Mutant il-2 fusions with immune cell specific binding proteins and methods of use thereof

Abstract

The present disclosure relates to fusion proteins comprising a mutant IL-2 polypeptide subunit and one or two antigen binding domains (e.g., VHH antibodies) that bind specifically to an antigen expressed on the surface of an immune cell, such as a tumor-reactive T cell expressing CD8 and / or PD1. The fusions can further comprise half-life extending proteins subunits, such as Ig1 Fc region monomer polypeptide. The disclosure also relates to uses of these fusion proteins as therapeutics, for example in pharmaceutical compositions for the treatment of cancers and autoimmune disorders.

Description

MUTANT IL-2 FUSIONS WITH IMMUNE CELL SPECIFIC BINDING PROTEINS AND METHODS OF USE THEREOF CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority of U.S. provisional patent application no.63 / 515,964, filed July 27, 2023, which is hereby incorporated by reference herein. FIELD

[0002] The present disclosure relates to fusion proteins comprising a mutant IL-2 polypeptide and one or two binding domains (e.g., VHH antibodies) that bind specifically to an antigen expressed on the surface of an immune cell, such as a tumor-reactive T cell expressing CD8 and / or PD1. The fusions can further comprise half-life extending proteins, such Fc monomer or Fc dimer, serum albumin polypeptides.  REFERENCE TO SEQUENCE LISTING

[0003] The official copy of the Sequence Listing is submitted concurrently with the specification via USPTO Patent Center as an WIPO Standard ST.26 formatted XML file with file name “17195-004PV1.xml”, a creation date of July 27, 2023, and a size of 114,715 bytes. This Sequence Listing filed via USPTO Patent Center is part of the specification and is incorporated in its entirety by reference herein.  BACKGROUND

[0004] Interleukin-2 (IL-2), also known as T-cell growth factor (TCGF), is a pluripotent cytokine produced mainly by activated T cells, in particular CD4+ T helper cells. IL-2 signaling is mediated through binding to three different receptor proteins: IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132). Immune cells express dimeric or trimeric complexes of the IL-2 receptor proteins. The dimeric receptor (IL-2Rβγ) is expressed on cytotoxic CD8+ T cells and natural killer cells (NK), whereas the trimeric receptor (IL-2Rαβγ) is expressed predominantly on activated lymphocytes and CD4+ CD25+ FoxP3+ suppressive regulatory T cells (Treg). Treg cells express high levels of IL-2Rα (CD25) and Treg proliferation is stimulated by IL-2. Effector T cells and NK cells in a resting state, however, do not have CD25 on the cell surface and are relatively insensitive to IL-2.

[0005] IL-2 binding to the three different receptor proteins varies significantly. IL-2 high affinity for the trimeric receptor with a KDof about 10 pM, an intermediate affinity for the dimeric receptor with a KDof about 1 nM, and low affinity for the monomeric IL-2Rα receptor, with a KDof about 10 nM. IL-2 signaling activity mediated by the different receptor complexes also varies significantly. Generally, it has been found that IL-2Rβ and IL-2Rγ are critical for IL-2 signaling, while IL-2Rα (CD25) is not essential.

[0006] IL-2 binding to the IL-2 receptor protein expressed on different cells mediates different immune responses. IL-2 can stimulate immune responses, such as: T cell proliferation and differentiation, cytotoxic T lymphocyte (CTL) production, B cell proliferation and differentiation, immunoglobulin synthesis, and production, proliferation, and activation of natural killer (NK) cells. IL-2 has been approved as an immunotherapeutic agent for the treatment of cancer and chronic viral infection. IL-2, however, can also promote the activation and proliferation of immunosuppressive CD4+ CD25+ Treg cells resulting in immunosuppression (Fontenot et al., Nature Immunol.6, 1142-51 (2005); D'Cruz and Klein, Nature Immunol.6, 1152-59 (2005); Maloy and Powrie, Nature Immunol.6, 1171-72 (2005)). In addition, IL-2 treatment is associated with vascular leak syndrome (VLS), and pulmonary edema in patients. It is believed that the pulmonary edema is due to the direct binding of IL-2 to trimeric receptors (IL-2Rαβγ) on lung endothelial cells (Krieg et al., Proc Nat Acad Sci USA 107, 11906-11 (2010)).

[0007] The engineering of IL-2 with mutations has been proposed to reduce these toxic side effects by altering the selectivity or preference of IL-2 for the different IL-2 receptor subunits and thereby improve its therapeutic effect. For example, it has been proposed that targeting IL- 2 to cells expressing IL-2Rβ but not IL-2Rα, can induce amplification of cell populations high in IL-2Rβ, which improves the therapeutic effect of IL-2 therapy (Boyman et al., Science 311, 1924-1927 (2006)). US Patent Publ.2018 / 0142037 A1 describes introducing mutations at IL-2 amino acid positions 42, 45, and 72, also for the purpose of reducing affinity of IL-2 for the IL- 2Rα receptor. Another mutant IL-2 called “IL-2H9” which includes the five mutations L80F, R81D, L85V, I86V, and I92F, exhibits enhanced binding to IL-2Rβ, resulting in the stimulation of CD25− cells (see, Levin et al., Nature, Vol 484, p 529-533, DOI: 10.1038 / nature10975). A mutant IL-2 protein “IL-23x,” has three mutations, R38D, K43E, and E61R that result in very low binding affinity for IL-2Rα (see, Rodrigo Vazquez-Lombardi et al., Nature Communications, 8:15373, DOI: 10.1038 / ncomms15373). However, the activation preference of IL-23X for CD25+ cells still exists, and the expression level of the mutant polypeptide is low, which is not conducive to subsequent large-scale drug production.

[0008] CD8 is a transmembrane glycoprotein that acts as a co-receptor with the T-cell receptor (TCR) to mediate T cell signaling that promotes cytotoxic T cell-antigen interactions. CD8 is expressed on the surface of cytotoxic T cells and binds to the major histocompatibility complex (MHC) class I protein. The extracellular domain of the CD8α isoform binds to the α3 portion of Class I MHC and this binding affinity keeps the cytotoxic T cell and the target cell bound closely during antigen-specific activation.

[0009] Cytotoxic T cells with CD8 surface protein are called CD8+ T cells. The CD8 co- receptor also facilitates T cell signaling via the cytoplasmic domain of the transmembrane CD8 receptor binding to Lck (lymphocyte-specific protein tyrosine kinase). Lck phosphorylates the cytoplasmic domain of the TCR complex which initiates a cascade of phosphorylation events resulting in activation of transcription factors including NFAT, NF-κB, and AP-1. CD8+ T cells  also have the ability to make some cytokines, such as TNF-α and IFN-γ, with antitumor and antimicrobial effects.

[0010] Cytotoxic CD8+ T cells are known play an important role in the immune response against cancer. Tumors, however, have mechanisms that can defeat the CD8+ T cell immune response, such as the production of immunosuppressive cytokines, or immune checkpoint molecules, such as PD-1, CTLA4, LAG3.

[0011] PD-1 (also known as, Programmed cell death protein 1, PDCD1, PD1) is a cell surface receptor expressed on T cells and at much lower level on NK cells, and Pro-B cells. On most naïve T cells, PD-1 is not expressed, or very low if any. But PD1 expression is induced on antigen-experienced T cells, including CD8+, CD4+, CD4+ / FOXP3+Tregs, gamma / delta T cells. PD-1 expression is especially high on exhausted T cells. PD-1 is a type I membrane protein of 288 amino acids that is a member of the extended CD28 / CTLA-4 family of T cell regulators. The protein's structure includes an extracellular IgV domain followed by a transmembrane region and an intracellular tail. The intracellular tail contains two phosphorylation sites located in an immunoreceptor tyrosine-based inhibitory motif and an immunoreceptor tyrosine-based switch motif, which suggests that PD-1 negatively regulates T-cell receptor TCR signals.

[0012] PD-1 binds two ligands, PD-L1 and PD-L2. PD-L1 protein is upregulated on macrophages and dendritic cells (DC) in response to LPS and GM-CSF treatment, and on T cells and B cells upon TCR and B cell receptor signaling. Human PD-L1 is overexpressed in a number of types of tumors from patients, including non-small cell lung cancer. PD-L1 is expressed on almost all murine tumor cell lines, including PA1 myeloma, P815 mastocytoma, and B16 melanoma upon treatment with IFN-γ. PD-L2 expression is more restricted and is expressed mainly by DCs and a few tumor lines.

[0013] It is well-known that PD-1 plays an important role in the ability of cancer to evade an immune response. Many tumor cells express the PD-1 ligand PD-L1, and it has been found that inhibition of the interaction between PD-1 and PD-L1 enhances T-cell responses that mediate the preclinical antitumor activity known as the immune checkpoint blockade. Many monoclonal antibodies have been or are being developed that target PD-1 for the treatment of cancer including Dostarlimab, Nivolumab, Pembrolizumab, Pidilizumab, Cemiplimab, and Toripalimab.

[0014] There remains a need for fusions of IL-2 with polypeptides that specifically bind CD-8 and / or PD-1, that can be used as immunotherapeutic agents to modulate the immune response mediated by activated T cells. SUMMARY

[0015] The present disclosure relates generally to fusion proteins that comprise a mutant interleukin-2 (IL-2) polypeptide and one or two antigen binding subunits comprising polypeptides that specifically bind to an antigen expressed on the surface of immune cells. The  disclosure also relates to pharmaceutical compositions comprising these fusion polypeptide molecules, and uses of these composition as therapeutics, for example in the treatment of cancer. This summary is intended to introduce the subject matter of the present disclosure, but does not cover each and every embodiment, combination, or variation that is contemplated and described within the present disclosure. Further embodiments are contemplated and described by the disclosure of the detailed description, drawings, and claims.

[0016] In at least one embodiment, the present disclosure provides a fusion protein comprising a first polypeptide chain, wherein the first polypeptide chain comprises: (a) an A1B subunit comprising a polypeptide that specifically binds a first antigen expressed on the surface of an immune cell; (b) an HLE subunit comprising a polypeptide with half-life extending activity; and (c) an IL2 subunit comprising a polypeptide having an amino acid sequence of at least 90% identity to SEQ ID NO: 10 and a set of amino acid differences relative to SEQ ID NO: 10 selected from: K35N and Y45R; E95N and K97T; E95N and K97S; K35N, Y45R, E95N, and K97S; K35N, Y45R, E95N, and K97T; K35N, Y45R, E61N, L63T, E95N, and K97T; and K35N, Y45R, E61N, L63T, E95N, and K97S.

[0017] In at least one embodiment of the fusion protein the first polypeptide chain further comprises: (d) an A2B subunit comprising a polypeptide that specifically binds a second antigen expressed on the surface of the immune cell.

[0018] In at least one embodiment of the fusion protein of the present disclosure, the IL2 subunit comprises a polypeptide having an amino acid sequence selected from SEQ ID NO: 11, 12, 13, 14, 15, 16, and 17.

[0019] In at least one embodiment of the fusion protein of the present disclosure, the A1B subunit and / or the A2B subunit are selected from an antibody, a Fab, a scFv, VHH antibody, and a nanobody. In at least one embodiment, the A1B subunit and / or A2B subunit comprise a VHH antibody that specifically binds to first and / or second antigens selected from PD1, CD8, CD39, and CD103.

[0020] In at least one embodiment of the fusion protein of the present disclosure, the A1B subunit and / or the A2B subunit comprise an VHH antibody, wherein the antibody: (a) specifically binds PD1 and comprises a CDR1 of SEQ ID NO: 19, a CDR2 of SEQ ID NO: 20, and a CDR3 of SEQ ID NO: 21; (b) specifically binds PD1 and comprises an amino acid sequence selected from SEQ ID NO: 20, 22, 23, 24, 25, and 26; (c) specifically binds CD8 and comprises a CDR1 of SEQ ID NO: 28, a CDR2 of SEQ ID NO: 29, and a CDR3 of SEQ ID NO: 30; and / or (d) specifically binds CD8 and comprises an amino acid sequence selected from SEQ ID NO: 27, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, and 52.

[0021] In at least one embodiment of the fusion protein of the present disclosure, the HLE subunit comprises an IgG1 Fc region polypeptide, wherein the IgG1 Fc region includes an amino acid sequence feature selected from: KK, DSDL, LALA, and N297G. In at least one  embodiment, the IgG1 Fc region polypeptide comprises an amino acid sequence selected from SEQ ID NO: 72, 73, 74, 75, 76, 77, 78, 79, 80, and 81.

[0022] In at least one embodiment of the fusion protein of the present disclosure, each of the subunits is covalently attached via a linker to at least one of the other subunits through its N- terminus and / or C-terminus. In at least one embodiment, the linkers between the subunits are the same or different. In at least one embodiment, the linkers between the subunits comprise an amino acid sequence selected from SEQ ID NO: 82-101; optionally, wherein the amino acid sequence is selected from (GGGGS)1(SEQ ID NO: 82), (GGGGS)2(SEQ ID NO: 83), (GGGGS)3(SEQ ID NO: 84), (GGGGS)4(SEQ ID NO: 85), (GGGGS)5(SEQ ID NO: 86), (GGGGS)6(SEQ ID NO: 87), and (GGGGS)3GGG (SEQ ID NO: 89).

[0023] In at least one embodiment of the fusion protein of the present disclosure, the N- terminal to C-terminal orientation of the subunits is selected from: (a) (A1B)-(HLE)-(IL2); (b) (A1B)-(IL2)-(HLE); (c) (IL2)-(A1B)-(HLE); (d) (IL2)-(HLE)-(A1B); (e) (HLE)-(A1B)-(IL2); and (f) (HLE)-(IL2)-(A1B).

[0024] In at least one embodiment of the fusion protein of the present disclosure, the fusion protein comprises an A2B subunit comprising a polypeptide that specifically binds a second antigen expressed on the surface of the immune cell, and the N-terminal to C-terminal orientation of the subunits is selected from: (a) (A1B)-(HLE)-(IL2)-(A2B); (b) (A1B)-(HLE)- (A2B)-(IL2); (c) (A1B)-(A2B)-(HLE)-(IL2); (d) (A2B)-(A1B)-(HLE)-(IL2); (e) (A1B)-(IL2)-(HLE)- (A2B); (f) (A1B)-(IL2)-(A2B)-(HLE); (g) (A1B)-(A2B)-(IL2)-(HLE); (h) (A2B)-(A1B)-(IL2)-(HLE); (i) (IL2)-(A1B)-(HLE)-(A2B); (j) (IL2)-(A1B)-(A2B)-(HLE); (k) (IL2)-(A2B)-(A1B)-(HLE); (l) (A2B)- (IL2)-(A1B)-(HLE); (m) (IL2)-(HLE)-(A1B)-(A2B); (n) (IL2)-(HLE)-(A2B)-(A1B); (o) (IL2)-(A2B)- (HLE)-(A1B); (p) (A2B)-(IL2)-(HLE)-(A1B); (q) (HLE)-(A1B)-(IL2)-(A2B); (r) (HLE)-(A1B)-(A2B)- (IL2); (s) (HLE)-(A2B)-(A1B)-(IL2); (t) (A2B)-(HLE)-(A1B)-(IL2); (u) (HLE)-(IL2)-(A1B)-(A2B); (v) (HLE)-(IL2)-(A2B)-(A1B); (w) (HLE)-(A2B)-(IL2)-(A1B); and (x) (A2B)-(HLE)-(IL2)-(A1B).

