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 specifically bind to an antigen expressed on the surface of immune cells such as tumor reactive T cells expressing CD8 and / or PD1. The fusion can also comprise a half-life extending protein subunit such as an Ig1 Fc region monomer polypeptide. The present disclosure also relates to the use of these fusion proteins as therapeutic agents, for example, in pharmaceutical compositions for treating cancer and autoimmune disorders.

Description

Cross-reference to related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 515,964, filed July 27, 2023, which is incorporated herein by reference in its entirety. Technical Field

[0002] This disclosure relates to fusion proteins comprising a mutant IL-2 peptide and one or two binding domains (e.g., VHH antibody) that specifically bind to antigens expressed on the surface of immune cells, such as tumor-reactive T cells expressing CD8 and / or PD1. The fusion may also comprise proteins with extended half-life, such as Fc monomers or Fc dimers, or serum albumin peptides.

[0003] References to sequence lists An official copy of the sequence list was submitted with this specification via the USPTO Patent Centre as an XML file in WIPO Standard ST.26 format, named "17195-004PV1.xml", created on July 27, 2023, and measuring 114,715 bytes. This sequence list, submitted via the USPTO Patent Centre, is an integral part of this specification and is incorporated herein by reference in its entirety. Background Technology

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

[0005] IL-2 binds to three different receptor proteins in significantly different ways. IL-2 has a high affinity for the trimer receptor, K... D It has a concentration of approximately 10 pM and moderate affinity for the dimer receptor, K D It is approximately 1 nM and has low affinity for the monomeric IL-2Rα receptor, K DThe concentration is approximately 10 nM. The IL-2 signaling activity mediated by different receptor complexes also varies significantly. Generally, IL-2Rβ and IL-2Rγ have been found to be crucial for IL-2 signaling, while IL-2Rα (CD25) is not essential.

[0006] IL-2 binding to IL-2 receptor proteins expressed on different cells mediates various 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 the production, proliferation, and activation of natural killer (NK) cells. IL-2 has been approved as an immunotherapeutic agent for the treatment of cancer and chronic viral infections. However, IL-2 can also promote the activation and proliferation of immunosuppressive CD4+CD25+ Treg cells, leading to immunosuppression (Fontenot et al., Nature Immunol. 6, 1142-51 (2005); D'Cruz and Klein, Nature Immunol. 6, 1152-59 (2005); Maloy and Powrie, Nature Immunol. 6, 1171-72 (2005)). Furthermore, IL-2 treatment is associated with vascular leakage syndrome (VLS) and pulmonary edema in patients. Pulmonary edema is believed to be caused by the direct binding of IL-2 to the trimeric receptor (IL-2Rαβγ) on pulmonary endothelial cells (Krieg et al., Proc Nat Acad Sci USA 107, 11906-11 (2010)).

[0007] The engineering of IL-2 through mutations has been proposed to reduce these toxic side effects and improve therapeutic efficacy by altering the selectivity or preference of IL-2 for different IL-2 receptor subunits. For example, it has been proposed that targeting cells expressing IL-2Rβ but not IL-2Rα with IL-2 can induce the expansion of a cell population with high IL-2Rβ, which improves the therapeutic effect of IL-2 therapy (Boyman et al., Science 311, 1924-1927 (2006)). US Patent Publication 2018 / 0142037 A1 describes the introduction of mutations at amino acid positions 42, 45, and 72 of IL-2, also to reduce the affinity of IL-2 for the IL-2Rα receptor. Another mutant IL-2, designated "IL-2H9," contains five mutations: L80F, R81D, L85V, I86V, and I92F, exhibiting enhanced binding to IL-2Rβ, thereby stimulating CD25- cells (see Levin et al., Nature, Vol. 484, pp. 529-533, DOI: 10.1038 / nature10975). The mutant IL-2 protein "IL-23x" possesses three mutations: R38D, K43E, and E61R, resulting in a very low 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 remains, and the mutant peptide is expressed at low levels, which is unfavorable for subsequent large-scale drug production.

[0008] CD8 is a transmembrane glycoprotein that acts as a co-receptor for 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 major histocompatibility complex (MHC) class I proteins. The extracellular domain of the CD8α isoform binds to the α3 portion of class I MHC, and this binding affinity maintains tight binding between cytotoxic T cells and target cells during antigen-specific activation.

