certain embodiments, the second polypeptide or protein complex includes a polypeptide having the sequences of SEQ ID NOs: 103 and 114, or SEQ ID NOs: 103 and 172. In some embodiments, the protein complex includes a polypeptide having the sequences of SEQ ID NOs: 103 and 114, or SEQ ID NOs: 103 and 172, and a polypeptide having a sequence selected from SEQ ID NOs: 124-128 and / or 190-193. In some embodiments, the protein complex includes a polypeptide having the sequences of SEQ ID NOs: 103, 114, and 124, SEQ ID NOs: 103, 114, and 125, SEQ ID NOs: 103, 114, and 126, SEQ ID NOs: 103, 114, and 127, SEQ ID NOs: 103, 114, and 128, SEQ ID NOs: 103, 172, and 190, SEQ ID NOs: 103, 172, and 191, SEQ ID NOs: 103, 172, and 192, or SEQ ID NOs: 103, 172, and 193.

[0098] In some embodiments, at least one additional molecule of the fusion polypeptide is a cytokine. In some embodiments, the cytokine is selected from interleukin 2, interleukin 15, interleukin 7, interleukin 10, and C-C motif chemokine ligand 19 (CCL19). In some embodiments, the additional molecule of the fusion polypeptide is a second modified IL2 polypeptide as described herein.

[0099] In some embodiments, at least one additional molecule of the fusion polypeptide is a half-life extending molecule. In some embodiments, the half-life extending molecule comprises a half-life extending polypeptide. In some embodiments, the half-life extending polypeptide comprises an Fc domain, human serum albumin (HSA), an HSA binding molecule or transferrin.

[0100] In some embodiments, the half-life extending molecule comprises polyethylene glycol (PEG) or polypropylene glycol (PPG).

[0101] Bifunctional protein In certain embodiments, bifunctional fusion proteins are disclosed herein. The bifunctional fusion protein can comprise a modified IL2 polypeptide disclosed herein and an antigen-binding portion. In some embodiments, the bifunctional fusion protein is a single protein sequence. In other embodiments, the bifunctional fusion protein is a heterodimer formed by at least two protein sequences. The bifunctional fusion protein can comprise an antigen-binding portion, which can include an immunoglobulin, Fab molecule, scFv, diabody, single domain antibody or VHH antibody. The antigen-binding portion can bind an antigen on the cell surface, such as, for example, a marker protein and / or a disease marker. The antigen-binding portion can bind, for example, PD-L1, PD-1, CTLA-4, TIM3, LAG3, B7-H2, B7-H3, CD4, CD8 or a cell marker. Examples of PD-L1 antigen-binding portions include atezolizumab, avelumab and durvalumab. Examples of PD-1 antigen-binding portions include nivolumab and pembrolizumab. In some embodiments, the bifunctional fusion protein can further comprise an IgG Fc domain. In some embodiments, the Fc domain is a human IgG heavy chain constant region. In some embodiments, the antibody heavy chain constant region is a human IgG1 heavy chain constant region. The IgG Fc domain can comprise a wild-type antibody constant region or a modified antibody constant region. Examples of modified antibody constant regions are provided herein and include, for example, knob mutations or hole mutations, and LALAPG mutations. In certain embodiments, the antibody heavy chain constant region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 137. In certain embodiments, the antibody heavy chain constant region comprises one or more mutations selected from L234A, L235A, P329G, Y349C, S354C, T366S, T366W, L368A, F405K, K409A and Y407V numbered according to the EU numbering system relative to SEQ ID NO: 137. In some embodiments, the antibody heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 123 or SEQ ID NO: 138.

[0102] The antigen-binding portion that binds to PD-L1 can be any of the anti-PD-L1 antigen-binding portions, antibodies, and / or scFv sequences disclosed herein. In some embodiments, the antigen-binding portion that binds to PD-L1 can include a Fab formed by the heavy and light chains of an antibody. In some embodiments, the antigen-binding portion that binds to PD-L1 can include an scFv.

[0103] In certain embodiments, the bifunctional fusion protein includes an antigen-binding portion that binds to PD-L1, and this antigen-binding portion includes (i) a heavy chain variable domain (VH) comprising a CDR1 comprising the sequence of SEQ ID NO: 141, a CDR2 comprising the sequence of SEQ ID NO: 142, and a CDR3 comprising the sequence of SEQ ID NO: 143, and (ii) a light chain variable domain (VL) comprising a CDR1 comprising the sequence of SEQ ID NO: 144, a CDR2 comprising the sequence of SEQ ID NO: 145, and a CDR3 comprising the sequence of SEQ ID NO: 146. In certain embodiments, the antigen-binding portion that binds to PD-L1 includes a light chain and a heavy chain, the light chain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 103, and the heavy chain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 104. In some embodiments, the bifunctional fusion protein has a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to one of SEQ ID NOs: 106-113 and / or SEQ ID NOs: 173-177. In some embodiments, the bifunctional fusion protein forms a heterodimer with a light chain comprising a polypeptide having the sequence of SEQ ID NO: 103.

[0104] In some embodiments, protein complexes (e.g., immunoconjugates) are disclosed herein that include a bifunctional fusion protein and a second antigen-binding portion disclosed herein. In some embodiments, the bifunctional fusion protein and the second antigen-binding portion have the same antigen-binding portion sequence. In some embodiments, the second antigen-binding portion binds to PD-L1. In some embodiments, the second antigen-binding portion includes (i) a heavy chain variable domain (VH) that includes a CDR1 comprising the sequence of SEQ ID NO: 141, a CDR2 comprising the sequence of SEQ ID NO: 142, and a CDR3 comprising the sequence of SEQ ID NO: 143, and (ii) a light chain variable domain (VL) that includes a CDR1 comprising the sequence of SEQ ID NO: 144, a CDR2 comprising the sequence of SEQ ID NO: 145, and a CDR3 comprising the sequence of SEQ ID NO: 146. In certain embodiments, the second antigen-binding portion that binds to PD-L1 includes a light chain and a heavy chain, where the light chain comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 103, and the heavy chain comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 104. In some embodiments, the protein complex includes a polypeptide having the sequence of SEQ ID NO: 104, 106 and 103, SEQ ID NO: 104, 107 and 103, SEQ ID NO: 104, 108 and 103, SEQ ID NO: 104, 109 and 103, SEQ ID NO: 104, 110 and 103, SEQ ID NO: 104, 111 and 103, SEQ ID NO: 104, 112 and 103, SEQ ID NO: 104, 113 and 103, SEQ ID NO: 172, 173 and 103, SEQ ID NO: 172, 174 and 103, SEQ ID NO: 172, 175 and 103, SEQ ID NO: 172, 176 and 103, or SEQ ID NO: 172, 177 and 103.

[0105] In some embodiments, the bifunctional fusion protein comprises an antigen-binding portion that binds to PD-1. Examples of PD-1 antigen-binding portions include nivolumab and pembrolizumab. In some embodiments, the bifunctional fusion protein comprises a sequence selected from the group consisting of comprising or consisting of SEQ ID NOs: 118-122 or 180-189. The bifunctional fusion protein may further comprise a light chain sequence comprising a polypeptide having the sequence of SEQ ID NO: 117 or SEQ ID NO: 178. The bifunctional fusion protein may be included in a protein complex (e.g., an immunoconjugate). In some embodiments, the protein complex may comprise a polypeptide having the sequence of SEQ ID NOs: 116, 117, and 118, SEQ ID NOs: 116, 117, and 119, SEQ ID NOs: 116, 117, and 120, SEQ ID NOs: 116, 117, and 121, or SEQ ID NOs: 116, 117, and 122. In some embodiments, the protein complex may comprise a polypeptide having the sequence of SEQ ID NOs: 178, 179, and 180, SEQ ID NOs: 178, 179, and 181, SEQ ID NOs: 178, 179, and 182, SEQ ID NOs: 178, 179, and 183, SEQ ID NOs: 178, 179, and 184, SEQ ID NOs: 178, 179, and 185, SEQ ID NOs: 178, 179, and 186, SEQ ID NOs: 178, 179, and 187, SEQ ID NOs: 178, 179, and 188, or SEQ ID NOs: 178, 179, and 189.

[0106] In some embodiments, a protein complex comprising a polypeptide having the sequence of SEQ ID NOs: 133, 134, and 135, or SEQ ID NOs: 133, 134, and 136 is disclosed herein.

[0107] Vectors, and methods for producing modified IL2 polypeptides In further embodiments, polynucleotides encoding the modified IL2, fusion polypeptides, protein complexes or bifunctional fusion proteins described above and herein are provided. In some embodiments, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NOs: 3-169, or a combination of polypeptides, or a nucleic acid sequence encoding a polypeptide selected from the group consisting of SEQ ID NOs: 3-211, or a combination of polypeptides, and comprises a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. In some embodiments, the polynucleotide(s) is selected from the group consisting of DNA, cDNA, RNA or mRNA. Further provided are expression cassette(s) comprising one or more regulatory sequences operably linked to the polynucleotide(s) described above and herein.

[0108] In some embodiments, there are provided polynucleotides, or vectors comprising an expression cassette, as described herein. In some embodiments, the vector is a plasmid vector or a viral vector. In some embodiments, the viral vector comprises a DNA virus or an RNA virus. In some embodiments, the viral vector is derived from a viral family selected from the group consisting of Adenoviridae (e.g., adenovirus), Arenaviridae (e.g., lymphocytic choriomeningitis mammarenavirus, Calomys callosus mammarenavirus (synonym, Pichinde mammarenavirus)), Poxviridae (e.g., vaccinia virus), Herpesviridae (e.g., herpesvirus, e.g., HSV-1), Parvoviridae (e.g., parvovirus H1), Reoviridae (e.g., reovirus), Retroviridae (e.g., lentivirus), Picornaviridae (e.g., coxsackievirus, Seneca Valley virus, poliovirus), Paramyxoviridae (e.g., measles virus, Newcastle disease virus (NDV)), Rhabdoviridae (e.g., vesicular stomatitis virus (VSV)), Togaviridae (e.g., alphavirus, Sindbis virus), Enteroviridae (e.g., echovirus). Further provided are lipid nanoparticles (LNPs) comprising the polynucleotides, expression cassette or vector described above and herein.

[0109] Modified IL2 polypeptides and / or modified IL2 fusion polypeptides can be prepared by genetic or chemical methods well known in the art and by the methods disclosed in the following examples. Genetic methods may include, for example, site-directed mutagenesis of the DNA sequence encoding the polypeptide, PCR, and gene synthesis. The intended nucleotide changes can be confirmed by sequencing. The nucleotide sequence of native IL2 has been described by Taniguchi et al. (Nature 302, 305-10 (1983)), and nucleic acids encoding native human IL2 are available, for example, from the American Type Culture Collection (Rockville, Maryland).

[0110] A modified IL2 polypeptide or a modified IL2 fusion polypeptide can be obtained, for example, by recombinant production or peptide solid-phase synthesis. For recombinant production, a polynucleotide encoding the modified IL2 polypeptide or the modified IL2 fusion polypeptide can be isolated and inserted into one or more vectors for cloning and / or expression in a host cell. Such polynucleotides can be readily isolated and sequenced by conventional procedures. In certain embodiments, vectors such as expression vectors are provided that contain one or more of the polynucleotides of the present disclosure. Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence modified IL2 polypeptide or modified IL2 fusion polypeptide, along with appropriate transcription / translation control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination. See, for example, the techniques described in Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL (FOURTH EDITION), Cold Spring Harbor Laboratory, N.Y. (2012), and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, N.Y (1993). The expression vector can be part of a plasmid, a virus, or can be a nucleic acid fragment. The expression vector contains an expression cassette, and the polynucleotide encoding the modified IL2 polypeptide or modified IL2 fusion polypeptide (i.e., the coding region) is operably associated with a promoter and / or other transcription or translation control elements and is cloned into this expression cassette. As used herein, a "coding region" is a portion of a nucleic acid consisting of codons that are translated into amino acids.A "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid and, if present, may be considered part of the coding region, but any adjacent sequences, such as a promoter, ribosome binding site, transcription terminator, intron, 5' and 3' untranslated regions, etc., are not part of the coding region. Two or more coding regions may be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. Further, any vector may contain a single coding region or two or more coding regions, e.g., the vectors disclosed herein may encode one or more polypeptides, which are cleaved proteolytically to yield the final protein after or simultaneously with translation. Also, the vectors, polynucleotides, or nucleic acids of the present disclosure may encode a heterologous coding region that is fused or not fused to a first or second polynucleotide encoding a polypeptide or a variant or derivative thereof disclosed herein. The heterologous coding region includes, but is not limited to, specialized elements or motifs such as a secretion signal peptide or a heterologous functional domain. An operable association is where the coding region of a gene product, e.g., a polypeptide, is associated with one or more control sequences such that the expression of the gene product is placed under the influence or control of the control sequence(s). Two DNA fragments, e.g., a polypeptide coding region and an associated promoter), are "operably associated" when induction of promoter function results in transcription of mRNA encoding the desired gene product and the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression control sequences to induce expression of the gene product and does not interfere with the ability of the DNA template to be transcribed. Thus, a promoter region is operably associated with a nucleic acid encoding a polypeptide when the promoter is capable of affecting the transcription of that nucleic acid. The promoter may be a cell-specific promoter that induces substantial transcription of DNA only in a given cell.In addition to promoters, other transcriptional control elements, such as enhancers, operators, repressors, and transcription termination signals, can be operably associated with a polynucleotide to induce cell-specific transcription. Suitable promoters and other transcriptional control regions are disclosed herein. These include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as promoters and enhancer segments derived from cytomegalovirus (e.g., the immediate early promoter combined with intron A), simian virus 40 (e.g., the early promoter), and retroviruses (e.g., Rous sarcoma virus). Other transcriptional control regions include transcriptional control regions derived from vertebrate genes (such as actin, heat shock protein, bovine growth hormone, and rabbit β-globin), as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcriptional control regions include tissue-specific promoters and enhancers, as well as inducible promoters (e.g., promoters that are inducible by tetracycline). Similarly, various translational control elements are known to those of skill in the art. These include, but are not limited to, ribosome binding sites, translation initiation codons, and translation termination codons, as well as elements derived from viral systems (in particular, internal ribosome entry sites or IRES, also referred to as CITE sequences). Expression cassettes may also include other features such as origins of replication, and / or chromosomal integration elements such as the long terminal repeats (LTRs) of retroviruses, or the inverted terminal repeats (ITRs) of adeno-associated virus (AAV).

