Engineered interleukin-2 receptor beta reduced binding agonists

Abstract

Provided herein are engineered IL2 polypeptide IL2R [beta] reduced binding agonists and fusion proteins thereof. Also provided are methods of modulating an immune response by administering an engineered IL2 polypeptide reduced binding agonist or a fusion protein thereof.

Description

[0001] Reference Electronic Sequence Listing

[0002] The contents of the electronic sequence listing (300096_403WO_SEQUENCE_LISTING.xml; size: 266,559 bytes; creation date: May 8, 2023) are incorporated herein by reference in their entirety. Background Art

[0003] Interleukin-2 (IL2) is a cytokine that regulates lymphocyte proliferation and activation. It has a length of 133 amino acids, and the structure includes four antiparallel amphipathic C-helices. IL2 mediates its effect by binding to IL2 receptors (IL2Rs), which include up to three separate subunits. The association of all three subunits (interleukin-2 receptor α chain (IL2Rα, or CD25), interleukin-2 receptor β chain (IL2Rβ, or CD122) and interleukin-2 receptor γ chain (IL2Rγ, or CD132)) produces a trimer IL2Rαβγ, which is a high-affinity receptor for IL2. The association of IL2Rβ with IL2Rγ subunits produces a dimer receptor IL2Rβγ, and is referred to as an intermediate affinity IL2R. IL2Rα subunits form monomeric low-affinity IL2 receptors. The expression of IL2Rα is involved in the expansion of immunosuppressive regulatory T cells (Treg); however, the dimer IL2Rβγ can lead to cell lytic CD8 + T cell and NK cell proliferation and killing. Summary of the invention

[0004] The present disclosure provides rationally designed engineered IL2 polypeptides having amino acid substitutions in IL2Rβ binding region 2 that have reduced binding to IL2Rβ compared to wild-type IL2.

[0005] In one aspect, the present disclosure provides an engineered interleukin-2 (IL2) polypeptide comprising an engineered IL2 receptor β (IL2Rβ) binding region 2 motif, wherein the engineered IL2 receptor β (IL2Rβ) binding region 2 motif comprises:

[0006] 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:

[0007] X1 comprises a residue selected from R, S, L, N, F, K or T;

[0008] X2 comprises a residue selected from A, F, S, L, R, T, I, H, P or N;

[0009] X3 comprises a residue selected from K, R, T, S, I or P;

[0010] X4 comprises a residue selected from G, D, R, A, Q, H, N, Y or E;

[0011] X5 comprises a residue selected from I, P, T, S, K, F, V or L;

[0012] X6 comprises a residue selected from I, R, V, M, T or L;

[0013] X7 comprises a residue selected from A, R, M, I, S, N, G or S;

[0014] X8 comprises a residue selected from E, N, H, T, K, Y, S, L, V, D or R;

[0015] X9 comprises a residue selected from I, V, A, T, L, T or M;

[0016] X 10 comprises a residue selected from N, G, V, Y, I, W, R, K, Q, A, D, S or D;

[0017] X 11 comprises a residue selected from F, G, V, N, T, I, R, E or A;

[0018] X 12 comprises a residue selected from I, S, R, V, P, G, T, L, M, F or Y;

[0019] X 13 comprises a residue selected from V, I, F, D, P, H, A, V or L;

[0020] X 14 comprises a residue selected from L, Q, R, E, P, K, H, W, F or V;

[0021] X 15 comprises a residue selected from A, E, L, K, V, D, Y, R or Q; and

[0022] X 16 comprises a residue selected from L or I, and

[0023] The engineered IL2Rβ binding region 2 motif does not comprise SEQ ID NO: 2, and the binding affinity of the engineered IL2 polypeptide to IL2Rβ is reduced compared to wild-type IL2.

[0024] In some aspects, the disclosure provides an engineered IL2 polypeptide comprising a sequence having at least 90% sequence identity to a sequence selected from the group consisting of: SEQ ID NOs: 46-102 and 147-169, and 203-211. In some aspects, the disclosure provides an engineered IL2 polypeptide comprising a sequence selected from the group consisting of: SEQ ID NOs: 46-102, 147-169, and 203-211.

[0025] In some aspects, the present disclosure provides a fusion polypeptide comprising a first polypeptide sequence and a second polypeptide sequence, wherein the first polypeptide sequence comprises an engineered IL2 polypeptide as provided herein. In some aspects, 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 a marker polypeptide, a targeting domain, a transport molecule, an immunotoxin, an NKG2D, a linker sequence, pegylation, a chemically connected small molecule, a nucleic acid, or any combination thereof. In some aspects, the second polypeptide sequence comprises an antibody heavy chain constant region. In some aspects, the antibody heavy chain constant region is a human IgG heavy chain constant region. In some aspects, 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.

[0026] In some aspects, the disclosure provides a monovalent engineered IL2-Fc fusion polypeptide complex comprising: (a) a first polypeptide comprising a fusion polypeptide described herein, and (b) a second polypeptide that forms a dimer with the first protein. In some aspects, the second polypeptide comprises a heavy chain constant region.

[0027] In some aspects, the present disclosure provides a protein complex comprising a first polypeptide and a second polypeptide, wherein the first polypeptide is a fusion polypeptide as described herein, and the second polypeptide comprises an antigen binding portion. In some aspects, 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.

[0028] In some aspects, the present disclosure provides a bifunctional fusion protein comprising: (a) an engineered IL2 polypeptide comprising a sequence of any one of claims 1-2524; and (b) an antigen binding portion. In some aspects, the antigen binding portion is capable of binding to PD-L1, PD-1, CTLA-4, TIM3, LAG3, B7-H2, B7-H3, CD4, CD8 or a cell marker.

[0029] In some aspects, the present disclosure provides isolated polynucleotides encoding at least one polypeptide disclosed herein. In some aspects, the present disclosure provides expression vectors comprising polynucleotides encoding at least one polypeptide disclosed herein. In some aspects, the present disclosure provides modified cells comprising isolated polynucleotides or expression vectors disclosed herein.

[0030] In some aspects, the present disclosure provides a pharmaceutical composition comprising an engineered IL2 polypeptide, fusion polypeptide, protein complex, bifunctional fusion protein, polynucleotide, vector or modified cell disclosed herein, and a pharmaceutically acceptable carrier.

[0031] In some aspects, the present disclosure provides methods of modulating an immune response in a subject in need thereof, comprising administering an effective amount of an engineered IL2 polypeptide, fusion polypeptide, protein complex, bifunctional fusion protein, polynucleotide, vector, modified cell, or pharmaceutical composition disclosed herein.

[0032] In some aspects, the present disclosure provides a method of treating a disease in a subject in need thereof, comprising administering to the subject an effective amount of an engineered IL2 polypeptide, fusion polypeptide, protein complex, bifunctional fusion protein, polynucleotide, vector, modified cell or pharmaceutical composition disclosed herein.

[0033] In some aspects, the present disclosure provides a cell culture medium comprising an engineered IL2 polypeptide, fusion polypeptide, protein complex, bifunctional fusion protein, polynucleotide, vector or cell disclosed herein. In some aspects, disclosed herein is a method of culturing cells, comprising incubating the cells with a culture medium comprising an engineered IL2 polypeptide, fusion polypeptide, protein complex, bifunctional fusion protein, polynucleotide, vector or cell disclosed herein.

[0034] In some aspects, the disclosure provides transgenic immune cells, which include engineered IL2 polypeptides, fusion polypeptides, protein complexes, bifunctional fusion proteins, polynucleotides or carriers as described herein. In some aspects, immune cells are CD4+T cells, CD8+T cells, γδT cells, NK cells, T regulatory cells or any combination thereof. In some aspects, immune cells also include chimeric antigen receptors (CAR). In some aspects, engineered IL2 polypeptides, fusion polypeptides, protein complexes or bifunctional fusion proteins are secreted or expressed / localized to the cell surface by transgenic immune cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Shown is SDS-PAGE of individual engineered IL2s after Ni column purification.

[0036] FIG. 2A to FIG. 2B Shown is the identification of IL2Rβ reduced binding mutations by ELISA. Figure 2A Binding to IL2Rα is shown. Figure 2B Binding to IL2Rβ is shown.

[0037] Figures 3A-3D Shown are the percentages of p-STAT5 positive cells upon titration of treatment with engineered IL2 compared to wild-type IL2. Figure 3A Results for CD4+ T cells are shown. Figure 3B Results for CD8+ T cells are shown. Figure 3C Results for NK cells are shown. Figure 3D Results for T reg cells are shown.

[0038] Figure 4 Shown is SDS-PAGE of individual engineered IL2s after Ni column purification.

[0039] Figure 5A-5B Binding of mammalian produced IL2 EP575 backmutant to IL2 receptor in ELISA is shown. Figure 5A Binding to IL2Rα is shown. Figure 5A Binding to IL2Rβ is shown.

[0040] Figures 6A-6D The effect of EP575 back mutation on pSTAT5 activity of human immune cells is shown. Fig. 6A Results for CD4+ T cells are shown. Figure 6B Results for CD8+ T cells are shown. Figure 6C Results for NK cells are shown. Fig.6D Results for T reg cells are shown.

[0041] Figures 7A-7D The effect of the back mutation of EP658 on the pSTAT5 activity of human immune cells is shown. Fig. 7A Results for CD4+ T cells are shown. Figure 7B Results for CD8+ T cells are shown. Figure 7C Results for NK cells are shown. Fig.7D Results for T reg cells are shown.

[0042] Figures 8A-8D The effect of the back mutation of EP661 on the pSTAT5 activity of human immune cells is shown. Fig. 8A Results for CD4+ T cells are shown. Figure 8B Results for CD8+ T cells are shown. Figure 8C Results for NK cells are shown. Fig.8D Results for T reg cells are shown.

[0043] Fig. 9Binding of engineered IL2 to cell surface IL2 receptors is shown.

[0044] Figures 10A-10C Diagrams of example IL2-Fc fusions and bifunctional proteins are shown. Fig. 10A An example of a monovalent IL2-Fc is shown. Fig. 10B An example of a protein complex of IL2-Fc and antibody Fab-Fc is shown. Fig. 10C Shown are examples of engineered IL2-antibody-antibody fusion proteins complexed with antibody Fab-Fc (eg, immunoconjugates).

[0045] Figures 11A-11C ELISA binding of engineered IL2 fused to anti-PD-L1 antibody Fab-Fc is shown. Fig.11A Shows binding to PD-L1. Fig. 11B Binding to IL2α was shown. Fig. 11C Binding to IL2β is shown.

[0046] Figures 12A-12D Shown is p-STAT5 activity in cells when treated with titrations of engineered IL2 fused to anti-PD-L1 antibody Fab-Fc. Fig. 12A Results for CD4+ T cells are shown. Fig. 12B Results for CD8+ T cells are shown. Fig. 12C Results for NK cells are shown. Fig.12D Results for T reg cells are shown.

[0047] Figures 13A-13B Shown are the results of a safety assessment in mice using an engineered IL2 (EP567) fused to an anti-PD-L1 antibody. Fig.13A Relative body weights are shown. Fig. 13B Lung weights are shown.

[0048] Figures 14A-14D The immunomodulatory activity of mice treated with a bifunctional protein comprising EP567 fused to an anti-PD-L1 antibody is shown. Fig.14A The percentages of live CD4+ T cells are shown. Fig. 14B The percentages of live CD8+ T cells are shown. Fig. 14C The percentages of live NK cells are shown. Fig.14D The percentages of live T reg cells are shown.

[0049] Figures 15A-15C The immunomodulatory activity of mice treated with a bifunctional protein comprising EP567 fused to an anti-PD-L1 antibody is shown. Fig.15A The percentages of Ki67+CD4+ T cells are shown. Fig. 15BThe percentages of Ki67+CD8+ T cells are shown. Fig. 15C The percentages of Ki67+ T reg cells are shown.

[0050] Figures 16A-16C The immunomodulatory activity of mice treated with a bifunctional protein comprising EP567 fused to an anti-PD-L1 antibody is shown. Fig.16A The percentages of CD69+CD4+ T cells are shown. Fig. 16B The percentages of CD69+CD8+ T cells are shown. Fig. 16C The percentages of CD69+ T reg cells are shown.

[0051] Figures 17A-17B Shown are the results of a safety assessment in mice using an engineered IL2 polypeptide fused to an anti-PD-L1 antibody. Fig.17A Body weight percentages are shown. Fig. 17B Lung weights are shown.

[0052] Figures 18A-18D Immunomodulatory activity of mice treated with engineered IL2 polypeptide fused to anti-PD-L1 antibody was shown. Fig.18A The percentages of CD8+ T cells are shown. Fig.18B The percentages of live CD4+ T cells are shown. Fig. 18C The percentages of NK cells are shown. Fig.18D The percentages of live T reg cells are shown.

[0053] Figures 19A-19D Immunomodulatory activity of mice treated with engineered IL2 polypeptide fused to anti-PD-L1 antibody was shown. Fig.19A The percentages of Ki67+CD4+ T cells are shown. Fig.19B The percentages of Ki67+CD8+ T cells are shown. Fig.19C The percentages of Ki67+ Treg cells are shown. Fig.19D The percentage of Ki67+ NK cells is shown.

[0054] Figures 20A-20B Subtype CD4 and CD8 T cell immunomodulatory activity in mice treated with engineered IL2 polypeptides fused to anti-PD-L1 antibodies was shown. Fig. 20A The percentages of subtype CD4+ T cells are shown. Fig. 20B The percentages of subtype CD8+ T cells are shown.

[0055] Fig.21 The pharmacokinetics of EP661 / anti-PD-L1 bispecific in a mouse model are shown.

[0056] Figures 22A-22C Shown is the anti-tumor activity of IL2 fused to an anti-PD-L1 bispecific in the B16F10-hPD-L1 mouse model. Fig.22A Mean tumor volumes are shown. Fig. 22B Relative body weight changes are shown. Fig. 22C Tumor volumes of individual mice treated with atezolizumab, EP658 / hPD-L1, EP661 / hPD-L1, or EP669 / hPD-L1 are shown.

[0057] Figures 23A-23D Peripheral immune cell activation in vivo was demonstrated. Fig.23A Activation of CD8+ T cells is shown. Fig. 23B NK cell activation is shown. Fig.23C Activation of CD4+ T cells is shown. Fig.23C Activation of T reg cells is shown.

[0058] Figures 24A-24B Peripheral immune cell activation in vivo was demonstrated. Fig.24A Raw counts of CD8+ T cells and T reg cells are shown. Fig. 24B Raw counts of NK cells and T reg cells are shown.

[0059] Figures 25A-25B Antitumor activity of engineered IL2 fused to anti-PD-L1 bispecific was shown in the B16F10-hPD-L1 mouse model. Fig.25A Tumor volume over time for mice treated with vehicle control, EP669 / PDL1 (3 mg / kg), or EP669 / PDL1 (10 mg / kg). Fig.25B Shown are body weights over time for mice treated with vehicle control, EP669 / PDL1 (3 mg / kg), or EP669 / PDL1 (10 mg / kg).

[0060] Figures 26A-26B Antitumor activity of engineered IL2 fused to anti-PD-1 bispecific in the B16F10-hPD-L1 mouse model is shown compared to vehicle control and the PD-1-targeted IL-2v antibody fusion protein RO7284755. Fig.26A Tumor volume over time in mice treated with vehicle control, EP661 / PD1 (3 mg / kg), or RO7284755 (1 mg / kg). Fig.26B Shown are body weights over time for mice treated with vehicle control, EP661 / PD1 (3 mg / kg), or RO7284755 (1 mg / kg).

[0061] Fig. 27 Anti-tumor activity of engineered IL2 fused to mouse anti-PD-1 bispecific (EP661 / mPD-1) compared to vehicle control and anti-mPD-1 treatment (10 mg / kg) in the MC38 mouse model is shown.

[0062] Figures 28A-28D Shown is peripheral immune cell activation in mice treated with EP661 / mPD-1 (1 mg / kg) compared to vehicle control and anti-mPD-1 treatment (10 mg / kg). Fig.28A The cell number of FoxP3-CD4+ T cells per mL is shown. Fig.28B The cell number of CD8+ T cells per mL is shown. Fig.28C The cell number of NK cells per mL is shown. Fig.28D The cell number of FoxP3+CD25+ regulatory T cells per mL is shown.

[0063] Figures 29A-29D Shown is the p-STAT5 activity in cells when cells were treated with titrations of engineered IL2 fused to anti-PD-L1 antibody Fab-Fc or anti-PD-1 antibody Fab-Fc, compared to WT IL2 fused to anti-PD-L1 antibody, anti-PD-L1 antibody, and PD-1-targeted IL-2v antibody fusion protein RO7284755. Fig.29A p-STAT5 activity in CD4+FoxP3- T cells is shown. Fig.29B p-STAT5 activity in CD8+ T cells is shown. Fig.29C p-STAT5 activity in NK cells is shown. Fig.29D p-STAT5 activity in regulatory T cells is shown.

[0064] Figures 30A-30B Immunohistochemical staining showing infiltrating CD8+ cells. Fig. 30A Tumor infiltrating cells from mice treated with vehicle control are shown. Fig. 30B Tumor infiltrating cells of mice treated with EP669 / PDL1 (3 mg / kg, Q4D) are shown.

[0065] Figures 31A-31C Fold changes in total white blood cells (WBC), lymphocytes, and neutrophils from pre-treatment (day 0) to day 2 are shown. Fig.31A WBC fold changes are shown. Fig.31B Lymphocyte fold changes are shown. Fig.31C Neutrophil fold changes are shown.

[0066] Figures 32A-32B Blood phenotypes are shown. Fig.32A The percentages of CD4+FoxP3- T cells are shown. Fig.32B The percentages of CD8+ T cells are shown.

[0067] Figures 33A-33B Anti-tumor activity of engineered IL2 fused to anti-PD-1 bispecifics (EP661 / PD-1, EP930 / PD-1, EP933 / PD-1, EP935 / PD-1) compared to vehicle control and RO7284775 treatment in the MC38 mouse model is shown. Fig.33A Changes in tumor volume over time are shown. Fig.33B Shown is the change in body weight over time.

[0068] Figures 34A-34B Blood phenotypes are shown. Fig.34A The percentages of CD4+FoxP3- T cells are shown. Fig.34B The percentages of CD8+ T cells are shown. DETAILED DESCRIPTION

[0069] Provided herein are rationally designed engineered IL2 polypeptides with amino acid substitutions in IL2Rβ binding region 2, which have reduced binding to IL2Rβ compared to wild-type IL2. The engineered IL2 polypeptide is an agonist with reduced binding to IL2Rβ, which has the advantage of stimulating NK cells and effector T cells compared to wild-type IL2, while providing improved safety and lasting immunomodulation. Therefore, engineered IL2Rβ agonists can be used to modulate or activate immune responses, such as for the treatment of cancer.