[0009] Cytotoxic T cells possessing the CD8 surface protein are called CD8+ T cells. The CD8 co-receptor also promotes T cell signaling through the cytoplasmic domain of the transmembrane CD8 receptor, which binds to Lck (a lymphocyte-specific protein tyrosine kinase). Lck phosphorylates the cytoplasmic domain of the TCR complex, initiating a cascade of phosphorylation events that lead to the activation of transcription factors, including NFAT, NF-κB, and AP-1. CD8+ T cells also have the ability to produce several cytokines with antitumor and antimicrobial effects, such as TNF-α and IFN-γ.

[0010] Cytotoxic CD8+ T cells are known to play a crucial role in the immune response against cancer. However, tumors possess mechanisms that can defeat the CD8+ T cell immune response, such as the production of immunosuppressive cytokines or immune checkpoint molecules, including PD-1, CTLA4, and LAG3.

[0011] PD-1 (also known as programmed cell death protein 1, PDCD1, or PD1) is a cell surface receptor expressed on T cells, but expressed at much lower levels on NK cells and Pro-B cells. On most naive T cells, PD-1 is not expressed, or if it is, it is expressed very little. However, PD1 expression is present on antigen-sensitive T cells (including CD8+ T cells). + CD4 + CD4 + / FOXP3 + PD-1 expression is induced on Treg (γ / δ T cells). It is particularly high on exhausted T cells. PD-1 is a 288-amino acid type I membrane protein and a member of the CD28 / CTLA-4 extended family of T cell regulatory factors. The protein structure includes an extracellular IgV domain, followed by a transmembrane region and an intracellular tail. The intracellular tail contains two phosphorylation sites located in both an immunoreceptor tyrosine-based inhibitory motif and an immunoreceptor tyrosine-based switching motif, suggesting that PD-1 negatively regulates T cell receptor TCR signaling.

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

[0013] It is well known that PD-1 plays a crucial role in cancer's ability to evade immune responses. Many tumor cells express the PD-1 ligand PD-L1, and it has been found that inhibiting the interaction between PD-1 and PD-L1 enhances T-cell responses that mediate preclinical antitumor activity known as immune checkpoint blockade. Numerous monoclonal antibodies targeting PD-1 have been developed or are under development for the treatment of cancer, including dostarlimab, nivolumab, pembrolizumab, pitilizumab, cimiplimab, and toripalimab.

[0014] There is still a need for fusions of IL-2 with peptides that specifically bind to CD-8 and / or PD-1, which could be used as immunotherapeutic agents to modulate immune responses mediated by activated T cells. Summary of the Invention

[0015] This disclosure generally relates to fusion proteins comprising a mutant interleukin-2 (IL-2) polypeptide and one or both antigen-binding subunits, wherein the antigen-binding subunits comprise polypeptides that specifically bind to antigens expressed on the surface of immune cells. This disclosure also relates to pharmaceutical compositions comprising these fusion polypeptide molecules, and the use of these compositions as therapeutic agents, such as in the treatment of cancer. This disclosure is intended to introduce the subject matter but does not cover every embodiment, combination, or variation contemplated and described within this disclosure. Further embodiments are contemplated and described through the detailed description, the accompanying drawings, and the claims.

[0016] In at least one embodiment, the present invention provides a fusion protein comprising a first polypeptide chain, wherein the first polypeptide chain comprises: (a) an A1B subunit comprising a polypeptide specifically binding to a first antigen expressed on the surface of an immune cell; (b) an HLE subunit comprising a polypeptide having a half-life-extending activity; and (c) an IL2 subunit comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 10 and a set of polypeptides differing from SEQ ID NO: 10 from the following: 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 containing a polypeptide that specifically binds to a second antigen expressed on the surface of an immune cell.

[0018] In at least one embodiment of the fusion protein disclosed herein, 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 disclosed herein, the A1B subunit and / or the A2B subunit are selected from antibodies, Fab, scFv, VHH antibodies, and nanobodies. In at least one embodiment, the A1B subunit and / or the A2B subunit comprises a VHH antibody that specifically binds to a first antigen and / or a second antigen selected from PD1, CD8, CD39, and CD103.