[0111] The polynucleotides and nucleic acid coding regions of the present disclosure may be associated with additional coding regions that encode a secretory peptide or signal peptide that directs the secretion of the polypeptide encoded by the polynucleotides of the present disclosure. For example, if secretion of a modified IL2 polypeptide or a modified IL2 fusion polypeptide is desired, DNA encoding a signal sequence may be placed upstream of the nucleic acid encoding the mature amino acids of the modified IL2 polypeptide or the modified IL2 fusion polypeptide. Those skilled in the art will recognize that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce the secreted or "mature" form of the polypeptide. For example, native human IL2 is translated with a 20 amino acid signal sequence at the N-terminus of the polypeptide, which is then cleaved to produce mature 133 amino acid human IL2. In some embodiments, a native signal peptide, such as the IL2 signal peptide or an immunoglobulin heavy or light chain signal peptide, or a functional derivative of that sequence that retains the ability to direct the secretion of a polypeptide operably associated therewith, is used. In some embodiments, the signal peptide comprises or consists of SEQ ID NO: 212.

[0112] In some embodiments, the polynucleotide encoding the modified IL2 polypeptide or the modified IL2 fusion polypeptide further comprises a DNA sequence encoding a sequence (e.g., a histidine tag) for facilitating purification or a sequence for labeling the modified IL2 polypeptide or the modified IL2 fusion polypeptide, within or at the end of the polynucleotide encoding the modified IL2 polypeptide or the modified IL2 fusion polypeptide.

[0113] In certain embodiments, a host cell is provided that contains one or more polynucleotides encoding a modified IL2 polypeptide or a modified IL2 fusion polypeptide. In certain embodiments, the host cell contains one or more vectors encoding a modified IL2 polypeptide or a modified IL2 fusion polypeptide. The host cell can be any type of cell line that can be used to produce a modified IL2 polypeptide or a modified IL2 fusion polypeptide. Such cells may be transfected or transduced as needed with a specific expression vector encoding a modified IL2 polypeptide or a modified IL2 fusion polypeptide, and a large number of vector-containing cells may be grown for seeding in a large-scale fermenter to obtain encoding of an amount of modified IL2 polypeptide or modified IL2 fusion polypeptide sufficient for clinical applications. Suitable host cells include prokaryotic microorganisms (such as E. coli) or various eukaryotic cells (such as Chinese hamster ovary cells (CHO), insect cells, etc.). For example, the polypeptide may be produced in bacteria, especially if glycosylation is not required. After expression, the polypeptide may be isolated from bacterial cells in the soluble fraction and further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts, including fungal and yeast strains, are suitable cloning or expression hosts for vectors encoding polypeptides, and their glycosylation pathways are "humanized," resulting in the production of polypeptides with a partial or complete human glycosylation pattern. Host cells suitable for the expression of (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. In particular, a number of baculovirus strains have been identified that can be used in combination with insect cells for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be utilized as hosts.For example, see U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants). Vertebrate cells may also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include the simian kidney CV1 line transformed by SV40 (COS-7); human fetal kidney lines (e.g., 293 or 293T cells described in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol Reprod 23, 243-251 (1980)), simian kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT060562), TRI cells (e.g., described in Mather et al., Annals N.Y.Acad Sci 383, 44-68 (1982)), MRC5 cells, and FS4 cells. Other useful mammalian host cell lines include dhfr. -Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003). Host cells include, but are not limited to, cultured cells such as mammalian cultured cells, yeast cells, insect cells, bacterial cells, and plant cells, as well as cells contained within transgenic animals, transgenic plants or cultured plants or animal tissues. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell such as Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, or lymphoid cells (e.g., Y0, NS0, Sp20 cells).

[0114] Standard techniques for expressing foreign genes in these systems are known in the art. Cells expressing a modified IL2 polypeptide fused to either the heavy or light chain of an antigen-binding portion such as an antibody can be modified to also express the other antibody chain so that the expressed modified IL2 fusion polypeptide comprises an antibody having both heavy and light chains.

[0115] In some embodiments, methods are provided for producing a modified IL2 polypeptide, a modified IL2 fusion polypeptide, or a protein complex described herein. In some embodiments, the method comprises culturing a host cell comprising a polynucleotide encoding a modified IL2 polypeptide, a modified IL2 fusion polypeptide, or a protein complex under conditions suitable for the expression of the modified IL2 polypeptide, the modified IL2 fusion polypeptide, or the protein complex, and optionally recovering and / or purifying the modified IL2 polypeptide, the modified IL2 fusion polypeptide, or the protein complex from the host cell (or, for example, the host cell culture medium if the host cell secretes the polypeptide).

[0116] In some embodiments, transgenic feeder cells comprising a modified IL2 polypeptide, a fusion polypeptide, a protein complex, a bifunctional fusion protein, a polynucleotide, or a vector disclosed herein are disclosed herein. Examples of feeder cells include K562 cells, 3T3 cells, fibroblasts (e.g., MEF) or antigen presenting cells.

[0117] In some embodiments, methods for in vitro or ex vivo expansion of immune cells are disclosed herein. The method comprises contacting a population of immune cells with an effective amount of a modified IL2 polypeptide, fusion polypeptide, protein complex, or bifunctional fusion protein disclosed herein under conditions sufficient to promote expansion of the population of immune cells, and culturing the population of immune cells for a time sufficient to at least double the number of immune cells. For example, the conditions sufficient to promote expansion may include one or more agents or ligands capable of activating the intracellular signaling domain of the TCR complex, examples of which include anti-CD3 antibodies or binding domains, anti-CD28 antibodies or binding domains, or combinations thereof. In some embodiments, the method for expanding immune cells further comprises culturing the population of immune cells with IL-7, IL-15, IL-21, or any combination thereof. In some embodiments, the population of immune cells is cultured in the presence of a population of feeder cells, such as the transgenic feeder cells disclosed herein.

[0118] Pharmaceutical composition Provided herein are pharmaceutical compositions comprising a modified IL2 polypeptide, a modified IL2 fusion polypeptide, a bifunctional fusion protein, a protein complex, a polynucleotide, a vector or a cell, and a pharmaceutically acceptable diluent(s), excipient(s) or carrier(s). In some embodiments, the pharmaceutical composition comprises a modified IL2 polypeptide, a modified IL2 fusion polypeptide, a bifunctional fusion protein, a protein complex, a polynucleotide, a vector or a cell, and an additional therapeutic agent (e.g., combination therapy). Non-limiting examples of such therapeutic agents are described hereinbelow. The modified IL2 polypeptide, the modified IL2 fusion polypeptide, the bifunctional fusion protein, the protein complex, the polynucleotide, the vector or the cell can be formulated in a conventional manner into a pharmaceutical composition using one or more pharmaceutically acceptable carriers including excipients and auxiliaries that facilitate processing of the modified IL2 polypeptide, the modified IL2 fusion polypeptide, the bifunctional fusion protein, the protein complex, the polynucleotide, the vector or the cell into preparations that can be used pharmaceutically. Suitable formulations depend on the chosen route of administration. The pharmaceutical compositions described herein can be formulated using any pharmaceutically acceptable technique, carrier and excipient as appropriate. Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999). Examples of IL-2 compositions are described in U.S. Patent Nos. 4,604,377 and 4,766,106, which are incorporated herein by reference.

[0119] As used herein, "pharmaceutically acceptable carrier" and "physiologically acceptable carrier" are used interchangeably and include any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweetening agents, flavoring agents, dyes, equivalent materials known to those of ordinary skill in the art, and combinations thereof, which are molecular entities and compositions. These molecular entities and compositions are generally non-toxic to the recipient at the dosages and concentrations employed, i.e., they do not cause adverse reactions, allergic reactions, or other undesirable reactions when administered to animals such as, for example, humans, as necessary (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, which is incorporated herein by reference). Therapeutic or pharmaceutical compositions are contemplated for use, except where any conventional carrier is incompatible with the active ingredient.

[0120] The pharmaceutical composition may contain different types of carriers depending on whether it is administered in solid, liquid, or aerosol form and whether it needs to be sterile for the route of administration such as injection. The modified IL2 polypeptide or modified IL2 fusion polypeptide (and any additional therapeutic agent) described herein can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrarenally, intrathoracically, intratracheally, intranasally, intravitreally, intravaginally, rectally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravascularly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation (e.g., aerosol inhalation), by injection, infusion, continuous infusion, local perfusion directly immersing target cells, via a catheter, via lavage, in a cream, in a lipid composition (e.g., liposome), or by any of the above-mentioned combinations or other methods known to those skilled in the art (see, e.g., Remington’s Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference). Parenteral administration, particularly intravenous injection, is most commonly used for administering polypeptide molecules, examples of which include the modified IL2 polypeptide or modified IL2 fusion polypeptide described herein.

[0121] Transgenic immune cells are provided herein. These transgenic immune cells express a modified IL2 polypeptide, a fusion polypeptide, a protein complex, a bifunctional fusion protein, a polynucleotide, or a vector disclosed herein. In some embodiments, the transgenic immune cells are CD4+ T cells, CD8+ T cells, γδ T cells, NK cells, regulatory T cells, or any combination thereof. In some embodiments, the transgenic immune cells further comprise a chimeric antigen receptor (CAR).

[0122] In some embodiments, the CAR comprises an antigen-binding domain capable of binding to a target / antigen produced by cancer cells. For example, the CAR comprises an antigen-binding domain capable of binding an antigen, examples of which include: CD19; CD123; CD22; CD30; CD171; CS-1 (also called CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGaip(1-4)bDGicp(1-1)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin 11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21 (testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine-protein kinase ERBB2 (Her2 / neu); mucin 1, cell surface-associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostase; prostate acid phosphatase (PAP); elongation factor 2 mutant (ELF2M); Ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (prosome, macropin) subunit, beta type, 9 (LMP2);Glycoprotein 100 (gp100); cancer gene fusion protein (bcr-abl) composed of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl); tyrosine kinase; Ephrin type-A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGicp(1-1)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); O-acetyl-GD2 ganglioside (OAcGD2); folate receptor beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRCSD); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide moiety of globoH glycosphingolipid (GloboH); breast differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor beta-3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternative reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1); ETS translocation variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member lA (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostain; survivin; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MART1);Rat sarcoma (Ras) mutants; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma apoptosis inhibitor (ML-IAP); ERG (membrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS, i.e., brother of regulator of imprinted sites), squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutant (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF) C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2) lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRLS); and immunoglobulin lambda-like polypeptide 1 (IGLL1).;

[0123] In some embodiments, the transgenic immune cells produce a modified IL2 polypeptide, a fusion polypeptide, a protein complex, or a bifunctional fusion protein, which are secreted by the transgenic immune cells. In other embodiments, the modified IL2 polypeptide, fusion polypeptide, protein complex, or bifunctional fusion protein comprises a transmembrane domain, or a cell surface anchor molecule is expressed on the surface of the cell and / or is localized on the cell surface. In some embodiments, the transgenic immune cells are engineered CAR T cells.

[0124] Methods of treatment and use In some embodiments of the present disclosure, methods are provided herein for modulating an immune response in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of a modified IL2 polypeptide, a modified IL2 fusion polypeptide, a bifunctional fusion protein, a protein complex, a polynucleotide, a vector, a cell, or a pharmaceutical composition thereof (see, e.g., SEQ ID NOs: 3-102, 147-169, 203-211, and Tables 2, 5, and 7). In certain embodiments, modulating the immune response comprises at least one of enhancing effector T cell activity, enhancing NK cell activity, and suppressing regulatory T cell activity. In some embodiments of the present disclosure, a modified IL2 polypeptide as described herein, a fusion polypeptide as described herein, a protein complex as described, a bifunctional fusion protein, a polynucleotide, a vector, a cell, and / or a pharmaceutical composition as described herein are provided herein for use in a method of modulating an immune response in a subject in need thereof.

[0125] In some embodiments of the present disclosure, provided herein are methods of treating a disease or condition in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of a modified IL2 polypeptide, a modified IL2 fusion polypeptide, a protein complex, a bifunctional fusion protein, a polynucleotide, a vector, a cell, or a pharmaceutical composition thereof, as described herein. In some embodiments, a modified IL2 polypeptide, a fusion polypeptide, a protein complex, a bifunctional fusion protein, a polynucleotide, a vector, a cell, and / or a pharmaceutical composition, as described herein, for use in a method of treating a disease of a subject. Non-limiting examples of diseases or conditions contemplated by this method include proliferative disorders such as cancer and immunosuppression.

[0126] In some embodiments, methods of treating a proliferative disorder are provided herein. The method includes administering to a subject a therapeutically effective amount of a modified IL2 polypeptide, a modified IL2 fusion polypeptide, a protein complex, or a pharmaceutical composition thereof, as described herein. In some embodiments, the proliferative disorder is cancer. In some embodiments, the cancer includes breast cancer, pancreatic cancer, lung cancer, glioblastoma, renal cell carcinoma, lymphoma, leukemia, head and neck cancer, liver cancer, gastric cancer, colorectal cancer, kidney cancer, bladder cancer, or melanoma. Non-limiting examples of cancers include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colorectal cancer, rectal cancer, gastric cancer, glioblastoma, prostate cancer, hematologic malignancies, skin cancer, squamous cell carcinoma, skin cancer, melanoma, bone cancer, renal cell carcinoma, and kidney cancer. Examples of hematologic malignancies include leukemia, lymphoma (e.g., non-Hodgkin lymphoma or Hodgkin lymphoma), multiple myeloma, myelodysplastic syndromes, myeloproliferative neoplasms (e.g., essential thrombocythemia, myelofibrosis, polycythemia vera). Also included are pre-cancerous conditions or lesions and cancer metastases. Other cell proliferation disorders include, but are not limited to, neoplasms located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testis, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thoracic region, and genitourinary system. Similarly, other cell proliferation disorders can be treated, examples of which include hypergammaglobulinemia, lymphoproliferative disorders, dysproteinemia, purpura, sarcoidosis, Sézary syndrome, Waldenström macroglobulinemia, Gaucher disease, histiocytosis, amyloidosis, and aplastic anemia, as well as any other cell proliferative diseases other than neoplasms located in the organ systems listed above.