[0070] definition

[0071] In this specification, unless otherwise stated, any concentration range, percentage range, ratio range or integer range should be understood to include any value or sub-range within the recited range. Unless otherwise stated, the term "about" as used herein means ±20% of the indicated range or value.

[0072] It should also be noted that the term "or" is generally employed in its sense including "and / or" (ie, to mean one, both, or any combination of the alternatives) unless content dictates otherwise.

[0073] Furthermore, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content dictates otherwise.

[0074] The terms "comprising," "having," "including," and variations thereof are used synonymously and should be construed as non-limiting.

[0075] As used herein, the term "combinations thereof" refers to all possible combinations of the items listed before the term. For example, "A, B, C, or a combination thereof" is intended to refer to any of the following: A, B, C, AB, AC, BC, or ABC. As used herein, the term "combinations thereof" refers to all possible combinations of the items listed before the term. For example, "A, B, C, and a combination thereof" is intended to refer to all of the following: A, B, C, AB, AC, BC, and ABC.

[0076] Unless otherwise indicated, the term "interleukin-2" or "IL2" as used herein refers to IL2 from any vertebrate source, including mammals such as humans or mice. The term encompasses precursor or unprocessed IL2, as well as any form of IL2 produced by cell processing. The term also encompasses naturally occurring variants of IL2, such as splice variants or allelic variants. "Wild type" or "native" refers to mature IL2 molecules (e.g., SEQ ID NO: 1) when used in reference to IL2. As used herein, the term "engineered IL2" or "engineered IL2 polypeptide" encompasses IL2 having at least one residue different from natural or wild-type IL2, and includes full-length IL2, truncated forms of IL2, and forms of IL2 connected or fused to another molecule such as another polypeptide. The various forms of engineered IL2 are characterized by having at least one amino acid substitution that affects the interaction of IL2 with IL2Rβ. The engineered IL2 mentioned herein can be an agonist that reduces binding to IL2Rβ. An agonist that reduces IL2Rβ binding has reduced binding to IL2Rβ compared to wild-type IL2 or, for example, an IL2 having T3A and C125S substitutions relative to SEQ ID NO: 1 (eg, SEQ ID NO: 171).

[0077] IL2Rβ binding region 1 and IL2Rβ binding region 2 are responsible for IL2 binding 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 provided in SEQ ID NO: 2.

[0078] IL2Rα binding region 1 and IL2Rα binding region 2 are responsible for 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.

[0079] As used herein, the terms "engineered", "recombinant" or "non-natural" refer to polypeptides / proteins, nucleic acid molecules, vectors, organisms, microorganisms or cells that include at least one genetic change or have been modified by the introduction of exogenous or heterologous nucleic acid molecules, wherein such changes or modifications are introduced by genetic engineering (i.e., human intervention). Genetic changes include, for example, modifications that introduce expressible nucleic acid molecules encoding functional RNA, proteins, fusion proteins or enzymes, or additions, deletions, substitutions or other functional disruptions of other nucleic acid molecules of the genetic material of the cell. Additional modifications include, for example, non-coding regulatory regions that alter the expression of polynucleotides, genes or operons.

[0080] As used herein, the term "substitution" or "residue substitution" refers to the replacement of 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. Mutation can result in several different types of sequence changes, including substitution, insertion or deletion of one or more nucleotides or one or more amino acids. Various identifiers may be used herein to represent the same residue substitution. For example, a substitution from threonine at position 3 to alanine may be represented as T3A or 3A.

[0081] As used herein, "nucleic acid molecule" or "polynucleotide" or "polynucleic acid" refers to a polymeric compound comprising covalently linked nucleotides, which may be composed of 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, any of which may be single-stranded or double-stranded. If single-stranded, the nucleic acid molecule may be a coding strand or a non-coding (antisense) strand. Nucleic acid molecules encoding amino acid sequences include all nucleotide sequences encoding the same amino acid sequence. Some versions of nucleotide sequences may also include one or more introns to the extent that the one or more introns will be removed by co-transcriptional or post-transcriptional mechanisms. In other words, due to the redundancy or degeneracy of the genetic code or by splicing, different nucleotide sequences can encode the same amino acid sequence.

[0082] As used herein, "protein" or "polypeptide" refers to a polymer of amino acid residues. "Protein" applies to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, and non-naturally occurring amino acid polymers. Variants of the proteins, peptides and polypeptides disclosed herein are also contemplated. In certain embodiments, the variant proteins, peptides and polypeptides comprise 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 an amino acid sequence as defined or a reference amino acid sequence as described herein.

[0083] "Fusion polypeptide" or "fusion protein" refers to a polypeptide encoded by at least two different DNA sequences corresponding to genes or fragments thereof, which are not naturally expressed by the same gene. An example of a fusion polypeptide is an engineered IL2-Fc fusion polypeptide, which comprises the amino acid sequence of an engineered IL2 polypeptide and the amino acid sequence of an Fc domain.

[0084] "Protein complex" or "multi-protein complex" refers to a group of two or more related polypeptide chains that interact to form a quaternary structure. The complex can be formed under energetically favorable conditions. For example, a protein complex can be formed due to ionic interactions and / or hydrophobic interactions. A protein complex can comprise two or more protein subunits linked by one or more disulfide bonds or disulfide bonds. An antibody comprising 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 can form a protein complex with the heavy and light chains of an antibody.

[0085] "Bifunctional fusion protein" or "bispecific" refers to a protein, fusion protein and / or heterodimeric protein pair comprising one or more functional domains. Examples of functional domains include antigen binding sites, antibody fragments (e.g., Fab, scFv, etc.), antibody heavy chains and light chains, and cytokines (e.g., IL-2, IL-15). Bifunctional fusion proteins or bispecifics may refer to antibodies fused to non-antibody polypeptides such as cytokines. For example, a bifunctional protein may include an antibody heavy chain and a light chain, wherein the heavy chain constant region is fused to an engineered IL-2. In addition, a bifunctional fusion protein may include an antibody heavy chain and a light chain, wherein the heavy chain constant region may form a heterodimer with a polypeptide or protein that does not comprise an antigen binding site. For example, a bifunctional fusion protein may include a heavy chain, a light chain, and an engineered IL-2 fusion protein, the fusion protein comprising an antibody Fc domain that can form a heterodimer with the Fc domain of an antibody heavy chain.

[0086] As used herein, "percentage of sequence identity" refers to the relationship between the two or more sequences as determined by comparing two or more sequences. The preferred method for determining sequence identity is designed to give the best match between the sequences being compared. For example, the sequences are aligned for the purpose of optimal comparison (for example, for optimal comparison, a gap can be introduced in one or both of the first and second amino acid or nucleic acid sequences). Further, for the purpose of comparison, non-homologous sequences can be ignored. Unless otherwise indicated, the percentage of sequence identity mentioned herein is calculated over the length of the reference sequence. The method for determining sequence identity and similarity can be found in publicly available computer programs. Sequence alignment and identity percentage calculations can be performed using BLAST programs (such as BLAST 2.0, BLASTP, BLASTN or BLASTX). The mathematical algorithm used in the BLAST program can be found in Altschul et al., Nucleic Acids Res.25:3389-3402,1997. In the context of the present disclosure, it should be understood that when sequence analysis software is used for analysis, the analysis results are based on the "default values" of the mentioned programs. "Default Values" means any set of values ​​or parameters that are initially loaded with the Software when first initialized.

[0087] The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if the original environment 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 separated from some or all of the coexisting materials in the natural system is isolated. Such a nucleic acid can be part of a vector and / or such a nucleic acid or polypeptide can be part of a composition (e.g., a cell lysate) and still be isolated because such a vector or composition is not part of the natural environment of the nucleic acid or polypeptide.

[0088] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., a receptor and a ligand). The affinity of a molecule-X for its partner Y can often be expressed in terms of the dissociation constant (K D ), which are the dissociation and association rate constants (k off and k on ). Thus, equivalent affinities can include different rate constants as long as the ratio of rate constants remains constant. Affinity can be measured by methods known to those skilled in the art, including those described herein.

[0089] "Immunoglobulin" refers to a protein with the structure of a naturally occurring antibody. For example, IgG class immunoglobulins are heterotetrameric glycoproteins with two light chains and two heavy chains bonded by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH) (also referred to as a variable heavy chain domain or a heavy chain variable domain), followed by three constant domains (CH1, CH2, and CH3) (also referred to as a heavy chain constant region). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL) (also referred to as a variable light chain domain or a light chain variable domain), followed by a constant light chain (CL) domain (also referred to as a light chain constant region). The heavy chains of immunoglobulins can be classified into one of five classes, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further divided into subclasses, for example, γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of immunoglobulins can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. Immunoglobulins include two Fab molecules and an Fc domain connected by an immunoglobulin hinge region.

[0090] As used herein, "Fc domain" or "Fc region" refers to a polypeptide derived from a polypeptide of the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of a constant region. The term includes polypeptides or variants thereof having a native sequence Fc region. Although the boundaries of the Fc region of an IgG heavy chain may be slightly different, the human IgG heavy chain Fc region is generally defined as extending from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) in 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 U.S. Patent No. 9,803,023 (all patents are incorporated by reference in their entirety).

[0091] The term "antibody" as used herein encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, bifunctional antibodies), antibody fusion proteins, antibodies in heterodimers in engineered proteins, and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0092] The term "antibody fragment" refers to a polypeptide or protein other than an intact antibody, which comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0093] "Antigen binding portion" or "antigen binding site" are used interchangeably herein and refer to sites (i.e., amino acid residues) of antigen binding molecules (e.g., antibodies) that provide interactions with antigen epitopes. The antigen binding portion may include one or more antibody variable domains (also referred to as antibody variable regions). In human antibodies, the antigen binding site is formed by amino acid residues of the N-terminal variable regions ("V") of the heavy chain ("H") and the light chain ("L"). "Hypervariable regions" are three highly divergent stretches (stretches) in the V region of the heavy and light chains inserted between the "framework regions" ("FR"), which are relatively conservative flanking stretches. The term "FR" refers to amino acid sequences naturally present between and adjacent to the hypervariable regions of immunoglobulins. In human antibody molecules, 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. The three hypervariable regions of each of the heavy ("H") and light ("L") chains are referred to as "complementarity determining regions" or "CDRs". Antigen binding sites can be present in intact antibodies, in antigen binding fragments of antibodies that retain an antigen binding surface, or in recombinant polypeptides such as scFvs, whereby a peptide linker is used to connect the heavy chain variable domains in a single polypeptide to the light chain variable domains. The antigen binding site may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Examples of antigen binding moieties include immunoglobulins, Fab molecules, scFvs, bispecific antibodies, diabodies, bispecific T-cell engagers, and nanobodies. Specific examples of antigen-binding portions include nivolumab, pembrolizumab, pidilizumab, atezolizumab, ipilimumab, tremelimumab, rituximab, ocrelizumab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, tositumomab, urituximab, and bevacizumab.

[0094] The numbering of CDRs and framework regions can be according to any known method or scheme, such as the Kabat, Chothia, EU, IMGT and AHo numbering schemes (see, e.g., Kabat et al., "Sequences of Proteins of Immunological Interest," USDept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); Lefranc et al., Dev. Comp. Immunol. 27:55, 2003; Honegger and Plückthun, J. Mol. Bio. 309:657-670 (2001)). The Antigen Receptor Numbering and Receptor Classification (ANARCI) software tool (2016, Bioinformatics 15:298-300) annotates equivalent residue positions and compares different molecules. The CDRs of an antigen binding site can be determined according to known methods, such as Kabat, Chothia, EU, IMGT and AHo as described above. CDRs determined according to these definitions generally include overlaps 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 different numbering conventions. For example, in certain embodiments, the heavy chain CDRs are defined according to Chothia (supra), and the light chain CDRs are defined according to Kabat (supra). CDRH1, CDRH2 and CDRH3 represent heavy chain CDRs, and CDRL1, CDRL2 and CDRL3 represent light chain CDRs.

[0095] A "Fab molecule" or "antigen-binding fragment" is an antigen-binding fragment of an antibody which comprises the variable domain and the constant domain of the light chain, and the variable domain and the CH1 domain of the heavy chain.

[0096] "Single-chain variable domain" or "scFv" refers to an antigen binding portion that includes the variable regions of the heavy and light chains connected by a linker peptide.

[0097] "Bispecific antibody" refers to an artificial antibody with two different antigen binding sites. Bispecific antibodies can refer to whole immunoglobulins with two different antigen binding sites, or can refer to other molecules with two antigen binding parts, such as a fusion protein containing two Fabs or two scFvs.

[0098] "Diabodies" refer to a class of bivalent and bispecific antigen-binding molecules. The fragments comprise a heavy chain variable domain (VH) connected 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 are forced to pair with the complementary domains of another chain and generate two antigen-binding sites.

[0099] "Bispecific T cell engager" refers to a type of bispecific antibody that has a first antigen binding portion that binds to a T cell (e.g., by binding CD3) and a second antigen binding portion that binds a different antigen (e.g., a tumor antigen).

[0100] "VHH antibody", "nanobody" or "single domain antibody" refers to an antigen-binding portion consisting of a single monomeric variable antibody domain.

[0101] "Transferrin" is an iron transport protein that can be used in fusion proteins to extend the half-life. The half-life of human transferrin in serum is 12 days.

[0102] As used herein, "cytokine" refers to a class of small (<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 (CC motif) ligand 2 (CCL2) and chemokine (CC motif) ligand 19 (CCL19).

[0103] As used herein, "half-life extension molecule" refers to a molecule that, when linked (e.g., covalently linked) to a second molecule, extends the half-life of the second molecule. Examples of half-life extension molecules include Fc domains, human serum albumin (HSA), HSA binding molecules, polyethylene glycol (PEG), and polypropylene glycol (PPG).

[0104] "Human serum albumin" or "HSA" refers to serum albumin found in human blood. The commonly used form of HSA has a molecular weight of 66.5 kDa and a half-life of approximately 20 days. Examples of HSA molecules are disclosed in U.S. Pat. No. 8,143,026 and U.S. Pat. No. 7,189,690, which are incorporated by reference in their entirety.

[0105] "HSA binding molecule" refers to a molecule that binds to human serum albumin (HSA), such as an antigen binding portion having an HSA binding domain.

[0106] "Polyethylene glycol" or "PEG", also known as polyethylene oxide or polyoxyethylene, is a polyether polymer that can be used to extend half-life.

[0107] "Polypropylene glycol" or "PPG", also known as polypropylene oxide, is a polymer of propylene glycol that can be used to extend half-life.

[0108] According to any of the above embodiments, a "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cattle, 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.

[0109] "Regulating an immune response" may include one or more of the following: general increase, increased effector T cell response (e.g., cytotoxicity against tumor cells and virus-infected cells), increased B cell activation, restoration of lymphocyte activation and proliferation, increased IL2 receptor expression, increased T cell response, increased natural killer cell activity or lymphokine-activated killer (LAK) cell activity, reduced response of regulatory T cells to other T cells, etc.

[0110] "Regulatory T cells" or "Treg cells" refer to a specialized type of CD4+ T cells that can inhibit 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 inducing and maintaining peripheral self-tolerance to antigens (including those expressed by tumors). The function, development, and induction of the suppressive properties of Treg cells require IL2.

[0111] "Effector T cells" refers to a population of T cells that respond to stimuli such as IL2. Effector T cells include CD8+ cytotoxic T cells and CD4+ helper T cells. As used herein, effector T cells do not include regulatory T cells.

[0112] "Natural Killer cells" or "NK cells" are components of the innate immune system and are cytotoxic lymphocytes that play a major role in the rejection of tumors and virus-infected cells.

[0113] "Chimeric antigen receptor" or "CAR" is an engineered antigen binding receptor that, when expressed in certain types of immune cells, activates immune cells when antigens bind. CAR typically comprises an extracellular domain containing 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). CAR can be expressed by, for example, T cells or NK cells, and can include an antigen binding portion that targets cancer antigens such as CD19 or ROR1.

[0114] "Treatment," "treating," or "ameliorating" refers to the medical management of a condition, disease, or disorder in a subject (e.g., a patient), which can be therapeutic, prophylactic / preventative, or a combination thereof.

[0115] An "effective amount" or "therapeutically effective amount" can refer to an amount of a therapeutic agent (e.g., an engineered IL2 polypeptide or engineered IL2 fusion polypeptide described herein) that provides a desired physiological change (e.g., an anti-cancer effect). The desired physiological change can be, for example, a reduction in disease symptoms, or a reduction in disease severity, or can be a reduction in disease progression. With respect to cancer, the desired physiological change can include, for example, tumor regression, a reduction in the rate of tumor progression, a reduction in the level of a cancer biomarker, a reduction in symptoms associated with the cancer, the prevention or delay of metastasis, or clinical remission.

[0116] As used herein, the term "inhibit" refers to a reduction in a specific activity (e.g., immune suppression or tumor growth). Unless otherwise specified, an activity is considered to be inhibited if it is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% as measured by methods disclosed herein or known in the art.

[0117] "Checkpoint inhibitors" refer to agents that reduce the activity of immune checkpoint proteins. Checkpoint inhibitors can be antigen binding moieties that bind to immune checkpoint proteins and reduce their 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).

[0118] "Tumor infiltrating lymphocytes" or "TILs" refer to lymphocytes that have been isolated from tumor tissue, manipulated in vitro (e.g., stimulated with cytokines such as interleukin-2), and then infused back into the patient such that the activated TILs return to the tumor site and induce tumor regression.

[0119] "Tumor microenvironment inhibitors" refer to agents that inhibit one or more conditions or cell types that promote tumor growth and are present in the local environment surrounding the tumor. For example, bevacizumab can inhibit the tumor microenvironment by reducing angiogenesis in the tumor microenvironment.

[0120] As used herein, PD-L1 (also known as "programmed 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.

[0121] 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 Sambrook et al., MolecuLar Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory, New York, 2001 and Ausubel et al., Current Protocols in MolecuLar Biology, John Wiley and Sons, Baltimore, MD, 1999.

[0122] Engineered interleukin-2 peptides

[0123] As described above, the IL2 polypeptides of the present disclosure include IL2Rβ-reducing binding agonists having an engineered IL2 receptor β (IL2Rβ) binding region 2. In some embodiments, the IL2Rβ binding region 2 comprises: 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

[0124] X1 comprises a residue selected from R, S, L, N, F, K or T;

[0125] X2 comprises a residue selected from A, F, S, L, R, T, I, H, P or N;

[0126] X3 comprises a residue selected from K, R, T, S, I or P;

[0127] X4 comprises a residue selected from G, D, R, A, Q, H, N, Y or E;

[0128] X5 comprises a residue selected from I, P, T, S, K, F, V or L;

[0129] X6 comprises a residue selected from I, R, V, M, T or L;

[0130] X7 comprises a residue selected from A, R, M, I, S, N, G or S;

[0131] X8 comprises a residue selected from E, N, H, T, K, Y, S, L, V, D or R;

[0132] X9 comprises a residue selected from I, V, A, T, L, T or M;

[0133] X 10 comprises a residue selected from N, G, V, Y, I, W, R, K, Q, A, D, S or D;

[0134] X 11 comprises a residue selected from F, G, V, N, T, I, R, E or A;

[0135] X 12 comprises a residue selected from I, S, R, V, P, G, T, L, M, F or Y;

[0136] X 13 comprises a residue selected from V, I, F, D, P, H, A, V or L;

[0137] X 14comprises a residue selected from L, Q, R, E, P, K, H, W, F or V;

[0138] X 15 comprises a residue selected from A, E, L, K, V, D, Y, R or Q; and

[0139] X 16 Contains a residue selected from L or I.