[0020] In at least one embodiment of the fusion protein disclosed herein, the A1B subunit and / or the A2B subunit comprises a VHH antibody, wherein the antibody: (a) specifically binds to PD1 and comprises CDR1 of SEQ ID NO: 19, CDR2 of SEQ ID NO: 20, and CDR3 of SEQ ID NO: 21; (b) specifically binds to PD1 and comprises an amino acid sequence selected from SEQ ID NO: 20, 22, 23, 24, 25, and 26; (c) specifically binds to CD8 and comprises CDR1 of SEQ ID NO: 28, CDR2 of SEQ ID NO: 29, and CDR3 of SEQ ID NO: 30; and / or (d) specifically binds to CD8 and comprises an amino acid sequence selected from SEQ ID NO: The amino acid sequences of 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 disclosed herein, the HLE subunit comprises an IgG1 Fc region polypeptide, wherein the IgG1 Fc region includes amino acid sequence features selected from the following: 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 this disclosure, each of the subunits is covalently attached to at least one of the other subunits via a linker through its N-terminus and / or C-terminus. In at least one embodiment, the linkers between the subunits may be the same or different. In at least one embodiment, the linker between the subunits comprises 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 disclosed herein, the N-terminal to C-terminal orientation of the subunit 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 disclosed herein, the fusion protein comprises: an A2B subunit comprising a polypeptide that specifically binds to a second antigen expressed on the surface of immune cells, and the N-terminal to C-terminal orientation of the subunit 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 disclosed herein, 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 this disclosure, the fusion protein further comprises a second polypeptide chain that dimers with the first polypeptide chain. In at least one embodiment, the fusion protein is a heterodimer, wherein the first polypeptide chain and the second polypeptide chain 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 this disclosure, the immune response expressing the antigen on its surface cells is tumor-reactive T cells or Treg cells. 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 disclosed herein, the fusion protein comprises: an A2B subunit containing a polypeptide that specifically binds to a second antigen expressed on the surface of an immune cell, wherein the first antigen and the second antigen are different; optionally, wherein the first antigen and the second antigen are CD8 and PD-1.

[0029] In at least one embodiment of the fusion protein disclosed herein, 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, this disclosure also provides a polynucleotide encoding the fusion protein of this disclosure. In at least one embodiment, this disclosure provides an expression vector comprising a polynucleotide encoding the fusion protein of this disclosure.

[0031] In another embodiment, this disclosure also provides isolated host cells containing polynucleotides or expression vectors encoding the fusion proteins of this disclosure; optionally, the host cells are mammalian cells or yeast cells. In at least one embodiment, the host cells are mammalian cells selected from: Chinese hamster ovary (CHO) cells, myeloma cells (e.g., Y0, NSO, Sp2 / O), monkey kidney cells (COS-7), human embryonic kidney cell line (293), young hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells, human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor cells, TR1 cells, Medical Research Council 5 (MRC 5) cells, and FS4 cells.

[0032] In another embodiment, this disclosure provides a method for generating the fusion protein of this disclosure, wherein the method includes culturing a host cell containing a polynucleotide encoding the fusion protein of this disclosure under conditions suitable for peptide expression.

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

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

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

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

[0037] In at least one embodiment of the method for treating a disease or condition in a subject, the disease or condition is a chronic viral infection; optionally, 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). Attached Figure Description

[0038] A better understanding of the novel features and advantages of this disclosure will be obtained by referring to the following detailed description of illustrative embodiments and accompanying drawings (also referred to herein as “Figures”), in which the principles of this disclosure are utilized, and in the accompanying drawings: Figures 1A-1N A schematic diagram of an exemplary mutant IL2 fusion protein of this disclosure is depicted.

[0039] Figures 2A-2I A schematic diagram depicts an exemplary mutant IL2 fusion protein of this disclosure having a homodimeric or heterodimeric structure comprising a single-chain mutant IL2 fusion polypeptide. Exemplary structures include homodimers or heterodimers of bispecific or trispecific single polypeptide chains, such as those depicted in Figure 1. Figure 3A , Figure 3B and Figure 3C SEC-HPLC spectra of the exemplary mutant IL2 fusion protein prepared and purified as described in Example 1 are depicted. Figure 3A SEC-HPLC spectra of exemplary mutant IL2 fusion proteins P401, P402, P404, P405, and P406 were depicted. Figure 3B and Figure 3C The SEC-HPLC chromatogram of the exemplary mutant IL2 fusion protein P703 after protein A affinity and AEX purification, as described in Example 1, is depicted.

[0040] Figure 4A , Figure 4B , Figure 4C and Figure 4D The results depict the IL2 activity of exemplary mutant IL2 fusion proteins P402, P404, P405, and P406 in STAT5 reporter gene assays performed with various HEK Blue cells expressing IL2 and CD8 and / or PD1, as described in Example 2.