[0127] In some embodiments, the method of treating or modulating an immune response further comprises administering to a subject a therapeutically effective amount of at least one additional therapeutic agent (e.g., combination therapy). In certain embodiments, the additional therapeutic agent is an anti-cancer agent. Examples of anti-cancer agents include checkpoint inhibitors (e.g., anti-PD1 antibodies), chemotherapeutic agents, agents that inhibit the tumor microenvironment, cancer vaccines (e.g., sipuleucel-T), oncolytic viruses (e.g., talimogene laherparepvec), immune cells expressing chimeric antigen receptors (CARs), and tumor infiltrating lymphocytes (TILs). In certain embodiments, the additional therapeutic agent is a molecule comprising an antigen-binding portion. In certain specific embodiments, the antigen-binding portion is selected from single domain antibodies, Fab molecules, scFvs, diabodies, nanobodies, bispecific T cell engagers, or immunoglobulins. In certain embodiments, the antigen-binding portion is specific for a tumor antigen (e.g., carcinoembryonic antigen, fibroblast activation protein-α, CD20) or a checkpoint protein (e.g., CTLA-4, PD-1, or PD-L1). In some embodiments, the additional therapeutic agent is a vaccine, gene therapy, cell therapy, or any combination thereof. In some embodiments, the additional therapeutic agent comprises immune cells expressing a chimeric antigen receptor, immune cells expressing a modified T cell receptor, or tumor infiltrating lymphocytes. In certain embodiments, a modified IL2 polypeptide, modified IL2 fusion polypeptide or protein complex may be encoded by a polynucleotide, which is introduced into immune cells expressing a chimeric antigen receptor, immune cells expressing a modified T cell receptor, or tumor infiltrating lymphocytes by transfection, transduction, or other methods. In such embodiments, the immune cells may be enhanced chimeric antigen receptor-expressing cells. In certain embodiments, the immune cells are T cells (e.g., CD4 and / or CD8 T cells) and / or NK cells.The polynucleotide may further encode a secretion signal (e.g., the native IL2 signal sequence, or a signal sequence derived from another protein) immediately upstream of the modified IL2 polypeptide coding sequence, enabling the cell to secrete the modified IL2 polypeptide or the modified IL2 fusion polypeptide.

[0128] Suitable routes of administration include, but are not limited to, intravenous, parenteral, transdermal, oral, rectal, aerosol, intraocular, pulmonary, transmucosal, intravaginal, otic, nasal, and topical administration. Additionally, by way of mere example, parenteral delivery includes intramuscular injection, subcutaneous injection, intravenous injection, intramedullary injection, as well as subdural injection, direct intraventricular injection, intraperitoneal injection, intralymphatic injection, and intranasal injection.

[0129] In certain embodiments, the modified IL2 polypeptide, IL2 fusion polypeptide or protein complex is administered systemically. In certain embodiments, the modified IL2 polypeptide, modified IL2 fusion polypeptide or protein complex as described herein is administered in a non-systemic, local manner, for example, by directly injecting the modified IL2 polypeptide, modified IL2 fusion polypeptide or protein complex into an organ, tissue or tumor. In some embodiments, the long-acting formulation is administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Further, in some embodiments, the drug is a targeted drug delivery system, for example, in liposomes coated with an organ-specific or cell-specific antibody. In such embodiments, the liposomes are targeted and selectively taken up by the organ. In some embodiments, the modified IL2 polypeptide, modified IL2 fusion polypeptide or protein complex as described herein is provided in the form of an immediate-release formulation, a sustained-release formulation or a controlled-release formulation, an intermediate-release formulation, or a depot preparation. In some embodiments, the modified IL2 polypeptide, modified IL2 fusion polypeptide or protein complex described herein is administered locally.

[0130] The appropriate dosage of a modified IL2 polypeptide, a modified IL2 fusion polypeptide, or a protein complex (used alone or in combination with one or more additional therapeutic agents) will depend on the type of disease or condition, the route of administration, the weight of the subject, the severity and progression of the disease, whether the polypeptide is administered for prophylactic or therapeutic purposes, previous or concurrent therapeutic interventions, the clinical history of the subject and the response to the modified IL2 polypeptide, modified IL2 fusion polypeptide, or protein complex, and the discretion of the attending physician. The physician involved in the administration will be able to determine the concentration of the active ingredient(s) in the composition and the appropriate dosage for the subject to be treated. Various dosage schedules are contemplated herein, including, but not limited to, single or multiple administrations at various time points, bolus administration, and pulse infusion.

[0131] A single administration of a modified IL2 polypeptide may be in the range of about 50,000 IU / kg to about 1,000,000 IU / kg or more of the modified IL2 polypeptide. This may be repeated several times a day (e.g., 2 to 4 times a day) for several days (e.g., 3 to 5 consecutive days), and then repeated one or more times after a rest period (e.g., 7 to 14 days). Thus, a therapeutically effective amount may include administration only, a single administration, or multiple administrations over a period of time (e.g., IL2 at about 600,000 IU / kg divided into about 10 to 30 individual administrations and each administered over a period of about 5 to 20 days). When administered in the form of a fusion polypeptide or protein complex, the therapeutic efficacy of the modified IL2 fusion polypeptide or protein complex may be lower than that of the non-fused modified IL2 polypeptide (e.g., 10,000 IU / kg to about 600,000 IU / kg). Similarly, the modified IL2 fusion polypeptide may be administered to a patient once or over the course of the series of treatments described above.

[0132] In certain embodiments, the daily dose of the modified IL2 polypeptide, modified IL2 fusion polypeptide, or protein complex ranges from about 1 μg / kg to about 100 mg / kg or more. For repeated administration over several days or more, depending on the condition, the treatment may be continued until the desired suppression of the symptoms of the disease (e.g., tumor shrinkage) occurs. In some embodiments, the single dose of the modified IL2 polypeptide, modified IL2 fusion polypeptide, or protein complex ranges from about 0.005 mg / kg to about 10 mg / kg. In some embodiments, the dose may be, per administration, about 1 μg / kg / body weight, about 5 μg / kg / body weight, about 10 μg / kg / body weight, about 50 μg / kg / body weight, about 100 μg / kg / body weight, about 200 μg / kg / body weight, about 350 μg / kg / body weight, about 500 μg / kg / body weight, about 1 mg / kg / body weight, about 5 mg / kg / body weight, about 10 mg / kg / body weight, about 50 mg / kg / body weight, about 100 mg / kg / body weight, about 200 mg / kg / body weight, about 350 mg / kg / body weight, about 500 mg / kg / body weight to about 1000 mg / kg / body weight, and any range derivable therein. Non-limiting examples of ranges derivable from the numerical values described herein include about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 micrograms / kg / body weight to about 500 milligrams / kg / body weight, etc., which can be administered based on the above numerical values. Such doses may be administered intermittently, for example, 2 to 3 times a day, weekly, or every three weeks. An initial higher loading dose followed by one or more lower doses may be administered. However, other dosing regimens may be useful.

[0133] The modified IL2 polypeptides and modified IL2 fusion polypeptides described herein may be used in an amount effective to achieve the intended purpose. For use in treating or preventing a medical condition, the modified IL2 polypeptide or modified IL2 fusion polypeptide, or a pharmaceutical composition thereof, is administered in a therapeutically effective amount. Determination of a therapeutically effective amount is within the capabilities of those skilled in the art, particularly in light of the details provided herein.

[0134] In systemic administration, a therapeutically effective amount can first be estimated from in vitro assays such as cell culture assays. Next, the dose can be formulated in an animal model to achieve a circulating concentration range that includes IC 50 (determined in cell culture). Such information can be used to more accurately determine a useful dose in humans. The initial dosage can also be estimated from in vivo data, such as animal models, using methods well known in the art. Administration to humans can be readily optimized by one of ordinary skill in the art based on animal data. The dosage and interval may each be adjusted to obtain plasma levels of the modified IL2 polypeptide and modified IL2 fusion polypeptide sufficient to maintain the therapeutic effect. Plasma levels may be measured, for example, by HPLC.

Example

[0135] In the examples provided below, IL2 labeled as "WT-IL2" refers to IL2 having the sequence of SEQ ID NO: 171, which includes the binding domains of wild-type IL2Rα and IL2Rβ and the T3A and C125S substitutions.

[0136] Example 1 Screening and Identification of IL2Rβ Binding-Decreasing Agonists Using in vitro mRNA display technology, IL2Rβ-binding decreased agonists were developed from three rationally designed IL2 mutant libraries. Briefly, the IL2 mutant DNA library was transcribed into an mRNA library, which was then translated into an mRNA-IL2 mutant fusion library by covalent attachment to a puromycin linker. The library was then purified and annealed to the corresponding single-stranded cDNA to stabilize the cytokine fusion mRNA library in the form of an mRNA / cDNA hybrid as reported in expired U.S. Patent No. 6,258,558. First, the fusion library was counterselected with human IgG (negative protein) over multiple rounds to remove non-specific binders, and then counterselected against recombinant human IL2Rβ-Fc protein captured on Protein G magnetic beads. Selection by gradient concentration was performed for a total of 2 - 4 rounds. In each round of selection, IL2Rβ-decreased binders were recovered and enriched by PCR amplification.

[0137] After 2 - 4 rounds of selection, the enriched IL2R-binding decreased library was cloned into the bacterial periplasmic expression vector pET22b and transformed into TOP10 competent E. coli cells. Each modified IL2 molecule was engineered to have a C-terminal flag and 6×HIS tags for purification and assay detection. Clones from TOP10 cells were pooled, mini-prep DNA was prepared, and subsequently transformed into E. coli Rosetta II strain for expression. Single clones were picked, grown, and induced with 0.25 mM IPTG in 96-well plates for expression. Supernatants were collected after 16 - 24 hours of induction at 30 °C for assays to identify binders.

[0138] The supernatant containing the modified IL2 mutant was evaluated by sandwich ELISA assay to screen for IL2Rα binding. Briefly, 96-well plates were immobilized with human Fc and human IL2Rα at a final concentration of 2 μg / mL in 1× PBS with a total volume of 50 μL per well. The plates were incubated overnight at 4°C and subsequently blocked with 200 μL of SuperBlock per well for 1 hour. 100 μL of the supernatant was added to both the Fc- and IL2Rα-immobilized wells and incubated for 1 hour with shaking. Binding of the modified IL2 mutant was detected by adding 50 μL of anti-Flag HRP diluted 1:5000 in 1× PBST. Between each step, the plates were washed three times with 1× PBST in a plate washer. The plates were then developed with 50 μL of TMB substrate for 5 minutes and stopped by adding 50 μL of 2N sulfuric acid. The plates were read at OD450nm using a Biotek plate reader to analyze binding and selectivity.

[0139] Next, single clones were screened for IL2Rβ binding. To identify individual modified IL2 mutants, an IL2Rβ binding screening ELISA was developed. Briefly, 96-well plates were immobilized with human Fc and human IL2Rβ at a final concentration of 2 μg / mL in 1× PBS with a total volume of 50 μL per well. The plates were incubated overnight at 4°C and subsequently blocked with 200 μL of SuperBlock per well for 1 hour. 100 μL of the supernatant was added to both the Fc- and IL2Rβ-immobilized wells and incubated for 1 hour with shaking. Binding of the modified IL2 mutant was detected by adding 50 μL of anti-Flag HRP diluted 1:5000 in 1× PBST. Between each step, the plates were washed three times with 1× PBST in a plate washer. The plates were then developed with 50 μL of TMB substrate for 5 minutes and stopped by adding 50 μL of 2N sulfuric acid. The plates were read at OD450nm using a Biotek plate reader to analyze binding and selectivity.

[0140] Clones that showed positive IL2Rα binding but reduced or very low IL2Rβ binding were identified from library 5 and further characterized. Multiple sequence alignment of the IL2Rβ-binding loop 2 (IL2Rβ-BL2) revealed both highly conserved and highly variable amino acids compared to IL2 WT, and also the clone sequences that were independently identified multiple times. No specific IL2Rβ-binding clones were identified from other libraries.

[0141] Furthermore, modified IL2 clones with unique sequences were produced in E. coli for further triage. Specifically, glycerol stocks of each modified agonist clone were inoculated into TB medium and grown overnight. The next day, the overnight cultured cells were inoculated into TB medium and grown until the cell density at OD 600 reached 0.6 - 0.8. IPTG was added to a final concentration of 1 mM to induce expression during overnight culture at 30 °C. The supernatant was collected by centrifugation. The agonist was purified by Ni-Sepharose (GE Healthcare) affinity column according to the manufacturer's protocol. The proteins were stored in 1x PBS buffer for binding and functional analysis, respectively. Since most clones in E. coli had low expression, the activity was evaluated by binding ELISA of IL2Rα and IL2Rβ as described above. Table 1 below lists the clones showing the corresponding OD450 values of receptor binding of IL2Rα and IL2Rβ measured by anti-Flag ELISA. Table 2 lists the sequences of modified IL2 molecules with mutations in the region of amino acids 81 - 96 (numbered based on the mature WT-IL2 sequence).

[0142]

Table 1

Table 2-1

Table 2-2

[0143] Example 2 Binding Activity of IL2Rβ Binding-Decreasing Agonists Using Surface Plasmon Resonance Method The binding reaction rates of WT IL2 produced by E. coli and modified clones were screened by surface plasmon resonance technology using a Biacore T200. The assay was performed using Biacore T200 control software version 2.0. In each cycle, 1 μg / mL of human IL2Rβ or IL2Rα was captured for 60 seconds at a flow rate of 10 μL / min in flow cell 2 in 1X HBSP buffer on a Protein A sensor chip. HIS-tag purified modified IL2 mutants at 100 nM and 20 nM were injected into both reference flow cell 1 and IL2Rβ or IL2Rα capture flow cell 2 at a flow rate of 30 μL / min for 150 seconds, followed by washing for 300 seconds. The flow cell was then regenerated with glycine at pH 2 at a flow rate of 30 μL / min for 60 seconds. Reaction rate data were analyzed using Biacore T200 evaluation software 3000. Specific binding response units were derived from the subtraction of binding to reference flow cell 1 from binding to target flow cell 2.

[0144] The binding protocol was verified using WT IL2 and the IL2Rβ-potentiating agonist EP596 (SEQ ID NO: 170) and included as a control for each run. The binding activities for IL2 produced by E. coli and IL2 produced by mammals are summarized in Table 3.

[0145] [Table 3]

[0146] Example 3 Characterization of IL2Rβ Binding-Decreasing Agonists Produced by Mammalian Cells For the production of IL2Rβ-binding decreasing agonists in mammalian cells, the DNA sequences corresponding to the amino acid sequences were codon-optimized, synthesized, and subcloned into pCDNA3.4 (Invitrogen). To decrease the IL2Rα-binding activity, point mutations of F35L, R38D, and F42R were introduced into each of the modified IL2 sequences. According to the standard protocol, each modified IL2 polypeptide was transiently expressed in ExpiHEK293-F cells in a FreeStyle system (Invitrogen). The cells were grown for 7 days under the above conditions before harvesting. The supernatant was collected by centrifugation and filtered through a 0.2 μm PES membrane. First, the agonist was purified on a Ni Sepharose Excel resin column (GE Healthcare) and buffer-exchanged into PBS at pH 7.4 containing 300 mM NaCl with a 7 kDa Zeba column. Then, each polypeptide was concentrated to 1 mL and purified by a Superdex 200 Increase 10 / 300 GL column (GE Healthcare) until homogeneous. The monomer peak fractions were pooled and concentrated. The protein was stored in 1x PBS / 300 mM NaCl buffer for analysis of binding, function, and mechanism. An example of the results of SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of individual IL2s after Ni column purification is shown in Figure 1.