[0025] In at least one embodiment of the fusion protein of the present disclosure, the fusion protein further comprises a second polypeptide chain that forms a dimer with the first polypeptide chain. In at least one embodiment, the fusion protein is a homodimer and the first and second polypeptide chains are selected from: (a) (A1B)-(HLE)-(IL2); (b) (A1B)-(IL2)-(HLE); (c) (IL2)-(A1B)-(HLE); (d) (IL2)-(HLE)-(A1B); (e) (HLE)-(A1B)-(IL2); (f) (HLE)-(IL2)-(A1B); (g) (A1B)-(HLE)-(IL2)-(A2B); (h) (A1B)-(HLE)-(A2B)-(IL2); (i) (A1B)-(A2B)-(HLE)-(IL2); (j) (A2B)- (A1B)-(HLE)-(IL2); (k) (A1B)-(IL2)-(HLE)-(A2B); (l) (A1B)-(IL2)-(A2B)-(HLE); (m) (A1B)-(A2B)- (IL2)-(HLE); (n) (A2B)-(A1B)-(IL2)-(HLE); (o) (IL2)-(A1B)-(HLE)-(A2B); (p) (IL2)-(A1B)-(A2B)- (HLE); (q) (IL2)-(A2B)-(A1B)-(HLE); (r) (A2B)-(IL2)-(A1B)-(HLE); (s) (IL2)-(HLE)-(A1B)-(A2B); (t) (IL2)-(HLE)-(A2B)-(A1B); (u) (IL2)-(A2B)-(HLE)-(A1B); (v) (A2B)-(IL2)-(HLE)-(A1B); (w) (HLE)- (A1B)-(IL2)-(A2B); (x) (HLE)-(A1B)-(A2B)-(IL2); (y) (HLE)-(A2B)-(A1B)-(IL2); (z) (A2B)-(HLE)- (A1B)-(IL2); (aa) (HLE)-(IL2)-(A1B)-(A2B); (bb) (HLE)-(IL2)-(A2B)-(A1B); (cc) (HLE)-(A2B)- (IL2)-(A1B); and (dd) (A2B)-(HLE)-(IL2)-(A1B).

[0026] In at least one embodiment of the fusion protein of the present disclosure, the fusion protein further comprises a second polypeptide chain that forms a dimer with the first polypeptide chain. In at least one embodiment, the fusion protein is a heterodimer wherein the first and second polypeptide chains are selected from: (a) (A1B)-(HLE)-(IL2) and (A1B)-(HLE); (b) (A1B)-(HLE)-(IL2) and (A2B)-(HLE); (c) (A1B)-(HLE)-(IL2)-(A2B) and (A1B)-(HLE); and (d) (A1B)-(HLE)-(A2B) and (A1B)-(HLE)-(IL2).

[0027] In at least one embodiment of the fusion protein of the present disclosure, the immune that expresses the antigen on its surface cell is a tumor-reactive T cell or a Treg cell. In at least one embodiment, the first antigen is selected from CD8, PD-1, CD39, and CD103. In at least one embodiment, the second antigen is selected from CD8, PD-1, CD39, and CD103.

[0028] In at least one embodiment of the fusion protein of the present disclosure, the fusion protein comprises an A2B subunit comprising a polypeptide that specifically binds a second antigen expressed on the surface of the immune cell, the first and second antigens are different; optionally, wherein the first and second antigens are CD8 and PD-1.

[0029] In at least one embodiment of the fusion protein of the present disclosure, the fusion protein comprises an amino acid sequence selected from SEQ ID NO: 53, 54, 55, 56, 57, 58, 59, 60, 61, 63, 64, 65, 66, 67, 68, 69, 70, and 71.

[0030] In another embodiment, the present disclosure also provides a polynucleotide encoding a fusion protein of the present disclosure. In at least one embodiment, the disclosure provides an expression vector comprising the polynucleotide encoding a fusion protein of the present disclosure.

[0031] In another embodiment, the present disclosure also provides an isolated host cell comprising the polynucleotide or the expression vector encoding a fusion protein of the present disclosure; optionally, wherein, the host cell is a mammalian cell or a yeast cell. In at least one embodiment, the host cell is a mammalian cell selected from a Chinese hamster ovary (CHO) cell, a myeloma cell (e.g.,Y0, NS0, Sp2 / 0), a monkey kidney cell (COS-7), a human embryonic kidney line (293), a baby hamster kidney cell (BHK), a mouse Sertoli cell (e.g., TM4), an African green monkey kidney cell (VERO-76), a human cervical carcinoma cell (HELA), a canine kidney cell, a human lung cell (W138), a human liver cell (Hep G2), a mouse mammary tumor cell, a TR1 cell, a Medical Research Council 5 (MRC 5) cell, and a FS4 cell.

[0032] In another embodiment, the present disclosure provides a method for producing a fusion protein of the present disclosure, wherein the method comprises culturing the host cell comprising a polynucleotide encoding a fusion protein of the present disclosure under a condition suitable for expressing the polypeptide.

[0033] In another embodiment, the present disclosure also provides a pharmaceutical composition comprising a fusion protein of the present disclosure and a pharmaceutically acceptable carrier.

[0034] In another embodiment, the present disclosure provides a method for treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of a fusion protein of the disclosure, or administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a fusion protein of the present disclosure.

[0035] In at least one embodiment of the method for treating a disease or disorder in a subject, the disease or disorder is cancer; optionally, wherein the cancer is selected from colorectal cancer, pancreatic cancer, ovarian cancer, liver cancer, renal cancer, breast cancer, lung cancer, esophageal and gastric cancer, head and neck cancer, cervical cancer, prostate cancer, melanoma, bladder cancer, oral cancer, or hematological malignancies.

[0036] In at least one embodiment of the method for treating a disease or disorder in a subject, the disease or disorder is an autoimmune disease; optionally, wherein the autoimmune disease is selected from Crohn’s disease, Ulcerative colitis, celiac disease, systemic lupus erythematosus, psoriatic arthritis, rheumatoid arthritis, Sjogren’s syndrome, type 1 diabetes, atopic dermatitis, psoriasis, multiple sclerosis.

[0037] In at least one embodiment of the method for treating a disease or disorder in a subject, the disease or disorder is a chronic viral infection; optionally, wherein the chronic viral infection is selected from hepatitis C virus (HCV), herpes simplex virus (HSV1 and HSV2), Epstein-Barr virus (EBV), Varicella virus, rubella virus, and cytomegalovirus (CMV). BRIEF DESCRIPTION OF THE DRAWINGS

[0038] A better understanding of the novel features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0039] FIGURES 1A-1N depict schematic representations of exemplary mutant IL2 fusion proteins of the present disclosure.

[0040] FIGURES 2A-2I depict schematic representations of exemplary mutant IL2 fusion proteins of the present disclosure that have homo-dimeric or hetero-dimeric structures that include single chain mutant IL2 fusion polypeptides. The exemplary structures include homo- or hetero-dimers of the bi-specific or tri-specific single polypeptide chains, such as those depicted in FIG.1

[0041] FIGURES 3A, 3B, and 3C depict SEC-HPLC profiles of the exemplary mutant IL2 fusion proteins prepared and purified as described in Example 1. FIG.3A depicts SEC-HPLC profiles of the exemplary mutant IL2 fusion proteins, P401, P402, P404, P405, and P406. FIG.3B and 3C depict SEC-HPLC profiles of the exemplary mutant IL2 fusion protein, P703, after Protein A affinity and AEX purification, as described in Example 1.

[0042] FIGURES 4A, 4B, 4C, and 4D depict plots of results showing IL2 activity of exemplary mutant IL2 fusion proteins P402, P404, P405, and P406 in STAT5 reporter assays with various HEK Blue cells that express IL2, and CD8, and / or PD1, as described in Example 2.

[0043] FIGURES 5A and 5B depicts plots of results of a study of the IL-2 stimulatory activity of mutant IL2 fusion proteins in PBMCs from two donors (Donor A and Donor B) using a Phospho- STAT5 and isotype control staining flow cytometry as described in Example 3.

[0044] FIGURE 6 depicts plots of results of a study of the thermostability of a mutant IL-2 fusion proteins, P703 and P709, using differential scanning fluorimetry (DSF), as described in Example 4.

[0045] FIGURE 7 depicts a plot of results of a study of the pharmacokinetics of the mutant IL2 fusion protein, P610 in mice, as described in Example 5.

[0046] FIGURES 8A and 8B depicts plots of results of a study of the in vivo anti-tumor efficacy of the mutant IL2 fusion protein, P701, in a syngeneic mouse tumor model, as described in Example 6. DETAILED DESCRIPTION

[0047] The present disclosure provides mutant IL-2 fusion proteins that comprise a IL2 subunit, comprising a mutant IL-2 polypeptide, fused to one or two antigen binding subunits comprising a polypeptide that specifically binds to an antigen (e.g., CD8 and / or PD1) expressed on the surface of an immune cell, such as a tumor-reactive T cell. The disclosure also relates to pharmaceutical compositions comprising these fusion polypeptide molecules, and uses of these composition as therapeutics, for example in the treatment of cancer or autoimmune diseases.

[0048] Overview of Terminology and Techniques

[0049] For the descriptions herein and the appended claims, the singular forms “a”, and “an” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a protein” includes more than one protein, and reference to “a compound” refers to more than one compound. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. The use of “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.”

[0050] Where a range of values is provided, unless the context clearly dictates otherwise, it is understood that each intervening integer of the value, and each tenth of each intervening integer of the value, unless the context clearly dictates otherwise, between the upper and lower  limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of these limits, ranges excluding (i) either or (ii) both of those included limits are also included in the invention. For example, “1 to 50,” includes “2 to 25,” “5 to 20,” “25 to 50,” “1 to 10,” etc.

[0051] Generally, the nomenclature used herein and the techniques and procedures described herein include those that are well understood and commonly employed by those of ordinary skill in the art, such as the common techniques and methodologies described in e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Vols.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2012 (hereinafter “Sambrook”); and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., originally published in 1987 in book form by Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., and regularly supplemented through 2011, and now available in journal format online as Current Protocols in Molecular Biology, Vols.00 - 130, (1987-2020), published by Wiley & Sons, Inc. in the Wiley Online Library (hereinafter “Ausubel”).

[0052] All publications, patents, patent applications, and other documents referenced in this disclosure are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference herein for all purposes.

[0053] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. It is to be understood that the terminology used herein is for describing particular embodiments only and is not intended to be limiting. For purposes of interpreting this disclosure, the following description of terms will apply and, where appropriate, a term used in the singular form will also include the plural form and vice versa.

[0054] “Fusion,” or “fusion protein,” as used herein, refers to two or more protein and / or polypeptide molecules that are linked (or “fused”) in a configuration that does not occur naturally. Fusion proteins of the present disclosure include fusions of a mutant IL2 polypeptide and one or more polypeptides that specifically bind an antigen expressed on the surface of an immune cell. The antigen binding polypeptides can include antibodies such an anti-PD-1 VHH and / or an anti-CD8 VHH covalently linked to the IL2 polypeptide through a polypeptide linker sequence and / or or another polypeptide, such as a half-life extending polypeptide.

[0055] “Polypeptide linker” or “linker sequence” as used herein refers to a chain of two or more amino acids with each end of the chain covalently attached to a different polypeptide molecule, thereby functioning to conjugate or fuse the different polypeptides. Typically, polypeptide linkers comprise polypeptide chains of 5 to 30 amino acids. A wide range of polypeptide linkers  are known in the art and can be used in the compositions and methods comprising the fusions of the anti-PD-1 VHH antibodies of the present disclosure. Exemplary polypeptide linkers include in the compositions and methods of the present disclosure include, but are not limited to (GGGGS)n, (SSSSG)n, (GGGG)(SGGGG)n, (EAAAK)n, (XP)n(wherein X can be any amino acid, preferably A, K, or E), ENLYFQ(-G / S), typically, where n is 1 to 10.

[0056] “IL-2” or “IL-2 polypeptide” as used herein refers to the cytokine, interleukin-2, and includes naturally occurring and recombinant forms of the interleukin-2 polypeptide from human, mouse, rat, or non-human primate, and in its unprocessed (with signal peptide) and processed forms (without signal peptide). In addition, this term includes naturally occurring IL-2 variants, such as allelic and splice variants, isotypes, homologs, and species homologs, and recombinant (i.e., man-made) IL-2 variants or mutants, including mutant IL-2 polypeptides having from 1-15 amino acid substitutions relative to the amino acid sequence of the naturally occurring IL-2. For example, the term encompasses the recombinant human IL-2 amino acid sequence of UniProt P60568 with an amino acid substitution at position C125, such as C125S, or C125A. This term is also intended to encompass IL-2 polypeptides that are covalently conjugated (or fused) to another polypeptide or protein. Exemplary IL-2 fusions of the present disclosure include a mutant IL-2 polypeptide fused to other cytokines (e.g., IL-15), or fused to a half-life extending polypeptide (e.g., monomeric Fc, dimeric Fc, or human serum albumin).

[0057] “IL-2 receptor” or “IL-2R,” as used herein refers to the heterotrimeric protein expressed on the surface of certain immune cells and endothelial cells and also encompasses each of the polypeptide subunits, IL-2Rα, IL-2Rβ, and IL-2Rγ (also known as cytokine receptor common subunit gamma), in their monomeric form, and in the dimeric form, such as IL-2Rβγ.

[0058] “PD-1” as used herein, refers to the “programmed cell death 1” protein that serves as a cell surface receptor for PD-L1 and PD-L2, and encompasses the full-length and portions of the full-length PD-1 proteins of human, cynomolgus monkey (herein referred to in some cases as “cyno”), rhesus monkey, and their various isoforms. An exemplary sequence of the human PD- L1 chain extracellular domain (ECD) is provided in Table 3 and the accompanying Sequence Listing.

[0059] “PD-1 mediated condition” or “PD-1 mediated disease,” as used herein, encompasses any medical condition associated with or effected by PD-1, or a therapeutic effect mediated by or afforded by cells that express PD-1 (including, but not limited to CD8+T cells, NK cells, NKT cells, CD4+T cells, CD4+ / FOXP3+Tregs, gamma / delta T cells). For example, specific binding to PD-1 expressed on cell surfaces can alter activation of CD8+ lymphocytes (e.g., T cells). Accordingly, PD-1 mediated diseases can include, but are not limited to, any disease or condition mediated by and / or responsive to agonists (or activators), or antagonists (or inhibitors) of PD-1 expressing cells, including but not limited to autoimmune disorders and cancers. Specific exemplary autoimmune disorders and cancers are provided elsewhere herein.