[0140] For example, in some embodiments, the engineered 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 -KGSETTFMCEYADETATI VEFLNRWITFSQSIISTLT(SEQ ID NO:45), where

[0141] X1 comprises a residue selected from R, S, L, N, F, K or T;

[0142] X2 comprises a residue selected from A, F, S, L, R, T, I, H, P or N;

[0143] X3 comprises a residue selected from K, R, T, S, I or P;

[0144] X4 comprises a residue selected from G, D, R, A, Q, H, N, Y or E;

[0145] X5 comprises a residue selected from I, P, T, S, K, F, V or L;

[0146] X6 comprises a residue selected from I, R, V, M, T or L;

[0147] X7 comprises a residue selected from A, R, M, I, S, N, G or S;

[0148] X8 comprises a residue selected from E, N, H, T, K, Y, S, L, V, D or R;

[0149] X9 comprises a residue selected from I, V, A, T, L, T or M;

[0150] X 10 comprises a residue selected from N, G, V, Y, I, W, R, K, Q, A, D, S or D;

[0151] X 11 comprises a residue selected from F, G, V, N, T, I, R, E or A;

[0152] X 12 comprises a residue selected from I, S, R, V, P, G, T, L, M, F or Y;

[0153] X 13 comprises a residue selected from V, I, F, D, P, H, A, V or L;

[0154] X 14 comprises a residue selected from L, Q, R, E, P, K, H, W, F or V;

[0155] X 15 comprises a residue selected from A, E, L, K, V, D, Y, R or Q; and

[0156] X 16 Contains a residue selected from L or I.

[0157] The engineered IL2 polypeptides disclosed herein do not comprise the wild-type IL2Rβ binding region 2 motif, i.e., SEQ ID NO: 2. The engineered IL2 polypeptides bind to IL2Rβ with reduced 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).

[0158] In some embodiments, the engineered IL2 polypeptide comprises an IL2Rβ binding region 2 selected from the group consisting of RFKALIIEINFIVQLL (SEQ ID NO:4), RSRQLISNMNGIILK L (SEQ ID NO:5), RLTHLRNVIGVILVQL (SEQ ID NO:6), LSLR EPIGNIVTSVRE (SEQ ID NO:7), NRTDLVGDVNATIKAL (SEQ ID NO:8), RNKGILGDISNIVLAL (SEQ ID NO:9), RSREVVSRIDA IILEL (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), NO:16), RNRGLISNISNIVLAL (SEQ ID NO:17), RNRGLISDINNIV LAL (SEQ ID NO:18), RNRGLISNINNIVLAL (SEQ ID NO:19), RNKGLISNINNIVLAL (SEQ ID NO:20), RPRGLIGDISNIVLAL (SEQ ID NO:21), RPKGLISDISNIVLAL (SEQ ID NO: 22), RPKGLIG NISNIVLAL (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: 27) 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), RPRGLISDISNIVL EL (SEQ IDNO: 34), RPRDLISDINNIVLAL (SEQ ID NO: 35), RPRDLISDISVIVLAL (SEQ ID NO: 36), RPRDLISDISNIVLEL (SEQ ID NO: 37), RPRGLISDINVIVLAL (SEQ ID NO: 38), RPRGLISDIN NIVLEL (SEQ ID NO: 39), RPRGLISDISVIVLEL (SEQ ID NO: 40), RPKDLISNISNIVLAL (SEQ ID NO: 41), RPKGLISNINNIVLAL (SEQ ID NO: 42), RPKGLISNISVIVLAL (SEQ ID NO: 43), RPKGLIS NISNIVLEL (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 IDNO: 198), RPRDLISRIDAIVL EL (SEQ ID NO: 199), RNRGLIGNINNIVLEL (SEQ ID NO: 200), RPKGLISEINNIVLEL (SEQ ID NO: 201) and RPKGLISRINNIVLEL (SEQ ID NO: 202).

[0159] In some embodiments, the engineered IL2 polypeptide comprises an IL2Rβ binding region 2 selected from the group consisting of RFKALIIEINFIVQLL (SEQ ID NO:4), RSRQLISNMNGIILK L (SEQ ID NO:5), RLTHLRNVIGVILVQL (SEQ ID NO:6), RNK GILGDISNIVLAL (SEQ ID NO:9), RSREVVSRIDAIILEL (SEQ ID NO:10), RPRGLISNIVLAL (SEQ ID NO:11), RPRGLIGDINN IVLAL (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:25), RNKGLISDINNIVLAL (SEQ ID NO:26), RNKGLISDINNIVLAL (SEQ ID NO:27), RNKGLISDINNIVLAL (SEQ ID NO:28), RNKGLISDINNIVLAL (SEQ ID NO:29), RNKGLISDINNIVLAL (SEQ ID NO:30), RNKGLISDINNIVLAL (SEQ ID NO:31), RNKGLISDINNIVLAL (SEQ ID NO:32), RNKGLISDINNIVLAL (SEQ ID NO:33), RNKGLISDINNIVLAL (SEQ ID NO:34), RNKGLISDINNIVLAL (SEQ ID NO:35), RNKGLISDINNIVLAL (SEQ ID NO:36), RNKGLISDINNIVLAL (SEQ ID NO:37), NO: 29), RPKGLISNISVIVLEL (SEQ ID NO: 194), RPKGLISNISVIV LEL (SEQ ID NO: 195), RPRDLISNISNIVLEL (SEQ ID NO: 196), RPKGLISNINNIVLEL (SEQ ID NO: 197), RPKGLISDINNIVLEL (SEQ ID NO:198), RPRDLISRIDAIVLEL (SEQ ID NO:199), RNRG LIGNINNIVLEL (SEQ ID NO:200), RPKGLISEINNIVLEL (SEQ ID NO:201) and RPKGLISRINNIVLEL (SEQ ID NO:202).

[0160] In some embodiments, the engineered IL2 polypeptide has reduced affinity for IL2Rβ compared to wild-type IL2 or IL2 having a sequence of SEQ ID NO: 171. In certain embodiments, the K of the engineered IL2 polypeptide binding to IL2Rβ is D Compared with wild-type IL2 or IL2 having the sequence of SEQ ID NO: 171, the K of the engineered IL2 polypeptide binding to IL2Rβ is reduced by at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or at least 30-fold. DCompared with wild-type IL2 or IL2 having the sequence of SEQ ID NO: 171, the affinity of the engineered IL2 polypeptide for IL2Rβ is reduced by at least 1 / 10, at least 1 / 15, at least 1 / 20, at least 1 / 25 or at least 1 / 30, compared with wild-type IL2 or IL2 having the sequence of SEQ ID NO: 171. The assay for detecting IL2 binding may include ELISA or surface plasmon resonance (SPR) detection.

[0161] In some embodiments, the engineered IL2 polypeptide has a reduced affinity for IL2 receptor alpha (IL2Rα) compared to wild-type IL2 or IL2 having the sequence of SEQ ID NO: 171. In certain embodiments, the engineered IL2 polypeptide has a reduced affinity for IL2Rα compared to wild-type IL2 or IL2 having the sequence of SEQ ID NO: 171 by at least 5%, at least 10%, at least 15%, or at least 20%. In some embodiments, the engineered IL2 polypeptide has no detectable binding to IL2Rα compared to wild-type IL2 or IL2 having the sequence of SEQ ID NO: 171. The assay for detecting IL2 binding may include ELISA or SPR detection.

[0162] In some embodiments, the engineered IL2 polypeptide has a similar affinity for IL2Rα as compared to wild-type IL2. In certain embodiments, the affinity of the engineered IL2 polypeptide for IL2Rα differs from the affinity of wild-type IL2 for IL2Rα by no more than + 20%, not more than ±15%, not more than ±10% or not more than ±5%.

[0163] Some embodiments of the present disclosure provide engineered IL2 polypeptides comprising an engineered IL2Rα binding region 1. The engineered IL2Rα binding region 1 may comprise a substitution selected from the group consisting of a substitution at position K35, a substitution at R38, a substitution at F42, a substitution at Y45, or a combination thereof. In some embodiments, the engineered IL2 polypeptide binds to IL2Rα with binding kinetics reduced by at least 1 / 2 compared to wild-type IL2.

[0164] In some embodiments, the engineered IL2 polypeptide may comprise a substitution at position K35. In some embodiments, the substitution at position K35 comprises a non-basic residue. In some embodiments, the substitution at position K35 comprises an uncharged residue or an acidic residue. In some embodiments, the substitution at position K35 is selected from the group consisting of: K35G, K35L, K35S, K35V, K35D, K35E, and K35C.

[0165] In some embodiments, the engineered IL2 polypeptide comprises a substitution at position R38. In some embodiments, the substitution at position R38 comprises a non-basic residue. In some embodiments, the substitution at position R38 comprises 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.

[0166] In some embodiments, the engineered IL2 polypeptide may comprise a substitution at position F42. In some embodiments, the substitution at position F42 comprises an uncharged residue. In some embodiments, the substitution at position F42 comprises a basic residue. In some embodiments, the substitution at position F42 is selected from the group consisting of: F42A, F42R, F42G, F42I, F42L, F42P, and F42H.

[0167] In some embodiments, the engineered IL2 polypeptide may comprise a substitution at position Y45. In some embodiments, the substitution at position Y45 comprises an uncharged residue. In some embodiments, the substitution at position Y45 comprises an uncharged polar residue or an uncharged non-polar residue. In some embodiments, the Y45 substitution is Y45S, Y45P, Y45A, Y45V, Y45C, Y45T, and Y45F.

[0168] In some embodiments, the engineered IL2 polypeptide may comprise a substitution at position K35 and a substitution at position R38. In some embodiments, the engineered IL2 polypeptide comprises a K35G substitution and a R38E substitution.

[0169] In some embodiments, the engineered IL2 polypeptide may comprise a substitution at position K35 and a substitution at position F42. In some embodiments, the engineered IL2 polypeptide comprises a K35S substitution and a F42G substitution.

[0170] In some embodiments, the engineered IL2 polypeptide may comprise a substitution at position K35, a substitution at position R38, and a substitution at position F42. In some embodiments, the engineered IL2 polypeptide comprises a K35L substitution, a R38D substitution, and a F42R substitution.

[0171] In some embodiments, the engineered IL2 polypeptide may comprise a substitution at position R38 and a substitution at position Y45S. In some embodiments, the engineered IL2 polypeptide comprises an R38D substitution and a Y45S substitution. In some embodiments, the engineered IL2 polypeptide comprises an R38V substitution and a Y45S substitution.

[0172] In some embodiments, the engineered IL2 polypeptide binds to IL2Rα with at least 10-fold reduced binding kinetics compared to wild-type IL2.

[0173] In some embodiments, the engineered IL2 is also modified to include a T3A substitution. The T3A substitution can be made for the wild-type sequence. In addition, the T3A substitution can be made for the engineered IL2 sequence disclosed herein. In some embodiments, the engineered IL2 is also modified to include a C125S substitution. The C125S substitution can be made for the wild-type sequence. In addition, the C125S substitution can be made for the engineered IL2 sequence disclosed herein. In some cases, the engineered IL2 sequence also includes T3A and C125S modifications.

[0174] In some embodiments, the engineered 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 residues outside the 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 engineered 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 residues outside the IL2Rβ binding region 2 of SEQ ID NO: 171 (i.e., residues 1-80 and 97-133), and binds to IL2Rβ.

[0175] In some embodiments, the engineered 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 engineered 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 residues outside of IL2Rβ binding region 2 (i.e., residues 1-80 and 97-133) of any one of SEQ ID NOs: 46-102, 147-169 and / or 203-211, and binds to IL2Rβ.

[0176] In some embodiments, the engineered IL2 polypeptide is selected from any one of SEQ ID NOs: 46-102, 147-169 and / or and 203-211, optionally including (or excluding) a C-terminal histidine tag. In some embodiments, the C-terminal histidine tag is replaced by a linker such as a Gly-Ser linker.

[0177] Engineered IL2 fusion peptides

[0178] Some embodiments of the present disclosure provide engineered IL2 fusion polypeptides. The engineered IL2 fusion polypeptides may include engineered IL2 polypeptides as described herein (see, e.g., SEQ ID NOs: 3-102, 147-169, 203-211 and Tables 2, 5 and 7), and at least one additional molecule covalently linked to the engineered IL2 polypeptide by a peptide bond or other chemical linkage. In certain embodiments, the engineered IL2 fusion polypeptides include engineered IL2 polypeptides that share 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 residues outside the 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 bind to IL2Rβ. In some embodiments, the engineered IL2 fusion polypeptide comprises a first polypeptide sequence and a second polypeptide sequence, wherein the first polypeptide sequence comprises an engineered IL2 polypeptide as described herein. In some embodiments, the second polypeptide sequence of the engineered IL2 fusion polypeptide comprises a polypeptide having a length of 5-500 amino acids, 5-400 amino acids, 5-300 amino acids, 5-250 amino acids, 5-200 amino acids, 50-500 amino acids, 50-400 amino acids, 50-300 amino acids, 50-250 amino acids, or 50-200 amino acids. In some embodiments, the second polypeptide sequence of the engineered IL2 fusion polypeptide comprises a polypeptide having a length of at least 5, at least 20, at least 40, at least 60, at least 80, at least 100, at least 150, at least 200, at least 250, or at least 300 amino acids. Non-limiting examples of polypeptides that may comprise the second polypeptide sequence of the fusion protein include Fc domains, antibodies, antigen binding moieties, cytokines, half-life extension molecules, tags or marker polypeptides, targeting domains, transport molecules, immunotoxins (e.g., diphtheria toxin), NKG2D, linker sequences, chemically linked small molecules, nucleic acids, pegylation, or any combination thereof. In some embodiments, the second polypeptide sequence includes a ligand or scaffold protein.

[0179] In certain embodiments, the engineered IL2 fusion polypeptide comprises an engineered 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 a specific embodiment, 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 relative 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 a LALAPG mutation. The LALAPG mutation refers to L234A, L235A and P329G changes 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, which are hereby incorporated by reference). In some embodiments, the IgG heavy chain constant region comprises a knob mutation or a hole mutation. Knob-into-hole mutation is a modification of the IgG heavy chain constant region that allows heterodimerization of IgG heavy chain constant regions comprising a knob mutation and a hole mutation, respectively. The knob mutation or the hole mutation allows the preferential formation of heterodimers with low levels of homodimer contaminants in vitro. Knob-into-hole mutations are disclosed in Merchant et al. Nat. Biotechnol. 16: 677–681, 1998; and Wei et al., Oncotarget. 8(31): 51037-51049, 2017, which are hereby incorporated by reference. Examples of knob mutations include S354C, T366W, and K409A mutations in the IgG heavy chain constant region.Examples of hole mutations include Y349C, T366S, L368A, F405K and Y407V mutations in the IgG heavy chain constant region. In some embodiments, the IgG heavy chain constant region comprises a LALAPG mutation and a hole mutation or a knob mutation. For example, in some embodiments, the IgG heavy chain constant region may comprise L234A, L235A, P329G, S354C, T366W and K409A mutations. In some embodiments, the IgG heavy chain constant region may comprise L234A, L235A, P329G, Y349C, T366S, L368A, F405K and Y407V mutations. In certain embodiments, the IgG heavy chain constant region comprises 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 comprising one or more mutations selected from S354C, T366W, and K409A as numbered according to the EU numbering system relative to SEQ ID NO: 137 and a second polypeptide comprising a second IgG heavy chain constant region comprising one or more mutations selected from Y349C, T366S, L368A, F405K, and Y407V as numbered according to the EU numbering system relative to SEQ ID NO: 137. 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 numbered according to the EU numbering system relative to SEQ ID NO: 137; 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 numbered according to the EU numbering system relative to SEQ ID NO: 137. In some embodiments, the IgG heavy chain constant region comprises at least one amino acid residue modification to extend serum half-life. Representative modifications to the IgG heavy chain constant region are described in US Pat. No. 7,317,091, US Pat. No. 8,735,545, US Pat. No. 7,371,826, US Pat. No. 7,670,600, and US Pat. No. 9,803,023.

[0180] In some embodiments, the engineered IL2 fusion polypeptide includes an engineered IL2 polypeptide connected to a fusion partner such as an IgG heavy chain constant region. A variety of joints are known in the art and can be used to covalently connect the engineered IL2 described herein to a fusion partner, such as an Fc region. "Joint", "joint sequence" means in this article a molecule or group of molecules (such as a monomer or polymer) that connects two molecules and is generally used to place two molecules in a preferred configuration. The joint may include amino acid residues that provide flexibility. Therefore, the joint peptide may mainly include the following amino acid residues: Gly, Ser, Ala or Thr. The length of the joint peptide should be sufficient to connect the two molecules in such a way that they take the correct conformation relative to each other so that they retain the desired activity. For this purpose, a suitable length includes at least one but no more than 30 amino acid residues. Preferably, the joint length is about 1 to 30 amino acids, wherein a joint 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 is preferred. Useful linkers include glycine-serine polymers (including, for example, (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. A bivalent fusion polypeptide may refer to a molecular complex comprising two copies of an engineered IL2 polypeptide, which may have the same sequence or different sequences. The molecular complex may be non-covalently bound. For example, a bivalent fusion polypeptide may include two Fc regions bound together by one or more disulfide bridges or by knobs-into-holes chemistry.

[0181] In some embodiments, the fusion polypeptide is a monovalent fusion polypeptide, such as a monovalent engineered IL2-Fc fusion polypeptide. A monovalent fusion polypeptide refers to a fusion polypeptide having one copy of an engineered IL2 polypeptide. In some embodiments, the fusion polypeptide disclosed herein comprises 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%, at least 99% or at least 100% sequence identity to a sequence selected from SEQ ID NOs: 124-128 and / or 190-193.

[0182] In some embodiments, the monovalent engineered IL2-Fc fusion polypeptide forms a complex with at least one additional protein. In some embodiments, the monovalent engineered IL2-Fc fusion polypeptide complex includes a first protein comprising a fusion polypeptide as described herein, and a second protein that forms a dimer 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 with a sequence of SEQ ID NO: 123. In certain embodiments, the monovalent engineered IL2-Fc fusion polypeptide complex includes a first polypeptide sequence selected from SEQ ID NO: 124-128 and / or 190-193 and a second polypeptide with a sequence of SEQ ID NO: 123. For example, a monovalent engineered IL2-Fc fusion polypeptide complex may comprise: SEQ ID NOs: 124 and 123; SEQ ID NOs: 125 and 123; SEQ ID NOs: 126 and 123; SEQ ID NOs: 127 and 123; SEQ ID NOs: 128 and 123; SEQ ID NOs: 190 and 123; SEQ ID NOs: 191 and 123; SEQ ID NOs: 192 and 123; and / or SEQ ID NOs: 193 and 123.