[0147] The agonists produced in mammalian cells were characterized by ELISA for their binding activities to the receptors of each of IL2Rβ and IL2Rα. Briefly, 384-well plates were immobilized with the Fc fusion proteins of human IL2Rα and IL2Rβ at a final concentration of 2 μg / mL in 1× PBS with a total volume of 25 μL per well. The plates were incubated overnight at 4°C and blocked for 1 hour with 80 μL of SuperBlock per well. The purified clones were added in duplicate in parallel as a titration at a maximum concentration of 1 μM to the IL2Rα or IL-2Rβ wells, followed by 10-fold serial dilution of the clones in 5 points. The binding activity was detected by adding 25 μL of anti-His HRP diluted 1:5000 in 1× PBST. Between each step, the plates were washed 3 times with 1× PBST using a plate washer. The plates were then developed with 25 μL of TMB substrate for 5 minutes and stopped by adding 25 μL of 2N sulfuric acid. The plates were read with a Biotek plate reader at OD450nm, and a bar graph at 1 μM of IL2 was generated with Prism8.1 software (Figure 2).

[0148] To further evaluate the immunomodulatory activity of the purified IL2 clones, human PBMCs were isolated from the peripheral blood of three separate donors, stained with a dead cell labeling reagent, and plated at 250,000 cells / well in a 96-well plate in 90 μL of medium. The cells were allowed to sit at 37 °C for 1 hour. The cells were stimulated with 10-fold concentrated human IL2 WT, IL2Rβ binding-enhanced EP596, and modified His-Flag-tagged IL2 in 10 μL for 20 minutes at 37 °C. The stimulated PBMCs were immediately fixed, permeabilized, stained for cell lineage markers (CD3, CD56, CD4, CD8, FOXP3) and p-STAT5, and visualized on an Attune flow cytometer. CD8+ T cells were defined as CD3+CD56-CD4-CD8+. NK cells were defined as CD3-CD56+. Regulatory T cells were defined as CD3+CD56-CD4+CD8-FOXP3+. The percentage of cells that were p-STAT5+ was determined and graphed for each IL2 titration (Figure 3). P-STAT5 activation was determined using Prism software and summarized in Table 4.

[0149]

Table 4-1

Table 4-2

Table 4-3

[0150] Example 4 Rational Generation of IL2Rβ EP575 Agonist-Returning Mutant Clones A strategy of reverse mutation of rationally designed IL2Rβ agonists was implemented to generate a series of IL2Rβ agonist candidate mutations. EP575 contains mutations of P82N, R83K, D84G, I85I, I86L, S87G, N88D, N90S, V91N, and E95A. I85 and L86 were reverse mutated to 85L and 86I of WT IL2, but the mutations of 87G, 91N, and 95A were retained for all mutations. Then, by applying overall reverse mutations to the other six residues, each reverse mutated IL2 finally contains a total of five mutations including the 87G, 91N, and 95A sites. A total of 20 combinations of reverse mutations were designed, and mutations were created by site-directed mutagenesis using EP575 as a template (Table 5). Reverse mutation clones of IL2Rβ agonists were sequence-verified after mutagenesis.

[0151]

Table 5

[0152] Example 5 Characterization of IL2Rβ agonist reverse mutation clones EP575 reverse mutation clones were expressed in mammalian cells as described. The clones were purified by Ni Sepharose column and Superdex 200 Increase 10 / 300 GL column (GE Healthcare) until homogeneous. The clones were concentrated to an appropriate concentration and stored in 1xPBS / 300 mM NaCl buffer for binding, function, and mechanism analysis. An example of the SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) results of individual IL2 after Ni column purification is shown in Figure 4.

[0153] The revertant mutations were characterized for their binding activity to the IL2Rβ and IL2Rα receptors by ELISA. Briefly, 384-well plates were immobilized with the Fc fusion proteins of human IL2Rα and IL2Rβ at a final concentration of 2 μg / mL in 1× PBS with a total volume of 25 μL per well. The plates were incubated overnight at 4°C and blocked for 1 hour with 80 μL of SuperBlock per well. Each of the purified EP575 revertant mutant clones was added in duplicate in parallel to the IL2Rα or IL-2Rβ wells to a final concentration of 10 μM. The binding of the IL2 mutants was detected by adding 25 μL of anti-His HRP diluted 1:5000 in 1X PBST. Between each step, the plates were washed 3 times with 1X PBST using a plate washer. 1 μM of WT IL2 and 1 μM of EP596 were used as reference IL2s. The plates were then developed with 25 μL of TMB substrate for 5 minutes and stopped by adding 25 μl of 2N sulfuric acid. The plates were read on a Biotek plate reader at OD450nm (Table 6), and the data were analyzed with Prism 8.1 software to generate bar graphs (Figure 5).

[0154] To further evaluate the immunomodulatory activity of the purified IL2 revertant mutants, human PBMCs were isolated from the peripheral blood of three separate donors, stained with a dead cell labeling reagent, and plated at 250,000 cells / well in a 96-well plate in 90 μL of medium. The cells were allowed to rest at 37 °C for 1 hour. The cells were stimulated with 10-fold concentrated human IL2 WT and modified His-tagged IL2 in 10 μL for 20 minutes at 37 °C. The stimulated PBMCs were immediately fixed, permeabilized, stained for cell lineage markers (CD3, CD56, CD4, CD8, FOXP3) and p-STAT5, and visualized on an Attune flow cytometer. CD8+ T cells were defined as CD3+CD56-CD4-CD8+. NK cells were defined as CD3-CD56+. Regulatory T cells were defined as CD3+CD56-CD4+CD8-FOXP3+. The percentage of cells that were p-STAT5+ was determined and graphed for each IL2 titration (Figure 6). P-STAT5 activation was determined using Prism software and summarized in Table 6. These data supported that the modified IL2 had reduced binding to the IL2Rα and IL2Rβ receptors. Similarly, the modified IL2 showed weak p-STAT5 phosphorylation activity with an incomplete curve or low activity at high concentrations compared to WT IL2 (Figure 6).

[0155]

Table 6

[0156] Example 6 Generation of revertant mutations of IL2Rβ EP658 and EP661 agonists EP658 and EP661 were employed as examples of another round of modification to further reduce the total number of mutated sites within modified IL2. EP658 contains the mutations D84G, N88D, N90S, V91N and E95A, and EP661 contains the mutations R83K, D84G, N90S, V91N and E95A. EP658 was applied to single-point and two-site revertant mutations to generate four and three mutated sites containing IL2 mutants. On the other hand, single-point mutagenesis was applied to EP661 to generate a four-point mutation containing an IL2 mutant. A total of 14 combinations of revertant mutations were designed, and mutations were created by site-directed mutagenesis using EP658 and EP661 as templates respectively (Table 7). These revertant mutant clones were sequence-verified after mutagenesis and produced in mammalian cells as described above for further characterization. Figures 7 and 8 show the immune cell p-STAT5 activities of the EP658 and EP661 revertant mutations after treating healthy human PBMC donors respectively.

[0157]

Table 7

[0158] Example 7 Binding of IL2Rβ-binding reduced agonists to cell surface receptors HEK Blue IL2 cells expressing IL2 receptor alpha, beta, and gamma were seeded at a density of 100,000 cells per well in a 96-well round-bottom plate in a total volume of 100 μL of medium. The cells were allowed to rest in the incubator at 37 °C for 1 hour. After 1 hour, the medium was replaced with 100 μL of medium containing a 7-point 4-fold titration of the IL2 mutant protein starting from 10 μM, and the plate was incubated at 4 °C for 30 minutes with shaking. After washing away the unbound IL2, the cells were resuspended in 100 μL of medium containing a 1:100 anti-His Dylight 650 secondary antibody and incubated with shaking at 4 °C for 30 minutes in the dark. The unbound secondary antibody was washed away, and the cells were resuspended in PBS containing a 1:800 Zombie Aqua dead cell labeling reagent and incubated at room temperature for 5 minutes. After washing the cells twice, they were analyzed using an Attune NxT flow cytometer. The percentage of HEK Blue IL2 cells with positive IL2 mutant protein binding was measured (Figure 9). Recombinant human CD22 was used as a negative control and WT IL2 was used as a reference. Consistent with the binding and p-STAT5 data, the agonist showed partial binding to the cell surface IL2 receptor compared to complete binding of WT-IL2.

[0159] Example 8 Design of IL2Rβ agonist Fc fusion protein and anti-PD-(L)1 bispecific antibody Modified IL2Rβ-binding reducing agonists can be further developed into Fc fusion proteins to further improve certain properties. To generate monovalent IL2-Fc fusion proteins, protein sequences encoding the modified IL2 polypeptides of EP567 (SEQ ID NO: 49), EP575 (SEQ ID NO: 57), EP581 (SEQ ID NO: 63), EP658 (SEQ ID NO: 69), EP660 (SEQ ID NO: 71), EP661 (SEQ ID NO: 72), EP669 (SEQ ID NO: 80) and EP676 (SEQ ID NO: 87) were fused to the N-terminal site of the constant framework sequence of the human IgG1 isotype to produce modified agonist-Fc fusion proteins (SEQ ID NO: 137). Knob mutations of S354C, T366W and K409A were introduced into the construct. Hole mutations of Y349C, T366S, L368A, F405K, Y407V were introduced into the CH2 and CH3 fragments of IgG1. The L234A, L235A and P329G mutations of human IgG1 were introduced to eliminate complement binding and Fc-γ-dependent antibody-dependent cell-mediated cytotoxicity (ADCC) effects (Lo et al., JBC 2017). Then, the DNA encoding the entire Fc fusion agonist protein was synthesized with codons optimized for mammalian cell expression and subcloned into pCDNA3.4 (Invitrogen).

[0160] The other party can develop a modified IL2Rβ-binding reducing agonist into a bifunctional antibody or a bispecific antibody in order to endow its immunomodulatory activity with synergistic effects with other therapeutic mechanisms. To generate an anti-PD-L1-IL2 fusion bispecific antibody, S354C, T366W and K409A mutations (Wei et al., Oncotarget, 2017; Xu et al., mAbs, 2015) were introduced into one heavy chain of the anti-PD-L1 antibody as a knob molecule (EP362, SEQ ID NO: 104). The C-terminus of another heavy chain of anti-PD-L1 was fused to human IL2 protein together with a (G4S)4 linker. S354C, Y349C, T366S, L368A, F405K, Y407V mutations were introduced to create the chain as a hole molecule (for example, EP643 fused with modified IL2 of EP575). Human IgG1 L234A, L235A and P329G mutations were introduced to eliminate the complement binding and Fc-γ-dependent antibody-dependent cell-mediated cytotoxicity (ADCC) effects (Lo et al., JBC 2017).

[0161] In another example, S354C, Y349C, T366S, L368A, F405K, Y407V mutations were introduced into the heavy chain of the anti-PD-L1 sequence to create the chain as a hole molecule (EP325; SEQ ID NO: 114). A knob molecule with an N-terminal IL2 fusion (for example, EP826) can form a monovalent (Fab format) anti-PD-L1-IL2 bispecific antibody together with that of EP325 and EP205, which are the light chains of the anti-PD-L1 antibody.

[0162] In another example, the variable heavy chain regions of an anti-mouse PD-L1 antibody or an anti-mouse PD-1 antibody were fused to the constant region of a human IgG1 antibody containing them, respectively, to generate a chimeric anti-mouse PD1 antibody or an anti-mouse PD-L1 antibody. A knob mutation and a hole mutation, as well as an ADCC silent mutation, were introduced into the heavy chain. By fusing a modified IL2 molecule to one end of the heavy chain arm, the binding site of the anti-mouse PD-1 or PD-L1 antibody was enabled to form a bispecific protein. Such a bispecific protein can serve as an alternative anti-PD-(L)1 / IL2 bispecific protein (e.g., a bifunctional fusion protein complex) for investigating the mechanism of action and efficacy in a mouse model.

[0163] Next, the DNA encoding the entire sequence designed above was synthesized with codons optimized for expression in mammalian cells and subcloned into pCDNA3.4 (Invitrogen). Figure 10 shows an example of a schematic diagram of the selected anti-PD-L1 antibody or anti-PD-1 antibody format.

[0164] Example 9 Production of IL2-Fc Protein and Bispecific Antibody For the production of the monovalent IL2-Fc fusion protein, the "knob" and "hole" constructs of each IgG1 backbone format were transfected into ExpiHEK293-F cells at a 1:1 ratio. The cells were grown for 5 days, and the supernatant was collected by centrifugation and filtered through a 0.2 μm PES membrane. First, the Fc fusion agonist was purified by MabSelect PrismA protein A resin (GE Health). The protein was eluted with 100 mM Gly pH 2.5 and 150 mM NaCl and rapidly neutralized with 20 mM citric acid pH 5.0 and 300 mM NaCl. The agonist protein was then concentrated to 1 mL and further purified by a Superdex 200 16 / 600 gel filtration column. The monomer peak fractions were pooled and concentrated. The final purified protein had endotoxin less than 10 EU / mg and was stored in 1xPBS. The purified monovalent IL2-Fc fusion agonist was run on an SDS gel (4-12% bis-tris bolt gel, using MES running buffer).

[0165] For the production of the monovalent (Fab format) anti-PD-L1-IL2 bispecific antibody (e.g., a bifunctional fusion protein), the "knob" and "hole" constructs of each IgG1 backbone format were transfected into ExpiHEK293-F cells in a FreeStyle system (Invitrogen) according to a standard protocol with a ratio of knob:hole:light chain of 1:4:4. After the cells were grown for 5 days, they were harvested. The supernatant was collected by centrifugation and filtered through a 0.2 μm PES membrane. The antibody was purified by MabSelect PrismA protein A resin (GE Health). The protein was eluted with 100 mM Gly pH 2.5 and 150 mM NaCl and rapidly neutralized with 20 mM histidine pH 5.0 + 150 mM NaCl. The antibody was then further purified by a Superdex 200 16 / 600 column. The monomer peak fractions were pooled and concentrated. The final purified protein had endotoxin less than 10 EU / mg and was stored in 20 mM histidine, 150 mM NaCl buffer.

[0166] For the production of bivalent anti-PD-L1-IL2 and / or anti-PD-1-IL2 bispecific antibodies (e.g., bifunctional fusion protein complexes), the "knob" and "hole" constructs in their respective IgG1 backbone formats were transfected into ExpiHEK293-F cells in a FreeStyle system (Invitrogen) according to a standard protocol with a knob:hole:light chain ratio of 1:2:2. After growing the cells for 5 days, they were harvested. The supernatant was collected by centrifugation and filtered through a 0.2 μm PES membrane. The antibody was purified by MabSelect PrismA protein A resin (GE Health). The protein was eluted with 100 mM Gly pH2.5 + 150 mM NaCl and rapidly neutralized with 20 mM histidine pH5.0 + 150 mM NaCl. The antibody was then further purified by a Superdex 200 16 / 600 column. The monomer peak fractions were pooled and concentrated. The finally purified protein had endotoxin less than 10 EU / mg and was stored in 20 mM histidine, 150 mM NaCl buffer.