[0041] Figure 5A and Figure 5BThe figure depicts the results of a study on the IL-2 stimulatory activity of the mutant IL2 fusion protein in PBMCs from two donors (donor A and donor B) using phosphorylated STAT5 and isotype control staining flow cytometry, as described in Example 3.

[0042] Figure 6 The figure depicts the results of a study on the thermal stability of mutant IL-2 fusion proteins P703 and P709 using differential scanning fluorescence (DSF) as described in Example 4.

[0043] Figure 7 The results of a pharmacokinetic study of the mutant IL2 fusion protein P610 in mice, as described in Example 5, are depicted.

[0044] Figure 8A and Figure 8B The figure depicts the results of an in vivo study of the antitumor efficacy of the mutant IL2 fusion protein P701 in a syngeneic mouse tumor model, as described in Example 6. Detailed Implementation

[0045] This disclosure provides a mutant IL-2 fusion protein comprising an IL2 subunit fused to one or two antigen-binding subunits, the IL2 subunit comprising a mutant IL-2 polypeptide, and the antigen-binding subunit comprising a polypeptide that specifically binds to an antigen (e.g., CD8 and / or PD1) expressed on the surface of immune cells (such as tumor-reactive T cells). This disclosure also relates to pharmaceutical compositions comprising these fusion polypeptide molecules, and the use of these compositions as therapeutic agents, such as in the treatment of cancer or autoimmune diseases.

[0046] Terminology and Technical Overview In the description herein and the appended claims, unless the context clearly indicates otherwise, the singular forms “a” and “an” include plural references. Thus, for example, reference to “a protein” includes more than one protein, and reference to “a compound” refers to more than one compound. It should be further noted that claims may be drafted to exclude any optional elements. Therefore, this description is intended to serve as an existing basis for the use of exclusive terms such as “only” or “just” in relation to references to claim elements, or for the use of negative limiting terms. The use of “comprise / comprises / comprising” and “include / includes / including” is interchangeable and not intended to be restrictive. It should also be understood that where the term “comprising / including” is used in the description of various embodiments, those skilled in the art will understand that in certain specific cases, embodiments may alternatively be described using the language “consistently of” or “comprises of”.

[0047] Where a range of values ​​is provided, unless the context explicitly indicates otherwise, it should be understood that, unless the context explicitly indicates otherwise, every intermediate integer and every tenth of every intermediate integer of the values ​​between the upper and lower limits of the range, as well as any other specified values ​​or intermediate values ​​within the specified range, are covered within this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller range and also covered within this invention, subject to any specifically excluded limits within the specified range. When a specified range includes one or both of these limits, the range excluding (i) one or (ii) both of the included limits is also included in this invention. For example, “1 to 50” includes “2 to 25”, “5 to 20”, “25 to 50”, “1 to 10”, etc.

[0048] Generally speaking, the nomenclature used in this article and the techniques and procedures described herein include those that are well understood and commonly used by those skilled in the art, such as common techniques and methodologies described in, for example, the following literature: Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Volumes 1-3, ColdSpring Harbor Laboratory, Cold Spring Harbor, NY, 2012 (hereinafter referred to as "Sambrook"); and Current Protocols in Molecular Biology Edited by FM Ausubel et al., it was originally published as a book in 1987 by Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., with regular updates since 2011, and is now available online as a journal. Current Protocols in Molecular Biology, Volumes 00-130 , (1987-2020), included in WileyOnline Library (hereinafter referred to as "Ausubel") by Wiley & Sons, Inc.

[0049] All publications, patents, patent applications and other documents cited in this disclosure are hereby incorporated in their entirety by reference for all purposes, just as each individual publication, patent, patent application or other document is individually indicated to the extent to which it is incorporated herein by reference for all purposes.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should be understood that the terminology used herein is for describing particular embodiments only and is not intended to be limiting. For the purposes of interpreting this disclosure, the following terminology will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0051] As used herein, a “fusion compound” or “fusion protein” refers to two or more protein and / or polypeptide molecules linked (or “fused”) in a conformation not naturally present in the body. Fusion proteins disclosed herein include fusions of mutant IL2 polypeptides and one or more polypeptides that specifically bind to antigens expressed on the surface of immune cells. Antigen-binding polypeptides may include: antibodies, such as anti-PD-1 VHH and / or anti-CD8 VHH, which are covalently linked to the IL2 polypeptide via a polypeptide linker sequence; and / or another polypeptide, such as a polypeptide with extended half-life.