[0183] In some embodiments, the fusion polypeptide disclosed herein includes an antigen binding portion. In some embodiments, the antigen binding portion includes an immunoglobulin, a Fab molecule, a scFv, a bispecific T cell conjugate, a double antibody, a single domain antibody or a VHH antibody (nanoantibody). The antigen binding portion can be combined with, 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 dactuximab, and an example of a CD20 antigen binding portion is rituximab.

[0184] In some embodiments, disclosed herein is a protein complex comprising a first polypeptide as a fusion polypeptide as disclosed herein and a second polypeptide or protein complex comprising an antigen binding portion. In certain embodiments, the second polypeptide or protein complex comprises a polypeptide having a sequence of SEQ ID NOs: 103 and 114 or SEQ ID NOs: 103 and 172. In some embodiments, the protein complex comprises a polypeptide having a sequence 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 comprises a polypeptide having the following sequences: 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.

[0185] 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 CC motif chemokine ligand 19 (CCL19). In some embodiments, the additional molecule of the fusion polypeptide is a second engineered IL2 polypeptide described herein.

[0186] 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.

[0187] In some embodiments, the half-life extending molecule comprises polyethylene glycol (PEG) or polypropylene glycol (PPG).

[0188] Bifunctional Protein

[0189] In certain embodiments, bifunctional fusion proteins are disclosed herein. Bifunctional fusion proteins may include engineered IL2 polypeptides disclosed herein and antigen binding moieties. 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 may include an antigen binding moiety comprising an immunoglobulin, a Fab molecule, a scFv, a double antibody, a single domain antibody, or a VHH antibody. The antigen binding moiety may bind, for example, to antigens on the cell surface, such as marker proteins and / or disease markers. The antigen binding moiety may 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 moieties include atezolizumab, avelumab, and durvalumab. Examples of PD-1 antigen binding moieties include nivolumab and pembrolizumab. In some embodiments, the bifunctional fusion protein may also include 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 may include a wild-type antibody constant region or a modified antibody constant region. Examples of modified antibody constant regions are provided herein, and the examples 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.

[0190] 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 comprise a Fab formed by an antibody heavy chain and a light chain. In some embodiments, the antigen binding portion that binds to PD-L1 can comprise an scFv.

[0191] In certain embodiments, the bifunctional fusion protein comprises an antigen binding portion that binds to PD-L1, the antigen binding portion comprising: (i) a heavy chain variable domain (VH) comprising CDR1, CDR2, and CDR3, the CDR1 comprising the sequence of SEQ ID NO: 141, the CDR2 comprising the sequence of SEQ ID NO: 142, and the CDR3 comprising the sequence of SEQ ID NO: 143; and (ii) a light chain variable domain (VL) comprising CDR1, CDR2, and CDR3, the CDR1 comprising the sequence of SEQ ID NO: 144, the CDR2 comprising the sequence of SEQ ID NO: 145, and the CDR3 comprising the sequence of SEQ ID NO: 146. In certain embodiments, the antigen binding portion that binds to PD-L1 comprises a light chain and a heavy chain, wherein 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 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 the light chain comprising a polypeptide having the sequence of SEQ ID NO:103.

[0192] In some embodiments, disclosed herein is a protein complex (e.g., an immunoconjugate) comprising a bifunctional fusion protein disclosed herein and a second antigen binding portion. 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 comprises: (i) a heavy chain variable domain (VH) comprising CDR1, CDR2, and CDR3, the CDR1 comprising a sequence of SEQ ID NO: 141, the CDR2 comprising a sequence of SEQ ID NO: 142, and the CDR3 comprising a sequence of SEQ ID NO: 143; and (ii) a light chain variable domain (VL) comprising CDR1, CDR2, and CDR3, the CDR1 comprising a sequence of SEQ ID NO: 144, the CDR2 comprising a sequence of SEQ ID NO: 145, and the CDR3 comprising a sequence of SEQ ID NO: 146. In certain embodiments, the second antigen binding moiety that binds PD-L1 comprises a light chain and a heavy chain, wherein 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 comprises a polypeptide having the following sequence: SEQ ID NOs: 104, 106 and 103; SEQ ID NOs: 104, 107 and 103; SEQ ID NOs: 104, 108 and 103; SEQ ID NOs: 104, 109 and 103; SEQ ID NOs: 104, 110 and 103; SEQ ID NOs: 104, 111 and 103; SEQ ID NOs: 104, 112 and 103; SEQ ID NOs: 104, 113 and 103; SEQ ID NOs: 172, 173 and 103; SEQ ID NOs: 172, 174 and 103; SEQ ID NOs: 172, 175 and 103; SEQ ID NOs: 172, 176 and 103; or SEQ ID NOs: 172, 177 and 103.

[0193] 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 SEQ ID NO: 118-122 or 180-189 or consisting of SEQ ID NO: 118-122 or 180-189. The bifunctional fusion protein may also include a light chain sequence comprising a polypeptide having a sequence of SEQ ID NO: 117 or SEQID NO: 178. The bifunctional fusion protein may be included in a protein complex (e.g., an immunoconjugate). In some embodiments, the protein complex may include a polypeptide having the following sequence: SEQ ID NO: 116, 117, and 118; SEQ ID NO: 116, 117, and 119; SEQ ID NO: 116, 117, and 120; SEQ ID NO: 116, 117, and 121; or SEQ ID NO: 116, 117, and 122. In some embodiments, the protein complex may include a polypeptide having the following sequence: 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.

[0194] In some embodiments, disclosed herein is a protein complex comprising a polypeptide having the following sequence: SEQ ID NOs: 133, 134, and 135; or SEQ ID NOs: 133, 134, and 136.

[0195] Vectors and methods for making engineered IL2 polypeptides

[0196] In a further embodiment, polynucleotides encoding the engineered IL2, fusion polypeptide, protein complex or bifunctional fusion protein described above and herein are provided. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a polypeptide or polypeptide combination selected from SEQ ID NO: 3-169, or a nucleic acid sequence encoding a polypeptide or polypeptide combination selected from SEQ ID NO: 3-211 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 or polynucleotides are selected from the group consisting of DNA, cDNA, RNA or mRNA. An expression cassette or multiple expression cassettes are also provided, which include one or more regulatory sequences operably connected to the polynucleotide or multiple polynucleotides described above and herein.

[0197] In some embodiments, a vector comprising one or more polynucleotides as described herein, or an expression cassette is provided. 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 from a family of viruses selected from the group consisting of: Adenoviridae (e.g., adenovirus), Arenaviridae (e.g., lymphocytic choriomeningitis mammalian arenavirus), Cali mammalian arenavirus (also known as Pichinde mammalian arenavirus), and Cali mammalian arenavirus. 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), and Enteroviridae (e.g., Echovirus). Also provided are lipid nanoparticles (LNPs) comprising one or more polynucleotides, expression cassettes, or vectors as described above and herein.

[0198] Engineered IL2 polypeptides and / or engineered IL2 fusion polypeptides can be prepared by genetic or chemical methods well known in the art and by methods disclosed in the following examples. Genetic methods can include, for example, site-specific mutagenesis, PCR and gene synthesis of the DNA sequence encoding the polypeptide. Expected nucleotide changes can be verified by sequencing. The nucleotide sequence of natural IL2 has been described by Taniguchi et al. (Nature 302, 305-10 (1983)), and nucleic acids encoding natural human IL2 can be obtained from, for example, the American Type Culture Collection (Rockville Md.).

[0199] The engineered IL2 polypeptide or engineered IL2 fusion polypeptide can be obtained, for example, by recombinant production or solid-state peptide synthesis. For recombinant production, polynucleotides encoding engineered IL2 polypeptides or engineered IL2 fusion polypeptides can be isolated and inserted into one or more vectors for cloning and / or expression in host cells. Such polynucleotides can be easily separated and sequenced by conventional procedures. In certain embodiments, a vector comprising one or more polynucleotides of the present disclosure is provided, such as an expression vector. Methods well known to those skilled in the art can be used to construct an expression vector containing a coding sequence encoding an engineered IL2 polypeptide or an engineered IL2 fusion polypeptide and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, synthetic technology, and in vivo recombination / genetic recombination. See, for example, Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL (4th edition), Cold Spring Harbor Laboratory, NY (2012); and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, NY (1993) described in the technology. The expression vector can be a part of a plasmid, a virus, or can be a nucleic acid fragment. The expression vector includes an expression cassette, and the polynucleotide (i.e., coding region) encoding the engineered IL2 polypeptide or the engineered IL2 fusion polypeptide can be cloned into the expression cassette and operably associated with a promoter and / or other transcription or translation control elements. As used herein, a "coding region" is a part of a nucleic acid composed of codons translated into amino acids. Although "stop codons" (TAG, TGA or TAA) are not translated into amino acids, if present, they can be considered as a part of the coding region, but any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, 5' and 3' untranslated regions, etc., are not a part of the coding region. Two or more coding regions can be present in a single polynucleotide construct, for example, on a single vector, or in a separate polynucleotide construct, for example, on a separate (different) vector. In addition, any vector may contain a single coding region, or may contain two or more coding regions, for example, the vectors disclosed herein may encode one or more polyproteins that are separated into final proteins after translation by proteolytic cleavage or separated into final proteins co-translationally by proteolytic cleavage. In addition, the vectors, polynucleotides or nucleic acids disclosed herein may encode heterologous coding regions that may or may not be fused to the first or second polynucleotide encoding a polypeptide disclosed herein or a variant or derivative thereof.Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as secretory signal peptides or heterologous functional domains. Operable association is when the coding region of a gene product (e.g., a polypeptide) is associated with one or more regulatory sequences in a manner that places the expression of the gene product under the influence or control of one or more regulatory sequences. If the induction of promoter function leads to the transcription of mRNA encoding the desired gene product, and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequence to direct the expression of the gene product or the ability of the DNA template to be transcribed, then the two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated". Therefore, if the promoter is able to affect the transcription of a nucleic acid encoding a polypeptide, then the promoter region will be operably associated with the nucleic acid. The promoter can be a cell-specific promoter that directs a large amount of transcription of DNA only in a predetermined cell. In addition to the promoter, other transcription control elements, such as enhancers, operators, repressors, and transcription termination signals can also be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein. A variety of transcription control regions are known to those skilled in the art. These include, but are not limited to, transcription control regions that function in vertebrate cells, such as, but not limited to, promoters and enhancer segments from cytomegalovirus (e.g., immediate early promoter, linked to intron-A), simian virus 40 (e.g., early promoter), and retroviruses (e.g., Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone, and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Other suitable transcription control regions include tissue-specific promoters and enhancers and inducible promoters (e.g., promoters inducible by tetracycline). Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly internal ribosome entry sites or IRES, also referred to as CITE sequences). The expression cassette may also include other features, such as an origin of replication, and / or chromosomal integration elements, such as retroviral long terminal repeats (LTRs), or adeno-associated virus (AAV) inverted terminal repeats (ITRs).

[0200] The polynucleotides and nucleic acid coding regions of the present disclosure may be associated with additional coding regions encoding secretory peptides or signal peptides that direct the secretion of the polypeptides encoded by the polynucleotides of the present disclosure. For example, if secretion of an engineered IL2 polypeptide or an engineered 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 engineered IL2 polypeptide or the engineered IL2 fusion polypeptide. It is known to those of ordinary skill in the art that polypeptides secreted by vertebrate cells typically have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce a secreted or "mature" form of the polypeptide. For example, natural human IL2 is translated with a 20 amino acid signal sequence at the N-terminus of the polypeptide, which is subsequently cleaved to produce a mature 133 amino acid human IL2. In some embodiments, a natural signal peptide, such as an IL2 signal peptide or an immunoglobulin heavy chain or light chain signal peptide, or a functional derivative of the sequence is used, which retains the ability to direct the secretion of a polypeptide operably associated therewith. In some embodiments, the signal peptide comprises or consists of SEQ ID NO: 212.

[0201] In some embodiments, the polynucleotide encoding the engineered IL2 polypeptide or engineered IL2 fusion polypeptide further comprises a DNA sequence encoding a sequence facilitating purification (e.g., a histidine tag) or for labeling the engineered IL2 polypeptide or engineered IL2 fusion polypeptide within or at the end of the polynucleotide encoding the engineered IL2 polypeptide or engineered IL2 fusion polypeptide.

[0202] In certain embodiments, a host cell comprising one or more polynucleotides encoding engineered IL2 polypeptides or engineered IL2 fusion polypeptides is provided. In certain embodiments, the host cell comprises one or more vectors encoding engineered IL2 polypeptides or engineered IL2 fusion polypeptides. The host cell can be any type of cell system that can be used to produce engineered IL2 polypeptides or engineered IL2 fusion polypeptides. Such cells can be transfected or transduced with specific expression vectors encoding engineered IL2 polypeptides or engineered IL2 fusion polypeptides, and a large number of cells containing vectors can be grown for inoculating large-scale fermentation tanks to obtain sufficient numbers of coding engineered IL2 polypeptides or engineered IL2 fusion polypeptides for clinical applications. Suitable host cells include prokaryotic microorganisms, such as Escherichia coli (E.Coli), or various eukaryotic cells, or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, etc. For example, polypeptides can be produced in bacteria, particularly when glycosylation is not required. After expression, the polypeptide can be separated from the bacterial cell in a soluble fraction and can be further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable for cloning or expression hosts of vectors encoding antibodies, including fungi and yeast strains whose glycosylation pathways have been "humanized" to produce polypeptides with partial or complete human glycosylation patterns. Suitable host cells for expressing (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plants and insect cells. Many baculovirus strains have been identified that can be used in combination with insect cells, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, for example, U.S. Patents Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 (which describe PLANTIBODIES for producing antibodies in transgenic plants). TMTechniques). Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 cell line transformed by SV40 (COS-7); human embryonic kidney line (such as 293 or 293T cells described in, e.g., Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (such as, e.g., TM4 cells described in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (such as, e.g., described in Mather et al., Annals NY Acad Sci 383, 44-68 (1982)), MRC 5 cells and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr - CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for protein production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). Host cells include cultured cells, e.g., mammalian cultured cells, yeast cells, insect cells, bacterial cells and plant cells, to name a few examples, also including cells contained in 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 a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell).

[0203] Standard techniques for expressing foreign genes in these systems are known in the art. Cells expressing an engineered IL2 polypeptide of a heavy or light chain fused to an antigen binding portion (such as an antibody) can be engineered to also express the other antibody chain so that the engineered IL2 fusion polypeptide expressed includes an antibody having both a heavy chain and a light chain.

[0204] In some embodiments, methods of producing an engineered IL2 polypeptide, an engineered IL2 fusion polypeptide, or a protein complex as described herein are provided. In some embodiments, the method comprises culturing a host cell comprising a polynucleotide encoding an engineered IL2 polypeptide, an engineered IL2 fusion polypeptide, or a protein complex provided herein under conditions suitable for the engineered IL2 polypeptide, the engineered IL2 fusion polypeptide, or the protein complex, and optionally recovering and / or purifying the engineered IL2 polypeptide, the engineered IL2 fusion polypeptide, or the protein complex from the host cell (or the host cell culture medium, e.g., if the host cell secretes the polypeptide).

[0205] In some embodiments, disclosed herein are transgenic feeder cells comprising an engineered IL2 polypeptide, fusion polypeptide, protein complex, bifunctional fusion protein, polynucleotide or vector disclosed herein. Examples of feeder cells include K562 cells, 3T3 cells, fibroblasts (e.g., MEFs) or antigen presenting cells.

[0206] In some embodiments, disclosed herein is a method for in vitro or ex vivo expansion of immune cells, including contacting the immune cell population with an effective amount of an engineered IL2 polypeptide, fusion polypeptide, protein complex, or bifunctional fusion protein disclosed herein under conditions sufficient to promote the expansion of the immune cell population, and culturing the immune cell population for a sufficient time to at least double the number of immune cells. For example, conditions sufficient to promote expansion can include one or more agents or ligands capable of activating the intracellular signaling domain of the TCR complex, such as anti-CD3 antibodies or binding domains, anti-CD28 antibodies or binding domains, or combinations thereof. In some embodiments, the method for expanding immune cells also includes culturing immune cell populations with IL-7, IL-15, IL-21, or any combination thereof. In some embodiments, the immune cell population is cultured in the presence of a feeder cell population (e.g., a transgenic feeder cell disclosed herein).

[0207] Pharmaceutical composition

[0208] Provided herein are pharmaceutical compositions comprising engineered IL2 polypeptides, engineered IL2 fusion polypeptides, bifunctional fusion proteins, protein complexes, polynucleotides, carriers or cells and pharmaceutically acceptable diluents, excipients or carriers as described herein. In some embodiments, the pharmaceutical composition comprises engineered IL2 polypeptides, engineered IL2 fusion polypeptides, bifunctional fusion proteins, protein complexes, polynucleotides, carriers or cells and other therapeutic agents (e.g., conjoint therapy) disclosed herein. Non-limiting examples of such therapeutic agents are described herein below. Pharmaceutical compositions can be prepared in a conventional manner using one or more pharmaceutically acceptable carriers, the carriers including excipients and adjuvants, the excipients and adjuvants help to process engineered IL2 polypeptides, engineered IL2 fusion polypeptides, bifunctional fusion proteins, protein complexes, polynucleotides, carriers or cells into pharmaceutically usable preparations. Appropriate preparations depend on the selected route of administration. Any pharmaceutically acceptable techniques, carriers and excipients are suitable for formulating the pharmaceutical compositions described herein: Remington: The Science and Practice of Pharmacy, 19th Edition (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, HA and Lachman, L., eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980 and Pharmaceutical Dosage Forms and Drμg Delivery Systems, 7th Edition (Lippincott Williams & Wilkins 1999). Examples of IL-2 compositions are described in U.S. Pat. Nos. 4,604,377 and 4,766,106, which are incorporated herein by reference.

[0209] As used herein, "pharmaceutically acceptable carriers" and "physiologically acceptable carriers" are used interchangeably and include any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, such and similar materials and combinations thereof as known to those of ordinary skill in the art, and are molecular entities and compositions that are generally non-toxic to recipients at the dosages and concentrations employed, i.e., do not produce adverse, allergic or other untoward reactions when administered to animals such as humans under appropriate circumstances (see, e.g., Remington's Pharmaceutical Sciences, 18th Edition Mack Printing Company, 1990, pp. 1289-1329, which is incorporated herein by reference). The use of carriers in therapeutic or pharmaceutical compositions is contemplated except in cases where any conventional carrier is incompatible with the active ingredient.