[0167] The anti-PD-L1 monoclonal antibody was transiently expressed in ExpiHEK293-F cells in a FreeStyle system (Invitrogen) according to a standard protocol with a ratio of heavy chain and light chain plasmid DNA of 1:2. After growing the cells for 5 days, they were harvested. The supernatant was collected by centrifugation and filtered through a 0.2 μm PES membrane. The antibody was purified by MabSelect PrismA protein A resin (GE Health). The protein was eluted with 100 mM Gly pH2.5 and 150 mM NaCl and rapidly neutralized with 20 mM citric acid pH5.0 and 300 mM NaCl. The antibody was then further purified by a Superdex 200 16 / 600 column. The monomer peak fractions were pooled and concentrated. The finally purified protein had endotoxin less than 10 EU / mg and was stored in 20 mM histidine pH6.0 and 150 mM NaCl.

[0168] Example 10 Characterization of IL2Rβ agonist Fc fusion antibodies and bispecific antibodies For anti-PD-L1 / IL2 and / or anti-PD-1-IL2 bispecific antibodies, goat anti-human Fc was added to the wells of a 384-well plate in 25 μL of 1X PBS and incubated overnight at 4 °C to coat the plate. The plate was washed three times with 0.05% Tween20 / 1X PBS. The plate was blocked with 100 μL of SuperBlock for 1 hour at room temperature and then washed three times with 0.05% Tween20 / 1X PBS. Anti-PD-L1 / IL2 mutant bispecific (e.g., bifunctional fusion protein complex) was diluted from 1000 nM to 0 nM in 0.05% Tween 20 / 1X PBS and added to the plate for 2 hours at room temperature. The plate was then washed six times with 0.05% Tween20 / 1X PBS. The captured protein was detected with biotinylated recombinant PD-(L)1, IL2Rα or IL2Rβ, washed, and visualized with streptavidin-HRP diluted 1:5000 in 0.05% Tween20 / 1X PBS. The plate was then washed six times with 0.05% Tween20 / 1X PBS, TMB was added, and the color was developed to blue. The reaction was stopped with 2N hydrogen sulfide, and the optical absorbance at 450 nm was read with a BioTek plate reader. The relationship between the absorbance of the binding of human PD-(L)1, IL2Rα and IL2Rβ and the IL2 concentration was graphed (Figure 11). An overview of the ELISA binding activities of PD-(L)1, IL2Rα and IL2Rβ is shown (Table 8). All constructs showed similar PD-L1 binding activity. For the fusion anti-PD-L1 / IL2 bispecifics (e.g., bifunctional fusion protein complex) of EP567, EP575, EP581, EP658, EP660, EP661 and EP669, no binding activity of IL2Rα and IL2Rβ was observed at the highest concentration of 1 μM, but the IL2Rβ agonist of the EP415 fusion bispecific showed strong IL2Rβ binding activity. The reference WT-IL2 fusion bispecific showed strong IL2Rα binding consistent with the reported data.

[0169] [Table 8]

[0170] Example 11 P-STAT5 Activation of Human PBMCs by an IL2Rβ Agonist Fc-Fusion Bispecific Antibody Human PBMCs were isolated from peripheral blood, stained with a dead cell labeling reagent, and plated at 250,000 cells / well in a 96-well plate in 90 μl of medium. The cells were allowed to sit at 37 °C for 1 hour. The cells were stimulated with 10-fold concentrated human IL2 WT and IL2Rβ agonist Fc-fusion protein in 10 μl at 37 °C for 20 minutes. The stimulated PBMCs were immediately fixed, permeabilized, stained for cell lineage markers (CD3, CD56, CD4, CD8, FOXP3) and p-STAT5, and visualized on an Attune flow cytometer. CD8+ T cells were defined as CD3+CD56-CD4-CD8+. NK cells were defined as CD3-CD56+. Regulatory T cells were defined as CD3+CD56-CD4+CD8-FOXP3+. The percentage of cells that were p-STAT5+ was determined and graphed for each IL2 titration (Figure 12).

[0171] Example 12 Safety and Immune Cell Activation of IL2Rβ Agonist EP567 / anti-PD-L1 in Mice Six- to eight-week-old female C57BL / 6 mice were administered either vehicle or EP567 / PD-L1 at a concentration of 2.5 mg / kg, 5 mg / kg, or 10 mg / kg on days 1 and 4. Body weight was monitored daily and expressed as relative body weight normalized to the pre-treatment body weight. On day 8, the mice were sacrificed and the lungs were harvested and measured.

[0172] On the 8th day of slaughter, blood was collected by cardiac puncture. After lysing red blood cells, the blood was stained with immune phenotype markers (CD3, CD4, CD8, NKp46, NK1.1, and FOXP3), as well as CD69, Ki67, and a survival rate marker. Cells were analyzed using an Attune NxT flow cytometer. The numbers of CD4+ FOXP3− T cells, CD8+ T cells, NK cells, and Tregs were calculated as the proportion of live cells. The proportions of CD4+ FOXP3− T cells, CD8+ T cells, and Tregs that were Ki67+ were calculated. The proportions of CD4+ FOXP3− T cells, CD8+ T cells, and Tregs that were CD69+ were calculated.

[0173] Figure 13A shows the change in mouse body weight. EP567 / anti-PD-L1 bispecificity began to induce weight loss at 5 mg / kg and induced severe weight loss and death at 10 mg / kg. Accordingly, the lung weight (Figure 13B) increased proportionally with the increase in the dose of EP567.

[0174] Peripheral immune cell profiling data confirmed that EP567 / anti-PD-L1 increased CD8 T cell levels and decreased Treg cell levels in a dose-dependent manner (Figure 14). Similarly, EP567 increased the proliferation of CD8 T cells at all doses and downregulated the proliferation of Treg cells (Figure 15). EP567 induced early activation of CD8 and CD4 T cells even at a high dose of 10 mg / kg (Figure 16). Collectively, these data confirmed that EP567 / anti-PD-L1 could stimulate the activation of immune cells, but it was shown that more modified IL2 needed to be evaluated because mice could not tolerate high concentrations of the bispecificity.

[0175] Example 13 Safety and immune cell activation of additional IL2 / anti-PD-L1 bispecificity in mice Female C57BL / 6 mice at 6 - 8 weeks of age were administered 10 mg / kg of IL2 / anti - PD - L1 bispecificity on day 0 and day 4. Body weight was monitored 5 times per week and expressed as relative body weight normalized to the body weight before administration. On day 8, the mice were sacrificed and the lungs were collected and measured.

[0176] During sacrifice on day 8, blood was collected by cardiac puncture. After lysing red blood cells, the blood was stained with immune phenotype markers (CD3, CD4, CD8, NKp46, NK1.1, FOXP3, CD44 and CD62L), as well as Ki67 and survival markers. Cells were analyzed using an Attune NxT flow cytometer. The concentrations of CD4+ FOXP3 - T cells, CD8+ T cells, NK cells, and Tregs in each sample were calculated. The percentages of CD4+ FOXP3 - T cells, CD8+ T cells, NK cells, and Tregs that were Ki67+ were calculated. The percentages of CD4+ FOXP3 - T cells and CD8+ T cells that were naive effector central memory were calculated.

[0177] Figure 17Aa shows the change in the body weight of mice. Consistent with previous results, EP567 / anti - PD - L1 bispecificity induced significant weight loss at 10 mg / kg and induced death with an increase in lung weight at 10 mg / kg (Figure 17B). In contrast, all other IL2 / PD - L1 bispecific antibodies tested, including EP575 / anti - PD - L1, EP581 / anti - PD - L1, EP658 / anti - PD - L1, EP660 / anti - PD - L1, EP661 / anti - PD - L1, and EP669 / anti - PD - L1, did not show weight loss (Figure 17A). Therefore, there was no obvious change in lung weight (Figure 17B).

[0178] Peripheral immune phenotype profiling was performed to evaluate whether these IL2 / anti-PD-L1 antibodies (e.g., bifunctional fusion protein complexes) can regulate the activation of immune cells. Only one mouse survived in the EP567 / anti-PD-L1 group. The profiling results of this group were consistent with previous data. Interestingly, increased CD8 T cells, NK cells, and decreased Treg cells were observed in this group (Figure 18). Among the remaining IL2 / anti-PD-L1 bispecifics, EP661 / anti-PD-L1 showed a significant increase in the activation of CD8 and NK cells along with a moderate increase in Tregs. EP575 / anti-PD-L1 and EP581 / anti-PD-L1 showed moderate activation of CD8 T cells, NK cells, and Treg cells (Figure 18). On the other hand, EP658 / anti-PD-L1, EP660 / anti-PD-L1, EP669 / anti-PD-L1, EP676 / anti-PD-L1 showed weak or minimal activation of CD8 T cells and NK cells. In these three groups, Treg cells increased moderately. Interestingly, immune cell proliferation decreased in the EP567 / anti-PD-L1 group (Figure 19). EP575 / anti-PD-L1, EP581 / anti-PD-L1, and EP658 / anti-PD-L1 showed a significant increase in CD8 T cell proliferation (Figure 19B). EP660 / anti-PD-L1, EP661 / anti-PD-L1, EP669 / anti-PD-L1, and EP676 / anti-PD-L1 showed moderate CD8 T cell proliferation (Figure 19B). Only EP575 / anti-PD-L1 showed a significant increase in NK cell proliferation (Figure 19D). From the results of subtype immune cell profiling, it was suggested that the EP567 / anti-PD-L1 group contains more effective CD4 and CD8 T cells, as well as a lower proportion of naive T cells. EP661 / anti-PD-L1 showed a pattern similar to EP567 / anti-PD-L1. EP575 / anti-PD-L1, EP581 / anti-PD-L1, and EP661 / anti-PD-L1 contain high levels of central memory CD8 T cells. EP660 / anti-PD-L1, EP669 / anti-PD-L1, and EP676 / anti-PD-L1 contain high levels of naive CD8 T cells similar to the vehicle, further suggesting that their systemic immune regulatory activities are weak or moderate (Figure 20).Table 9 lists the mean fold changes in the concentrations of immune cells in the peripheral blood of IL2 / anti-PD-L1 treated mice compared to those in the vehicle group mice.

[0179]

Table 9

[0180] Example 14 Pharmacokinetic Analysis of EP661 / anti-PD-L1 Agonist in Mice Six- to eight-week-old female C57BL / 6 mice were given a single intravenous (iv) bolus dose of 200 μg of EP661 / anti-PD-L1. The mice were sacrificed by terminal cardiac puncture at the time points before dosing, 1 hour, 2 hours, 6 hours, 1 day, 3 days, 7 days, and 10 days after the first dose. Blood was collected into lithium heparin tubes and centrifuged, and plasma was collected and stored at -80°C until analysis.

[0181] Once all time points were collected, the plasma was thawed for analysis. Anti-human Fc was diluted with 25 μL of 1X PBS and added to the wells of a 384-well immunosorbent plate, and the plate was incubated overnight at 4°C to coat the plate. The plate was blocked with 100 μL of SuperBlock for 1 hour at room temperature. Plasma samples were diluted 1:1000 or 1:2000 in PBS and added in duplicate to the wells. Standards were prepared by serially diluting known concentrations of EP661 / anti-PD-L1 in PBS containing 0.1% mouse plasma and added in duplicate to the wells. Samples and standards were incubated for 1 hour at room temperature. Bound EP661 / anti-PD-L1 was detected by sandwiching with biotinylated recombinant PD-L1 and adding 25 μL of streptavidin HRP diluted 1:5000 in 1X PBST. Between each step, the plate was washed 3 times with 1X PBST using a plate washer. The plate was then developed with 25 μL of TMB substrate for 5 minutes and stopped by adding 25 μl of 2N sulfuric acid. The plate was read on a Biotek plate reader at OD450nm.

[0182] The OD values from the standard were plotted against the known concentrations in Microsoft Excel, and the best-fit straight line was drawn by performing a simple linear regression. The EP661 / anti-PD-L1 concentration in the sample was calculated from the OD value based on the equation of the standard calibration curve and multiplied by the initial 1:1000 dilution factor. The protein concentration was plotted against time. The PK parameters were calculated using PKSolver software and Microsoft Excel and are shown in Figure 21. Table 10 shows the calculated PK parameters.

[0183]

Table 10

[0184] Example 15 IL2Rβ agonist anti-tumor activity in a mouse tumor model 500,000 B16-F10-hPD-L1 cells were subcutaneously injected into the flanks on the back of 6-8-week-old female C57BL / 6 mice. Tumors were measured with calipers. When the average volume reached approximately 50 mm 3 the mice were treated with 10 mg / kg of IL2 / anti-PD-L1 bispecific (e.g., bifunctional fusion protein complex) or atezolizumab every 4 days. Tumors were measured three times per week and body weights were measured five times per week.

[0185] Peripheral blood was collected on day 8. After lysing the red blood cells, the blood was stained with immunophenotype markers (CD3, CD4, CD8, NKp46, NK1.1, and FOXP3), as well as viability markers. The cells were analyzed using an Attune NxT flow cytometer. The numbers of CD4+ FOXP3− T cells, CD8+ T cells, NK cells, and Tregs in each sample were calculated. The ratios of CD8+ T cells and NK cells to the number of Tregs were calculated.

[0186] Figure 22A shows the tumor growth curves of EP658 / anti-PD-L1, EP661 / anti-PD-L1, and EP669 / anti-PD-L1 (every 4 days, 200 μg), compared with atezolizumab (every 4 days, 200 μg) and vehicle (1xPBS, every 4 days). Figure 22B shows the change in body weight of the mice during the treatment. Consistent with previous investigations, all mice tolerated the repeated dosing in all investigated groups well. All IL2 / anti-PD-L1 treatment groups showed stronger tumor growth inhibitory activity than atezolizumab. In the IL2 / anti-PD-L1 groups, some mice experienced complete tumor regression. Among them, no tumor was seen in one mouse in the EP658 / anti-PD-L1 group, in two mice in the EP661 / anti-PD-L1 group, and in three mice in the EP669 / anti-PD-L1 group. EP669 / anti-PD-L1 showed the strongest anti-tumor activity with 69% tumor growth inhibition. Figure 22C shows the tumor growth curves of individual mice. In addition to the consistently improved safety, this initial tumor model investigation supported the possibility that IL2 with reduced IL2Rβ binding exerts a synergistic effect to improve anti-tumor activity in the bispecificity with anti-PD-L1.