[0060] “CD8” as used herein, refers to the cluster of differentiation 8 (CD8) transmembrane glycoprotein that serves as a co-receptor for the T cell receptor, and encompasses the full- length and portions of the full-length CD8 proteins of human, cynomolgus monkey (herein referred to in some cases as “cyno”), rhesus monkey, and their various isoforms. The human CD8 protein is a dimer, consisting of a pair of CD8 chains, including CD8 alpha (or “CD8A”) and CD8 beta (or “CD8B”) chains. The term “human CD8” encompasses CD8A / CD8A homodimer, CD8A / CD8B heterodimer, CD8A chain, CD8B chain, or portions thereof, such as extracellular domains. “CD8A” and “CD8a” are used interchangeably herein, and “CD8B” and “CD8b” are used interchangeably herein. An exemplary sequence of the human CD8A chain extracellular domain (ECD) is provided in Table 3 and the accompanying Sequence Listing.

[0061] “CD8 mediated condition” or “CD8 mediated disease,” as used herein, encompasses any medical condition associated with or effected by CD8, or a therapeutic effect mediated by or afforded by CD8+ cells (including, but not limited to CD8+ T cells, NK cells, NKT cells). For example, specific binding to CD8 expressed on cell surfaces can alter activation of CD8+ lymphocytes (e.g., T cells). Accordingly, CD8 mediated diseases can include, but are not limited to, any disease or condition mediated by and / or responsive to agonists (or activators), or antagonists (or inhibitors) of CD8 expressing cells, including but not limited to autoimmune disorders and cancers. Specific exemplary autoimmune disorders and cancers are provided elsewhere herein.

[0062] “Antibody,” as used herein, refers to a molecule comprising one or more polypeptide chains that specifically binds to, or is immunologically reactive with, a particular antigen. Exemplary antibodies include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies (e.g., single-arm antibodies), multivalent antibodies, single- chain antibodies, heavy chain (only) antibodies (e.g., sdAb, or VHH, IgNAR, nanobody (or nanoAb)), antigen-binding fragments (e.g., Fab′, F(ab′)2, Fab, Fv, rIgG, and scFv fragments), antibody fusions, and synthetic antibodies (or antibody mimetics).

[0063] “Anti-PD-1 antibody” or “antibody that binds PD-1” refers to an antibody that binds PD-1 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting PD-1. In some embodiments, the extent of binding of an anti-PD-1 specific antibody to an unrelated, non-PD-1 antigen is less than about 20%, less than about 15%, less than about 10%, or less than about 5% of the binding of the antibody to PD-1 as measured, e.g., by a radioimmunoassay (RIA) or surface plasmon resonance (SPR). In some embodiments, an antibody that binds to PD-1 has a dissociation constant (KD) of < 1 μΜ, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 1 pM (e.g., 10-8M or less, e.g., from 10-8M to 10-13M, e.g., from 10-9M to 10-13M).

[0064] “Anti-CD8 antibody” or “antibody that binds CD8” refers to an antibody that binds CD8 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in  targeting CD8. In some embodiments, the extent of binding of an anti-CD8 specific antibody to an unrelated, non-CD8 antigen is less than about 20%, less than about 15%, less than about 10%, or less than about 5% of the binding of the antibody to CD8 as measured, e.g., by a radioimmunoassay (RIA) or surface plasmon resonance (SPR). In some embodiments, an antibody that binds to CD8 has a dissociation constant (KD) of < 1 μΜ, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 1 pM (e.g., 10-8M or less, e.g., from 10-8M to 10-13M, e.g., from 10-9M to 10-13M).

[0065] “Full-length antibody,” “intact antibody,” or “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0066] “Antibody fragment” refers to a portion of a full-length antibody which is capable of binding the same antigen as the full-length antibody. Examples of antibody fragments include, but are not limited to, VHH, single-domain antibodies, Fv, Fab, Fab', Fab'-SH, F(ab')2fragments, diabodies; linear antibodies; monovalent, or single-armed antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0067] “Heavy chain antibody,” “heavy chain-only antibody,” or “HCAb,” as used herein, refers to a functional antibody, which comprises two heavy chains, but lacks two light chains usually found in 4-chain antibodies. Camelid animals (such as camels, llamas, or alpacas) are known to produce HCAbs.

[0068] “Single-domain antibody” or “sdAb,” as used herein, refers to a single antigen-binding domain having three complementarity determining regions (CDRs). The sdAb alone is capable of binding to the antigen without pairing with a corresponding CDR-containing polypeptide. A camelid sdAb is one of the smallest known antigen-binding antibody fragments (see, e.g., Hamers-Casterman et al., Nature 363:446-8 (1993); Greenberg et al., Nature 374:168-73 (1995); Hassanzadeh-Ghassabeh et al., Nanomedicine (Lond), 8:1013-26 (2013)). “VHHs” (defined below) are a type of single-domain antibody that are engineered from camelid HCAbs.

[0069] “VHH,” or “variable domain of the heavy chain of a heavy chain antibody” refers to a single chain including a heavy chain variable domain of an antibody. A VHH typically has the following structure from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3- FR4, in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3. VHH molecules can be derived from antibodies raised in Camelidae species, for example, camel, llama, vicuna, dromedary, alpaca, and guanaco, or generated from synthetic libraries.

[0070] “VHH antibody” or “heavy chain antibody” or “heavy chain only antibody” refers to a single chain including a heavy chain variable domain of an antibody and an Fc region, with a hinge or other linker of amino acids with natural or synthetic sequence in between. The “single chain” could form dimer, such as when fused with Fc region that is a dimer.

[0071] “Class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these are further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0072] “Variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VHand VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt et al., Kuby Immunology, 6thed., W.H. Freeman and Co., page 91). A single VHor VLdomain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VHor VLdomain from an antibody that binds the antigen to screen a library of complementary VLor VHdomains, respectively (see, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0073] “Hypervariable region” or “HVR,” as used herein, refers to each of the regions of an antibody variable domain which are hypervariable in sequence and / or form structurally defined loops ("hypervariable loops"). Generally, native antibodies comprise four chains with six HVRs; three in the heavy chain variable domain, VH(HVR-H1, HVR-H2, HVR-H3), and three in the light chain variable domain, VL(HVR-L1, HVR-L2, HVR-L3). The HVRs generally comprise amino acid residues from the hypervariable loops and / or from the “complementarity determining regions” (CDRs). A number of hypervariable region delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol.196:901-917 (1987)). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. The “contact” hypervariable regions are based on an analysis of the available complex crystal structures. The residues from each of these hypervariable regions are noted in the Table 1 below.

[0074] TABLE 1 Loop Kabat AbM Chothia Contact  H3 H95-H102 H95-H102 H96-H101 H93-H101 1 Kabat numbering 2 Chothia numberin [ main (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0076] Hypervariable regions, as used herein, may include extended or alternative hypervariable regions as follows: 26-35 or 30-35 (H1), 50-61, 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VHdomain; and 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89- 97 or 89-96 (L3) in the VLdomain. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.

[0077] “Complementarity determining region,” or “CDR,” as used herein, refers to the regions within the HVRs of the variable domain which have the highest sequence variability and / or are involved in antigen recognition. Generally, native antibodies comprise four chains with six CDRs; three in the heavy chain variable domains, VH(H1, H2, H3), and three in the light chain variable domains, VL(L1, L2, L3). Generally, a VHH antibody comprises just the CDRs of the heavy chain (VH(H1, H2, H3). Exemplary CDRs (numbered according to Kabat et al., supra) occur at the following amino acid residue positions of the VH and VL domains: CDR-H1 at 31- 35; CDR-H2 at 50-61; CDR-H3 at 95-102; CDR-L1 at 24-34; CDR-L2 at 50-56; and CDR-L3 at 89-97.

[0078] “Framework” or “FR” refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH(or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0079] “Native antibody” refers to a naturally occurring immunoglobulin molecule. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light chains and two identical heavy chains that are disulfide- bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0080] “Monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies (e.g., variant antibodies contain mutations that occur naturally or arise during production of a monoclonal antibody, and generally are present in minor amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different  determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the term “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage- display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

[0081] “Chimeric antibody” refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0082] “Humanized antibody” refers to a chimeric antibody comprising amino acid sequences from non-human HVRs and amino acid sequences from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0083] “Human antibody” refers to an antibody which possesses an amino acid sequence corresponding to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

[0084] “Human consensus framework” is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VLor VHframework sequences. Generally, the selection of human immunoglobulin VLor VHsequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91- 3242, Bethesda MD (1991), vols.1-3. In some embodiments, for the VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In some embodiments, for the VH, the subgroup is subgroup III as in Kabat et al., supra.

[0085] “Acceptor human framework” as used herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it  may contain amino acid sequence changes. In some embodiments, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VLacceptor human framework is identical in sequence to the VLhuman immunoglobulin framework sequence or human consensus framework sequence.

[0086] “Fc region,” or “fragment crystallizable region” refers to a dimer complex comprising the C-terminal polypeptide sequences of an immunoglobulin heavy chain, wherein a C-terminal polypeptide sequence is that which is obtainable by papain digestion of an intact antibody. The Fc region of an immunoglobulin generally includes the heavy chain CH2 and CH3 domains, and optionally the CH4 domain. The Fc region may comprise native or variant Fc sequences. Although the boundaries of the Fc sequence of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc sequence is usually defined to stretch from an amino acid residue at about position Cys226, or from about position Pro230, to the carboxyl-terminus of the Fc sequence.

[0087] “Fc receptor” or “FcR,” refers to a receptor that binds to the Fc region of an antibody. In some embodiments, an FcR is a native human FcR. In some embodiments, an FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of those receptors. FcγRII receptors include FcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain, (see, e.g., Daeron, Annu. Rev. Immunol.15:203-234 (1997)). FcR, as used herein, also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al, J. Immunol.117:587 (1976) and Kim et al, J. Immunol.24:249 (1994)) and regulation of homeostasis of immunoglobulins. FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al, Immunomethods 4:25-34 (1994); and de Haas et al, J. Lab. Clin. Med.126:330-41 (1995).

[0088] “Multivalent antibody,” as used herein, is an antibody comprising three or more antigen binding sites. The multivalent antibody is preferably engineered to have the three or more antigen binding sites and is generally not a native sequence IgM or IgA antibody.

[0089] “Multispecific antibody” is an antibody having at least two different binding sites, each site with a different binding specificity. A multispecific antibody can be a full-length antibody or an antibody fragment, and the different binding sites may bind each to a different antigen or the different binding sites may bind to two different epitopes of the same antigen.

[0090] “Fv fragment” refers to an antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight association, which can be covalent in nature, for example in scFv. It is  in this configuration that the three HVRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six HVRs or a subset thereof confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although usually at a lower affinity than the entire binding site.

[0091] “Fab fragment’ refers to an antibody fragment that contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CH1) of the heavy chain. “F(ab')2fragments” comprise a pair of Fab fragments which are generally covalently linked near their carboxy termini by hinge cysteines between them. Other chemical couplings of antibody fragments also are known in the art.

[0092] “Antigen binding arm,” as used herein, refers to a component of an antibody that has an ability to specifically bind a target molecule of interest. Typically the antigen binding arm is a complex of immunoglobulin polypeptide sequences, e.g., HVR and / or variable domain sequences of an immunoglobulin light and heavy chain.

[0093] “Single-chain Fv” or “scFv” refer to antibody fragments comprising the VHand VLdomains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, an Fv polypeptide further comprises a polypeptide linker between the VHand VLdomains which enables the scFv to form the desired antigen binding structure.

[0094] “Diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VHand 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 create two antigen-binding sites.

[0095] “Linear antibodies" refers to the antibodies described in Zapata et al., Protein Eng., 8(10): 1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (VH- CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

[0096] “Naked antibody” refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel.

[0097] “Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an IL-2 polypeptide) and its binding partner (e.g., an IL-2 receptor). “Binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. “Binds specifically” or “specific binding” refers to binding of IL-2 polypeptide to its receptor with an affinity value of no more than about 1 x 10-7M. Illustrative and exemplary embodiments for measuring binding affinity and / or specific binding are described elsewhere herein including the Examples.

[0098] “Binds specifically” or “specific binding” refers to binding of an antibody to an antigen with an affinity value of no more than about 1 x 10-7M. In some embodiments, an antibody may have a secondary affinity for an antigen other than the antigen to which it binds specifically, where “secondary affinity” will generally refer to binding of an antibody to a secondary antigen with an affinity value of more than about 10 nM as described elsewhere herein. Where an antibody may have a secondary affinity for a secondary antigen, such an antibody will nevertheless bind specifically to the primary antigen.

[0099] “Affinity matured” antibody refers to an antibody with one or more alterations in one or more HVRs, compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.

[0100] “Functional antigen binding site” of an antibody is one which is capable of binding a target antigen. The antigen binding affinity of the antigen binding site is not necessarily as strong as the parent antibody from which the antigen binding site is derived, but the ability to bind antigen must be measurable using any one of a variety of methods known for evaluating antibody binding to an antigen.

[0101] “Isolated antibody” refers to an antibody which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic methods (e.g., ion exchange or reverse phase HPLC). For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87.

[0102] “Substantially similar” or “substantially the same,” as used herein, refers to a sufficiently high degree of similarity between two numeric values (for example, one associated with a test antibody and the other associated with a reference antibody), such that one of skill in the art would consider the difference between the two values to be of little or no biological and / or statistical significance within the context of the biological characteristic measured by said values (e.g., KDvalues).

[0103] “Substantially different,” as used herein, refers to a sufficiently high degree of difference between two numeric values (generally one associated with a molecule and the other associated with a reference molecule) such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values (e.g., KDvalues).

[0104] “Effector functions” refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody- dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0105] “Host cell” as used herein refers to a cell capable of being functionally modified with recombinant nucleic acids and functioning to express recombinant products, including polypeptides and compounds produced by activity of the polypeptides.

[0106] “Nucleic acid,” or “polynucleotide” as used herein interchangeably to refer to two or more nucleosides that are covalently linked together. The nucleic acid may be wholly comprised ribonucleosides (e.g., RNA), wholly comprised of 2'-deoxyribonucleotides (e.g., DNA) or mixtures of ribo- and 2'-deoxyribonucleosides. The nucleoside units of the nucleic acid can be linked together via phosphodiester linkages (e.g., as in naturally occurring nucleic acids), or the nucleic acid can include one or more non-natural linkages (e.g., phosphorothioester linkage). Nucleic acid or polynucleotide is intended to include single- stranded or double-stranded molecules, or molecules having both single-stranded regions and double-stranded regions. Nucleic acid or polynucleotide is intended to include molecules composed of the naturally occurring nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), or molecules comprising that include one or more modified and / or synthetic nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc.

[0107] “Protein,” “polypeptide,” and “peptide” are used herein interchangeably to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristilation, ubiquitination, etc.). As used herein “protein” or “polypeptide” or “peptide” polymer can include D- and L-amino acids, and mixtures of D- and L-amino acids.

[0108] “Naturally-occurring” or “wild-type” as used herein refers to the form as found in nature. For example, a naturally occurring nucleic acid sequence is the sequence present in an organism that can be isolated from a source in nature, and which has not been intentionally modified by human manipulation.

[0109] “Recombinant,” “engineered,” or “non-naturally occurring” when used herein with reference to, e.g., a cell, nucleic acid, or polypeptide, refers to a material, or a material corresponding to the natural or native form of the material, that has been modified in a manner that would not otherwise exist in nature, or is identical thereto but is produced or derived from synthetic materials and / or by manipulation using recombinant techniques. Non-limiting examples include, among others, recombinant cells expressing genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise expressed at a different level.