[0210] The pharmaceutical composition may contain different types of carriers depending on whether it is administered in solid, liquid or aerosol form, and whether sterility is required for administration routes such as injection. The engineered IL2 polypeptides or engineered IL2 fusion polypeptides described herein (and any additional therapeutic agent) can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrasplenicly, intrarenally, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intracapsularly, transmucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, by catheter, lavage directly into the local perfusion bathing target cells, in a cream, in a lipid composition (e.g., liposomes), or by other methods as known to one of ordinary skill in the art or any combination of the foregoing methods (see, e.g., Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, incorporated herein by reference). Parenteral administration, particularly intravenous injection, is most commonly used to administer polypeptide molecules, such as the engineered IL2 polypeptides or engineered IL2 fusion polypeptides described herein.

[0211] Provided herein are transgenic immune cells expressing engineered IL2 polypeptides, fusion polypeptides, protein complexes, bifunctional fusion proteins, polynucleotides or vectors disclosed herein. In some embodiments, transgenic immune cells are CD4+T cells, CD8+T cells, γδT cells, NK cells, T regulatory cells or any combination thereof. In some embodiments, transgenic immune cells also include chimeric antigen receptors (CAR).

[0212] In some embodiments, CAR comprises an antigen binding domain capable of binding to a target / antigen produced by a cancer cell. For example, CAR comprises an antigen binding domain capable of binding to the following antigens: CD19; CD123; CD22; CD30; CD171; CS-1 (also known as CD2 subgroup 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); Testisin or PRSS21; vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine protein kinase ERBB2 (Her2 / neu); cell surface-associated mucin 1 (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostatic enzyme; prostatic 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 (macropin) subunit, beta type, 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein composed of breakpoint cluster region (BCR) and Abelson murine leukemia virus oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; 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 β; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); tight junction protein 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRCS D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide moiety of globoH glycoceramide (GloboH); mammary differentiation antigen (NY-BR-1); uroplaque protein 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor β3 (ADRB3); ubiquitin ligase 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, K 9 locus (LY6K); olfactory receptor 51E2 (OR51E2); TCRγ alternate 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 located on chromosome 12p 6 (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (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 mutants; prostaglandins; 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 breakpoints; melanoma apoptosis inhibitor (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian granulocytoma viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P4501B1 (CYP1B1);CCCTC-binding factor (zinc finger protein)-like (brother of BORIS or imprinted site regulator), squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosomal enzyme binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchoring protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); kidney ubiquitous 1 (RU1); kidney ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPVE6); 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); mucin-like hormone receptor-like 2 containing an EGF-like module (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRLS); and immunoglobulin lambda-like polypeptide 1 (IGLL1). ;

[0213] In some embodiments, the transgenic immune cell produces an engineered IL2 polypeptide, fusion polypeptide, protein complex, or bifunctional fusion protein that is secreted by the transgenic immune cell. In other embodiments, the engineered IL2 polypeptide, fusion polypeptide, protein complex, or bifunctional fusion protein comprising a transmembrane domain or a cell surface anchoring molecule is expressed and / or localized on the cell surface. In some embodiments, the transgenic immune cell is an armored CAR T cell.

[0214] Methods of treatment and use

[0215] In some embodiments of the present disclosure, provided herein is a method for regulating the immune response of a subject in need thereof, comprising administering to a subject a therapeutically effective amount of an engineered IL2 polypeptide, an engineered IL2 fusion polypeptide, a bifunctional fusion protein, a protein complex, a polynucleotide, a vector, a cell, or a pharmaceutical composition thereof as described herein (see, e.g., SEQ ID NO: 3-102, 147-169, 203-211 and Tables 2, 5, and 7). In certain embodiments, regulating the immune response comprises at least one of: enhancing effector T cell activity, enhancing NK cell activity, and inhibiting regulatory T cell activity. In some embodiments of the present disclosure, provided herein is an engineered 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 for regulating the immune response of a subject in need thereof.

[0216] In some embodiments of the present disclosure, the embodiment is a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an engineered IL2 polypeptide, engineered IL2 fusion polypeptide, protein complex, bifunctional fusion protein, polynucleotide, vector, cell, or pharmaceutical composition thereof as described herein. In some embodiments, the engineered IL2 polypeptide, fusion polypeptide, protein complex, bifunctional fusion protein, polynucleotide, vector, cell, and / or pharmaceutical composition described herein are used in a method of treating a disease in a subject. Non-limiting examples of diseases or disorders contemplated in the method include proliferative disorders (such as cancer) and immunosuppression.

[0217] In some embodiments, the embodiment is a method of treating a proliferative disorder, comprising administering to a subject a therapeutically effective amount of an engineered IL2 polypeptide as described herein, an engineered IL2 fusion polypeptide, a protein complex, or a pharmaceutical composition thereof. In some embodiments, the proliferative disorder is cancer. In some embodiments, the cancer comprises breast cancer, pancreatic cancer, lung cancer, glioblastoma, renal cell carcinoma, lymphoma, leukemia, head and neck cancer, liver cancer, gastric cancer, colon / colorectal cancer, kidney cancer, bladder cancer, or melanoma. Non-limiting examples of cancer include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, glioblastoma, prostate cancer, hematological malignancies, skin cancer, squamous cell carcinoma, skin cancer, melanoma, bone cancer, renal cell carcinoma, and kidney cancer. Examples of hematological malignancies include leukemias, lymphomas (e.g., non-Hodgkin's lymphoma or Hodgkin's lymphoma), multiple myeloma, myelodysplastic syndrome, myeloproliferative neoplasms (e.g., essential thrombocythemia, myelofibrosis, and polycythemia vera). Precancerous conditions or lesions and cancer metastasis are also included. Other cell proliferative disorders include, but are not limited to, tumors located in the abdomen, bones, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary glands, testicles, ovaries, thymus, thyroid gland), eyes, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thoracic region, and genitourinary system. Similarly, other cell proliferative disorders such as hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sezary syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, amyloidosis and aplastic anemia, as well as any other cell proliferative disease other than neoplasia located in the organ systems listed above, may also be treated.

[0218] In some embodiments, the method for treating or regulating an immune response also includes administering to the subject at least one additional therapeutic agent (e.g., combination therapy) of a therapeutically effective amount. In certain embodiments, the additional therapeutic agent is an anticancer agent. Examples of anticancer agents include checkpoint inhibitors (e.g., anti-PD1 antibodies), chemotherapeutic agents, agents that inhibit tumor microenvironment, cancer vaccines (e.g., Sipuleucel-T), oncolytic viruses (e.g., talimogenelaherparepvec), immune cells expressing chimeric antigen receptors (CARs), and tumor infiltrating lymphocytes (TILs). In certain embodiments, the additional therapeutic agent is a molecule including an antigen binding moiety. In certain specific embodiments, the antigen binding moiety is selected from a single domain antibody, a Fab molecule, a scFv, a double antibody, a nanobody, a bispecific T cell conjugate, or an immunoglobulin. In certain embodiments, the antigen binding moiety is specific to tumor antigens (e.g., carcinoembryonic antigen, fibroblast activation protein-α, CD20) or checkpoint proteins (e.g., CTLA-4, PD-1, or PD-L1). In some embodiments, additional therapeutic agents are vaccines, gene therapy, cell therapy or any combination thereof. In some embodiments, additional therapeutic agents include immune cells expressing chimeric antigen receptors, immune cells expressing engineered T cell receptors or tumor infiltrating lymphocytes. In certain embodiments, engineered IL2 polypeptides, engineered IL2 fusion polypeptides or protein complexes can be transfected, transduced or otherwise introduced into immune cells expressing chimeric antigen receptors, immune cells expressing engineered T cell receptors or polynucleotide encodings in tumor infiltrating lymphocytes. In such embodiments, immune cells can be cells expressing armored chimeric antigen receptors (armored chimeric antigen receptor-expressing cell). In certain embodiments, immune cells are T cells (e.g., CD4 and / or CD8 T cells) and / or NK cells. Polynucleotides can also encode secretion signals (e.g., natural IL2 signal sequences or signal sequences derived from another protein) directly upstream of engineered IL2 polypeptide coding sequences to allow cells to secrete engineered IL2 polypeptides or engineered IL2 fusion polypeptides.

[0219] Suitable routes of administration include, but are not limited to, intravenous, parenteral, transdermal, oral, rectal, aerosol, ocular, pulmonary, transmucosal, vaginal, aural, nasal, and topical administration. In addition, parenteral delivery includes, by way of example only, intramuscular, subcutaneous, intravenous, intramedullary injection, as well as intradural, direct intraventricular, intraperitoneal, intralymphatic, and intranasal injection.

[0220] In certain embodiments, engineered IL2 polypeptides, IL2 fusion polypeptides or protein complexes are administered systemically. In certain embodiments, engineered IL2 polypeptides, engineered IL2 fusion polypeptides or protein complexes described herein are administered locally rather than systemically, for example, by injecting engineered IL2 polypeptides, engineered IL2 fusion polypeptides or protein complexes directly into organs, tissues or tumors. In some embodiments, long-acting preparations are administered by implantation (e.g., subcutaneous or intramuscular) or by intramuscular injection. In addition, in some embodiments, the drug is delivered in a targeted drug delivery system, for example, in a liposome coated with an organ-specific or cell-specific antibody. In such embodiments, the liposome is targeted to an organ and selectively absorbed by the organ. In some embodiments, engineered IL2 polypeptides, engineered IL2 fusion polypeptides or protein complexes described herein are provided in the form of a rapid release preparation, an extended or sustained release preparation, an intermediate release preparation or a depot preparation. In some embodiments, engineered IL2 polypeptides, engineered IL2 fusion polypeptides or protein complexes described herein are administered locally.

[0221] The appropriate dosage of the engineered IL2 polypeptide, engineered IL2 fusion polypeptide or protein complex (used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease or condition, the route of administration, the subject's weight, the severity and progression of the disease, whether the polypeptide is administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the subject's clinical history and response to the engineered IL2 polypeptide, engineered IL2 fusion polypeptide or protein complex, and the judgment of the attending physician. The physician responsible for administration will be able to determine the concentration of one or more active ingredients in the composition and the appropriate dosing for the subject to be treated. Various dosing regimens including, but not limited to, single or multiple administrations at different time points, bolus administrations, and pulse infusions are contemplated herein.

[0222] The single application range of engineered IL2 polypeptide can be about 50,000IU / kg to about 1,000,000IU / kg or more engineered IL2 polypeptide. This can be repeated several times a day (for example, 2-4 times a day), for several days (for example, 3-5 consecutive days), and then can be repeated once or many times after a period of rest (for example, 7-14 days). Therefore, the therapeutically effective amount can include only one application or multiple applications within a period of time (for example, about 10 to 30 single applications in about 5-20 days, each time giving about 600,000IU / kg of IL2). When applied in the form of fusion polypeptide or protein complex, the therapeutically effective amount of engineered IL2 fusion polypeptide or protein complex can be lower than non-fusion engineered IL2 polypeptide (for example, 10,000IU / kg to about 600,000IU / kg). Similarly, as described above, the engineered IL2 fusion polypeptide can be applied to the patient once or by a series of treatments.

[0223] In certain embodiments, the daily dose of the engineered IL2 polypeptide, engineered 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 longer (depending on the condition), treatment will generally be maintained until the desired suppression of disease symptoms occurs (e.g., tumor shrinkage). In some embodiments, a single dose of the engineered IL2 polypeptide, engineered IL2 fusion polypeptide, or protein complex ranges from about 0.005 mg / kg to about 10 mg / kg. In some embodiments, the dosage can be 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 ranges derivable therefrom. In non-limiting examples of ranges derivable from the numbers listed herein, a range of about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 micrograms / kg / body weight to about 500 mg / kg / body weight, etc. can be administered based on the above numbers. Such dosages may be administered intermittently, for example 2-3 times a day, once a week, or once every three weeks. An initial higher loading dose may be administered, followed by one or more lower doses. However, other dosing regimens may also be used.

[0224] The engineered IL2 polypeptides and engineered IL2 fusion polypeptides described herein can be used in an amount effective to achieve the intended purpose. For use in treating or preventing a disease condition, the engineered IL2 polypeptide or engineered IL2 fusion polypeptide or a pharmaceutical composition thereof is administered in a therapeutically effective amount. The determination of a therapeutically effective amount, especially in light of the details provided herein, is within the capabilities of those skilled in the art.

[0225] For systemic administration, the therapeutically effective amount can be estimated initially from in vitro assays such as cell culture assays. A dose can then be formulated in animal models to achieve an IC including that determined in cell culture. 50 The circulating plasma concentration range of the IL2 polypeptide and the engineered IL2 fusion polypeptide can be adjusted to provide plasma levels of the engineered IL2 polypeptide and the engineered IL2 fusion polypeptide sufficient to maintain the therapeutic effect. The levels in plasma can be measured, for example, by HPLC.

[0226] Example

[0227] In the examples provided below, IL2 labeled "WT-IL2" refers to IL2 having the sequence of SEQ ID NO: 171, which includes the wild-type IL2Rα and IL2Rβ binding domains and the T3A and C125S substitutions.

[0228] Example 1

[0229] Screening and identification of agonists that reduce IL2RB binding

[0230] In vitro mRNA display technology was used to develop IL2Rβ-reduced binding agonists from three rationally designed IL2 mutagenesis libraries. Briefly, the IL2 mutant DNA library was transcribed into an mRNA library, which was then translated into an mRNA-IL2 mutant fusion library via covalent coupling via a puromycin linker. These libraries were then purified and coupled to the corresponding single-stranded cDNA to stabilize the cytokine fusion mRNA library in the form of mRNA / cDNA hybrids, similar to the procedure reported in expired U.S. Patent No. 6,258,558. The fusion library was first subjected to multiple rounds of counter-selection with human IgG (negative protein) to remove non-specific binders, followed by counter-selection against recombinant human IL2Rβ-Fc protein captured on protein G magnetic beads. A total of 2-4 rounds of gradient concentration selection were performed. In each round of selection, IL2Rβ-reduced binders were recovered and enriched by PCR amplification.

[0231] After 2-4 rounds of selection, the enriched IL2Rβ-reduced binding library was cloned into the bacterial periplasmic expression vector pET22b and transformed into TOP10 competent E. coli cells. Each engineered IL2 molecule was engineered to have a C-terminal flag tag and a 6xHIS tag for purification and assay detection. Clones from TOP10 cells were pooled, miniprep DNA was prepared, and subsequently transformed into E. coli Rosetta II strain for expression. Individual clones were picked, grown, and induced for expression with 0.25 mM IPTG in 96-well plates. Supernatants were collected after 16 to 24 hours of induction at 30°C for assays to identify binders.

[0232] Supernatants containing engineered IL2 mutants were evaluated using a sandwich ELISA assay to screen for IL2Rα binding. Briefly, 96-well plates were immobilized with human Fc and human IL2Rα in a total volume of 50 μL per well at a final concentration of 2 μg / mL in 1x PBS, respectively. The plates were incubated overnight at 4°C and then blocked with 200 μL of superblock per well for 1 hour. 100 μl of supernatant was added to the Fc and IL2Rα immobilized wells and incubated with shaking for 1 hour. The engineered IL2 mutant binding was detected by adding 50 μL of anti-Flag HRP diluted 1:5000 in 1x PBST. Between each step, the plates were washed three times with 1x PBST in a plate washer. The plates were then developed with 50 μL of TMB substrate for 5 minutes and development was stopped by adding 50 μL of 2N sulfuric acid. The plates were read at OD450 nm using a Biotek plate reader and analyzed for binding and selectivity.

[0233] Next, individual clones were screened for IL2Rβ binding. An IL2Rβ binding screening ELISA was developed to identify individual engineered IL2 mutants. Briefly, 96-well plates were immobilized with human Fc and human IL2Rβ in a total volume of 50 μL per well at a final concentration of 2 μg / mL in 1x PBS, respectively. The plates were incubated overnight at 4°C and then blocked with 200 μL of superblock per well for 1 hour. 100 μL of supernatant was added to the Fc and IL2Rβ immobilized wells and incubated with shaking for 1 hour. Engineered IL2 mutant binding was detected by adding 50 μL of anti-Flag HRP diluted 1:5000 in 1x PBST. Between each step, the plates were washed three times with 1x PBST in a plate washer. The plates were then developed with 50 μL of TMB substrate for 5 minutes and development was stopped by adding 50 μL of 2N sulfuric acid. The plates were read at OD450 nm using a Biotek plate reader and analyzed for binding and selectivity.

[0234] Clones that exhibited positive IL2Rα binding but had reduced or negligible IL2Rβ binding were identified from library 5 for further characterization. Multiple sequence alignment of IL2Rβ binding loop 2 (IL2Rβ-BL2) compared to IL2 WT revealed highly conserved and highly variable amino acids, as well as clone sequences that were independently identified multiple times. No specific IL2Rβ binding clones were identified from the other libraries.

[0235] For further sorting, engineered IL2 clones with unique sequences were generated in E. coli. Briefly, a glycerol stock of each engineered agonist clone was inoculated into TB medium for overnight growth. The next day, cells from the overnight culture were inoculated into TB medium and grown to an OD of 600 The cell density was 0.6 to 0.8. IPTG was added until the final concentration was 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 each stored in 1x PBS buffer for binding and functional analysis. Due to the low expression of most clones in E. coli, the activity was evaluated by the above-mentioned IL2Rα and IL2Rβ binding ELISA. Table 1 below lists clones showing OD450 values ​​of corresponding IL2Rα and IL2Rβ receptor binding, respectively, as measured by anti-flag ELISA. Table 2 lists the sequences of engineered IL2 molecules mutated in the amino acid region from 81 to 96 (numbered according to the mature WT-IL2 sequence).

[0236] Table 1. Receptor binding activity of engineered IL2 clones produced in E. coli.

[0237]

[0238]

[0239] Table 2. List of IL2 mutation sequences in the region 81 to 96 compared to WT-IL2.

[0240]

[0241]

[0242] Example 2

[0243] Detection of IL2R using surface plasmon resonance Β Reduces the binding activity of bound agonists

[0244] The binding kinetics of WT IL2 produced in E. coli and engineered clones were screened using Biacore T200 surface plasmon resonance technology. The assay was run using Biacore T200 control software version 2.0. For each cycle, 1 μg / mL of human IL2Rβ or IL2Rα was captured on flow cell 2 at a flow rate of 10 μL / min in 1X HBSP buffer on a protein A sensor chip for 60 seconds. 100nM and 20nM of HIS-tagged purified engineered IL2 mutants were injected onto reference flow cell 1 and IL2Rβ or IL2Rα captured flow cell 2 at a flow rate of 30 μL / min for 150 seconds, followed by a 300 second wash. The flow cell was then regenerated with glycine pH 2 at a flow rate of 30 μL / min for 60 seconds. The kinetic data were analyzed using Biacore T200 Evaluation Software 3000. The specific binding response units were obtained by subtracting the binding to the reference flow cell 1 from the target flow cell 2.