[0187] Analysis of the immunophenotype of peripheral blood collected on the 8th day (before the third dose) suggested results consistent with previous investigations. Marked activation of CD8 T cells and NK cells was observed with EP661 / anti-PD-L1, while EP658 / anti-PD-L1 and EP669 / anti-PD-L1 induced moderate activation of CD8 and NK cells. In these two groups, Treg cells were also moderately activated. Consistent with previous investigations, EP661 / anti-PD-L1 decreased Treg cells and CD4 cells (Figure 23). EP661 / anti-PD-L1 showed a significant increase in the CD8:Treg and NK:Treg ratios in peripheral blood (Figure 24), while the increase in the ratio in the EP658 / anti-PD-L1 and EP669 / anti-PD-L1 groups was minimal. Table 11 shows the fold changes in the mean raw counts of immune cells in the peripheral blood of the IL2 / anti-PD-L1 treatment group compared to the vehicle group. Immunophenotype analysis further confirmed that EP661 is a strong CD8 and NK cell activation modulator, while EP669 and EP658 are mild to moderate systemic CD8 and NK cell activation modulators.

[0188]

Table 11

[0189] Example 16 Antitumor Activity of IL2 / PD1 and IL2 / PD-L1 Bispecificity in Tumor Models To evaluate the antitumor activity of IL-2 / PD1 and PD-L1 bispecificity (e.g., bifunctional fusion protein complexes), 500,000 B16-F10-hPD-L1 cells were subcutaneously injected into the posterior flanks of 6- to 8-week-old female C57BL / 6 mice. Tumors were measured with calipers. The mean volume was approximately 50 mm 3Once the mice reached that point, they were treated with either 3 or 10 mg / kg of EP669 / PDL1 (both every 4 days), 3 mg / kg of EP661 / PD-1 once a week, or 1 mg / kg of RO7284755 once a week. The body weights of the mice were measured twice a week and plotted against time for each group. The mean tumor volume and individual tumor volumes were plotted against time for each group (Figures 25 - 26).

[0190] Female C57Bl / 6 mice (6 - 8 weeks old) were subcutaneously inoculated in the right posterior flank with 2E5 MC38 tumor cells suspended in 200 μl of PBS. Tumor volume was measured using calipers, and when the tumor volume reached approximately 50 mm 3 the mice were randomized into three treatment groups. The mice were treated with vehicle (PBS), 1 mg / kg of EP661 / mPD-1, or 10 mg / kg of anti-mPD-1. The treatment was administered by intraperitoneal injection once a week for two weeks. During the course of the investigation, the tumor volume was measured twice a week. On day 17, the percentage of tumor growth inhibition (TGI) relative to vehicle was calculated, and animals showing complete regression (CR) were determined (Figure 27, Table 12). Peripheral blood was collected on day 11 of the investigation. After lysing the red blood cells, the remaining cells were stained with immunophenotypic markers (CD3, CD4, CD8, NKp46, NK1.1, FOXP3) and viability markers. The cells were analyzed using an Attune NxT flow cytometer. The concentrations of CD4+ FOXP3- T cells, CD8+ T cells, NK cells, and CD4+ FOXP3+ TReg in each sample were calculated and plotted (Figures 28A - 28D).

[0191]

Table 12

[0192] Example 17 IL2Rβ agonist immune cell regulatory activity To characterize the immunomodulatory activity of reduced-activity IL2 mutants bispecificities (e.g., bifunctional fusion protein complexes) compared to WT IL2 and RO7284755, human PBMCs were isolated from peripheral blood, stained with a dead cell labeling reagent, and plated at 250,000 cells / well in 96-well plates in 90 μL of medium. Cells were allowed to rest at 37 °C for 1 hour. Cells were stimulated at 37 °C for 20 minutes with titrations of 10-fold concentrated WT IL2 bispecificity and reduced IL2 mutant bispecificity in 10 μL. Stimulated PBMCs were immediately fixed, permeabilized, stained for cell lineage markers (CD3, CD56, CD4, CD8, FOXP3) and p-STAT5, and visualized on an Attune flow cytometer. CD4+ FOXP3- T cells were defined as CD3+CD56-CD4+CD8-FOXP3-. CD8+ T cells were defined as CD3+CD56-CD4-CD8+. NK cells were defined as CD3-CD56+. Regulatory T cells were defined as CD3+CD56-CD4+CD8+FOXP3+. The percentage of cells that were p-STAT5+ was determined and graphed for each IL2 titration (Figure 29). p-STAT5 activation was determined using Prism software. These data supported that the modified IL2 had reduced binding to the receptors of IL2Rα and IL2Rβ. Similarly, the modified IL2 showed weak p-STAT5 phosphorylation activity with an incomplete curve or low activity at high concentrations compared to WT IL2 and RO7284755 (Figure 29).

[0193] Example 18 IL2Rβ agonist activity against tumor infiltrating cells 500,000 B16-F10-hPD-L1 cells were subcutaneously injected into the posterior flanks of 6- to 8-week-old female C57BL / 6 mice. Tumors were measured with calipers. The average volume was approximately 50 mm 3Once reached, mice were treated with vehicle or EP669 / PDL1 (3 mg / kg, every 4 days). At the end of the study, animals were euthanized by CO2 asphyxiation and tumors were excised. Tumors were fixed in formalin and embedded in paraffin. Tumor blocks were then sectioned at 5 μm thickness on glass slides and processed for IHC staining. Slides were blocked with BLOXALL (Vector, SP-6000-100) and 2.5% normal horse serum (Vector S-2012-50), then incubated with CD8α antibody (CST, 98941) diluted 1:1000 in Signal Stain (CST, 8112L) for 1 hour at room temperature. After washing with TBS-T, slides were incubated with goat anti-rabbit IgG Amplifier (Vector, MP-7601-50) for 1 hour. After washing the slides with TBS-T, they were incubated with ImmPress HRP horse anti-goat IgG polymer reagent (Vector, MP-7601-50). After washing, ImmPACT DAB EqV chromogen solution (Vector, MP-7601-50) was applied and the slides were incubated. All incubations were performed at room temperature. Slides were rinsed in TBS-T and distilled water, counterstained with Gill II Hematoxylin (StatLab, SL94-1), dehydrated and covered with a coverslip. Slides were then imaged and representative areas of untreated and treated tumors were magnified to visualize CD8 positive cells (Figure 30).

[0194] Example 19 IL2Rβ agonist activity on systemic immune cells To evaluate the activity of IL2 bispecificity (e.g., bifunctional fusion protein complex) against systemic immune cells, blood was collected from naïve female C57Bl / 6 mice (6 - 8 weeks old) by submandibular bleeding, and complete blood counts were measured using a VETSCAN hematology analyzer (Abaxis). The mice were then treated with vehicle (PBS), RO7284755, or an IL2 mutant bispecific molecule (EP877 / EP923 / EP740; EP877 / EP930 / EP740; EP877 / EP933 / EP740; EP876 / EP928 / EP205; EP876 / EP934 / EP205; or EP876 / EP935 / EP205). For the treatment, it was administered by intraperitoneal injection at a volume of 200 μl at a dosing concentration of 200 μg / mouse. Two days after treatment, blood was collected again from all animals for analysis of complete blood counts. The fold changes in total white blood cells (WBC), lymphocytes, and neutrophils from pre-treatment (day 0) to day 2 blood samples were graphed (Figure 31). The data confirmed that RO7284755 had a stronger lymphocyte-depleting effect than the IL2 mutant bispecific molecules disclosed herein.

[0195] Peripheral blood was collected on day 5. After lysing red blood cells, the remaining cells were stained with immune phenotype markers (CD3, CD4, CD8, NKp46, NK1.1, FOXP3, CD44, CD62L), and a viability marker. The cells were analyzed using an Attune NxT flow cytometer. CD4+ FOXP3- T cells were identified as CD3+, NKp46-, NK1.1-, CD8-, CD4+, FOXP3-. CD8+ T cells were identified as CD3+, NKp46-, NK1.1-, CD8+, CD4-. From these two populations, the proportions of naïve (CD44-, CD62L+), central memory (CD44+, CD62L+), and effector (CD44+, CD62L-) of the parental population were determined and plotted (Figure 32).

[0196] Example 20 Anti-tumor activity of IL2 / PD1 bispecificity in a tumor model Female C57Bl / 6 mice (6 - 8 weeks old) were subcutaneously inoculated with 2E5 MC38 tumor cells suspended in 200 μl of PBS in the right posterior flank. Tumor volume was measured using calipers, and when the tumor volume reached approximately 50 mm 3 3 , the mice were randomized into three treatment groups. The mice were treated with vehicle (PBS), RO7284755, EP661 / mPD-1, EP930 / PD-1, EP933 / PD-1, or EP935 / PD-1 at 1 mg / kg. The treatments were administered by intraperitoneal injection once a week for two weeks. During the period of the investigation, tumor volume was measured twice a week. On day 17, the percentage of tumor growth inhibition (TGI) relative to vehicle was calculated, and animals showing complete regression (CR) were determined (Figure 33). Peripheral blood was collected on day 11 of the investigation. After lysing red blood cells, the remaining cells were stained with immunophenotypic markers (CD3, CD4, CD8, NKp46, NK1.1, FOXP3) and viability markers. The cells were analyzed using an Attune NxT flow cytometer. CD4+ FOXP3- T cells were identified as CD3+, NKp46-, NK1.1-, CD8-, CD4+, FOXP3-. CD8+ T cells were identified as CD3+, NKp46-, NK1.1-, CD8+, CD4-. From these two populations, the percentages of naive (CD44-, CD62L+), central memory (CD44+, CD62L+), and effector (CD44+, CD62L-) of the parental population were determined and plotted (Figure 34).

[0197] Additional embodiments can be provided by combining the various embodiments described above. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications (including U.S. Provisional Patent Application No. 63 / 340,294 filed on May 10, 2022 and U.S. Provisional Patent Application No. 63 / 391,243 filed on July 21, 2022) mentioned herein and / or listed in the application data sheet are hereby incorporated by reference in their entirety. Embodiment aspects can be modified and additional embodiments can be provided by adopting concepts from various patents, applications, and publications as needed.

[0198] In light of the above detailed description, these and other modifications can be made to the embodiments. Generally, the terms used in the following claims should not be construed as limiting the claims to the specific embodiments disclosed in this specification and the claims, but should be construed to include all possible embodiments, as well as the full scope of equivalents given to such claims. Accordingly, the claims are not limited by the disclosure.

Claims

**Claim 1** A modified interleukin-2 (IL2) polypeptide comprising a modified interleukin-2 receptor beta (IL2Rβ) binding region 2 motif, wherein said modified IL2Rβ binding region 2 motif is X １ -X ２ -X ３ -X ４ -X- ５ -X ６ -X ７ -X ８ -X ９ -X １０ -X １１ -X １２ -X １３ -X １４ -X １５ -X １６ including (array number 3) X １ comprises a residue selected from the group consisting of R, S, L, N, F, K or T, X ２ contains residues selected from A, F, S, L, R, T, I, H, P or N, X ３ comprises residues selected from K, R, T, S, I or P, X ４ comprises residues selected from G, D, R, A, Q, H, N, Y or E, X ５ comprises residues selected from the group consisting of I, P, T, S, K, F, V or L, X ６ comprises a residue selected from I, R, V, M, T or L, X ７ comprises residues selected from A, R, M, I, S, N, G or S, X ８ comprises residues selected from E, N, H, T, K, Y, S, L, V, D or R, X ９ comprises a residue selected from the residues I, V, A, T, L, T or M, X １０ comprises a residue selected from residues N, G, V, Y, I, W, R, K, Q, A, D, S or D, X １１ comprises residues selected from F, G, V, N, T, I, R, E or A, X １２ comprises a residue selected from the group consisting of I, S, R, V, P, G, T, L, M, F or Y, X １３ comprises residues selected from V, I, F, D, P, H, A, V or L, X １４ comprises residues selected from L, Q, R, E, P, K, H, W, F or V, X １５ comprises a residue selected from the group consisting of A, E, L, K, V, D, Y, R or Q, X １６ comprises a residue selected from L or I, said modified IL2Rβ binding region 2 motif does not include SEQ ID NO: 2, and said modified IL2 polypeptide binds to IL2Rβ with a decreased affinity as compared to wild-type IL2, said modified IL2 polypeptide. **Claim 2** The modified IL2Rβ binding region 2 is RFKALIIEINFIVQLL (SEQ ID NO: 4), RSRQLISNMNGLIIKL (SEQ ID NO: 5), RLTHLRNVIGVILVQL (SEQ ID NO: 6), LSLREPIGNIVTSVRE (SEQ ID NO: 7), NRTLVGDV NATIKAL (SEQ ID NO: 8), RNKGILGDISNIVLAL (SEQ ID NO: 9), RSREVVSRIDAIILEL (SEQ ID NO: 10), RPRGLISDISNIVLAL (SEQ ID NO: 11), RPRGLIGNISNIVLAL (SEQ ID NO: 12), RPRGLIGDINNIVLAL (SEQ ID NO: 13), RPKG LISNISNIVLAL (SEQ ID NO: 14), RPKG LISDINNIVLAL (SEQ ID NO: 15), RPKG LIGNINNIVLAL (SEQ ID NO: 16), RNRGLISNISNIVLAL (SEQ ID NO: 17), RNRGLISDINNIVLAL (SEQ ID NO: 18), RNRGLIGNINNIVLAL (SEQ ID NO: 19), RNKG LISNINNIVLAL (SEQ ID NO: 20), RPRGLIGDISNIVLAL (SEQ ID NO: 21), RPKG LISDISNIVLAL (SEQ ID NO: 22), RPKG LIGNISNIVLAL (SEQ ID NO: 23), RPKG LIGDINNIVLAL (SEQ ID NO: 24), RNRGLISDISNIVLAL (SEQ ID NO: 25), RNRGLIGNISNIVLAL (SEQ ID NO: 26), RNRGLIGDINNIVLAL (SEQ ID NO: 27), RNKG LISNISNIVLAL (SEQ ID NO: 28), RNKG LISDINNIVLAL (SEQ ID NO: 29), RNKG LIGNINNIVLAL (SEQ ID NO: 30), RPRDLISDISNIVLAL (SEQ ID NO: 31), RPRGLISDINNIVLAL (SEQ ID NO: 32), RPRGLISDISVIVLAL (SEQ ID NO: 33), RPRGLISDISNIVLEL (SEQ ID NO: 34), RPRDLISDINNIVLAL (SEQ ID NO: 35), RPRDLISDISVIVLAL (SEQ ID NO: 36), RPRDLISDISNIVLEL (SEQ ID NO: 37), RPRGLISDINVIVLAL (SEQ ID NO: 38), RPRGLISDINNIVLEL (SEQ ID NO: 39), RPRGLISDISVIVLEL (SEQ ID NO: 40), RPKDLISNISNIVLAL (SEQ ID NO: 41), RPKG LISNINNIVLAL (SEQ ID NO: 42),The modified IL2 polypeptide according to claim 1, comprising or consisting of a group selected from RPKGLISNISVIVLAL (SEQ ID NO: 43), RPKGLISNISNIVLEL (SEQ ID NO: 44), RPKGLISNISVIVLEL (SEQ ID NO: 194), RPRGLISNISVIVLEL (SEQ ID NO: 195), RPRDLISNISNIVLEL (SEQ ID NO: 196), RPKGLISNINNIVLEL (SEQ ID NO: 197), RPKGLISDINNIVLEL (SEQ ID NO: 198), RPRDLISRIDAIEL (SEQ ID NO: 199), RNRGLIGNINNIVLEL (SEQ ID NO: 200), RPKGLISEINNIVLEL (SEQ ID NO: 201), and RPKGLISRINNIVLEL (SEQ ID NO: 202). **Claim 3** Said modified IL2Rβ binding region 2 is selected from the group consisting of or comprising RFKALIIEINFIVQLL (SEQ ID NO: 4), RSRQLISNMNGLIILK (SEQ ID NO: 5), RLTHLRNVIGVILVQL (SEQ ID NO: 6), RNKGILGDISNIVLAL (SEQ ID NO: 9), RSREVVSRIDAIILEL (SEQ ID NO: 10), RPRGLISDISNIVLAL (SEQ ID NO: 11), RPRGLIGDINNIVLAL (SEQ ID NO: 13), RPKGGLISNISNIVLAL (SEQ ID NO: 14), RPRGLIGDISNIVLAL (SEQ ID NO: 21), RPKGGLISDISNIVLAL (SEQ ID NO: 22), RPKGGLIGDINNIVLAL (SEQ ID NO: 24), RNKGGLISNISNIVLAL (SEQ ID NO: 28), RNKGGLISDINNIVLAL (SEQ ID NO: 29), RPKGGLISNISVIVLEL (SEQ ID NO: 194), RPRGLISNISVIVLEL (SEQ ID NO: 195), RPRDLISNISNIVLEL (SEQ ID NO: 196), RPKGGLISNINNIVLEL (SEQ ID NO: 197), RPKGGLISDINNIVLEL (SEQ ID NO: 198), RPRDLISRIDAIIVLEL (SEQ ID NO: 199), RNRGLIGNINNIVLEL (SEQ ID NO: 200), RPKGGLISEINNIVLEL (SEQ ID NO: 201), and RPKGGLISRINNIVLEL (SEQ ID NO: 202); the modified IL2 polypeptide according to claim 1 or 2. **Claim 4** The modified IL2 polypeptide according to any one of claims 1 to 3, wherein said modified IL2 polypeptide has at least a 1 / 2 decrease in affinity for IL2Rβ as compared to wild-type IL2 or IL2 having the sequence of SEQ ID NO:

171. **Claim 5** The modified IL2 polypeptide according to any one of claims 1 to 4, wherein the affinity for IL2Rα is reduced as compared with wild-type IL2 or IL2 having the sequence of SEQ ID NO:

171.

6. The modified IL2 polypeptide according to any one of claims 1 to 4, wherein the modified IL2 polypeptide has a similar affinity for IL2Rα as compared with wild-type IL2 or IL2 having the sequence of SEQ ID NO:

171.

7. Comprising a modified IL2 receptor α (IL2Rα) binding region 1 containing at least one substitution at a position selected from K35, R38, F42 and Y45, i) the substitution at position K35 is selected from K35G, K35L, K35S, K35V, K35D, K35E and K35C, ii) the substitution at position R38 is selected from R38V, R38D, R38E, R38S, R38I, R38A, R38Y, R38G, R38C or R38N, iii) the substitution at position F42 is selected from F42A, F42R, F42G, F42I, F42L, F42P and F42H, iv) the substitution at position Y45 is Y45S, Y45P, Y45A, Y45V, Y45C, Y45T and Y45F, The modified IL2 polypeptide according to any one of claims 1 to 6.

8. The modified IL2 polypeptide according to claim 7, wherein the substitution is at least two, at least three, or all four of positions K35, R38, F42 and Y45.

9. The modified IL2 polypeptide according to claim 7, wherein the substitution is K35L, R38D and F42R.

10. The modified IL2 polypeptide according to any one of claims 1 to 9, wherein the modified IL2 polypeptide binds to IL2Rα with a binding reaction rate reduced by at least one tenth as compared with wild-type IL2 or IL2 having the sequence of SEQ ID NO:

171.

11. Sequence number 45 (APASSSSTKKTQLQLLEHLLLLDLQMILNINGINNYKNPL LTDMLTRKFYMPKKATELKHLQCL EEEE LKPL EEVLNL AQSKNFHL-X １ -X ２ -X ３ -X ４ -X- ５ -X ６ -X ７ -X ８ -X ９ -X １０ -X １１ -X １２ -X １３ -X １４ -X １５ -X １６ -KGSETTFMCEYADETATIVEFLNRWITFSQSIISTL) and having at least 90% sequence identity, a modified interleukin-2 (IL2) polypeptide comprising X １ comprises a residue selected from R, S, L, N, F, K or T, X ２ comprises residues selected from A, F, S, L, R, T, I, H, P or N, X ３ comprises a residue selected from K, R, T, S, I or P, X ４ comprises a residue selected from the group consisting of G, D, R, A, Q, H, N, Y or E, X ５ comprises a residue selected from the group consisting of I, P, T, S, K, F, V or L, X ６ comprises a residue selected from the group consisting of I, R, V, M, T or L, X ７ comprises residues selected from A, R, M, I, S, N, G or S, X ８ comprises a residue selected from the group consisting of E, N, H, T, K, Y, S, L, V, D or R, X ９ comprises residues selected from I, V, A, T, L, T or M, X １０ comprises residues selected from N, G, V, Y, I, W, R, K, Q, A, D, S or D, X １１ comprises residues selected from F, G, V, N, T, I, R, E or A, X １２ comprises a residue selected from the group consisting of I, S, R, V, P, G, T, L, M, F or Y, X １３ comprises a residue selected from the group consisting of V, I, F, D, P, H, A, V or L, X １４ comprises residues selected from L, Q, R, E, P, K, H, W, F or V, X １５ comprises a residue selected from residues A, E, L, K, V, D, Y, R or Q, X １６ contains a residue selected from L or I, The modified IL2 polypeptide, wherein the modified IL2Rβ binding region 2 motif does not include SEQ ID NO: 2, and the modified IL2 polypeptide binds to IL2Rβ with a reduced affinity as compared with wild-type IL2.

12. The IL2Rβ binding region 2 is RFKALIIEINFIVQLL (SEQ ID NO: 4), RSRQLISNMN GIILK L (SEQ ID NO: 5), RLTHLRNVIGVILVQL (SEQ ID NO: 6), LSLREPIGNIVTSVRE (SEQ ID NO: 7), NRT DLVGDVNATIKAL (SEQ ID NO: 8), RNKGILGDISNIVLAL (SEQ ID NO: 9), RSREVVSRIDAI ILEL (SEQ ID NO: 10), RPRGLISDISNIVLAL (SEQ ID NO: 11), RPRGLIGNISNIVLAL (SEQ ID NO: 12), RPRGLIGDINNIVLAL (SEQ ID NO: 13), RP KGLISNISNIVLAL (SEQ ID NO: 14), RP KGLISDINNIVLAL (SEQ ID NO: 15), RP KGLIGNINNIVLAL (SEQ ID NO: 16), RNRGLISNISNIVLAL (SEQ ID NO: 17), RNRGLISDINNIVLAL (SEQ ID NO: 18), RNRGLIGNINNIVLAL (SEQ ID NO: 19), RNKGLISNINNIVLAL (SEQ ID NO: 20), RPRGLIGDISNIVLAL (SEQ ID NO: 21), RP KGLISDISNIVLAL (SEQ ID NO: 22), RP KGLIGNISNIVLAL (SEQ ID NO: 23), RP KGLIGDINNIVLAL (SEQ ID NO: 24), RNRGLISDISNIVLAL (SEQ ID NO: 25), RNRGLIGNISNIVLAL (SEQ ID NO: 26), RNRGLIGDINNIVLAL (SEQ ID NO: 27), RNKGLISNISNIVLAL (SEQ ID NO: 28), RNKGLISDINNIVLAL (SEQ ID NO: 29), RNKGLIGNINNIVLAL (SEQ ID NO: 30), RPRDLISDISNIVLAL (SEQ ID NO: 31), RPRGLISDINNIVLAL (SEQ ID NO: 32), RPRGLISDISVIVLAL (SEQ ID NO: 33), RPRGLISDISNIVLEL (SEQ ID NO: 34), RPRDLISDINNIVLAL (SEQ ID NO: 35), RPRDLISDISVIVLAL (SEQ ID NO: 36), RPRDLISDISNIVLEL (SEQ ID NO: 37), RPRGLISDINVIVLAL (SEQ ID NO: 38), RPRGLISDINNIVLEL (SEQ ID NO: 39), RPRGLISDISVIVLEL (SEQ ID NO: 40), RP KD LISNISNIVLAL (SEQ ID NO: 41), RP KGLISNINNIVLAL (SEQ ID NO: 42),The modified IL2 polypeptide according to claim 11, which is a sequence selected from the group consisting of or comprising RPKGLISNISVIVLAL (SEQ ID NO: 43), RPKGLISNISNIVLEL (SEQ ID NO: 44), RPKGLISNISVIVLEL (SEQ ID NO: 194), RPRGLISNISVIVLEL (SEQ ID NO: 195), RPRDLISNISNIVLEL (SEQ ID NO: 196), RPKGLISNINNIVLEL (SEQ ID NO: 197), RPKGLISDINNIVLEL (SEQ ID NO: 198), RPRDLISRIDAI VLEL (SEQ ID NO: 199), RNRGLIGNINNIVLEL (SEQ ID NO: 200), RPKGLISEINNIVLEL (SEQ ID NO: 201), and RPKGLISRINNIVLEL (SEQ ID NO: 202).

13. The IL2Rβ binding region 2 is a sequence selected from the group consisting of or comprising RFKALIIEINFIVQLL (SEQ ID NO: 4), RSRQLISNMNGLIILK (SEQ ID NO: 5), RLTHLRNVIGVILVQL (SEQ ID NO: 6), RNKGILGDISNIVLAL (SEQ ID NO: 9), RSREVVSRIDAIILEL (SEQ ID NO: 10), RPRGLISDISNIVLAL (SEQ ID NO: 11), RPRGLIGDINNIVLAL (SEQ ID NO: 13), RPKGLISNISNIVLAL (SEQ ID NO: 14), RPRGLIGDISNIVLAL (SEQ ID NO: 21), RPKGLISDISNIVLAL (SEQ ID NO: 22), RPKGLIGDINNIVLAL (SEQ ID NO: 24), RNKGLISNISNIVLAL (SEQ ID NO: 28), RNKGLISDINNIVLAL (SEQ ID NO: 29), RPKGLISNISVIVLEL (SEQ ID NO: 194), RPRGLISNISVIVLEL (SEQ ID NO: 195), RPRDLISNISNIVLEL (SEQ ID NO: 196), RPKGLISNINNIVLEL (SEQ ID NO: 197), RPKGLISDINNIVLEL (SEQ ID NO: 198), RPRDLISRIDAIIVLEL (SEQ ID NO: 199), RNRGLIGNINNIVLEL (SEQ ID NO: 200), RPKGLISEINNIVLEL (SEQ ID NO: 201), and RPKGLISRINNIVLEL (SEQ ID NO: 202), the modified IL2 polypeptide according to claim 11.

14. The modified IL2 polypeptide according to any one of claims 1 to 13, comprising a sequence selected from the group consisting of or comprising SEQ ID NOs: 46 to 102, 147 to 169, and 203 to 211.

15. A modified IL2 polypeptide comprising a sequence having at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 46 to 102, 147 to 169, and 203 to 211.

16. The modified IL2 polypeptide comprising a sequence selected from the group consisting of or comprising SEQ ID NOs: 46 to 102, 147 to 169, and 203 to 211.

17. A fusion polypeptide comprising a first polypeptide sequence and a second polypeptide sequence, wherein the first polypeptide sequence comprises the modified IL2 polypeptide according to any one of claims 1 to 16, the fusion polypeptide.

18. The second polypeptide sequence of the fusion protein includes an Fc domain, an antibody, an antigen-binding portion, a cytokine, a half-life extension molecule, a tag or marker polypeptide, a target domain, a transport molecule, an immunotoxin, NKG2D, a linker sequence, PEGylation, a chemically conjugated small molecule, a nucleic acid, or any combination thereof, the fusion polypeptide according to claim 17.

19. The second polypeptide sequence includes a constant region of an antibody heavy chain, the fusion polypeptide according to claim 17 or 18.

20. The constant region of the antibody heavy chain is a human IgG heavy chain constant region, the fusion polypeptide according to claim 19.

21. The constant region of the antibody heavy chain is a human IgG1 heavy chain constant region, the fusion polypeptide according to claim 19 or 20.

22. The constant region of the antibody heavy chain includes an amino acid sequence that is at least 90% identical to SEQ ID NO: 137, the fusion polypeptide according to any one of claims 19 to 21.

23. The constant region of the antibody heavy chain includes one or more mutations selected from L234A, L235A, P329G, Y349C, S354C, T366S, T366W, L368A, F405K, K409A, and Y407V numbered according to the EU numbering system with respect to SEQ ID NO: 137, the fusion polypeptide according to any one of claims 19 to 22.

24. The constant region of the antibody heavy chain includes the amino acid sequence of SEQ ID NO: 123 or SEQ ID NO: 138, the fusion polypeptide according to any one of claims 19 to 23.

25. The fusion protein includes a sequence having at least 90% sequence identity with a sequence selected from SEQ ID NOs: 124 to 128 and 190 to 193, the fusion polypeptide according to any one of claims 19 to 24.

26. A monovalent modified IL2-Fc fusion polypeptide complex, a. A first polypeptide comprising the fusion polypeptide according to any one of claims 17 to 25, and b. A second polypeptide that forms a dimer with the first protein The monovalent modified IL2-Fc fusion polypeptide complex comprising.

27. The second polypeptide includes a constant region of a heavy chain, the monovalent modified IL2-Fc fusion polypeptide complex according to claim 26.

28. The monovalent modified IL2-Fc fusion polypeptide complex according to claim 26 or 27, wherein the second polypeptide comprises the sequence of SEQ ID NO:

123.

29. a. SEQ ID NO: 124 and 123, b. SEQ ID NO: 125 and 123, c. SEQ ID NO: 126 and 123, d. SEQ ID NO: 127 and 123, e. SEQ ID NO: 128 and 123, f. SEQ ID NO: 190 and 123, g. SEQ ID NO: 191 and 123, h. SEQ ID NO: 192 and 123, or i. SEQ ID NO: 193 and 123 The monovalent modified IL2-Fc fusion polypeptide complex according to any one of claims 26 to 28, comprising first and second polypeptides having the sequences of.