[0110] “Nucleic acid derived from” as used herein refers to a nucleic acid having a sequence at least substantially identical to a sequence of found in naturally in an organism. For example, cDNA molecules prepared by reverse transcription of mRNA isolated from an organism, or nucleic acid molecules prepared synthetically to have a sequence at least substantially identical to, or which hybridizes to a sequence at least substantially identical to a nucleic sequence found in an organism.

[0111] “Coding sequence” refers to that portion of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein.

[0112] “Heterologous nucleic acid” as used herein refers to any polynucleotide that is introduced into a host cell by laboratory techniques and includes polynucleotides that are removed from a host cell, subjected to laboratory manipulation, and then reintroduced into a host cell.

[0113] “Codon optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, the polynucleotides encoding the imine reductase enzymes may be codon optimized for optimal production from the host organism selected for expression.

[0114] “Preferred, optimal, high codon usage bias codons” refers to codons that are used at higher frequency in the protein coding regions than other codons that code for the same amino acid. The preferred codons may be determined in relation to codon usage in a single gene, a set of genes of common function or origin, highly expressed genes, the codon frequency in the aggregate protein coding regions of the whole organism, codon frequency in the aggregate protein coding regions of related organisms, or combinations thereof. Codons whose frequency increases with the level of gene expression are typically optimal codons for expression. A variety of methods are known for determining the codon frequency (e.g., codon usage, relative synonymous codon usage) and codon preference in specific organisms, including multivariate analysis, for example, using cluster analysis or correspondence analysis, and the effective number of codons used in a gene (see GCG CodonPreference, Genetics Computer Group Wisconsin Package; CodonW, John Peden, University of Nottingham; McInerney, J. O, 1998, Bioinformatics 14:372-73; Stenico et al., 1994, Nucleic Acids Res.222437-46; Wright, F., 1990, Gene 87:23-29). Codon usage tables are available for a growing list of organisms (see for example, Wada et al., 1992, Nucleic Acids Res.20:2111-2118; Nakamura et al., 2000, Nucl. Acids Res.28:292; Duret, et al., supra; Henaut and Danchin, "Escherichia coli and Salmonella," 1996, Neidhardt, et al. Eds., ASM Press, Washington D.C., p.2047-2066. The data source for obtaining codon usage may rely on any available nucleotide sequence capable of coding for a protein. These data sets include nucleic acid sequences actually known to encode expressed proteins (e.g., complete protein coding sequences-CDS), expressed sequence tags (ESTS), or predicted coding regions of genomic sequences (see for example, Mount, D., Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring  Harbor, N.Y., 2001; Uberbacher, E. C., 1996, Methods Enzymol.266:259-281; Tiwari et al., 1997, Comput. Appl. Biosci.13:263-270).

[0115] “Control sequence” as used herein refers to all sequences, which are necessary or advantageous for the expression of a polynucleotide and / or polypeptide as used in the present disclosure. Each control sequence may be native or foreign to the nucleic acid sequence encoding a polypeptide. Such control sequences include, but are not limited to, a leader, a promoter, a polyadenylation sequence, a pro-peptide sequence, a signal peptide sequence, and a transcription terminator. At a minimum, control sequences typically include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide.

[0116] “Operably linked” as used herein refers to a configuration in which a control sequence is appropriately placed (e.g., in a functional relationship) at a position relative to a polynucleotide sequence or polypeptide sequence of interest such that the control sequence directs or regulates the expression of the sequence of interest.

[0117] “Promoter sequence” refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences, which mediate the expression of a polynucleotide of interest. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0118] “Percentage of sequence identity,” “percent sequence identity,” “percentage homology,” or “percent homology” are used interchangeably herein to refer to values quantifying comparisons of the sequences of polynucleotides or polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (or gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage values may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are  many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math.2:482, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol.48:443, by the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)). Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1990, J. Mol. Biol.215: 403-410 and Altschul et al., 1977, Nucleic Acids Res. 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:10915). Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison Wis.), using default parameters provided.

[0119] “Reference sequence” refers to a defined sequence used as a basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length nucleic acid or polypeptide sequence. A reference sequence typically  is at least 20 nucleotide or amino acid residue units in length but can also be the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity. “Comparison window” refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (or gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.

[0120] “Substantial identity” or “substantially identical” refers to a polynucleotide or polypeptide sequence that has at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95 % sequence identity, or at least 99% sequence identity, as compared to a reference sequence over a comparison window of at least 20 nucleoside or amino acid residue positions, frequently over a window of at least 30-50 positions, wherein the percentage of sequence identity is calculated by comparing the reference sequence to a sequence that includes deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.

[0121] “Corresponding to,” “reference to,” or “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of an engineered imine reductase, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned.

[0122] “Isolated” as used herein in reference to a molecule means that the molecule (e.g., cannabinoid, polynucleotide, polypeptide) is substantially separated from other compounds that naturally accompany it, e.g., protein, lipids, and polynucleotides. The term embraces nucleic acids which have been removed or purified from their naturally occurring environment or expression system (e.g., host cell or in vitro synthesis).

[0123] “Substantially pure” refers to a composition in which a desired molecule is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition) and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight.

[0124] “Treatment,” “treat” or “treating” refers to clinical intervention in an attempt to alter the natural course of a disorder in the individual being treated and can be performed either for prophylaxis or during the course of clinical pathology. Desired results of treatment can include, but are not limited to, preventing occurrence or recurrence of the disorder, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disorder, preventing metastasis, decreasing the rate of progression, amelioration or palliation of a disease state, and remission or improved prognosis. For example, treatment can include administration of a therapeutically effective amount of pharmaceutical formulation comprising an IL-2 mutant polypeptide to a subject to delay development or slow progression of a disease or condition mediated by IL-2R or a disease or condition in which IL-2R may play a role in the pathogenesis and / or progression.

[0125] “Pharmaceutical formulation” refers to a preparation in a form that allows the biological activity of the active ingredient(s) to be effective, and which contain no additional components which are toxic to the subjects to which the formulation is administered. A pharmaceutical formulation may include one or more active agents. For example, a pharmaceutical formulation may include a mutation IL-2 polypeptide as the sole active agent of the formulation or may include a mutant IL-2 polypeptide and one or more additional active agents, such as e.g., an immune checkpoint inhibitor.

[0126] “Pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to the subject to whom it is administered. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0127] “Therapeutically effective amount” refers to the amount of an active ingredient or agent (e.g., a pharmaceutical formulation) to achieve a desired therapeutic or prophylactic result, e.g., to treat or prevent a disease, disorder, or condition in a subject. In the case of an IL-2 mediated disease or condition, the therapeutically effective amount of the therapeutic agent is an amount that reduces, prevents, inhibits, and / or relieves to some extent one or more of the symptoms associated with the disease, disorder, or condition. For cancer therapy, efficacy in vivo can, for example, be measured by assessing the growth of a primary tumor, occurrence and / or growth of secondary tumor(s), occurrence and / or number of metastases, duration, severity, and / or recurrence of symptoms, the response rate (RR), duration of response, and / or quality of life.

[0128] “Individual” or “subject” refers to a mammal, including but not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0129] IL-2 Receptors

[0130] IL-2 signaling is mediated through binding to three different IL-2 receptor protein subunits: IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132). Immune cells express dimeric or trimeric IL-2 receptors. The dimeric receptor is expressed on cytotoxic CD8+ T cells and natural killer cells (NK), whereas the trimeric receptor is expressed predominantly on activated lymphocytes and CD4+ CD25+ FoxP3+ suppressive regulatory T cells (Treg) (see, Byman et al., J. Nat. Rev. Immunol.12, 180-190 (2012)). Effector T cells and NK cells in a resting state do not have CD25 on the cell surface and thus, are relatively insensitive to IL-2. Treg cells, however, express high levels of CD25, and thus, Treg proliferation is stimulated by IL-2.

[0131] The trimeric receptor, IL-2Rαβγ, formed by the combination of IL-2Ra, IL-2Rβ, and IL- 2Rγ, is an IL-2 high affinity receptor with a KDof about 10 pM. The dimeric receptor (IL-2Rβγ) is an intermediate affinity receptor with a KDof about 1 nM. The monomeric IL-2Rα receptor is a low affinity IL-2 receptor. IL-2 signaling activity mediated by the receptors and their complexes also varies significantly. Generally, it has been found that IL-2Rβ and IL-2Rγ are critical for IL-2 signaling, while IL-2Rα (CD25) is not essential for signaling, but the presence of IL-2Rα enables high affinity IL-2 binding to the receptor complex (see e.g., Krieg et al., Proc Natl Acad Sci 107, 11906-11 (2010)).

[0132] The amino acid sequence of the IL-2Rα subunit can be found at UniProt P01589 and is set forth herein as SEQ ID NO: 2 with encoding polynucleotide sequence as SEQ ID NO: 1. The amino acid sequence of the IL-2Rβ subunit can be found at UniProt P14784 and is set forth herein as SEQ ID NO: 4 with encoding polynucleotide sequence as SEQ ID NO: 3. The amino acid sequence of the IL-2Rγ subunit can be found at UniProt 31785 and is set forth herein as SEQ ID NO: 6 with encoding polynucleotide sequence as SEQ ID NO: 5. Table 2 below provides a summary description of the sequences of the various IL-2R polypeptides of the present disclosure, and their sequence identifiers. The sequences also are included in the accompanying Sequence Listing.

[0133] TABLE 2: IL-2R protein subunits SEQ ID N N :  Human IL-2Rβ MAAPALSWRLPLLILLLPLATSWASAAVNGTSQFTCFYNSRANISCVW 4 UniProt P14784 SQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQ KLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVE

[0134] Mutant IL-2 Polypeptides

[0135] The present disclosure provides mutant IL2 fusion proteins that include a polypeptide subunit that is a mutant IL-2 polypeptide (or IL-2 mutein). Generally, the mutant IL2 subunit polypeptides used in the fusion proteins of the present disclosure comprise mutations that alter glycosylation of the polypeptide and affect various physicochemical and functional characteristics of the IL-2 including recombinant expression titer, solubility, and binding affinity to the IL-2R chains IL-2Rα, IL-2Rβ, and IL-2Rγ, in the monomeric, dimeric, and trimeric forms. The altered binding characteristics of the mutant IL-2 polypeptides with the different IL-2R chains allows for the inhibit, decrease, and / or fully block the function of the IL-2R receptor, particularly its function as a cell surface receptor in mediating immune regulation. Accordingly, it is contemplated that any of the mutation IL2 fusion protein compositions or formulations of the present disclosure can be used as therapeutics for treatment of diseases mediated by the function of IL-2R or its cognate ligand, IL-2, such as treatment of cancers and autoimmune disorders. Further, as described elsewhere herein, it is contemplated that the mutant IL-2 fusion proteins of the present disclosure can be used as a therapeutic in combination with other therapeutics, such as antibodies that target immune checkpoint molecules.

[0136] The naturally occurring human IL-2 is a 153 amino acid polypeptide sequence (Uniprot: P60568; disclosed herein as SEQ ID NO: 8 with encoding polynucleotide sequence as SEQ ID NO: 7) that includes a 20 amino acid N-terminal signal peptide: MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMP KKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI VEFLNRWITFCQSIISTLT (SEQ ID NO: 8)

[0137] The structure of the IL-2 polypeptide includes four antiparallel and amphipathic α- helices, which form a quaternary structure essential for its function (see e.g., Smith, Science 240, 1169-76 (1988); Bazan, Science 257, 410-413 (1992)). The 153 amino acid precursor IL- 2 of SEQ ID NO: 8 is processed to remove the signal peptide resulting in the mature secretory IL-2 polypeptide of 133 amino acids of SEQ ID NO: 9 shown below: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 9)

[0138] The mature IL-2 polypeptide of SEQ ID NO: 9 has been engineered for human pharmaceutical use by removing the cysteine residue at position C125, thereby reducing aggregation of the polypeptide. The C125S IL-2 mutant polypeptide, which is disclosed herein as SEQ ID NO: 10, is the active ingredient in aldesleukin, a drug which has been approved for use in humans in the treatment of cancers. APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 10)

[0139] The mutations in the IL-2 polypeptide subunit of the fusion proteins of the present disclosure are mutations relative to amino acid sequence of wild-type IL-2 or C125S IL-2. These mutations alter glycosylation, and thereby affect the functional properties of the fusion proteins that include these IL-2 polypeptides. The altered functional properties include increasing expression titer, increasing solubility, and / or altering binding affinity to the IL-2 receptor complexes. The altered glycosylation and functional properties resulting from the mutations in the IL-2 amino acid sequence relative to the parent IL-2 polypeptide (also referred to as amino acid substitutions or differences) result in IL-2 polypeptides with improved properties for use in pharmaceutical compositions for treatment of IL-2 mediated diseases, such as cancer and autoimmune disorders.

[0140] The mutant IL-2 polypeptides used in the fusion proteins of the present disclosure are derived from this parent C125S IL-2 polypeptide of SEQ ID NO: 10. The mutant IL-2 polypeptides have amino acid substitutions that result in new N-glycosylation sites on the expressed mutant IL-2 polypeptide. The engineering of N-glycosylation motifs in proteins is well-known in the art. Generally, the three amino acid sequence motifs, aspargine-X-serine (N- X-S) and asparagine-X-threonine (NXT), where X can be any amino acid except proline, provide result in a potential N-glycosylation site on the polypeptide. Depending on the sequence of the polypeptide, an N-glycosylation site can be introduced into a polypeptide by engineering amino acid substitutions at one, two, or three positions of an amino acid sequence.

[0141] In at least one embodiment of the present disclosure, the fusion protein can include a mutant IL-2 polypeptide subunit having a set of at least 2 mutations relative to SEQ ID NO: 10, wherein the set of mutations can include but is not limited to: K35N and Y45R; E95N and K97T;  E95N and K97S; K35N, Y45R, E95N, and K97T; or K35N, Y45R, E95N, and K972. Table 3 (below) provides seven exemplary IL-2 mutant polypeptides each of which includes one of these sets mutation relative to SEQ ID NO: 10.

[0142] TABLE 3: Exemplary mutant IL2 polypeptides Mutations Amino Acid Sequence of Exemplary IL-2 Mutant SEQ Relative to SEQ ID Polypeptide ID NO: 10 NO:p yp p , , , , , , , ange of additional exemplary mutant IL-2 polypeptides useful in the fusion proteins of the present disclosure are described in US Provisional Patent Appl. No.63 / 385,610, filed November 30, 2022, which is hereby incorporated by reference herein for all purposes. Table 4 (below) provides a summary of the additional sets of amino acid differences relative to the C125S IL-2 polypeptide of SEQ ID NO: 10 of exemplary mutant IL-2 polypeptides disclosed in US Provisional Patent Appl. No.63 / 385,610 that may be useful in the mutant IL-2 polypeptide subunits of the fusion proteins of the present disclosure.