[0245] WT IL2 and the IL2Rβ enhanced binding agonist EP596 (SEQ ID NO: 170) were used to validate the binding protocol and were included in each run as controls. Table 3 summarizes the binding activity of E. coli produced IL2 and mammalian produced IL2.

[0246] Table 3. Summary of IL2R binding kinetics of E. coli produced IL2Rβ agonist clones.

[0247]

[0248]

[0249] Due to the low expression of agonists in E. coli, clones with positive IL2Rα binding indicate reasonable expression of clones in the system. Weak binding: A weak sensorgram signal can be observed in SPR (Rmax ≥ 5RU). ND: No binding detected (Rmax < 5RU).

[0250] Example 3

[0251] IL2R produced by mammalian cells Β Characterization of agonists that reduce binding

[0252] In order to produce IL2Rβ-reducing binding agonists in mammalian cells, the DNA sequences corresponding to the amino acid sequences were codon optimized, synthesized and subcloned into pCDNA3.4 (Invitrogen). In order to reduce IL2Rα binding activity, point mutations of F35L, R38D and F42R were introduced into each engineered IL2 sequence. According to the standard protocol, each engineered IL2 polypeptide was transiently expressed in ExpiHEK293-F cells in a free system (Invitrogen). The cells were grown under the above conditions for seven days and then harvested. The supernatant was collected by centrifugation and filtered through a 0.2μm PES membrane. The agonists were first purified by a Ni Sepharose Excel resin column (GE Healthcare), and then the buffer was exchanged into PBS with a pH of 7.4 containing 300mM NaCl using a 7k Da Zeba column. Each polypeptide was then concentrated to 1 mL and purified to homogeneity by a Superdex 200Increase10 / 300GL column (GE Healthcare). Monomer peak fractions were pooled and concentrated. Proteins were stored in 1x PBS / 300 nM NaCl buffer for binding, functional and mechanistic analyses. Figure 1 An example of SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) results of single IL2 after Ni column purification is shown.

[0253] The binding activity of mammalian cell-produced agonists to the respective IL2Rβ and IL2Rα receptors was characterized by ELISA. Briefly, 384-well plates were immobilized with human IL2Rα and IL2Rβ Fc fusion proteins at a final concentration of 2 μg / mL in 1x PBS in a total volume of 25 μL / well. The plates were incubated overnight at 4°C and blocked with 80 μL of superblock per well for 1 hour. Purified clones were added to IL2Rα or IL-2Rβ wells in parallel in duplicate as titrations, with a maximum concentration of 1 μM, and then a 10-point 5-fold serial dilution of the clones was performed. Binding activity was detected by adding 25 μL of anti-hFc HRP diluted 1:5000 in 1x PBST. Between each step, the plates were washed three times with 1x PBST using a plate washer. The plates were then developed with 25 μL of TMB substrate for 5 minutes and development was stopped by adding 25 μL of 2N sulfuric acid. The plates were read at OD 450 nm using a Biotek plate reader and a bar graph of 1 μM IL2 was generated using Prism 8.1 software ( FIG. 2 ).

[0254] To further evaluate the immunomodulatory activity of the purified IL2 clones, human PBMCs were isolated from the peripheral blood of three different donors, stained with viability dye, and plated at a density of 250,000 cells / well in 90 μL of medium in a 96-well plate. The cells were allowed to rest at 37 °C for 1 hour. Cells were stimulated for 20 min with 10x concentrations of human IL2 WT, IL2Rβ-binding enhanced EP596, and engineered His-Flag-tagged IL2 in 10 μL 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+. T regulatory cells were defined as CD3+CD56-CD4+CD8-FOXP3+. The % of p-STAT5+ cells was determined and plotted against each IL2 titer (Figure 3). p-STAT5 activation was determined using Prism software and summarized in Table 4.

[0255] Table 4. Overview of IL2R binding activity of IL2Rβ agonist clones produced in mammals.

[0256]

[0257] WT IL2 and EP596 were used as references for ELISA binding assays. Strong: OD450 > 1.5; Moderate: 0.7 < OD450 < 1.5; Weak: 0.3 < OD450 < 0.7; Very weak: 0.1 < OD450 < 0.3; No binding: OD450 < 0.1. For p-STAT5 assays, very weak activity was defined as above baseline readings at the highest concentration in the assay. ND: Not detected; NA: Not applicable.

[0258] Example 4

[0259] Rational generation of IL2RΒ EP575 agonist revertant clones

[0260] A rationally designed IL2Rβ agonist back mutation strategy was performed to generate a series of IL2Rβ agonist candidate mutations. EP575 contains P82N, R83K, D84G, I85I, I86L, S87G, N88D, N90S, V91N and E95A mutations. I85 and L86 were back mutated to 85L and 86I of WT IL2, while 87G, 91N and 95A mutations were retained for all mutations. The other 6 residues were then systematically back mutated so that each back-mutated IL2 would eventually contain a total of 5 mutations, including 87G, 91N and 95A sites. A total of 20 back mutation combinations were designed, and the mutations were generated by site-directed mutagenesis using EP575 as a template (Table 5). After mutagenesis, the IL2Rβ agonist back mutation clones were sequence verified.

[0261] Table 5. List of EP575 back mutations. Residues highlighted in bold are mutations from WT-IL2.

[0262] Constructs 82 83 84 85 86 87 88 89 90 91 92 93 94 95 WT-IL2 P R D L I S N I N V I V L E EP575 N K G I L G D I S N I V L A EP658 P R G L I S D I S N I V L A EP659 P R G L I G N I S N I V L A EP660 P R G L I G D I N N I V L A EP661 P K G L I S N I S N I V L A EP662 P K G L I S D I N N I V L A EP663 P K G L I G N I N N I V L A EP664 N R G L I S N I S N I V L A EP665 N R G L I S D I N N I V L A EP666 N R G L I G N I N N I V L A EP667 N K G L I S N I N N I V L A EP668 P R G L I G D I S N I V L A EP669 P K G L I S D I S N I V L A EP670 P K G L I G N I S N I V L A EP671 P K G L I G D I N N I V L A EP672 N R G L I S D I S N I V L A EP673 N R G L I G N I S N I V L A EP674 N R G L I G D I N N I V L A EP675 N K G L I S N I S N I V L A EP676 N K G L I S D I N N I V L A EP677 N K G L I G N I N N I V L A

[0263] Example 5

[0264] Characterization of IL2RB agonist revertant clones

[0265] EP575 revertant clones were expressed in mammalian cells as described above. Clones were purified to homogeneity using Ni Sepharose columns and Superdex 200Increase 10 / 300GL columns (GE Healthcare). Clones were concentrated to appropriate concentrations and stored in 1xPBS / 300nM NaCl buffer for binding, functional, and mechanistic analyses. Figure 4 An example of SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) results of single IL2 after Ni column purification is shown.

[0266] The binding activity of the reverse mutations to the IL2Rβ and IL2Rα receptors was characterized by ELISA. Briefly, 384-well plates were immobilized with human IL2Rα and IL2Rβ Fc fusion proteins at a final concentration of 2μg / mL in 1x PBS in a total volume of 25μL / well. The plates were incubated overnight at 4°C and blocked with 80μL of superblock per well for 1 hour. Each purified EP575 reverse mutant clone was added to the IL2Rα or IL-2Rβ wells in parallel in duplicate at a final concentration of 10μM. IL2 mutant binding was detected by adding 25μL of anti-His HRP diluted 1:5000 in 1X PBST. Between each step, the plates were washed three times with 1XPBST using a plate washer. 1μM WT IL2 and 1μM EP596 were used as reference IL2. The plates were then developed with 25μL TMB substrate for 5 minutes and development was stopped by adding 25μl 2N sulfuric acid. Plates were read at OD450 nm using a Biotek plate reader (Table 6) and data were analyzed using Prism 8.1 software to generate a bar graph (Figure 5).

[0267] To further evaluate the immune cell regulatory activity of the purified IL2 reverse mutant clones, human PBMCs were isolated from peripheral blood of three different donors, stained with viability dyes, and plated in 90 μL of culture medium in 96-well plates at a density of 250,000 cells / well. The cells were allowed to rest at 37°C for 1 hour. The cells were stimulated with 10X concentrations of human IL2wt and engineered His-tagged IL2 in 10 μL at 37°C for 20 min. 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+. T regulatory cells were defined as CD3+CD56-CD4+CD8-FOXP3+. The percentage of p-STAT5+ cells was determined and plotted against each IL2 titer (Figure 6). P-STAT5 activation was determined using Prism software and summarized in Table 6. These data demonstrate that engineered IL2 has reduced binding to IL2Rα and IL2Rβ receptors. Accordingly, engineered IL2 exhibits weak p-STAT5 phosphorylation activity, with incomplete curves or low activity at high concentrations compared to WT IL2 (Figure 6).

[0268] Table 6. Summary of IL2Rα and IL2Rβ binding and p-STAT5 activity of EP575 backmutants.

[0269]

[0270] ND: Not detectable due to incomplete curve or very low activity. NA: Not available.

[0271] Example 6

[0272] Generation of IL2RBEP658 and EP661 agonist reversion mutations

[0273] Another round of engineering was carried out with EP658 and EP661 as examples to further reduce the total number of mutation sites in engineered IL2. EP658 comprises D84G, N88D, N90S, V91N and E95A mutations, while EP661 comprises R83K, D84G, N90S, V91N and E95A mutations. EP658 was subjected to single-point and double-site reverse mutations to produce IL2 mutants containing 4 and 3 mutation sites. EP661 was subjected to single-point mutagenesis to produce IL2 mutants containing 4 point mutations. A total of 14 reverse mutation combinations were designed, and mutations were produced by site-directed mutagenesis using EP658 and EP661 as templates (Table 7). These reverse mutation clones were sequence verified after mutagenesis, and were produced in mammalian cells as described above to further characterize. Fig. 7 and Fig. 8 show the p-STAT5 activity of immune cells after EP658 and EP661 reverse mutation treatment of healthy human PBMC donors, respectively.

[0274] Table 7. List of back mutations of EP658 and EP661. Residues highlighted in bold are mutations from WT-IL2.

[0275] Constructs 82 83 84 85 86 87 88 89 90 91 92 93 94 95 WT-IL2 P R D L I S N I N V I V L E EP575 N K G I L G D I S N I V L A EP658 P R G L I S D I S N I V L A EP661 P K G L I S N I S N I V L A EP722 P R D L I S D I S N I V L A EP723 P R G L I S D I N N I V L A EP724 P R G L I S D I S V I V L A EP725 P R G L I S D I S N I V L E EP726 P R D L I S D I N N I V L A EP727 P R D L I S D I S V I V L A EP728 P R D L I S D I S N I V L E EP729 P R G L I S D I N V I V L A EP730 P R G L I S D I N N I V L E EP731 P R G L I S D I S V I V L E EP732 P K D L I S N I S N I V L A EP733 P K G L I S N I N N I V L A EP734 P K G L I S N I S V I V L A EP735 P K G L I S N I S N I V L E

[0276] Example 7

[0277] IL2R Β Reduces binding of bound agonists to cell surface receptors

[0278] HEK Blue IL2 cells expressing IL2 receptor α, β, and γ were plated 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 left in a 37°C incubator for 1 hour. After 1 hour, the medium was replaced with 100 μL of medium containing 7-point 4x titrations of IL2 mutant proteins starting at 10 M, and the plates were incubated at 4°C with shaking for 30 minutes. After washing away unbound IL2, the cells were resuspended in 100 μL of medium containing 1:100 anti-His Dylight 650 secondary antibody and incubated at 4°C with shaking for 30 minutes in the dark. Unbound secondary antibody was washed away, and the cells were resuspended in PBS containing 1:800 Zombie Aqua viability dye and incubated at room temperature for 5 minutes. The cells were washed twice and analyzed using an Attune NxT flow cytometer. The percentage of HEK Blue IL2 cells positive for IL2 mutant protein binding was determined ( Fig. 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.

[0279] Example 8

[0280] IL2R Β Agonist F C Design of fusion proteins and anti-PD-(L)1 bispecific antibodies

[0281] The engineered IL2Rβ-reduced binding agonists can be further developed into Fc fusion proteins to further improve certain of their properties. To generate a monovalent IL2-Fc fusion protein, protein sequences encoding engineered 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 corresponding human IgG1 isotype to generate an engineered agonist-Fc fusion protein (SEQ ID NO: 137). Knob mutations of S354C, T366W, and K409A were introduced into the construct. Hole mutations of Y349C, T366S, L368A, F405K, and Y407V were introduced into the CH2 and CH3 fragments of IgG1. L234A, L235A, and P329G mutations were introduced into human IgG1 to eliminate complement binding and Fc-γ-dependent antibody-dependent cell-mediated cytotoxicity (ADCC) effects (Lo et al., JBC 2017). DNA encoding the complete Fc-fused agonist protein was then synthesized using codons optimized for mammalian cell expression and subcloned into pCDNA3.4 (Invitrogen).

[0282] On the other hand, engineered IL2Rβ-reducing agonists can be developed into bifunctional or bispecific antibodies to synergize their immunomodulatory activity with other therapeutic mechanisms. To generate anti-PD-L1-IL2 fusion bispecific antibodies, S354C, T366W and K409A mutations (Wei et al., Oncotarget, 2017; Xu et al., mAbs, 2015) were introduced as a knob molecule (EP362; SEQ ID NO: 104) to one heavy chain of the anti-PD-L1 antibody. The C-terminus of the other heavy chain of anti-PD-L1 was fused to the human IL2 protein with a (G4S)4 linker. S354C, Y349C, T366S, L368A, F405K, Y407V mutations were introduced to make the chain a hole molecule (e.g., EP643, in which EP575 of engineered IL2 was fused). The L234A, L235A, and P329G mutations in human IgG1 were introduced to abolish complement fixation and Fc-γ-dependent antibody-dependent cell-mediated cytotoxicity (ADCC) (Lo et al., JBC 2017).

[0283] In another embodiment, S354C, Y349C, T366S, L368A, F405K, Y407V mutations are introduced into the heavy chain of the anti-PD-L1 sequence, making the chain a hole molecule (EP325; SEQ ID NO: 114). The knob molecule (e.g., EP826) with an N-terminal IL2 fusion can form a monovalent (Fab format) anti-PD-L1-IL2 bispecific antibody with EP325 and the anti-PD-L1 antibody light chain EP205.

[0284] In another embodiment, the variable heavy chain region of an anti-mouse PD-L1 or anti-mouse PD-1 antibody is fused to the constant region of a human IgG1 antibody containing the antibody to produce a chimeric anti-mouse PD1 or anti-mouse PD-L1 antibody, respectively. Knob mutations and hole mutations as well as ADCC silent mutations are introduced into the heavy chain. The engineered IL2 molecule is fused to the end of one heavy chain arm so that the anti-mouse PD-1 or PD-L1 antibody binding site can form a bispecific protein. This bispecific protein can be used as an alternative anti-PD-(L)1 / IL2 bispecific protein (e.g., a bifunctional fusion protein complex) to study the mechanism of action and efficacy in mouse models.

[0285] DNA encoding the entire above designed sequence was then synthesized using codons optimized for mammalian cell expression and subcloned into pCDNA3.4 (Invitrogen). Figure 10 shows an example of a schematic representation of the format of selected anti-PD-L1 or anti-PD-1 antibodies.

[0286] Example 9

[0287] IL2-F C Production of proteins and bispecific antibodies

[0288] For monovalent IL2-Fc fusion protein production, the "knob" and "hole" constructs in the respective IgG1 main chain formats were transfected into ExpiHEK293-F cells at a ratio of 1: 1. Cells were grown for five days, and the supernatant was then collected by centrifugation and filtered through a 0.2 μm PES membrane. The Fc fusion agonist was first purified by MabSelect PrismA protein A resin (GE Health). The protein was eluted with 100mM Gly pH 2.5 + 150mM NaCl, and quickly neutralized with 20mM citrate pH 5.0 + 300mM NaCl. The agonist protein was then concentrated to 1mL and further purified by Superdex 200 16 / 600 gel filtration column. The monomer peak fractions were collected and concentrated. The final purified protein had an endotoxin level less than 10EU / mg and was stored in 1xPBS. Purified monovalent IL2-Fc fusion agonist was run on an SDS gel (4-12% Bis-Tris Bolt gel with MES running buffer).

[0289] For the production of monovalent (Fab format) anti-PD-L1-IL2 bispecific antibodies (e.g., bifunctional fusion proteins), "knob" and "hole" constructs in the respective IgG1 main chain formats were transfected into ExpiHEK293-F cells in a free style system (Invitrogen) according to standard protocols with a knob:hole:light chain ratio of 1:4:4. The cells were grown for five days and then harvested. The supernatant was collected by centrifugation and filtered through a 0.2 μm PES membrane. The antibodies were purified by MabSelect PrismA protein A resin (GE Health). The protein was eluted with 100mM Gly pH 2.5 and 150mM NaCl and rapidly neutralized with 20mM histidine pH 5.0+150mM NaCl. The antibodies were then further purified using a Superdex 200 16 / 600 column. The monomer peak fractions were pooled and concentrated. The final purified protein had less than 10 EU / mg endotoxin and was stored in 20 mN histidine, 150 mM NaCl buffer.

[0290] 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 the respective IgG1 main chain formats were transfected into ExpiHEK293-F cells in a free style system (Invitrogen) according to standard protocols with a knob:hole:light chain ratio of 1:2:2. The cells were grown for five days and then harvested. The supernatant was collected by centrifugation and filtered through a 0.2 μm PES membrane. The antibodies were purified by MabSelect PrismA protein A resin (GE Health). The protein was eluted with 100mM Gly pH 2.5+150mM NaCl and rapidly neutralized with 20mM histidine pH 5.0+150mM NaCl. The antibodies were then further purified using a Superdex 200 16 / 600 column. The monomer peak fractions were pooled and concentrated. The final purified protein had less than 10 EU / mg endotoxin and was stored in 20 mN histidine, 150 mM NaCl buffer.

[0291] Anti-PD-L1 monoclonal antibodies were transiently expressed in ExpiHEK293-F cells in the Free style system (Invitrogen) according to standard protocols, with a ratio of 1:2 for plasmid DNA of heavy and light chains. The cells were grown for five days and then 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 100mM Gly pH 2.5+150mM NaCl and rapidly neutralized with 20mM citrate pH 5.0+300mM NaCl. The antibody was then further purified using a Superdex 200 16 / 600 column. The monomer peak fractions were pooled and concentrated. The final purified protein had a final endotoxin level of less than 10EU / mg and was stored in 20mM histidine pH 6.0+150mM NaCl.