30. The fusion polypeptide according to any one of claims 17 to 29, wherein the fusion polypeptide further comprises an antigen-binding portion.

31. The fusion polypeptide according to claim 17, wherein the second polypeptide comprises an antigen-binding portion.

32. The fusion polypeptide according to claim 31, wherein the antigen-binding portion comprises an immunoglobulin.

33. The fusion polypeptide according to claim 31 or 32, wherein the antigen-binding portion comprises a Fab molecule, scFv, bispecific T cell engager, diabody, single domain antibody or VHH antibody (nanobody).

34. The fusion polypeptide according to any one of claims 30 to 33, wherein the antigen-binding portion binds to PD-L1, PD-1, CTLA-4, TIM3, LAG3, B7-H2, B7-H3, CD4, CD8 or a cell marker.

35. A protein complex comprising a first polypeptide that is the fusion polypeptide according to any one of claims 17 to 34 and a second polypeptide comprising an antigen-binding portion.

36. The protein complex according to claim 35, wherein the second polypeptide comprises at least one polypeptide having the sequences of SEQ ID NO: 103 and 114, or SEQ ID NO: 103 and 172.

37. a. SEQ ID NO: 103, 114 and 124, b. SEQ ID NO: 103, 114 and 125, c. SEQ ID NO: 103, 114 and 126, d. SEQ ID NO: 103, 114 and 127, e. SEQ ID NO: 103, 114 and 128, f. SEQ ID NO: 103, 172 and 190, g. SEQ ID NO: 103, 172 and 191, h. SEQ ID NO: 103, 172 and 192, or i. SEQ ID NO: 103, 172 and 193 The protein complex according to claim 36, comprising a polypeptide having the array of

38. (a) a modified IL2 polypeptide comprising the array according to any one of claims 1 to 25; and (b) an antigen-binding moiety and comprising a bifunctional fusion protein.

39. The bifunctional fusion protein according to claim 38, wherein the antigen-binding moiety comprises an immunoglobulin, a Fab molecule, a scFv, a diabody, a single domain antibody or a VHH antibody.

40. The bifunctional fusion protein according to claim 38 or 39, wherein the antigen-binding moiety binds to PD-L1, PD-1, CTLA-4, TIM3, LAG3, B7-H2, B7-H3, CD4, CD8 or a cell marker.

41. The antigen-binding moiety binds to PD-L1, (i) a light chain comprising the sequence of SEQ ID NO: 103; and (ii) a heavy chain comprising the sequence of SEQ ID NO: 104 and comprising the bifunctional fusion protein according to any one of claims 38 to 40.

42. The bifunctional fusion protein according to any one of claims 38 to 41, further comprising an antibody heavy chain constant region.

43. The bifunctional fusion protein according to claim 42, wherein the antibody heavy chain constant region is a human IgG heavy chain constant region.

44. The bifunctional fusion protein according to claim 43, wherein the antibody heavy chain constant region is a human IgG1 heavy chain constant region.

45. The bifunctional fusion protein according to any one of claims 42 to 44, wherein the antibody heavy chain constant region comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

137.

46. The bifunctional fusion protein according to any one of claims 42 to 45, wherein the antibody heavy chain constant region comprises one or more mutations selected from L234A, L235A, P329G, Y349C, S354C, T366S, T366W, L368A, F405K, K409A and Y407V numbered according to the EU numbering system with respect to SEQ ID NO:

137.

47. The bifunctional fusion protein according to any one of claims 42 to 46, wherein the antibody heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 123 or SEQ ID NO:

138.

48. The bifunctional fusion protein according to any one of claims 42 to 47, comprising a sequence selected from the group consisting of SEQ ID NOs: 106 to 113 and SEQ ID NOs: 173 to 177 or consisting of the same.

49. The bifunctional fusion protein according to any one of claims 42 to 48, further comprising a light chain sequence comprising a polypeptide having the sequence of SEQ ID NO:

103.

50. A protein complex comprising the bifunctional fusion protein according to any one of claims 42 to 49 and a second antigen-binding portion.

51. The protein complex according to claim 50, wherein the second antigen-binding portion comprises at least one polypeptide having the sequences of SEQ ID NO: 103 and 104, or SEQ ID NO: 103 and 172.

52. a. SEQ ID NO: 104, 106 and 103, b. SEQ ID NO: 104, 107 and 103, c. SEQ ID NO: 104, 108 and 103, d. SEQ ID NO: 104, 109 and 103, e. SEQ ID NO: 104, 110 and 103, f. SEQ ID NO: 104, 111 and 103, g. SEQ ID NO: 104, 112 and 103, h. SEQ ID NO: 104, 113 and 103, i. SEQ ID NO: 172, 173 and 103, j. SEQ ID NO: 172, 174 and 103, k. SEQ ID NO: 172, 175 and 103, l. SEQ ID NO: 172, 176 and 103, or m. SEQ ID NO: 172, 177 and 103 The protein complex according to claim 51, comprising a polypeptide having the sequence of.

53. The bifunctional fusion protein according to claim 38, wherein the antigen-binding portion binds to PD-1.

54. A protein complex comprising the bifunctional fusion protein according to any one of claims 38 to 53 and a second antigen-binding portion.

55. The protein complex according to claim 54, wherein the second antigen-binding portion binds to PD-1.

56. a. SEQ ID NO: 133, 134 and 135, or b. SEQ ID NO: 133, 134 and 136 The protein complex comprising a polypeptide having the sequence of.

57. The fusion polypeptide according to claim 18, wherein the half-life extending molecule comprises an Fc domain, human serum albumin (HSA), an HSA-binding molecule or transferrin.

58. The fusion polypeptide according to claim 18, wherein the half-life extending molecule comprises polyethylene glycol (PEG) or polypropylene glycol (PPG).

59. The fusion polypeptide according to claim 17 or 18, wherein the second polypeptide comprises interleukin-2, interleukin-15, interleukin-7, interleukin-10, or C-C motif chemokine ligand 19 (CCL19).

60. The fusion polypeptide according to claim 17 or 18, wherein the second polypeptide comprises a ligand or a scaffold protein.

61. An isolated polynucleotide encoding at least one polypeptide according to any one of claims 1 to 60.

62. An expression vector comprising the polynucleotide according to claim 61.

63. A modified cell comprising the isolated polynucleotide according to claim 61, or the expression vector according to claim 62.

64. A pharmaceutical composition comprising a modified IL2 polypeptide according to any one of claims 1 to 16, a fusion polypeptide according to any one of claims 17 to 34 and 58 to 60, a protein complex according to any one of claims 35 to 37 and 56 to 57, a bifunctional fusion protein according to any one of claims 38 to 55, the polynucleotide according to claim 61, the vector according to claim 62, or the modified cell according to claim 63, and a pharmaceutically acceptable carrier.

65. For use in a method of modulating an immune response in a subject in need thereof, a modified IL2 polypeptide according to any one of claims 1 to 16, a fusion polypeptide according to any one of claims 17 to 34 and 58 to 60, a protein complex according to any one of claims 35 to 37 and 56 to 57, a bifunctional fusion protein according to any one of claims 38 to 55, the polynucleotide according to claim 61, the vector according to claim 62, or the modified cell according to claim 63 or the pharmaceutical composition according to claim 64.

66. A method for modulating an immune response in a subject in whom it is necessary to modulate the immune response, comprising administering to the subject an effective amount of the modified IL2 polypeptide according to any one of claims 1 to 16, the fusion polypeptide according to any one of claims 17 to 34 and 58 to 60, the protein complex according to any one of claims 35 to 37 and 56 to 57, the bifunctional fusion protein according to any one of claims 38 to 55, the polynucleotide according to claim 61, the vector according to claim 62, or the modified cell according to claim 63 or the pharmaceutical composition according to claim 64.

67. The use or method according to claim 65 or 66, wherein said modulating the immune response comprises at least one of enhancing effector T cell activity, enhancing NK cell activity, and suppressing regulatory T cell activity.

68. The modified IL2 polypeptide according to any one of claims 1 to 16, the fusion polypeptide according to any one of claims 17 to 34 and 58 to 60, the protein complex according to any one of claims 35 to 37 and 56 to 57, the bifunctional fusion protein according to any one of claims 38 to 55, the polynucleotide according to claim 61, the vector according to claim 62, or the modified cell according to claim 63 or the pharmaceutical composition according to claim 64 for use in a method of treating a disease in a subject in need thereof.

69. A method of treating a disease in a subject in need thereof, comprising administering to the subject an effective amount of the modified IL2 polypeptide according to any one of claims 1 to 16, the fusion polypeptide according to any one of claims 17 to 34 and 58 to 60, the protein complex according to any one of claims 35 to 37 and 56 to 57, the bifunctional fusion protein according to any one of claims 38 to 55, the polynucleotide according to claim 61, the vector according to claim 62, or the modified cell according to claim 63 or the pharmaceutical composition according to claim 64.

70. The use or method according to claim 68 or 69, wherein said disease comprises cancer or immunosuppression.

71. The cancer of claim 70, the use or method thereof, includes hematological malignancies such as lymphoma, leukemia, multiple myeloma, and / or solid tumors such as breast cancer, pancreatic cancer, lung cancer, glioblastoma, renal cell carcinoma, head and neck cancer, liver cancer, gastric cancer, colorectal cancer, kidney cancer, bladder cancer or melanoma.

72. The use or method according to claims 65 to 71, wherein the subject is treated with an additional therapeutic agent.

73. The use or method according to claim 72, wherein the additional therapeutic agent is a vaccine, gene therapy, cell therapy, or any combination thereof.

74. The use or method according to claim 73, wherein the cell therapy is immune cells expressing a chimeric antigen receptor, immune cells expressing a modified T cell receptor, tumor infiltrating lymphocytes, or any combination thereof.

75. A cell culture medium comprising the modified IL2 polypeptide according to any one of claims 1 to 16, the fusion polypeptide according to any one of claims 17 to 34 and 58 to 60, the protein complex according to any one of claims 35 to 37 and 56 to 57, the bifunctional fusion protein according to any one of claims 38 to 55, the polynucleotide according to claim 61, the vector according to claim 62, or the modified cell according to claim 63.

76. A method for culturing cells, comprising incubating the cells in the culture medium according to claim 75.

77. The method according to claim 76, wherein the cells are present in a blood sample.

78. The method according to claim 76, wherein the cells are present in a sample containing PBMC.

79. The method according to claim 76, wherein the cells are T cells, NK cells, regulatory T cells, or any combination thereof.

80. The method according to claim 79, wherein the T cells are CD4+ T cells, CD8+ T cells, gamma delta (γδ) T cells, or any combination thereof.

81. The method according to any one of claims 76 to 79, wherein the modified IL2 polypeptide, the fusion polypeptide, the protein complex or the bifunctional fusion protein biases the growth, proliferation or persistence of the cell type.

82. A transgenic immune cell comprising the modified IL2 polypeptide according to any one of claims 1 to 16, the fusion polypeptide according to any one of claims 17 to 34 and 58 to 60, the protein complex according to any one of claims 35 to 37 and 56 to 57, the bifunctional fusion protein according to any one of claims 38 to 55, the polynucleotide according to claim 61, or the vector according to claim 62.

83. The transgenic immune cell according to claim 82, wherein the immune cell is a CD4+ T cell, a CD8+ T cell, a γδ T cell, an NK cell, a regulatory T cell, or any combination thereof.

84. The transgenic immune cell according to claim 82 or 83, wherein the immune cell further comprises a chimeric antigen receptor (CAR).

85. The transgenic immune cell according to claim 84, wherein the CAR targets an antigen produced by cancer cells.

86. The transgenic immune cell according to any one of claims 82 to 85, wherein the modified IL2 polypeptide, the fusion polypeptide, the protein complex or the bifunctional fusion protein is secreted by the transgenic immune cell.

87. The transgenic immune cell according to any one of claims 82 to 85, wherein the modified IL2 polypeptide, the fusion polypeptide, the protein complex or the bifunctional fusion protein comprises a transmembrane domain or a cell surface anchor molecule and is localized on the surface of the cell.

88. The transgenic immune cell according to any one of claims 85 to 87, wherein the transgenic immune cell is an enhanced CAR T cell.

89. The transgenic immune cell according to any one of claims 82 to 88, for use in a method of treating a disease in a subject in need thereof.

90. A method of treating a disease in a subject in need thereof, comprising administering an effective amount of the transgenic immune cell according to any one of claims 82 to 88.

91. The use or method according to claim 89 or 90, wherein the disease comprises cancer or immunosuppression.

92. The cancer in the use or method according to claim 91 includes hematological malignancies such as lymphoma, leukemia, multiple myeloma, and / or solid tumors such as breast cancer, pancreatic cancer, lung cancer, glioblastoma, renal cell carcinoma, head and neck cancer, liver cancer, gastric cancer, colon / rectal cancer, kidney cancer, bladder cancer or melanoma.

93. A transgenic feeder cell comprising a modified IL2 polypeptide according to any one of claims 1 to 16, a fusion polypeptide according to any one of claims 17 to 34 and 58 to 60, a protein complex according to any one of claims 35 to 37 and 56 to 57, a bifunctional fusion protein according to any one of claims 38 to 55, a polynucleotide according to claim 61, or a vector according to claim 62.

94. The transgenic feeder cell according to claim 93, wherein the feeder cell is a K562 cell, a 3T3 cell, a fibroblast or an antigen-presenting cell.

95. A method for in vitro or ex vivo proliferation of immune cells, comprising contacting a population of immune cells with an effective amount of a modified IL2 polypeptide according to any one of claims 1 to 16, a fusion polypeptide according to any one of claims 17 to 34 and 58 to 60, a protein complex according to any one of claims 35 to 37 and 56 to 57, or a bifunctional fusion protein according to any one of claims 38 to 55 under conditions sufficient to promote the proliferation of the population of immune cells, and culturing the population of immune cells for a time sufficient to at least double the number of immune cells.

96. The method according to claim 95, wherein the conditions sufficient to promote proliferation include one or more agents or ligands capable of activating the intracellular signaling domain of the TCR complex.

97. The agent capable of activating the intracellular signaling domain of the TCR complex according to claim 96 is an anti-CD3 antibody or binding domain, an anti-CD28 antibody or binding domain, or a combination thereof.

98. The method according to any one of claims 95 to 97, further comprising culturing the population of immune cells with IL-7, IL-15, IL-21, or any combination thereof.

99. The method according to any one of claims 95 to 98, wherein the population of immune cells is cultured in the presence of a population of feeder cells.

100. The method according to claim 99, wherein the feeder cells comprise or consist essentially of the transgenic feeder cells according to claim 93 or 94.

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