[0144] TABLE 4: Exemplary IL-2 polypeptide subunit mutations relative to SEQ ID NO: 10 E61N, L63T; E61N L63SF42N, F44S K35N, P65N, E67T; K35N P65N E67S;K35N, E61N, L63S, E95N, K97T; K35N, E61N, L63S, E95N, K97S; K35N E61N L63T E95N K97T N90S;  R81N, P82A, R83S, E61N, L63S, E95N, K97S; R81N, P82A, R83S, E61N, L63S, E95N, K97T; R81N P82A R83S E61N L63T E95N K97S;proteins of the present disclosure can result in technical effects such as increased fusion protein expression titer during preparation in mammalian cell culture, increased solubility, and altered binding affinity for the trimeric and dimeric IL-2R complexes formed by the IL-2Rα subunit of SEQ ID NO: 2, IL-2Rβ subunit of SEQ ID NO: 4, and the IL-2Rγ subunit of SEQ ID NO: 6. For example, it has been observed that engineering N-glycosylation mutations in a recombinant gene encoding an IL-2 polypeptide can result in a mutant IL-2 polypeptide that exhibits altered binding affinity for the different IL-2R receptors subunits, IL-2Rα, IL-2Rβ, and IL-2Rγ, in the monomeric, dimeric, and trimeric complex forms. It is believed that such mutant IL-2 polypeptide that exhibit reduced binding affinity for IL-2Rα can provide an improved therapeutic compound for treatment of cancers, e.g., due to reduced or no stimulation of immunosuppressive CD25+ cells. Additionally, is believed that mutant IL-2 polypeptides that exhibit reduced binding affinity for IL-2Rβγ with little or no reduction in binding affinity for IL-2Rα can provide an improved therapeutic compound for treatment of autoimmune disorders, e.g., due to the preferential stimulation of immunosuppressive CD25+ cells.

[0146] As described elsewhere herein, in at least one embodiment, the fusion proteins of the present disclosure comprise a mutant IL-2 polypeptide subunit (e.g., IL-2 mutant of SEQ ID NO: 11, 12, 13, 14, 15, 16, 17) that results reduced affinity of the fusion protein for IL-2Rα without little or no loss in binding affinity for IL-2β, IL-2γ, or the IL-2βγ dimer complex.

[0147] Specific Antigen Binding Subunits

[0148] The first and second antigen binding subunits (e.g., A1B and A2B) of the mutant IL2 fusion proteins are capable of binding with high affinity to the target antigen expressed on the surface of an immune cell. This affinity for its target antigen allows the fused IL2 subunit to bind to an IL-2 receptor provide a combined effect on a targeted immune cell such as tumor-reactive T cell that expresses the target antigen.

[0149] Generally, the antigen binding subunits in the fusion proteins should have an equilibrium dissociation constant (KD) for binding to PD-1 of < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., 10-8M or less, from 10-8M to 10-13M, e.g., from 10-9M to 10-13M). The binding affinity of the antigen binding subunit to its target antige can be determined using any of a variety of assays and expressed in terms of a variety of quantitative values. Specific binding affinity assays useful in determining affinity of the antigen binding subunit polypeptides are disclosed in the Examples herein. Additionally, antigen binding assays are known in the art and can be used herein including without limitation any direct or competitive binding assays using techniques such as western blots, radioimmunoassays, enzyme-linked immunoabsorbent assay (ELISA), “sandwich” immunoassays, surface plasmon resonance based assay (such as the BIAcore assay as described in WO2005 / 012359), immunoprecipitation assays, fluorescent immunoassays, protein A immunoassays, flow cytometric and fluorescence activated cell sorting (FACS) assays, and the like. Accordingly, in some embodiments, the binding affinity is expressed as KDvalues and reflects intrinsic binding affinity (e.g., with minimized avidity effects).

[0150] Antigen Binding Subunits Targeting PD-1

[0151] It is contemplated that an antigen binding subunit (e.g., A1B or A2B) of a mutant IL2 fusion protein of the present disclosure can comprise a polypeptide that specifically binds to the target antigen, PD-1. Accordingly, in at least one embodiment, the antigen binding subunit can comprise an anti-PD-1 antibody. Anti-PD-1 antibodies useful as antigen binding subunits in the fusion proteins can include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies (e.g., single-arm antibodies), multivalent antibodies, single-chain antibodies, heavy chain (only) antibodies (e.g., sdAb, or VHH, IgNAR, nanobody (or nanoAb)), antigen-binding fragments (e.g., Fab′, F(ab′)2, Fab, Fv, rIgG, and scFv fragments), antibody fusions, and synthetic antibodies (or antibody mimetics).

[0152] Generally, the anti-PD-1 antibodies used in the fusion proteins should have an equilibrium dissociation constant (KD) for binding to PD-1 of < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., 10-8M or less, from 10-8M to 10-13M, e.g., from 10-9M to 10-13M). Binding affinity can measured by equilibrium dissociation constant (KD) to the hu-PD-1 ECD polypeptide. In some embodiments, the anti-PD-1 antibodies used in the fusion proteins as disclosed herein are capable of high-affinity binding to cyno-PD-1, and / or to both hu-PD-1 and cyno-PD-1. In some embodiments, the anti-PD-1 antibodies of the present disclosure bind to cyno-PD-1 with a binding affinity of 1 x 10-8M or less, 1 x 10-9M or less, 1 x 10-10M or less, or 1 x 10-11M or less.

[0153] In at least one embodiment, an anti-PD1 antibody useful as an antigen binding subunit in the fusion proteins can have a single heavy chain variable domain (VH) defined in terms of  the amino acid and encoding nucleotide sequences of the various well-known immunoglobulin features (e.g., CDRs, VH domain). Table 5 below provides a summary description of anti-PD-1 antibody sequences of the present disclosure, and their sequence identifiers. The sequences are included in the accompanying Sequence Listing.

[0154] TABLE 5: Anti-PD-1 VHH antibody sequences SEQ ID Name Amino acid se uence NO:antibodies, which are useful in the fusion proteins of the present disclosure are described in US Provisional Patent Appl. No.63 / 488,176, filed March 3, 2023, which is hereby incorporated by reference herein for all purposes.

[0156] The anti-PD-1 antibodies provided herein for use as antigen binding subunits in mutant IL2 fusion proteins specifically target and bind to PD-1 expressed on T cells, without decreasing, inhibiting, or blocking the immune regulatory effects mediated by T cells, including the activation of T cells in response to a specific MHC antigen binding. In some embodiments, the anti-PD-1 antibodies provided herein specifically target and bind to PD-1 expressed on T cells and inhibit the immune regulatory and / or immune signaling pathways mediated by an activated T cell binding. The effects of antibody binding on the PD-1 mediated immune response can be assayed in vitro using known cell-based assays including the cell-based assays described in the Examples of the present disclosure. Accordingly, in some embodiments, the anti-PD-1 antibodies of the present disclosure are characterized by one or more of following functional properties based on the ability to alter activation of T cells and the associated immune response.

[0157] In at least one embodiment, the anti-PD-1 antibody used as an antigen binding subunit in a fusion protein of the present disclosure is capable of blocking binding to hu-PD-1 ECD measured by ELISA with an IC50of 10 nM or less, 7 nM or less, 5 nM or less, 2 nM or less, 1 nM or less, 0.5 nM or less, or 0.25 nM or less. In at least one embodiment, the anti-PD-1 antibody blocks binding to hu-PD-1 ECD expressed on a cell with an IC50of 2.5 nM or less, 1 nM or less, 0.5 nM or less, or 0.25 nM or less; optionally, wherein the cell is a HEKBlue cell stably expressing PD-1 ECD. In at least one embodiment, the anti-PD-1 antibody subunit of the fusion protein can block binding to hu-PD-1 ECD expressed on a human T cell with an IC50of 5 nM or less, 2.5 nM or less, 1 nM or less, 0.5 nM or less, or 0.25 nM or less; optionally, wherein the cell is a human CD8+T-cell.

[0158] Antigen Binding Subunits Targeting CD8

[0159] It is contemplated that an antigen binding subunit (e.g., A1B or A2B) of a mutant IL2 fusion protein of the present disclosure can comprise a polypeptide that specifically binds to the target antigen, CD8. Accordingly, in at least one embodiment, the antigen binding subunit can comprise an anti-CD8 antibody. Anti-CD8 antibodies useful as antigen binding subunits in the fusion proteins can include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies (e.g., single-arm antibodies), multivalent antibodies, single-chain antibodies, heavy chain (only) antibodies (e.g., sdAb, or VHH, IgNAR, nanobody (or nanoAb)), antigen-binding fragments (e.g., Fab′, F(ab′)2, Fab, Fv, rIgG, and scFv fragments), antibody fusions, and synthetic antibodies (or antibody mimetics).

[0160] Generally, the anti-CD8 antibodies used in the fusion proteins should have an equilibrium dissociation constant (KD) for binding to CD8 of < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., 10-8M or less, from 10-8M to 10-13M, e.g., from 10-9M to 10-13M). Binding affinity can measured by equilibrium dissociation constant (KD) to the hu-CD8 ECD polypeptide. In some embodiments, the anti-CD8 antibodies used in the fusion proteins as disclosed herein are capable of high-affinity binding to cyno-CD8, and / or to both hu-CD8 and cyno-CD8. In some embodiments, the anti-CD8 antibodies of the present disclosure bind to cyno-CD8 with a binding affinity of 1 x 10-8M or less, 1 x 10-9M or less, 1 x 10-10M or less, or 1 x 10-11M or less.

[0161] In at least one embodiment, an anti-CD8 antibody useful as an antigen binding subunit in the fusion proteins can have a single heavy chain variable domain (VH) defined in terms of the amino acid and encoding nucleotide sequences of the various well-known immunoglobulin features (e.g., CDRs, VH domain). Table 6 below provides a summary description of anti-CD8 antibody sequences of the present disclosure, and their sequence identifiers. The sequences are included in the accompanying Sequence Listing.

[0162] TABLE 6: Anti-CD8 VHH antibody sequencesSEQ ID Name Amino acid sequence NO:  Clone hu3A8-v1- EVQLVESGGGLVQPGGSLRLSCAASGFTFDEYAIGWFRQAPGKER 47 Y98L VHH EGVSCLRVSDGRTYYPDSVKGRFTISRDNSKNTVYLQMNSLRAED TAVYYCASGSYLGCTVDDYDFWGQGTQVTVSSantibodies, which are useful in the fusion proteins of the present disclosure are described in US Provisional Patent Appl. No.63 / 477,529, filed December 28, 2022, which is hereby incorporated by reference herein for all purposes.

[0164] The anti-CD8 antibodies provided herein for use as antigen binding subunits in mutant IL2 fusion proteins specifically target and bind to CD8 expressed on T cells, without decreasing, inhibiting, or blocking the immune regulatory effects mediated by T cells, including the activation of T cells in response to a specific MHC antigen binding. In some embodiments, the anti-CD8 antibodies provided herein specifically target and bind to CD8 expressed on T cells and inhibit the immune regulatory and / or immune signaling pathways mediated by an activated T cell binding. The effects of antibody binding on the CD8 mediated immune response can be assayed in vitro using known cell-based assays including the cell-based assays described in the Examples of the present disclosure. Accordingly, in some embodiments, the anti-CD8 antibodies of the present disclosure are characterized by one or more of following functional properties based on the ability to alter activation of T cells and the associated immune response.

[0165] In at least one embodiment, the anti-CD8 antibody used as an antigen binding subunit in a fusion protein of the present disclosure is capable of blocking binding to hu-CD8 ECD measured by ELISA with an IC50of 10 nM or less, 7 nM or less, 5 nM or less, 2 nM or less, 1 nM or less, 0.5 nM or less, or 0.25 nM or less. In at least one embodiment, the anti-CD8 antibody blocks binding to hu-CD8 ECD expressed on a cell with an IC50of 2.5 nM or less, 1 nM or less, 0.5 nM or less, or 0.25 nM or less; optionally, wherein the cell is a HEKBlue cell stably expressing CD8 ECD. In at least one embodiment, the anti-CD8 antibody subunit of the fusion protein can block binding to hu-CD8 ECD expressed on a human T cell with an IC50of 5  nM or less, 2.5 nM or less, 1 nM or less, 0.5 nM or less, or 0.25 nM or less; optionally, wherein the cell is a human CD8+T-cell.

[0166] Mutant IL-2 Fusions with Antigen Binding Polypeptide Subunits

[0167] FIGS.1A-1J depict schematic representations that illustrate the fused single-chain polypeptide structures of exemplary mutant IL2 fusion proteins of the present disclosure. FIGS. 1A, 1B, 1C, 1D, and 1E depict exemplary tri-specific fusion proteins. These fusion proteins can bind to IL-2R, as well as a first antigen and second antigen expressed on the surface of an immune cell. These fusions proteins that include a single polypeptide chain that includes a mutant IL2 subunit, a first and a second antigen binding subunit, A1B and A2B, which have different antigen binding specificity (e.g., AB1 binds PD1 and AB2 binds CD8), and a half-life extending polypeptide subunit, HLE, (e.g., IgG1 Fc region). Additionally, polypeptide linkers (L1, L2, and L3) connect the C-terminus to N-terminus of adjacent polypeptide subunits.

[0168] FIGS.1F, 1G, 1H, 1I, and 1J depict exemplary bi-specific fusion proteins that include a single polypeptide chain including a mutant IL2 polypeptide subunit that binds IL-2R, and only a single antigen binding subunit (e.g., A1B or A2B). The bi-specific fusion proteins also include a half-life extending polypeptide subunit, HLE, and polypeptide linkers connecting the C-terminus to N-terminus of adjacent polypeptide subunits.

[0169] The present disclosure also provides dimeric complexes of the mutant IL2 fusion proteins described in FIG.1A-1J. FIGS.2A-2I depict schematic representations of exemplary mutant IL2 fusion proteins that have homo-dimeric or hetero-dimeric structures comprising a pair single chain mutant IL2 fusion polypeptides. These exemplary structures include homo- or hetero-dimers of the bi-specific or tri-specific single polypeptide chains, such as those depicted in FIG.1A-1J. In at least one embodiment, it is contemplated that formation of the dimeric complexes can be facilitated by including a HLE subunit comprising “knob” (e.g., Fc-Kb) and “hole” (e.g., Fc-Ho) amino acid sequence features. Such knob and hole features are well- known and commonly used in forming bi-specific antibodies. As described elsewhere herein, hese same knob and hole features can be used to form the dimeric complexes of mutant IL2 protein fusions of the present disclosure, such as those illustrated in FIG.2A-2I.

[0170] A range of exemplary polypeptides useful in the mutant IL2 fusion proteins of the present disclosure are provided in Table 7 (below).

[0171] TABLE 7: Exemplary fusion protein polypeptides SEQ ID :DLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGKGGGGSGGGGSAPT SSSTKKTQLQLEHLLLDLQMILNGINNYKNPNLTRMLTFKFRMPKKATELKH LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLNLTGSETTFMCEYDWRDLNYDSRGQGTLVTVSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREP645 (Light DIVMTQGTLPNPVPSGESVSITCRSSKSLLYSDGKTYLNWYLQRPGQSPQLL 64 chain of IYWMSTRASGVSDRFSGSGSGTDFTLKISGVEAEDVGIYYCQQGLEFPTFGG P701) GTKLELKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDG  P577 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIW 70 [(P709 YDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWG nivolumab QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSthat can be used in preparing exemplary mutant IL2 fusion proteins of the present disclosure, such as those single-chain structures illustrated in FIGS.1A-1J, and the dimeric complexes illustrated in FIGS.2A-2I. The schematic fusion structures of the polypeptides of Table 7 are summarized in Table 8 (below).