[0292] Example 10

[0293] IL2R Β Agonist F C Characterization of fusion and bispecific antibodies

[0294] 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 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 3 times with 0.05% Tween20 / 1X PBS. The anti-PD-L1 / IL2 mutant bispecific (e.g., bifunctional fusion protein complex) was diluted from 1000 nM to 0 nM in 0.05% Tween20 / 1X PBS and added to the plate for 2 hours at room temperature. The plate was then washed 6 times with 0.05% Tween20 / 1X PBS. The captured proteins were detected using biotinylated recombinant PD-(L)1, IL2Rα or IL2Rβ, and after washing, they were visualized with streptavidin-HRP diluted 1:5000 in 0.05% Tween20 / 1X PBS. The plates were then washed 6 times with 0.05% Tween20 / 1X PBS, and TMB was added to develop blue. The reaction was stopped with 2N hydrogen sulfide, and the absorbance was read at 450nm on a BioTek plate reader. The absorbance of human PD-(L)1, IL2Rα and IL2Rβ binding was plotted against IL2 concentration (Figure 11). A summary of PD-(L)1, IL2Rα and IL2Rβ ELISA binding activity is shown (Table 8). All constructs showed similar PD-L1 binding activity. For the anti-PD-L1 / IL2 bispecifics (e.g., bifunctional fusion protein complexes) fused to EP567, EP575, EP581, EP658, EP660, EP661, and EP669, no IL2Rα and IL2Rβ binding activity was observed at the highest concentration of 1 μM, while the IL2Rβ agonist of the EP415-fused bispecific exhibited strong IL2Rβ binding activity. The reference WT-IL2 fusion bispecific exhibited strong IL2Rα binding, which is consistent with the reported data.

[0295] Table 8. Summary of PD-(L)1, IL2Rα and IL2Rβ binding analysis of IL2 Fc fusion proteins

[0296]

[0297] Embodiment 11

[0298] IL2R Β Agonist F C Activation of P-STAT5 in human PBMCs by fusion bispecific antibody

[0299] Human PBMCs were isolated from peripheral blood, stained with viability dyes, and plated in 96-well plates at 250,000 cells / well in 90 μL of culture medium. The cells were left to stand at 37°C for 1 hour. The cells were stimulated with 10X concentrations of human IL2 wt and IL2Rβ agonist Fc-fusion proteins in 10 μl at 37°C for 20 min. 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+. T regulatory cells were defined as CD3+CD56-CD4+CD8-FOXP3+. The percentage of p-STAT5+ cells was determined and plotted against each IL2 titer (Figure 12).

[0300] Example 12

[0301] IL2R Β Safety and immune cell activation of agonist EP567 / anti-PD-L1 in mice

[0302] Female C57BL / 6 mice aged 6-8 weeks were dosed with vehicle or EP567 / PD-L1 at concentrations 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 pre-dose body weight. On day 8, mice were sacrificed and lungs were removed and measured.

[0303] During the 8th day of sacrifice, blood was collected by cardiac puncture. After erythrocyte lysis, blood was stained for immunophenotypic markers (CD3, CD4, CD8, NKp46, NK1.1 and FOXP3) as well as CD69, Ki67 and viability markers. Cells were analyzed using an Attune NxT flow cytometer. The number of CD4+FOXP3-T cells, CD8+T cells, NK cells and Tregs was calculated as a percentage of live cells. The percentages of CD4+FOXP3-T cells, CD8+T cells and Tregs for Ki67+ were calculated. The percentages of CD4+FOXP3-T cells, CD8+T cells and Tregs for CD69+ were calculated.

[0304] Fig.13A The weight changes of mice are shown. EP567 / anti-PD-L1 bispecific began to induce weight loss at 5 mg / kg and led to severe weight loss and death at 10 mg / kg. Therefore, lung weight ( Fig. 13B ) increased proportionally with the increase of EP567 dose.

[0305] Peripheral immune cell profiling data support that EP567 / anti-PD-L1 increases CD8 T cell levels and reduces Treg cell levels in a dose-dependent manner (Figure 14). Accordingly, EP567 increased the proliferation of CD8 T cells and downregulated the proliferation of Treg cells at all doses (Figure 15). EP567 also induced early activation of CD8 and CD4 T cells at a high dose of 10 mg / kg (Figure 16). In summary, the data support that EP567 / anti-PD-L1 can stimulate immune cell activation, however, mice cannot tolerate high concentrations of bispecifics, indicating that more engineered IL2s should be evaluated.

[0306] Example 13

[0307] Safety and immune cell activation of additional IL2 / anti-PD-L1 bispecifics in mice

[0308] Female C57BL / 6 mice aged 6-8 weeks were dosed with 10 mg / kg IL2 / anti-PD-L1 bispecific on days 0 and 4. Body weight was monitored 5 times per week and expressed as relative body weight, normalized to pre-dose body weight. On day 8, mice were sacrificed and lungs were removed and measured.

[0309] During the sacrifice on day 8, blood was collected by cardiac puncture. After erythrocyte lysis, the blood was stained for immunophenotypic markers (CD3, CD4, CD8, NKp46, NK1.1, FOXP3, CD44, and CD62L) as well as Ki67 and viability markers. Cells were analyzed using an Attune NxT flow cytometer. The concentrations of CD4+FOXP3-T cells, CD8+T cells, NK cells, and Tregs were calculated in each sample. The percentages of CD4+FOXP3-T cells, CD8+T cells, NK cells, and Ki67+ Tregs were calculated. The percentages of CD4+FOXP3-T cells and CD8+T cells as initial, central memory effectors were calculated.

[0310] Fig.17A The weight changes of mice are shown. Consistent with previous results, EP567 / anti-PD-L1 bispecific caused significant weight loss at 10 mg / kg, death at 10 mg / kg, and increased lung weight ( Fig. 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, showed no weight loss ( Fig.17A Therefore, there was no significant change in lung weight ( Fig. 17B ).

[0311] Peripheral immune phenotyping was performed to assess whether these IL2 / anti-PD-L1 antibodies (e.g., bifunctional fusion protein complexes) can modulate immune cell activation. Only one mouse survived in the EP567 / anti-PD-L1 group. The analysis results of this group are consistent with previous data. This group observed a significant increase in CD8 T cells, NK cells, and a decrease in Treg cells (Figure 18). Among the remaining IL2 / anti-PD-L1 bispecifics, EP661 / anti-PD-L1 showed a significant increase in CD8 and NK cell activation, while Treg increased moderately. EP575 / anti-PD-L1 and EP581 / anti-PD-L1 showed moderate activation of CD8 T cells, NK cells, and Treg cells (Figure 18). EP658 / anti-PD-L1, EP660 / anti-PD-L1, EP669 / anti-PD-L1, and EP676 / anti-PD-L1 showed weak or minimal activation of CD8 T cells and NK cells. Treg cells in all three groups increased moderately. Interestingly, the EP567 / anti-PD-L1 group showed a decrease in immune cell proliferation (Figure 19). EP575 / anti-PD-L1, EP581 / anti-PD-L1, and EP658 / anti-PD-L1 showed a significant increase in CD8 T cell proliferation ( Fig.19B EP660 / anti-PD-L1, EP661 / anti-PD-L1, EP669 / anti-PD-L1, and EP676 / anti-PD-L1 showed moderate CD8 T cell proliferation ( Fig.19B Only EP575 / anti-PD-L1 showed significantly increased NK cell proliferation ( Fig.19D ). The results of the subtype immune cell analysis spectrum showed that the EP567 / anti-PD-L1 group contained more effective CD4 and CD8 T cells and a small percentage of initial T cells. EP661 / anti-PD-L1 showed a similar pattern to EP567 / anti-PD-L1. EP575 / anti-PD-L1, EP581 / anti-PD-L1 and EP661 / anti-PD-L1 contained high levels of central memory CD8 T cells. EP660 / anti-PD-L1, EP669 / anti-PD-L1 and EP676 / anti-PD-L1 contained high levels of initial CD8 T cells similar to the vehicle, further indicating that they have weak or moderate systemic immunomodulatory activity (Figure 20). Table 9 lists the average concentration fold changes of peripheral blood immune cells in IL2 / anti-PD-L1 treated mice relative to mice in the vehicle group.

[0312] Table 9. Fold changes in the concentration of immune cells in the peripheral blood of mice compared with the vehicle group.

[0313]

[0314] Embodiment 14

[0315] Pharmacokinetic analysis of EP661 / anti-PD-L1 agonist in mice

[0316] Female C57BL / 6 mice aged 6-8 weeks were given a single intravenous (iv) bolus of 200 μg EP661 / anti-PD-L1. Mice were sacrificed by terminal cardiac puncture at the following 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, centrifuged, and plasma was collected and stored at -80°C until analysis.

[0317] Once all time points were collected, plasma was thawed for analysis. Anti-human Fc was diluted in 25 μL 1X PBS and added to the wells of a 384-well immunoadsorbent plate and 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 to the wells in duplicate. Standards were serially diluted with known concentrations of EP661 / anti-PD-L1 in PBS containing 0.1% mouse plasma and added to the wells in duplicate. Samples and standards were incubated for 1 hour at room temperature. Bound EP661 / anti-PD-L1 was sandwiched between biotinylated recombinant PD-L1 and detected by adding 25 μL of streptavidin HRP diluted 1:5000 in 1X PBST. Between each step, the plate was washed three times with 1X PBST using a plate washer. The plates were then developed with 25 μl of TMB substrate for 5 minutes and stopped by the addition of 25 μl of 2N sulfuric acid. The plates were read using an OD 450 nm Biotek plate reader.

[0318] The OD values ​​of the standards were plotted against the known concentrations in Microsoft Excel and a line of best fit was drawn by performing a simple linear regression. The EP661 / anti-PD-L1 concentration in the samples was calculated from the OD values ​​based on the standard fit line equation and multiplied by the initial 1:1000 dilution factor. Protein concentration was plotted against time. PK parameters were calculated using PKSolver software and Microsoft Excel and are displayed in Fig.21 Table 10 shows the calculated PK parameters.

[0319] Table 10. PK parameters of EP661 / anti-PD-L1 in mice.

[0320] parameter 10mg / kg T1 / 2(h) 82.56 Cmax(ng / ml) 19903.87 Cl_obs(mg / kg / h) 5.49107E-06 Vss_obs(mg / kg) 0.000732028

[0321] Embodiment 15

[0322] IL2R ΒAgonist antitumor activity in mouse tumor models

[0323] 500,000 B16-F10-hPD-L1 cells were injected subcutaneously into the dorsal flank of 6-8 week old female C57BL / 6 mice. Tumors were measured with calipers. When the average volume reached approximately 50 mm 3 Mice were treated with 10 mg / kg IL2 / anti-PD-L1 bispecific (e.g., bifunctional fusion protein complex) or atezolizumab Q4D. Tumors were measured 3 times per week and body weight was measured 5 times per week.

[0324] On day 8, peripheral blood was collected. After erythrocyte lysis, the blood was stained for immunophenotypic markers (CD3, CD4, CD8, NKp46, NK1.1, and FOXP3) as well as viability markers. Cells were analyzed using an Attune NxT flow cytometer. The number of CD4+FOXP3- T cells, CD8+ T cells, NK cells, and Tregs in each sample was calculated. The ratio of CD8+ T cells and NK cells relative to the number of Tregs was calculated.

[0325] Fig.22A Shown are tumor growth curves of EP658 / anti-PD-L1, EP661 / anti-PD-L1, and EP669 / anti-PD-L1 (Q4D, 200 μg) compared to atezolizumab (Q4D, 200 μg) and vehicle (1×PBS, Q4D). Fig. 22B The weight changes of mice during treatment are shown. Consistent with previous studies, mice tolerated repeated doses well in all study groups. All IL2 / anti-PD-L1 treatment groups showed stronger tumor growth inhibition activity than atezolizumab. Some mice in the IL2 / anti-PD-L1 group experienced complete tumor regression. Among them, 1 mouse in the EP658 / anti-PD-L1 group was tumor-free, 2 mice in the EP661 / anti-PD-L1 group were tumor-free, and 3 mice in the EP669 / anti-PD-L1 group were tumor-free. EP669 / anti-PD-L1 showed the strongest anti-tumor activity, with a tumor growth inhibition rate of 69%. Fig. 22C A single mouse tumor growth curve is shown. In addition to the continued improved safety profile, this early tumor model study supports that IL2 and anti-PD-L1 binding to IL2Rβ in the bispecific can synergize to enhance anti-tumor activity.

[0326] Immunophenotyping of peripheral blood collected on day 8 (before the third dose) showed results consistent with previous studies. Significant CD8 T cell and NK cell activation was observed for EP661 / anti-PD-L1, while EP658 / anti-PD-L1 and EP669 / anti-PD-L1 induced moderate CD8 and NK cell activation. Treg cells in both groups were also moderately activated. Consistent with previous studies, EP661 / anti-PD-L1 reduced Treg and CD4 cells (Figure 23). EP661 / anti-PD-L1 showed a significant increase in the ratio of CD8:Treg and NK:Treg in peripheral blood (Figure 24), while the ratio of EP658 / anti-PD-L1 and EP669 / anti-PD-L1 groups increased minimally. Table 11 shows the average original count fold change of immune cells in peripheral blood of the IL2 / anti-PD-L1 treatment group relative to the vehicle group. Immunophenotyping further demonstrated that EP661 was a potent modulator of CD8 and NK cell activation, whereas EP669 and EP658 were mild to moderate modulators of systemic CD8 and NK cell activation.

[0327] Table 11. Fold changes in raw counts of immune cells in peripheral blood in the IL2 / anti-PD-L1 treatment group compared to the vehicle group.

[0328]

[0329] Example 16

[0330] Antitumor activity of IL2 / PD1 and IL2 / PD-L1 bispecifics in tumor models

[0331] To evaluate the anti-tumor activity of IL-2 / PD1 and PD-L1 bispecifics (e.g., bifunctional fusion protein complexes), 500,000 B16-F10-hPD-L1 cells were injected subcutaneously into the flank of 6-8 week old female C57BL / 6 mice. Tumors were measured with a caliper. When the average volume reached approximately 50 mm 3 At 4 d, mice were treated with 3 or 10 mg / kg EP669 / PDL1 (all Q4D), EP661 / PD-1 3 mg / kg QW, or RO7284755 1 mg / kg QW. The body weight of mice was measured twice a week and plotted against time for each group. The mean tumor volume and individual tumor volume of each group were plotted against time (Figures 25-26).

[0332] 2E5 MC38 tumor cells suspended in 200 μl PBS were subcutaneously inoculated into the right flank of female C57Bl / 6 mice (6-8 weeks old). Tumor volume was measured using a caliper and was determined when the tumor volume reached approximately 50 mm 3Mice were randomly divided into three treatment groups. Mice were treated with vehicle (PBS), 1 mg / kg EP661 / mPD-1, or 10 mg / kg anti-mPD-1. Treatment was administered by intraperitoneal injection once a week for 2 weeks. Tumor volume was measured twice a week during the study. The percentage of tumor growth inhibition (TGI) relative to vehicle was calculated on day 17, and animals that showed complete regression (CR) were identified ( Fig. 27 , Table 12). On day 11 of the study, peripheral blood was collected. After erythrocyte lysis, the remaining cells were stained for immunophenotypic markers (CD3, CD4, CD8, NKp46, NK1.1, FOXP3) and viability markers. 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 ).

[0333] Table 12. Tumor Growth Inhibition

[0334] TGI (%) CR vehicle - 0 / 10 Anti-mPD1 43 0 / 10 EP661 / mPD1 99 8 / 10

[0335] Embodiment 17

[0336] IL2R Β Agonist immune cell modulatory activity

[0337] To characterize the immune cell modulatory activity of IL2 mutant bispecifics (e.g., bifunctional fusion protein complexes) with reduced activity compared to WT IL2 and RO7284755, human PBMCs were isolated from peripheral blood, stained with viability dyes, and plated in 90 μL of culture medium in 96-well plates at a density of 250,000 cells / well. The cells were left to rest at 37°C for 1 hour. The cells were stimulated with 10X concentrations of WT IL2 bispecific and reduced IL2 mutant bispecific titrations 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. CD4+FOXP3-T cells were defined as CD3+CD56-CD4+CD8-FOXP3-. CD8+T cells were defined as CD3+CD56-CD4-CD8+. NK cells are defined as CD3-CD56+. T regulatory cells are defined as CD3+CD56-CD4+CD8-FOXP3+. The % of p-STAT5+ cells were determined and plotted against each IL2 titer (Figure 29). p-STAT5 activation was determined using Prism software. These data support that engineered IL2 shows reduced binding to IL2Rα and IL2Rβ receptors. Accordingly, engineered IL2 exhibits weak p-STAT5 phosphorylation activity, with incomplete curves or low activity at high concentrations compared to WT IL2 and RO7284755 (Figure 29).

[0338] Embodiment 18

[0339] IL2R Β Agonist activity on tumor-infiltrating cells

[0340] 500,000 B16-F10-hPD-L1 cells were injected subcutaneously into the flank of 6-8 week old female C57BL / 6 mice. Tumors were measured with calipers. When the average volume reached approximately 50 mm 3Mice were treated with vehicle or EP669 / PDL1 (3 mg / kg, Q4D) at 4 pm. At the end of the study, animals were euthanized by CO2 asphyxiation and tumors were removed. Tumors were fixed in formalin and embedded in paraffin. Tumor blocks were then cut into 5 μm thickness on glass slides and stained for IHC. The slides were blocked with BLOXALL (Vector, SP-6000-100) and 2.5% normal horse serum (Vector S-2012-50) and 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, the slides were incubated with goat anti-rabbit IgG amplifier (Vector, MP-7601-50) for 1 hour. The slides were washed in TBS-T and then incubated with ImmPress HRP horse anti-goat IgG polymer reagent (Vector, MP-7601-50). After washing, ImmPACT DAB EqV chromogen (Vector, MP-7601-50) was added and the slides were incubated. All incubations were performed at room temperature. The slides were rinsed in TBS-T and distilled water, counterstained with Gill II hematoxylin (StatLab, SL94-1), dehydrated and covered with a coverslip. The slides were then imaged and representative areas of untreated and treated tumors were enlarged to observe CD8 positive cells (Figure 30).

[0341] Embodiment 19

[0342] IL2R Β Agonist activity on systemic immune cells

[0343] To evaluate the activity of IL2 bispecific antibodies (e.g., bifunctional fusion protein complexes) on systemic immune cells, blood was collected by submandibular bleeding from naive female C57Bl / 6 mice (6-8 weeks old), and complete blood counts were measured using a VETSCAN hematology analyzer (Abaxis). Mice were then treated with vehicle (PBS), RO7284755, or IL2 mutant bispecific molecules (EP877 / EP923 / EP740; EP877 / EP930 / EP740; EP877 / EP933 / EP740; EP876 / EP928 / EP205; EP876 / EP934 / EP205; or EP876 / EP935 / EP205). Treatment was administered by intraperitoneal injection with a dose volume of 200 μl and a dose concentration of 200 μg / mouse. Two days after treatment, blood was collected again from all animals for complete blood count analysis. The fold changes in total white blood cells (WBC), lymphocytes, and neutrophils in blood samples from pre-treatment (Day 0) to Day 2 were plotted (Figure 31). The data support that RO7284755 has a more potent lymphopenic effect than the IL2 mutant bispecific molecules disclosed herein.