[0052] As used herein, a "peptide linker" or "linker sequence" refers to a chain of two or more amino acids, each end of which is covalently attached to a different polypeptide molecule, thereby enabling the conjugation or fusion of different polypeptides. Typically, a peptide linker comprises a polypeptide chain of 5 to 30 amino acids. A wide variety of peptide linkers are known in the art and can be used in compositions and methods incorporating fusions of the anti-PD-1 VHH antibody of this disclosure. Exemplary peptide linkers included in the compositions and methods of this disclosure include, but are not limited to, (GGGGS). n (SSSSG) n (GGGG)(SGGGG) n (EAAAK) n (XP) n (where X can be any amino acid, preferably A, K or E), ENLYFQ(-G / S), typically, where n is 1 to 10.

[0053] As used herein, “IL-2” or “IL-2 polypeptide” refers to the cytokine interleukin-2 and includes naturally occurring and recombinant forms of interleukin-2 polypeptides derived from humans, mice, rats, or non-human primates, as well as their unprocessed (with signal peptide) and processed (without signal peptide) forms. Furthermore, the term includes naturally occurring IL-2 variants, such as allelic and splice variants, isotypes, homologs, and species homologs, as well as recombinant (i.e., artificial) IL-2 variants or mutants, including mutant IL-2 polypeptides with 1-15 amino acid substitutions relative to the amino acid sequence of naturally occurring IL-2. For example, the term covers the recombinant human IL-2 amino acid sequence of UniProt P60568, which has an amino acid substitution at position C125, such as C125S or C125A. The term is also intended to cover IL-2 polypeptides covalently conjugated (or fused) to another polypeptide or protein. Exemplary IL-2 fusions disclosed herein include mutant IL-2 peptides fused with other cytokines (e.g., IL-15) or with peptides that have extended half-life (e.g., monomeric Fc, dimer Fc, or human serum albumin).

[0054] As used herein, “IL-2 receptor” or “IL-2R” refers to a heterotrimeric protein expressed on the surface of certain immune cells and endothelial cells, and also encompasses each of the polypeptide subunits IL-2Rα, IL-2Rβ and IL-2Rγ (also known as the common subunit γ of cytokine receptors), in both monomeric and dimeric forms (such as IL-2Rβγ).

[0055] As used herein, “PD-1” refers to the “programmed cell death 1” protein that acts as the cell surface receptor for PD-L1 and PD-L2, and encompasses the full length and portions of the full-length PD-1 protein in humans, cynomolgus monkeys (referred to as “cyno” in some contexts herein), and rhesus monkeys, as well as their various isotypes. Exemplary sequences of the extracellular domain (ECD) of the human PD-L1 chain are provided in Table 3 and the attached sequence listing.

[0056] As used herein, “PD-1-mediated symptoms” or “PD-1-mediated diseases” encompass any medical condition that is PD-1-related or affected by PD-1, or caused by cells expressing PD-1 (including but not limited to CD8 cells). + T cells, NK cells, NKT cells, CD4 + T cells, CD4 + / FOXP3 +Therapeutic effects mediated or provided by Treg (γ / δ T cells). For example, specific binding to PD-1 expressed on the cell surface can alter the activation of CD8+ lymphocytes (e.g., T cells). Therefore, PD-1-mediated diseases can include, but are not limited to, any disease or condition mediated and / or responsive to an agonist (or activator) or antagonist (or inhibitor) of PD-1-expressing cells, including, but not limited to, autoimmune diseases and cancers. Specific exemplary autoimmune diseases and cancers are provided elsewhere in this document.

[0057] As used herein, “CD8” refers to the group 8 (CD8) transmembrane glycoprotein, which acts as a co-receptor for T-cell receptors, and encompasses the full length and portions of the full-length CD8 protein in humans, cynomolgus monkeys (referred to in some contexts as “cyno”), and rhesus monkeys, as well as their various isotypes. The human CD8 protein is a dimer consisting of a pair of CD8 chains, comprising the CD8 α (or “CD8A”) and CD8 β (or “CD8B”) chains. The term “human CD8” encompasses the CD8A / CD8A homodimer, the CD8A / CD8B heterodimer, the CD8A chain, the CD8B chain, or portions thereof, such as the extracellular domain. “CD8A” and “CD8a” are used interchangeably herein, and “CD8B” and “CD8b” are used interchangeably herein. Exemplary sequences of the extracellular domain (ECD) of the human CD8A chain are provided in Table 3 and the attached sequence listing.