[0173] TABLE 8: Exemplary fusion protein structures Fusion SEQ ID Name NO: Fusion Structure  P582 (Hole 51 (A1B)-(L1)-(Fc-Ho)-(L2)-(IL2)-(L3)-(A2B) of P610)(below).

[0175] TABLE 9: Exemplary fusion protein subunits Fusion Name A1B L1 HLE L2 Mutant IL2 L3 A2B  P703 3H9 IgG1 Fc IgG1 Fc LALA (G4S)2K35N+Y45R (G4S)13A8 Natural Knob into Hole +E95N+K97T linker[ ] u u oypep es

[0177] As shown by the exemplary structures of the mutant IL2 fusion proteins of the present disclosure, the mutant IL-2 fusion proteins include an HLE (half-life extending) subunit that can comprise an Fc polypeptide, such as the wild-type monomeric human IgG1 Fc lower hinge region polypeptide of SEQ ID NO: 72 as shown below: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSPGK (SEQ ID NO: 72).

[0178] In at least one embodiment, the HLE subunit used in mutant IL-2 fusion proteins comprises a variant of the wild-type human IgG1 Fc polypeptide of SEQ ID NO: 72 that includes the “KK” variant of SEQ ID NO: 73: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYG STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVS  LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQEGNVFSCSVLHEA LHNHYTQKSLSLSPGK (SEQ ID NO: 73)

[0179] In at least one embodiment, the HLE subunit used in mutant IL-2 fusions comprises a variant of the wild-type human IgG1 Fc polypeptide of SEQ ID NO: 72 that includes the “DSDL” variant of SEQ ID NO: 74, both of which are shown below: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYG STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLSSDLTVDKSRWQEGNVFSCSVLHEA LHNHYTQKSLSLSPGK (SEQ ID NO: 74).

[0180] In addition to the three human IgG1 Fc lower hinge region polypeptides are described above, it is contemplated that the mutant IL-2 fusion proteins of the present disclosure can be prepared using HLE subunits comprising other IgG1 Fc region fragments and variants that are known to provide improved properties when conjugated to polypeptides, such as cytokines, for therapeutic use. For example, an Fc polypeptide variant can be used that removes effector function, such as an Fc region with the amino acid substitutions L234A / L235A (“LALA”) (Woodle, E. Steve et al., Transplantation, 68(5): 608-616 (1999)). Other effectorless Fc region mutations are well known in the art, such as L234A / L235A / P329G (“LALAPG”) (see e.g., Schlothauer, T. et al., “Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions”, Protein Eng. Des. Sel., 29(10): 457– 466 (2016)), or when the Fc is of isotype IgG2 or IgG4, the amino acid substitutions S228P and / or L235E.

[0181] In addition, HLE subunits useful in the mutant IL-2 fusion proteins can comprise IgG1 hinge and Fc region amino acid sequences that provide a “knob” or a “hole” functionality, thereby allowing fusions comprising a “knob” HLE subunit and “hole” HLE subunit to be used in forming dimeric complexes of the mutant IL-2 fusion proteins of the present disclosure. Exemplary IgG1 hinge and Fc region amino acid sequences useful as HLE subunits in the fusion proteins of the present disclosure are provided in Table 10 (below).

[0182] TABLE 10: Exemplary IgG1 hinge and Fc region sequences SEQ ID :  NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKN QVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0184] In at least one embodiment, it is contemplated that the mutant IL-2 fusion proteins of the present disclosure are conjugated between the subunit polypeptides via linkers (e.g., L1, L2, L3). Any of the wide range of synthetic chain-like molecules useful as linkers between biomolecules that are known in the art can be used to fuse the subunits of the mutant IL-2 fusion proteins. In at least one embodiment, a polypeptide linker may be used. Such polypeptide linkers comprise a chain of amino acids with each end of the chain covalently attached to one of the two different polypeptides, and thereby functioning to conjugate or fuse them. Typically, such polypeptide linkers comprise a chain of 5 to 30 amino acids.

[0185] A wide range of polypeptide linkers are known in the art and can be used in the mutant IL-2 polypeptide fusions of the present disclosure. Polypeptides known in the art that may be can include, but are not limited to: (GGGGS)n(n is 1-10), (GRPGS)n(n is 1-4), (GEPGS)n(n is 1-4), (GDPGS)n(n is 1-4), (SSSSG)n(n is 1-10), (GGGG)(SGGGG)n(n is 1-10), (EAAAK)n(n is 1-10), (XP)n(n is 1-10), and ENLYFQ(-G / S). Exemplary polypeptide linkers useful in the IL-2 fusions of the present disclosure include but are not limited to the linkers provided in Table 11 (below).

[0186] TABLE 11: Exemplary linkers SEQ ID  (G4S)3GGGGSGGGGSGGGGS84(G4S)4GGGGSGGGGSGGGGSGGGGS85(G4S)5GGGGSGGGGSGGGGSGGGGSGGGGS86

[0188] The fusion proteins of the present disclosure can be produced using recombinant methods and materials well-known in the art of polypeptide and protein production. In some embodiments, the present disclosure provides an isolated nucleic acid encoding a mutant IL-2 polypeptide. The nucleic acid can encode an amino acid sequence comprising the IL-2 polypeptide alone or as a fusion with another polypeptide, such as a monomeric Fc polypeptide. In some embodiments, one or more vectors (e.g., expression vectors) comprising nucleic acid sequences encoding a mutant IL-2 polypeptide of the present disclosure are provided. In some embodiments, a host cell comprising nucleic acid sequences encoding a mutant IL-2 polypeptide of the present disclosure are provided. In one embodiment, the host cell has been transformed with a vector comprising a nucleic acid that encodes an amino acid sequence comprising the mutant IL-2 polypeptide. In some embodiments, the host cell used is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell, or a lymphoid cell (e.g., Y0, NS0, Sp20).

[0189] In at least one embodiment, a method of making a mutant IL-2 polypeptide is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the polypeptide, as provided above, under conditions suitable for expression of the polypeptide, and optionally recovering the polypeptide from the host cell (or host cell culture medium). Briefly, recombinant production of a mutant IL-2 polypeptide is carried out by synthesizing or isolating a nucleic acid encoding the mutant IL-2 polypeptide (e.g., as described herein) and inserting this nucleic acid into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids are readily isolated and sequenced using conventional procedures well-known in the art (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding IL-2. Suitable host cells and culturing methods for  cloning or expressing the IL-2 polypeptide-encoding vectors are well-known in the art and include prokaryotic or eukaryotic cells. Typically, after expression, the mutant IL-2 polypeptide may be isolated from cell paste in a soluble fraction and further purified. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for the vectors.

[0190] Examples of suitable mammalian host cell lines useful for the production of the mutant IL-2 fusion proteins of the present disclosure include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (see e.g., Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); myeloma cell lines such as Y0, NS0 and Sp2 / 0; monkey kidney CVl line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CVl); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TR1 cells (see e.g., in Mather et al., Annals N Y. Acad. Sci.383:44-68 (1982) and US 6,235,498); Medical Research Council 5 (MRC 5) cells (such as e.g., those available from ATCC and also referred to as CCL-171); and Foreskin 4 (FS-4) cells (see e.g., in Vilcek et al. Ann. N. Y. Acad. Sci.284:703-710 (1977), Gardner & Vilcek. J. Gen. Virol.44:161-168 (1979), and Pang et al. Proc. Natl. Acad. Sci. U.S.A.77:5341- 5345 (1980)).

[0191] Pharmaceutical Compositions and Formulations of Mutant IL-2 Fusion Proteins

[0192] The present disclosure also provides pharmaceutical compositions and pharmaceutical formulations comprising a mutant IL-2 polypeptide. In some embodiments, the present disclosure provides a pharmaceutical formulation comprising a mutant IL-2 polypeptide as described herein and a pharmaceutically acceptable carrier. In some embodiments, the mutant IL-2 polypeptide is the sole active agent of the pharmaceutical composition. Such pharmaceutical formulations can be prepared by mixing a mutant IL-2 polypeptide, having the desired degree of purity, with one or more pharmaceutically acceptable carriers. Typically, such mutant IL-2 polypeptide formulations can be prepared as an aqueous solution or as a lyophilized formulation.

[0193] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed. A wide range of such pharmaceutically acceptable carriers are well-known in the art (see e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Exemplary pharmaceutically acceptable carriers useful in the formulations of the present disclosure can include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl  or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m- cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn- protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0194] Pharmaceutically acceptable carriers useful in the formulations of the present disclosure can also include interstitial drug dispersion agents, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP) (see e.g., US Pat. Publ. Nos.2005 / 0260186 and 2006 / 0104968), such as human soluble PH-20 hyaluronidase glycoproteins (e.g., rHuPH20 or HYLENEX®, Baxter International, Inc.).

[0195] It is also contemplated that the formulations disclosed herein may contain active ingredients in addition to the mutant IL-2 polypeptide, as necessary for the particular indication being treated in the subject to whom the formulation is administered. Preferably, any additional active ingredient has activity complementary to that of the IL-2 activity and the activities do not adversely affect each other.

[0196] As disclosed elsewhere herein, including the Examples, it has been shown that the mutant IL-2 polypeptide of the present disclosure can be used as a fusion to a Fc polypeptide to provide improved therapeutic effect in treating autoimmune disorders and / or cancers.

[0197] As described elsewhere herein, in some embodiments the present disclosure provides a pharmaceutical composition or formulation for use in a therapeutic method wherein the composition comprises a mutant IL-2 polypeptide fused to an antigen binding subunit polypeptide and a half-life extending subunit polypeptide. In some embodiments, this pharmaceutical composition or formulation can comprise a mutant IL-2 protein fusion comprises linker polypeptides between the subunit polypeptides, such as a polypeptide linker of amino acid sequence of SEQ ID NO: 82-89. Examples demonstrating such mutant IL-2 protein fusions, and their use in pharmaceutical compositions for reducing treatment of cancer or autoimmune disorders as described elsewhere herein.

[0198] In some embodiments, the pharmaceutical composition can comprise a mutant IL-2 protein fusion of the present disclosure and an additional active agent for treatment of cancer, such as an immune checkpoint inhibitor. Checkpoint inhibitors useful in such embodiments include, but are not limited to, an antibody comprising a specificity for an antigen that is an immune checkpoint molecule, such as, PD1, LAG3, CTLA-4, A2AR, TIM-3, BTLA, CD276, CD328, VTCN1, KIR, NOX2, VISTA, OX40, CD27, CD28, CD40, CD122, CD137, GITR, or ICOS.

[0199] Generally, pharmaceutical composition active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0200] In some embodiments, the formulation of a mutant IL-2 protein fusion can be a sustained-release preparation of the polypeptide, and / or other active ingredients. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the mutant IL-2 protein fusion, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0201] Typically, the formulations of the present disclosure to be administered to a subject are sterile. Sterile formulations may be readily prepared using well-known techniques, e.g., by filtration through sterile filtration membranes.

[0202] Uses and Methods of Treatment

[0203] It is contemplated that any of the compositions or formulations comprising a mutant IL-2 fusion protein of the present disclosure can be used for any methods or uses, such as in therapeutic methods, that utilize the ability of the polypeptides to specifically bind to IL-2 receptor protein. The binding of IL-2 to the IL-2 receptor protein as it is expressed on different cells mediates different immune responses. IL-2 binding can stimulate an immune response, such as T cell proliferation and differentiation, cytotoxic T lymphocyte (CTL) production, B cell proliferation and differentiation, immunoglobulin synthesis, and production, and proliferation and activation of NK cells. The IL-2 polypeptide, such as the C125S IL-2 (Proleukin), has been approved as an immunotherapeutic agent for the treatment of cancer and chronic viral infection. The IL-2 polypeptide, however, can also promote the activation and proliferation of immunosuppressive CD4+ CD25+ Treg cells resulting in immunosuppression. Accordingly, there are a range of diseases, disorders, and conditions that can potentially be treated by altering the immune regulatory and / or immune signaling activity of IL-2 binding to IL-2 receptor proteins, particularly, the effect of IL-2 on tumor progression. Diseases, disorders, and conditions include, but are not limited to, cancers, including but not limited to colon cancer, pancreatic cancer, ovarian cancer, liver cancer, renal cancer, breast cancer, lung cancer, gastric cancer, head and neck cancer, or oral cancer. It is contemplated that any of the compositions or formulations comprising a mutant IL-2 fusion protein of the present disclosure can be used in a method or use for the treatment of any of the above-listed cancers. Thus, in at least one embodiment, the present disclosure provides a method of treating cancer in a subject, wherein the method comprises administering to the subject in need thereof a therapeutically effective amount of a mutant IL-2 fusion protein of the present disclosure or  administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising a mutant IL-2 fusion protein of the present disclosure and a pharmaceutically acceptable carrier.

[0204] As disclosed herein, including in the Examples below, the mutant IL-2 fusion proteins of the present disclosure have the ability to specifically bind to IL-2 receptors proteins differentially, and thereby differentially alter the immune signaling pathways mediated by IL-2 binding to IL-2 receptor proteins expressed on different cells. Accordingly, in some embodiments, the present disclosure provides a method of treating an IL-2 mediated disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of a mutant IL-2 fusion protein of the present disclosure or administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a mutant IL-2 fusion protein of the present disclosure and a pharmaceutically acceptable carrier. Similarly, in some embodiments, the present disclosure provides a method of treating a disease mediated by IL-2 binding to IL-2 receptor proteins expressed on cells in a subject, the method comprising administering to the subject, the method comprising administering to the subject a therapeutically effective amount of a mutant IL-2 fusion protein of the present disclosure or administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a mutant IL-2 fusion protein of the present disclosure and a pharmaceutically acceptable carrier.

[0205] Administration of the mutant IL-2 fusion protein, composition, or pharmaceutical formulation in accordance with the method of treatment provides an antibody-induced therapeutic effect that protects the subject from and / or treats the progression of an IL-2- mediated disease in a subject. In some embodiments, the method of treatment can further comprise administration of one or more additional therapeutic agents or treatments known to those of skill in the art to prevent and / or treat the IL-2-mediated disease or condition. Such methods comprising administration of one or more additional agents can encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the mutant IL-2 fusion protein composition or formulation can occur prior to, simultaneously, and / or following, administration of the additional therapeutic agent.

[0206] In some embodiments of the methods of treatment of the present disclosure, the mutant IL-2 fusion protein or pharmaceutical formulation comprising a mutant IL-2 fusion protein is administered to a subject by any mode of administration that delivers the agent systemically, or to a desired target tissue. Systemic administration generally refers to any mode of administration of the antibody into a subject at a site other than directly into the desired target site, tissue, or organ, such that the antibody or formulation thereof enters the subject's circulatory system and, thus, is subject to metabolism and other like processes. Accordingly, modes of administration useful in the methods of treatment of the present disclosure can  include, but are not limited to, injection, infusion, instillation, and inhalation. Administration by injection can include intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion.