[0344] On day 5, peripheral blood was collected. After erythrocyte lysis, the remaining cells were stained for immunophenotypic markers (CD3, CD4, CD8, NKp46, NK1.1, FOXP3, CD44, CD62L) and viability markers. 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 cells (CD44-, CD62L+), central memory cells (CD44+, CD62L+), and effector cells (CD44+, CD62L-) in the parental population were determined and plotted (Figure 32).

[0345] Embodiment 20

[0346] Antitumor activity of IL2 / PD1 bispecifics in tumor models

[0347] 2E5 MC38 tumor cells suspended in 200 μl PBS were subcutaneously inoculated into the right flank of female C57Bl / 6 mice (6-8 weeks old). Tumor volume was measured using a caliper and was determined when the tumor volume reached approximately 50 mm 3When, mice were randomly divided into three treatment groups. Mice were treated with 1mg / kg of vehicle (PBS), RO7284755, EP661 / mPD-1, EP930 / PD-1, EP933 / PD-1 or EP935 / PD-1. Treatment was administered by intraperitoneal injection once a week for 2 weeks. During the study, tumor volume was measured twice a week. The tumor growth inhibition percentage (TGI) relative to the vehicle was calculated on the 17th day, and animals showing complete regression (CR) were determined (Figure 33). On the 11th day of the study, peripheral blood was collected. After erythrocyte lysis, the remaining cells were stained with immunophenotypic markers (CD3, CD4, CD8, NKp46, NK1.1, FOXP3) and viability markers. Cells were analyzed using 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 cells (CD44-, CD62L+), central memory cells (CD44+, CD62L+), and effector cells (CD44+, CD62L-) in the parental population were determined and plotted (Figure 34).

[0348] The various embodiments described above can be combined to provide additional embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications cited in this specification and / or listed in the application data sheet, 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, are incorporated herein by reference in their entirety. If necessary, various aspects of the embodiments can be modified to provide additional embodiments using the concepts of various patents, applications, and disclosures.

[0349] These and other changes can be made to the embodiments in light of the above detailed description. In general, in the appended claims, the terms used should not be interpreted to limit the claims to the specific embodiments disclosed in the specification and claims, but should be interpreted to include all possible embodiments and the full range of equivalents to which these claims are entitled. Therefore, the claims are not limited by the present disclosure.

Claims

1. An engineered interleukin-2 (IL2) polypeptide comprising an engineered IL2 receptor β (IL2Rβ) binding region 2 motif, wherein the engineered IL2 receptor β (IL2Rβ) binding region 2 motif comprises: 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; and X 16 comprises a residue selected from L or I, and The engineered IL2Rβ binding region 2 motif does not comprise SEQ ID NO: 2, and the engineered IL2 polypeptide has reduced binding affinity to IL2Rβ compared to wild-type IL2.

2. The engineered IL2 polypeptide of claim 1, wherein the engineered IL2Rβ binding region 2 is selected from the group comprising or consisting of: RFKALIIEINFIVQLL (SEQ ID NO:4), RSRQLISNMNGIILKL (SEQ ID NO:5), RLTHL RNVIGVILVQL (SEQ ID NO:6), LSLREPIGNIVTSVRE (SEQ ID NO:7), NRTDLVGDVNATIKAL (SEQ ID NO:8), RNKGILGDISNI VLAL (SEQ ID NO:9), RSREVVSRIDAIILEL (SEQ ID NO:10), RPRGLISNIVLAL (SEQ ID NO:11), RPRGLIGNISNIVLAL (SEQ ID NO:12), RPRGLIGDINNIVLAL (SEQ ID NO:13), RPKGLISNIVLAL (SEQ ID NO:14), RPKGLISDINNIVLAL (SEQ ID NO:15), RPKGLIGNINNIVLAL (SEQ ID NO:16), NO: 16), RNRGLISNISNIVLA L (SEQ ID NO: 17), RNRGLISDINNIVLAL (SEQ ID NO: 18), RN RGLIGNINNIVLAL (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:27) NO:28), RNKGLISDINNIVLAL (SEQ ID NO:29), RNKGLIGN INNIVLAL (SEQ ID NO: 30), RPRDLISDISNIVLAL (SEQ ID NO: 31), RPRGLISDINNIVLAL (SEQ ID NO: 32), RPRGLISDISVIVLA L (SEQ ID NO: 33), RPRGLISDISNIVLAL (SEQ ID NO: 33), RPRGLISDISNIVLAL (SEQ ID NO: 32)ID NO: 34), RPR DLISDINNIVLAL (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), RPKGLISNISVIVLAL (SEQ ID NO: 43), RPKGLISNISNIVLEL (SEQ ID NO: 44), RPKGLISNISVIVLEL (SEQ ID NO: 194), RPRGLISNISVIVL EL (SEQ ID NO: 195), RPRDLISNISNIVLEL (SEQ ID NO: 196), RPKGLISNINNIVLEL (SEQ ID NO: 197), RPKGLISDINNIVLEL (SEQ ID NO: 198), RPRDLISRIDAIVLEL (SEQ ID NO: 199), RN RGLIGNINNIVLEL (SEQ ID NO: 200), RPKGLISEINNIVLEL (SEQ ID NO: 201) and RPKGLISRINNIVLEL (SEQ ID NO: 202).

3. The engineered IL2 polypeptide of claim 1 or 2, wherein the engineered IL2Rβ binding region 2 is selected from the group comprising or consisting of: RFKALIIEINFI VQLL (SEQ ID NO:4), RSRQLISNMNGIILKL (SEQ ID NO:5), RLTHLRNVIGVILVQL (SEQ ID NO:6), RNKGILGDISNIVLAL (SEQ ID NO:9), RSREVVSRIDAIILEL (SEQ ID NO:10), RPRGLISDI SNIVLAL (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:25), RNKGLISNISNIVLAL (SEQ ID NO:26), RNKGLISNISNIVLAL (SEQ ID NO:27), RNKGLISNISNIVLAL (SEQ ID NO:28), ID NO: 29), RPKGLISNISV IVLEL (SEQ ID NO: 194), RPKGLISNISVIVLEL (SEQ ID NO: 195), RPRDLISNISNIVLEL (SEQ ID NO: 196), RPKGLISNINNIVLEL (SEQ ID NO: 197), RPKGLISDINNIVLEL (SEQ ID NO: 198), RPRDL ISRIDAIVLEL (SEQ ID NO: 199), RRNRGLIGNINIVLEL (SEQ ID NO: 200), RPKGLISEINNIVLEL (SEQ ID NO: 201), and RPKGLISRINNIVLEL (SEQ ID NO: 202).

4. The engineered IL2 polypeptide of any one of claims 1-3, wherein the affinity of the engineered IL2 polypeptide for IL2Rβ is reduced by at least 1 / 2 compared to the wild-type IL2 or the IL2 having the sequence of SEQ ID NO:

171.

5. The engineered IL2 polypeptide of any one of claims 1-4, wherein the engineered IL2 polypeptide has reduced affinity for IL2Rα compared to wild-type IL2 or IL2 having the sequence of SEQ ID NO:

171.

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

171.

7. The engineered IL2 polypeptide of any one of claims 1-6, comprising an engineered IL2 receptor alpha (IL2Rα) binding region 1, wherein the IL2Rα binding region 1 comprises a substitution at at least one position selected from K35, R38, F42 and Y45, wherein: i) the substitution at position K35 is selected from the group consisting of: 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 the group consisting of: F42A, F42R, F42G, F42I, F42L, F42P and F42H; and iv) the substitutions at position Y45 are Y45S, Y45P, Y45A, Y45V, Y45C, Y45T and Y45F.

8. The engineered IL2 polypeptide of claim 7, wherein the substitutions are at least 2, at least 3, or all 4 of positions K35, R38, F42, and Y45.

9. The engineered IL2 polypeptide of claim 7, wherein the substitutions are K35L, R38D and F42R.

10. The engineered IL2 polypeptide of any one of claims 1-9, wherein the engineered IL2 polypeptide binds to IL2Rα with at least 10-fold reduced binding kinetics compared to wild-type IL2 or IL2 having the sequence of SEQ ID NO:

171.

11. An engineered interleukin-2 (IL2) polypeptide comprising the sequence of SEQ ID NO:45 (APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPLLTDMLT RKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHL-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) having at least 90% sequence identity, 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; and X 16 comprises a residue selected from L or I, and The engineered IL2Rβ binding region 2 motif does not comprise SEQ ID NO: 2, and the engineered IL2 polypeptide has reduced binding affinity to IL2Rβ compared to wild-type IL2.

12. The engineered IL2 polypeptide of claim 11, wherein the IL2Rβ binding region 2 is a sequence selected from the group consisting of: RFKALIIEINFIVQ LL (SEQ ID NO:4), RSRQLISNMNGIILKL (SEQ ID NO:5), RLT HLRNVIGVILVQL (SEQ ID NO:6), LSLREPIGNIVTSVRE (SEQ ID NO:7), NRTDLVGDVNATIKAL (SEQ ID NO:8), RNKGILGDIS NIVLAL (SEQ ID NO:9), RSREVVSRIDAIILEL (SEQ ID NO:10), RPRGLISNIVLAL (SEQ ID NO:11), RPRGLIGNISNIVLAL (SEQ ID NO:12), RPRGLIGDINNIVLAL (SEQ ID NO:13), RPKGLISNIVLAL (SEQ ID NO:14), RPKGLISDINNIVLAL (SEQ ID NO:15), NO:15), RPKGLIGNINNIVLAL (SEQ ID NO:16), RNRGLISNISNIVLAL (SEQ ID NO:17), RNRGLISDINNIVLAL (SEQ ID NO:18), RNKGLIGNINNIVLAL (SEQ ID NO:19), RNKGLISNINNIVLAL (SEQ ID NO:20), RPRGLIGDISNIVLAL (SEQ ID NO:19) 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 (SE Q ID NO:28), RNKGLISDINNIVLAL (SEQ ID NO:29), RNKGLIG NINNIVLAL (SEQ ID NO:30), RRPDLISDISNIVLAL (SEQ ID NO:31), RRPGLISDINNIVLAL (SEQ ID NO:32), RRPGLISDISVIVLAL (SEQ IDNO:33), RPRGLISDISNIVLEL(SEQ ID NO:34), RP RDLISDINNIVLAL(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), RPKGLISNISVIVLAL(SEQ ID NO:43), RPKGLISNISNIVLEL(SEQ ID NO:44), RPKGLISNISVIVLEL(SEQ ID NO:194), RPRGLISNISVIVL EL(SEQ ID NO:195), RPRDLISNISNIVLEL(SEQ ID NO:196), RPKGLISNINNIVLEL(SEQ ID NO:197)、RPKGLISDINNIVLEL(SEQ ID NO:198)、RPRDLISRIDAIVLEL(SEQ ID NO:199)、RN RGLIGNINNIVLEL(SEQ ID NO:200)、RPKGLISINNIVLEL(SEQ ID NO:201)、RPKGLISRINNIVLEL(SEQ ID NO:202)。 13. The engineered IL2 polypeptide of claim 11, wherein the IL2Rβ binding region 2 is a sequence selected from the group consisting of: RFKALIIEINFIVQ LL (SEQ ID NO:4), RSRQLISNMNGIILKL (SEQ ID NO:5), RLT HLRNVIGVILVQL (SEQ ID NO:6), RNKGILGDISNIVLAL (SEQ ID NO:9), RSREVVSRIDAIILEL (SEQ ID NO:10), RPRGLISNISNIVLAL (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:25), NO:28), RNKGLISDINNIVLAL (SEQ ID NO:29), RPKGLISNISVIVL EL (SEQ ID NO:194), RPRGLISNISVIVLEL (SEQ ID NO:195), RPKGLISNISNIVLEL (SEQ ID NO:196), RPKGLISNINNIVLEL (SEQID NO:197), RPKGLISDINNIVLEL (SEQ ID NO:198), RPRDLI SRIDAIVLEL (SEQ ID NO:199), RRNRGLIGNINIVLEL (SEQ ID NO:200), RPKGLISEINNIVLEL (SEQ ID NO:201) and RPKGLISRINNIVLEL (SEQ ID NO:202).

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

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

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

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

18. A fusion polypeptide as described in claim 17, wherein 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 linked small molecule, a nucleic acid, or any combination thereof.

19. The fusion polypeptide of claim 17 or 18, wherein the second polypeptide sequence comprises an antibody heavy chain constant region.

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

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

137.

23. A fusion polypeptide as described in any one of claims 19-22, 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 relative to SEQ ID NO:

137.

24. The fusion polypeptide of any one of claims 19-23, wherein the antibody heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 123 or SEQ ID NO:

138.

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

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

27. The monovalent engineered IL2-Fc fusion polypeptide complex of claim 26, wherein the second polypeptide comprises a heavy chain constant region.

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

123.

29. The monovalent engineered IL2-Fc fusion polypeptide complex of any one of claims 26 to 28, comprising a first and a second polypeptide having the following sequence: 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.

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

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

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

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

34. A fusion polypeptide according to any one of claims 30 to 33, wherein the antigen binding portion binds 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 and a second polypeptide, wherein the first polypeptide is the fusion polypeptide of any one of claims 17-34, and the second polypeptide comprises an antigen binding portion.

36. The protein complex of claim 35, wherein the second polypeptide comprises at least one polypeptide having the sequence of SEQ ID NOs: 103 and 114 or SEQ ID NOs: 103 and 172.

37. The protein complex of claim 36, comprising a polypeptide having the following sequence: 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.

38. A bifunctional fusion protein comprising: (a) an engineered IL2 polypeptide comprising the sequence of any one of claims 1-25; and (b) Antigen binding portion.

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

40. The bifunctional fusion protein of claim 38 or 39, 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.

41. The bifunctional fusion protein of any one of claims 38-40, wherein the antigen binding portion binds PD-L1, and comprises: (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.

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

43. The bifunctional fusion protein of claim 42, wherein the antibody heavy chain constant region is a human IgG heavy chain constant region. The bifunctional fusion protein of claim 43 , wherein the antibody heavy chain constant region is a human IgG1 heavy chain constant region.

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

137.

46. ​​A bifunctional fusion protein as described in 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 relative to SEQ ID NO:

137.

47. The bifunctional fusion protein of any one of claims 42-46, 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 of any one of claims 42-47, comprising a sequence selected from the group consisting of SEQ ID NOs: 106-113 and SEQ ID NOs: 173-177 or consisting thereof.

49. The bifunctional fusion protein of any one of claims 42-48, 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 of any one of claims 42-49, and a second antigen binding portion.

51. The protein complex of claim 50, wherein the second antigen binding portion comprises at least one polypeptide having the sequence of SEQ ID NOs: 103 and 104 or SEQ ID NOs: 103 and 172.

52. The protein complex of claim 51, comprising a polypeptide having the following sequence: 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; 1. SEQ ID NO: 172, 176 and 103; or m. SEQ ID NO: 172, 177 and 103.

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

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

55. The protein complex of claim 54, wherein the second antigen binding moiety binds PD-1.

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

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

59. The fusion polypeptide of 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 of claim 17 or 18, wherein the second polypeptide comprises a ligand or a scaffold protein.

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

62. An expression vector comprising the polynucleotide of claim 61.

63. A modified cell comprising the isolated polynucleotide of claim 61 or the expression vector of claim 62.

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

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

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

67. The use or method of claim 65 or 66, wherein the regulating immune response comprises at least one of: enhancing effector T cell activity, enhancing NK cell activity, and inhibiting regulatory T cell activity.

68. The engineered IL2 polypeptide of any one of claims 1-16, the fusion polypeptide of any one of claims 17-34 and 58-60, the protein complex of any one of claims 35-37 and 56-57, the bifunctional fusion protein of any one of claims 38-55, the polynucleotide of claim 61, the vector of claim 62, or the modified cell of claim 63 or the pharmaceutical composition of 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 an engineered IL2 polypeptide of any one of claims 1-16, a fusion polypeptide of any one of claims 17-34 and 58-60, a protein complex of any one of claims 35-37 and 56-57, a bifunctional fusion protein of any one of claims 38-55, a polynucleotide of claim 61, a vector of claim 62, or a modified cell of claim 63, or a pharmaceutical composition of claim 64.

70. The use or method of claim 68 or 69, wherein the disease comprises cancer or immunosuppression.

71. The use or method of claim 70, wherein the cancer comprises a hematological malignancy such as lymphoma, leukemia, multiple myeloma and / or a solid tumor such as breast cancer, pancreatic cancer, lung cancer, glioblastoma, renal cell carcinoma, head and neck cancer, liver cancer, gastric cancer, colon / colorectal cancer, kidney cancer, bladder cancer or melanoma.

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

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

74. The use or method of claim 73, wherein the cell therapy is an immune cell expressing a chimeric antigen receptor, an immune cell expressing an engineered T cell receptor, a tumor infiltrating lymphocyte, or any combination thereof.

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

76. A method of culturing cells, comprising incubating the cells with the culture medium of claim 75.

77. The method of claim 76, wherein the cells are in a blood sample.

78. The method of claim 76, wherein the cells are in a sample comprising PBMCs.

79. The method of claim 76, wherein the cell is a T cell, a NK cell, a T regulatory cell, or any combination thereof.

80. The method of 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 of any one of claims 76-79, wherein the engineered IL2 polypeptide, fusion polypeptide, protein complex, or bifunctional fusion protein biases the growth, expansion, or persistence of a cell type.

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

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

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

85. A transgenic immune cell as described in claim 84, wherein the CAR targets an antigen produced by cancer cells.

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

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

88. The transgenic immune cell of any one of claims 85-87, wherein the transgenic immune cell is an armored CAR T cell.

89. The transgenic immune cell of any one of claims 82-88, 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 of any one of claims 82-88.

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

92. The use or method of claim 91, wherein the cancer comprises a hematological malignancy such as lymphoma, leukemia, multiple myeloma and / or a solid tumor such as breast cancer, pancreatic cancer, lung cancer, glioblastoma, renal cell carcinoma, head and neck cancer, liver cancer, gastric cancer, colon / colorectal cancer, kidney cancer, bladder cancer or melanoma.

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

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

95. A method for expanding immune cells in vitro or ex vivo, comprising contacting the immune cell population with an effective amount of an engineered IL2 polypeptide of any one of claims 1-16, a fusion polypeptide of any one of claims 17-34 and 58-60, a protein complex of any one of claims 35-37 and 56-7, or a bifunctional fusion protein of any one of claims 38-55 under conditions sufficient to promote expansion of the immune cell population, and culturing the immune cell population for a sufficient time to at least double the number of the immune cells.

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

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

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

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

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

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