[0207] In at least one embodiment, a pharmaceutical formulation of the mutant IL-2 fusion protein is formulated such that the IL-2 is protected from inactivation in the gut. Accordingly, the method of treatments can comprise oral administration of the formulation.

[0208] In at least one embodiment, use of the compositions or formulations comprising a mutant IL-2 fusion protein of the present disclosure as a medicament are also provided. Additionally, in some embodiments, the present disclosure also provides for the use of a composition or a formulation comprising a mutant IL-2 fusion protein in the manufacture or preparation of a medicament, particularly a medicament for treating an IL-2 mediated disease. In a further embodiment, the medicament is for use in a method for treating disease comprising administering to an individual having a disease an effective amount of the medicament. In certain embodiments, the medicament further comprises an effective amount of at least one additional therapeutic agent, or treatment.

[0209] As disclosed elsewhere herein, it is also contemplated that additional therapeutic agents or treatments that can be used in such medicaments with a mutant IL-2 fusion protein of the present disclosure. Generally, it is contemplated that the mutant IL-2 fusion proteins of the present disclosure can be used with any therapeutic agent or treatment, such as a therapeutic antibody, that specifically targets a cell surface receptor on an immune cell, a tumor cell, or a myeloid cell. In at least one embodiment, the additional therapeutic agent can include, but is not limited to a therapeutic antibody that specifically binds to an immune checkpoint molecule, such as PD1, PD-L1, LAG3, CTLA-4, A2AR, TIM-3, BTLA, CD276, CD328, VTCN1, IDO, KIR, NOX2, VISTA, OX40, CD27, CD28, CD40, CD122, CD137, GITR, ICOS.

[0210] In a further embodiment, the medicament is for use in treating an IL-2-mediated disease, such as a cancer, in a subject comprising administering to the subject an amount effective of the medicament to treat, inhibit or prevent the IL-2-mediated disease. The appropriate dosage of the mutant IL-2 fusion protein contained in the compositions and formulations of the present disclosure (when used alone or in combination with one or more other additional therapeutic agents) will depend on the specific disease or condition being treated, the severity and course of the disease, whether the dosage is administered for preventive or therapeutic purposes, the previous therapy administered to the patient, the patient's clinical history and response to the mutant IL-2 fusion protein composition, and the discretion of the attending physician. It is contemplated that the mutant IL-2 fusion protein included in the compositions and formulations described herein, can be suitably administered to the patient at one time, or over a series of treatments. Various dosing schedules including but  not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

[0211] Depending on the type and severity of the disease, about 1 µg / kg to 30 mg / kg of a mutant IL-2 fusion protein in a formulation of the present disclosure is an initial candidate dosage for administration to a human subject, whether, for example, by one or more separate administrations, or by continuous infusion. Generally, the administered dosage of the antibody would be in the range from about 0.05 mg / kg to about 10 mg / kg. In some embodiments, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to a patient.

[0212] Dosage administration can be maintained over several days or longer, depending on the condition of the subject, for example, administration can continue until the IL-2-mediated disease is sufficiently treated, as determined by methods known in the art. In some embodiments, an initial higher loading dose may be administered, followed by one or more lower doses. However, other dosage regimens may be useful. The progress of the therapeutic effect of dosage administration can be monitored by conventional techniques and assays. Accordingly, in some embodiments of the methods of the present disclosure, the administration of the mutant IL-2 fusion protein comprises a daily dosage from about 0.05 mg / kg to about 50 mg / kg, at least about 0.5 mg / kg to about 30 mg / kg, or at least about 1 mg / kg to about 25 mg / kg. In some embodiments, the dosage of mutant IL-2 fusion protein comprises a daily dosage of at least about 0.1 mg / kg, at least about 1 mg / kg, at least about 5 mg / kg, at least about 10 mg / kg, at least about 20 mg / kg, at least about 30 mg / kg, at least about 40 mg / kg, or at least about 50 mg / kg. EXAMPLES

[0213] Various features and embodiments of the disclosure are illustrated in the following representative examples, which are intended to be illustrative, and not limiting. Those skilled in the art will readily appreciate that the specific examples are only illustrative of the invention as described more fully in the claims which follow thereafter. Every embodiment and feature described in the application should be understood to be interchangeable and combinable with every embodiment contained within.  Example 1: Cloning, Expression, and Purification of Mutant IL-2 Fusions with Anti-PD1 VHH and / or anti-CD8 VHH Antibodies

[0214] This example illustrates the design, cloning, expression, and purification of exemplary mutant IL2 fusion proteins of the present disclosure.

[0215] A. Design and cloning of fusion protein constructs

[0216] Bispecific fusions of mutant IL-2 and anti-PD1 VHH , or mutant IL-2 and anti-CD8 VHH, or trispecific fusions of mutant IL-2 and anti-PD1 VHH and anti-CD8 VHH antibodies were  designed having polypeptide chain structures of the general form depicted schematically in FIGS.1A – 1J. The proteins fuse the VHH to the N-terminus of the hinge and Fc region of human IgG1, including a human constant heavy chain 2 (CH2) and constant heavy chain 3 (CH3) domains of SEQ ID NO: 57 (Uniprot ID: P01857). Polynucleotides encoding these bispecific and trispecific fusion protein structures were cloned in pcDNA3.1(+) vector with standard molecular biology techniques. Hetero-dimeric versions of the exemplary fusion proteins having structures of the of the general form depicted schematically in FIGS.2A – 2I were prepared using the single-chain fusion polypeptides. Schematic structures and polypeptide sequences used in the exemplary IL2 fusion proteins prepared in this Example are provided in Table 12 below.

[0217] B. Expression of fusion proteins in CHO cells

[0218] The cloned mutant IL2 fusion protein constructs were expressed in Chinese hamster ovary (CHO) cells using ExpiCHO expression system (Thermo Fisher Scientific). ExpiCHO cells (Thermo Fisher Scientific) were maintained at 37 °C, 5% CO2and 130 rpm in 60 mL of ExpiCHO expression medium in Erlenmeyer flasks. Transfection was performed according to instructions provided with the ExpiCHO™ Expression System Kit (ThermoFisher – Catalog number A29133). Briefly, when cells reached Viable Cell Density (VCD) of 6×106cells / mL and doubling time of 18–20 hrs, 1 μg plasmid DNA per 106cells and ExpiFectamine CHO was mixed by repeated inversions, diluted with cold OptiPRO serum free medium, and finally complexed to the diluted plasmid DNA at room temperature. After 5 min, the mix was added drop by drop to the cell culture at room temperature. After transfection, cells were cultured at 130 RPM for 25 mL or large flasks. After 7-12 days post transfection, culture media were centrifuged at 25 °C and 500×g for 5 min to pellet cells and the supernatant was further centrifuged at 4 °C and 4500×g for 30 min. The clarified supernatant was filtered by 0.45 μm filtration and the VHH antibodies were purified with protein A affinity column chromatography.

[0219] C. Purification of IL-2 fusion proteins by Protein A affinity chromatography

[0220] The CHO cell-expressing various IL-2 fusion constructs were purified by affinity chromatography with protein-A resin (Atmosphere A3, JSR Life Sciences). Dripping columns packed with protein A resin (column volume = 0.2 mL) were equilibrated with 5 column volume (CV) 1X PBS buffer and 4 mL supernatant was directly loaded. Unbound protein was removed by washing with 10 CV 1X PBS buffer, and the protein of interest was eluted with100 mM triethanolamine at pH 11.5 or 100 mM Na2HPO4-NaOH buffer at pH 11.5. The eluted fractions were collected, and pH was adjusted to 9.5 using 1M Tris buffer, pH 8.0.

[0221] The expression titer of each sample was quantitated in HPLC with POROSTMA column as manufactory protocol (ThermoFisher, catalog# 2100100). The expression levels of exemplary IL-2 fusion proteins are shown in Table 12 (below).

[0222] TABLE 12  Fusion Expression Name Fusion Structure (mg / L)

[0223] D. SEC-HPLC characterization of IL-2 fusion proteins

[0224] Size exclusion-high-performance liquid chromatography (SEC-HPLC) analysis was performed on an Agilent 1100 HPLC system (Santa Clara, California) with 1X PBS as a running buffer. Purified IL-2 fusion samples were injected into a prepacked Superose 12-300 column (Cytiva) which was equilibrated with 1 X PBS buffer. The flow rate was 0.65 mL / min and the total running time was 40 min.

[0225] Exemplary SEC-HPLC profiles showing protein conformations of the purified fusion proteins are shown in FIG.3A.

[0226] E. AEX ion exchange chromotography

[0227] Protein A purified IL-2 fusions were diluted with 20 mM Tris Propane buffer (Buffer A, pH 9.5), and then loaded on AEX column packed with Q-FF resin (column volume =5 mL). After 5-column volume washing with buffer A, the target protein was eluted with 20-column volume buffer B ( 20 mM Tris Propane + 1M NaCl, pH9.5) with a linear gradient from 0 to 60%.

[0228] Comparative SEC-HPLC profiles demonstrating the further purification of IL2 fusion protein P703 by AEX chromatography are shown in FIG.3B (no AEX step) and FIG.3C (with AEX step). Example 2: Generation of stable cell lines and IL-2 reporter assay for IL-2 fusion protein

[0229] This Example illustrates a study of the IL-2 stimulatory activity of mutant IL2 fusion proteins using an IL-2 inducible STAT5 reporter assay using HEKBlue cell lines.

[0230] Materials and methods

[0231] A. Stable cell line generation

[0232] 1) HEKBlue-PD1+, HEKBlue CD122 / CD132+ cell line: HEKBlue CD122 / CD132 cells (Invivogen Cat# hkb-il2bg) were infected at MOI 20 with PD1 (PDCD1) (NM_005018) Human Tagged ORF Clone Lentiviral Particle (Origene Cat# RC210364L4V). The transduced cells were about 60% PD1 expressed (captured with GFP tag by flow). Through 2 rounds of single cell cloning (limiting dilutions at 0.3 cells / well), cell clones above 95% positive for PD-1 were picked for amplifying and used in the later cell-based assay.

[0233] 2) HEKBlue-CD8+ HEKBlue CD122 / CD132 cell line: HEKBlue-IL2 cells (Invivogen Cat# hkb-il2) were infected at MOI 20 with CD8A (NM_001768) Human Tagged ORF Clone Lentiviral Particle (Origene Cat# RC206608L3V). About 60% CD8 positive (measured by flow with BV421-CD8 antibody) transduced cells went through 2 rounds of single cell cloning (limiting dilutions at 0.3 cells / well), cell clones above 95% positive for CD8 were picked for amplifying and used in the later cell-based assay.

[0234] 3) HEKBlue-PD1+CD8+ Cell Line: HEKBlue-PD1+ cells (in house made) were infected at MOI 20 with CD8A (NM_001768) Human Tagged ORF Clone Lentiviral Particle (Origene Cat# RC206608L3V). About 40% CD8 positive (measured by flow with BV421-CD8 antibody) transduced cells went through 2 rounds of single cell cloning (limiting dilutions at 0.3 cells / well), The cell clones above 95% positive for CD8 and PD-1 were picked for amplifying and used in the later cell-based assay.

[0235] B. Reporter assay of IL-2 induced STAT5 activity

[0236] HEK293 stable cell lines (prepared as above) which express IL-2 receptor subunits (CD122 / CD132) and STAT5-inducible SEAP reporter were transfected with either PD-1, CD8A, or PD-1+CD8A genes, respectively, and used in a cell based functional assay of the IL-2 fusion proteins. Briefly, cell suspensions above 90% viability were prepared. Test articles at the  concentration indicated in each experiment were prepared by serial dilution in DMEM+10% heat inactivated FBS, added to cell suspension at 50K / well in a flat bottom 96-well plate, incubated at 37C for 20-24 hours. Next day, QuantI-Blue solution was prepared following the instructions from Invivogen.20 μl of induced HEKBlue cell supernatant per well were added to a flat-bottom 96-well plate, then 100ul of resuspended QUANTI-Blue Solution was added to each well, and the plate was incubated at 37C for 1-3 h SEAP levels were determined by reading absorbance at 630 nm (OD630) using spectrophotometer.

[0237] Results

[0238] FIG.4A shows exemplary results demonstrating that the A2B antigen binding polypeptide had more significant effects on decreasing the IL-2 inducible STAT5 activities of the fusion proteins than A1B antigen binding polypeptide did in the absence of the A1B and / or A2B antigens. However, in the cell lines expressed with PD-1 (FIG.4C and FIG.4D) or CD8A (FIG. 4B and FIG.4D), the presence of the A1B and A2B polypeptide subunits in the fusion protein was able to be fully or partially rescue the lost IL2 activity of the fusion proteins. Example 3: IL-2 stimulation by IL-2 fusion proteins in PBMCs

[0239] This Example illustrates a study of the IL-2 stimulatory activity of mutant IL2 fusion proteins in PBMCs using a Phospho-STAT5 and isotype control staining flow cytometric study.

[0240] Materials and methods

[0241] A. PBMC preparation

[0242] Peripheral blood mononuclear cells (PBMCs) were isolated from fresh buffy coat ordered from a Blood Center by Ficoll-Histopaque 1077 (Sigma H8889) density centrifugation. Briefly, The buffy coat was diluted 1:1 with PBS, then overlaid 15ml ficoll with 30ml diluted buffy coat, spined at 600xg for 30min with brake off. Harvest The PBMC layer was harvested and washed with RPMI1640 medium (Sigma R0883) twice at 500xg for 10min, counted cell number and viability. The cells with 99% viability were used in the following experiment.

[0243] B. PBMCs stimulation and Phospho-STAT5 and isotype control staining

[0244] The PBMCs were seeded into 96-well cell culture plates at 106cells / well in 100ul and stimulated with IL2 bispecific antibody drugs at concentrations indicated at 37oC for 30 min, then immediately fixed with 2% PFA (Paraformaldehyde Electron Microscopy Science Cat#: 15710) on ice for 30min. The fixed cells were washed with HBSS twice at 500xg for 7min at 10oC and treated with prechilled (at -80oC) 90% methanol 30min on ice, then washed cells sequentially with HBSS+10%FBS and FACs buffer. Split the cells into 2 replicates for isotype control and experiment staining, respectively. Then, the cells were pre-incubated with 20 μL of anti-human Fc receptor binding inhibitor antibody per 100 µL for 10–20 minutes at 2–25°C, then before staining. Resuspend in 50µL of eBioscience Flow Cytometry Staining Buffer at room temperature for 2 hours. Details of antibodies used are provided in Table 13 below).

[0245] TABLE 13 Marker Fluorochrome Vendor Cat# BD AF700 PE-Cy7 Biolegend 300526 CD8 APC-Cy7 Biolegend 301016

[0246] The s flex flowcytometer.

[0247] C. Data collection and analysis

[0248] Data were collected on a Cytoflex and analyzed using CytExpert software (Beckman). Tregs were gated as CD3+ CD4+ CD25high cells, CD8+ T cells were gated as CD3+ CD8+ cells, and TConv cells were gated as CD3+ CD4+ CD25- cells. pSTAT5 dose-response curves were fitted to a logistic model and EMax and EC50 values were calculated using GraphPad Prism data analysis software after subtraction of the MFI of unstimulated cells and normalization to the maximum signal intensity.

[0249] Results

[0250] Assays were carried out using the exemplary tri-specific mutant IL-2 fusion protein, P402, which has the 3H9 anti-PD1 VHH as A1B, the 3A8 anti-CD8 VHH as A2B, and the mutant IL2 subunit polypeptide. As shown by the results for PBMC assay of IL-2 stimulation in samples collected from two blood donors, Donor A and Donor B, depicted in FIG.5A and FIG. 5B, respectively. As shown in the depicted results, p402 has much less stimulatory activity on Treg (Kd=620 nM), CD4+ (Kd<1000nM), and NK (Kd=1 nM) than wild type IL-2 and non ^IL-2, p132. It indicated that p402 would have much less immune suppression and toxicities. However, in the presence of binding targets for A1B and A2B VHH on CD8+ T cells, p402 has much stronger stimulatory activities (Kd= 0.31nM) as wild type IL-2 does, which is required for tumor killing. It was proposed that the binding of p402 with its A1B and A2B VHH targets on CD8+ T cells caused protein conformation changes, which re-opened the binding sites of IL-2 for IL-2 ^ ^ receptors. The avidities of both A1B / A2B VHHs to its targets and IL2- ILR ^ ^ ^interactions further increased CD8+ stimulation as wild type IL-2 did. The ratio of stimulation on CD8+ to Treg is considered as the therapeutic window for a drug candidate. For p402, the therapeutic window could reach to 2000 folds. However, non ^ (p132) and wild type IL-2 had the therapeutic windows around 2 and <0.05 folds, respectively. Example 4: Differential Scanning Fluorimetry (DSF)

[0251] This Example illustrates a study of the thermostability of a mutant IL-2 fusion proteins, P703 and P709, the latter of which includes a nivolumab antigen binding subunit.

[0252] Materials and methods

[0253] The thermostability of mutant IL2 fusion protein samples was analyzed by Differential Scanning Fluorimetry (DSF) using StepOne™ Real-Time PCR System (ThermoFisher) aSypro Orange as a fluorescent dye. Samples were buffer exchanged into 1 mg / mL with 1X PBS. The suitable amount of 5000x Sypro Orange stock solution was spiked in to achieve a dye concentration of 20 X. Samples with Sypro Orange dye were added into 0.1 mL PCR tubes and heated from 25oC to 95oC in 90 min. The temperature-dependent fluorescent signal curve was recorded and melting temperature (Tm) was determined by the first derivative curve method.

[0254] Results

[0255] The differential scanning fluorimetry (DSF) profiles of exemplary fusion proteins, P703, and P709, and the nivolumab antibody alone are shown on FIG.6. P709 includes a mutant IL2 polypeptide with mutations T3A, F42A Y45A, and L72G fused with the nivolumab antibody. As shown by the Tm values are summarized in Table 14 (below), the P703 fusion protein shows surprising thermostability comparable to that of Nivolumab monoclonal antibody.

[0256] TABLE 14: Differential Scanning Fluorimetry (DSF) results o Tm ( C) Proteinxampe 5: armaco netcs o - mutant uson protens n mce

[0257] This Example illustrates a study of pharmacokinetics of the mutant IL2 fusion protein, P610 in mice.

[0258] Materials and methods

[0259] Six C57BL6 female mice were selected as a study group for testing pharmacokinetics (PK) of the mutant IL-2 fusion protein, P610, at a specific dose (mg / kg). The test article was intravenously injected into mouse tails and collected at around 0.150 mL of blood sample from 2 mice from each group at time points (5 min, 1 hr, 4 hr, 8 hr, 24 hr, 48 hr, 4 day, 7 day, 10 day, and 14 day). Serum was separated from the blood cells and stored in a new tube. Samples from each mouse contributed 3-4 time points. The concentration of the IL-2 fusion protein test article was detected using ELISA according to manufacturer’s protocol.

[0260] Results

[0261] The pharmacokinetics profile of determined for the exemplary IL-2 fusion protein, p610, is shown in FIG.7 with the results summarized in Table 15 (below).

[0262] TABLE 15 P610 Dose (mg / kg) 10Example 6: Efficacy of Mutant IL2 Fusion Protein Inhibition of Tumor Growth in a Syngeneic Mouse Tumor Model

[0263] This Example illustrates a study of the in vivo anti-tumor efficacy of a mutant IL2 fusion protein, P701, in a syngeneic mouse tumor model.

[0264] Materials and methods

[0265] 5x105cells of mouse tumor cell line MC-38 were implanted to the left flank of C57BL / 6 mice, under IACUC guidelines. Tumor volumes were measured by caliper and tumor volume calculated as Lx(W / 2)2. After tumor volumes reached 80-120 mm3, mice were randomized into treatment groups. The mutant IL2 fusion protein surrogate, P701, was dosed intraperitoneally on day 1 and once per week at 0.3 mg / kg, 1.0 mg / kg, and 30 mg / kg. Mouse body weights were also measured on days indicated.

[0266] Results

[0267] FIG.8A depicts a plot of results of the study showing significant inhibition of tumor growth in mice treated with mutant IL2 fusion protein, P701 at all three dosages. FIG.8B depicts plots of mouse body weight showing that that there was no significant body weight loss in the treated mice.

[0268] While the foregoing disclosure of the present invention has been described in some detail by way of example and illustration for purposes of clarity and understanding, this disclosure including the examples, descriptions, and embodiments described herein are for illustrative purposes, are intended to be exemplary, and should not be construed as limiting the present disclosure. It will be clear to one skilled in the art that various modifications or changes to the examples, descriptions, and embodiments described herein can be made and are to be included within the spirit and purview of this disclosure and the appended claims. Further, one of skill in the art will recognize a number of equivalent methods and procedure to those described herein. All such equivalents are to be understood to be within the scope of the present disclosure and are covered by the appended claims.

[0269] Additional embodiments of the invention are set forth in the following claims.

[0270] The disclosures of all publications, patent applications, patents, or other documents mentioned herein are expressly incorporated by reference in their entirety for all purposes to the same extent as if each such individual publication, patent, patent application or other document were individually specifically indicated to be incorporated by reference herein in its entirety for all purposes and were set forth in its entirety herein. In case of conflict, the present specification, including specified terms, will control.

Claims

CLAIMS What is claimed is:

1. A fusion protein comprising a first polypeptide chain, wherein the first polypeptide chain comprises: (a) an A1B subunit comprising a polypeptide that specifically binds a first antigen expressed on the surface of an immune cell; (b) an HLE subunit comprising a polypeptide with half-life extending activity; (c) an IL2 subunit comprising a polypeptide having an amino acid sequence of at least 90% identity to SEQ ID NO: 10 and a set of amino acid differences relative to SEQ ID NO: 10 selected from: K35N and Y45R; E95N and K97T; K35N, Y45R, E95N, and K97T; E95N and K97S; K35N, Y45R, E95N, and K97S; K35N, Y45R, E61N, L63T, E95N, and K97T; and K35N, Y45R, E61N, L63T, E95N, and K97S; and optionally, (d) an A2B subunit comprising a polypeptide that specifically binds a second antigen expressed on the surface of the immune cell.

2. The fusion protein of claim 1, wherein the IL2 subunit comprises a polypeptide having an amino acid sequence selected from SEQ ID NO: 14, 11, 12, 13, 15, 16, and 17.

3. The fusion protein of claim 1, wherein the A1B subunit and / or the A2B subunit are selected from a VHH antibody, an antibody, a Fab, a scFv, and a nanobody.

4. The fusion protein of claim 3, wherein the A1B subunit and / or A2B subunit comprise a VHH antibody that specifically binds to first and / or second antigens selected from CD8, PD1, CD39, and CD103.

5. The fusion protein of claim 4, wherein the A1B subunit and / or A2B subunit VHH antibody: (a) specifically binds CD8 and comprises a CDR1 of SEQ ID NO: 28, a CDR2 of SEQ ID NO: 29, and a CDR3 of SEQ ID NO: 30; (b) specifically binds CD8 and comprises an amino acid sequence selected from SEQ ID NO: 27, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, and 52; (c) specifically binds PD1 and comprises a CDR1 of SEQ ID NO: 19, a CDR2 of SEQ ID  NO: 20, and a CDR3 of SEQ ID NO: 21; and / or (d) specifically binds PD1 and comprises an amino acid sequence selected from SEQ ID NO: 20, 22, 23, 24, 25, and 26.

6. The fusion protein of claim 1, wherein the HLE subunit comprises an IgG1 Fc region polypeptide, wherein the IgG1 Fc region includes an amino acid sequence feature selected from: KK, DSDL, LALA, and N297G.

7. The fusion protein of claim 6, wherein the IgG1 Fc region polypeptide comprises an amino acid sequence selected from SEQ ID NO: 72, 73, 74, 75, 76, 77, 78, 79, 80, and 81.

8. The fusion protein of claim 1, wherein the fusion protein further comprises a second polypeptide chain that forms a dimer with the first polypeptide chain.

9. The fusion protein of claim 8, wherein the fusion protein is a heterodimer and the first and second polypeptide chains are selected from: (a) (A1B)-(HLE)-(IL2)-(A2B) and (A1B)-(HLE); (b) (A1B)-(HLE)-(IL2) and (A1B)-(HLE); (c) (A1B)-(HLE)-(IL2) and (A2B)-(HLE); and (d) (A1B)-(HLE)-(A2B) and (A1B)-(HLE)-(IL2).

10. The fusion protein of claim 8, wherein the fusion protein is a homodimer and the first and second polypeptide chains are selected from: (a) (A1B)-(HLE)-(IL2)-(A2B); (b) (A1B)-(HLE)-(IL2); (c) (A1B)-(IL2)-(HLE); (d) (IL2)-(A1B)-(HLE); (e) (IL2)-(HLE)-(A1B); (f) (HLE)-(A1B)-(IL2); (g) (HLE)-(IL2)-(A1B); (h) (A1B)-(HLE)-(A2B)-(IL2); (i) (A1B)-(A2B)-(HLE)-(IL2); (j) (A2B)-(A1B)-(HLE)-(IL2); (k) (A1B)-(IL2)-(HLE)-(A2B); (l) (A1B)-(IL2)-(A2B)-(HLE); (m) (A1B)-(A2B)-(IL2)-(HLE); (n) (A2B)-(A1B)-(IL2)-(HLE); (o) (IL2)-(A1B)-(HLE)-(A2B); (p) (IL2)-(A1B)-(A2B)-(HLE); (q) (IL2)-(A2B)-(A1B)-(HLE); (r) (A2B)-(IL2)-(A1B)-(HLE); (s) (IL2)-(HLE)-(A1B)-(A2B); (t) (IL2)-(HLE)-(A2B)-(A1B); (u) (IL2)-(A2B)-(HLE)-(A1B); (v) (A2B)-(IL2)-(HLE)-(A1B);  (w) (HLE)-(A1B)-(IL2)-(A2B); (x) (HLE)-(A1B)-(A2B)-(IL2); (y) (HLE)-(A2B)-(A1B)-(IL2); (z) (A2B)-(HLE)-(A1B)-(IL2); (aa) (HLE)-(IL2)-(A1B)-(A2B); (bb) (HLE)-(IL2)-(A2B)-(A1B); (cc) (HLE)-(A2B)-(IL2)-(A1B); and (dd) (A2B)-(HLE)-(IL2)-(A1B).

11. The fusion protein of claim 1, wherein the fusion protein comprises an amino acid sequence selected from SEQ ID NO: 60, 61, 53, 54, 55, 56, 57, 58, 59, 63, 64, 65, 66, 67, 68, 69, 70, and 71.

12. The fusion protein of claim 1, wherein each of the subunits is covalently attached via a linker to at least one of the other subunits through its N-terminus and / or C-terminus.

13. The fusion protein of claim 12, wherein the linkers between the subunits are the same or different; optionally, wherein the linkers comprise an amino acid sequence selected from SEQ ID NO: 82-101; optionally, wherein the amino acid sequence is selected from (GGGGS)1(SEQ ID NO: 82), (GGGGS)2(SEQ ID NO: 83), (GGGGS)3(SEQ ID NO: 84), (GGGGS)4(SEQ ID NO: 85), (GGGGS)5(SEQ ID NO: 86), (GGGGS)6(SEQ ID NO: 87), and (GGGGS)3GGG (SEQ ID NO: 89).

14. The fusion protein of any one of claims 1-13, wherein: (a) the immune cell is a tumor-reactive T cell or a Treg cell; (b) the first antigen is selected from CD8, PD-1, CD39, and CD103; (c) the second antigen is selected from CD8, PD-1, CD39, and CD103; and / or (d) the first and second antigens are different; optionally, wherein the first and second antigens are CD8 and PD-1.

15. A polynucleotide encoding a fusion protein of claim 1.

16. An expression vector comprising the polynucleotide of claim 15.

17. An isolated host cell comprising the polynucleotide of claim 15 or the vector of claim 16; optionally, wherein, the host cell is a mammalian cell or a yeast cell.

18. The isolated host cell of claim 17, wherein the host cell is a mammalian cell selected from a Chinese hamster ovary (CHO) cell, a myeloma cell (e.g.,Y0, NS0, Sp2 / 0), a monkey kidney cell (COS-7), a human embryonic kidney line (293), a baby hamster kidney cell (BHK), a mouse Sertoli cell (e.g., TM4), an African green monkey kidney cell (VERO-76), a human  cervical carcinoma cell (HELA), a canine kidney cell, a human lung cell (W138), a human liver cell (Hep G2), a mouse mammary tumor cell, a TR1 cell, a Medical Research Council 5 (MRC 5) cell, and a FS4 cell.

19. A method for producing a fusion protein of claim 1 comprising culturing the host cell of any one of claim 17 or 18 under a condition suitable for expressing the polypeptide.

20. A pharmaceutical composition comprising a fusion protein of claim 1 and a pharmaceutically acceptable carrier.

21. A method for treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of a fusion protein of claim 1, or administering to the subject a therapeutically effective amount of a pharmaceutical composition of claim 20.

22. The method of claim 21, wherein the disease or disorder is cancer.

23. The method of claim 22, wherein the cancer is selected from colorectal cancer, pancreatic cancer, ovarian cancer, liver cancer, renal cancer, breast cancer, lung cancer, esophageal and gastric cancer, head and neck cancer, cervical cancer, prostate cancer, melanoma, bladder cancer, oral cancer, or hematological malignancies.

24. The method of claim 21, wherein the disease or disorder is an autoimmune disease.

25. The method of claim 24, wherein the autoimmune disease is selected from Crohn’s disease, Ulcerative colitis, celiac disease, systemic lupus erythematosus, psoriatic arthritis, rheumatoid arthritis, Sjogren’s syndrome, type 1 diabetes, atopic dermatitis, psoriasis, multiple sclerosis.

26. The method of claim 21, wherein the disease or disorder is a chronic viral infection.

27. The method of claim 26, wherein the chronic viral infection is selected from hepatitis C virus (HCV), herpes simplex virus (HSV1 and HSV2), Epstein-Barr virus (EBV), Varicella virus, rubella virus, and cytomegalovirus (CMV).

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