[0058] As used herein, “CD8-mediated condition” or “CD8-mediated disease” encompasses any medical condition related to or affected by CD8, or therapeutic effects mediated or provided by CD8+ cells (including, but not limited to, CD8+ T cells, NK cells, and NKT cells). For example, specific binding to CD8 expressed on a cell surface can alter the activation of CD8+ lymphocytes (e.g., T cells). Therefore, CD8-mediated disease may include, but is not limited to, any disease or condition mediated and / or responsive to an agonist (or activator) or antagonist (or inhibitor) of CD8-expressing cells, including, but not limited to, autoimmune diseases and cancer. Specific exemplary autoimmune diseases and cancers are provided elsewhere in this document.

[0059] As used herein, an "antibody" is a molecule comprising one or more polypeptide chains that specifically binds to or triggers an immune response to a specific 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, (only) heavy-chain antibodies (e.g., sdAb or VHH, IgNAR, nanobodies (or nanoAbs)), antigen-binding fragments (e.g., Fab′, F(ab′)2, Fab, Fv, rIgG, and scFv fragments), antibody fusions, and synthetic antibodies (or antibody mimics).

[0060] "Anti-PD-1 antibody" or "PD-1-binding antibody" refers to an antibody that binds to PD-1 with sufficient affinity, such that the antibody can be used as a diagnostic and / or therapeutic agent targeting PD-1. In some embodiments, such as those measured by radioimmunoassay (RIA) or surface plasmon resonance (SPR), the binding degree of the anti-PD-1 specific antibody to unrelated non-PD-1 antigens is less than about 20%, less than about 15%, less than about 10%, or less than about 5% of the antibody's binding to PD-1. In some embodiments, the dissociation constant (Ki) of the PD-1-binding antibody is... D <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <1 pM (e.g., 10 μM). -8 M or lower, such as 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 M).

[0061] "Anti-CD8 antibody" or "CD8-binding antibody" refers to an antibody that binds to CD8 with sufficient affinity, making it usable as a diagnostic and / or therapeutic agent targeting CD8. In some embodiments, such as those measured by radioimmunoassay (RIA) or surface plasmon resonance (SPR), the binding degree of the anti-CD8 specific antibody to unrelated non-CD8 antigens is less than about 20%, less than about 15%, less than about 10%, or less than about 5% of the antibody's binding to CD8. In some embodiments, the dissociation constant (Ki) of the CD8-binding antibody is... D <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <1 pM (e.g., 10 μM). - 8 M or lower, such as 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 M).

[0062] The terms “full-length antibody,” “complete antibody,” or “all antibody” are used interchangeably in this document and refer to antibodies that have a structure substantially similar to that of natural antibodies or that have a heavy chain containing an Fc region as defined herein.

[0063] An "antibody fragment" refers to a portion of a full-length antibody that is capable of binding to 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')2 fragments, biantibodies, linear antibodies, monovalent or single-arm antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0064] As used herein, “heavy chain antibody,” “heavy chain-only antibody,” or “HCAb” refers to a functional antibody that contains two heavy chains but lacks the two light chains typically found in tetrachain antibodies. Cameloideas (such as camels, llamas, or alpacas) are known to produce HCAbs.

[0065] As used herein, a “single-domain antibody” or “sdAb” refers to a single antigen-binding domain having three complementarity-determining regions (CDRs). A standalone sdAb can bind to an antigen without pairing with the corresponding CDR-containing peptide. Cameloidea sdAbs are among the smallest known antigen-binding antibody fragments (see, for example, 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)). “VHH” (defined below) is a class of single-domain antibodies engineered from Cameloidea HCAbs.

[0066] "VHH" or "variable domain of heavy chain antibody" refers to a single chain containing the variable domain of the antibody's heavy chain. VHH typically has the following structure from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to frame regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3. VHH molecules can be derived from... Camelidae Antibodies produced in or generated from synthetic libraries in species such as camels, llamas, dromedary camels, alpacas, and guanacos.

[0067] "VHH antibody," "heavy chain antibody," or "heavy chain-only antibody" refers to a single chain comprising a heavy chain variable domain and an Fc region of the antibody, with hinges or other linkers containing amino acids of a natural or synthetic sequence. The "single chain" can form a dimer, such as when fused with the Fc region, which is a dimer.

[0068] An antibody's "class" refers to the type of constant domain or constant region possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes are further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are designated as α, δ, ε, γ, and μ, respectively.

[0069] "Variable regions" or "variable domains" refer to the structural domains of the antibody heavy or light chain that participate in antibody-antigen binding. The variable domains of the heavy and light chains of natural antibodies (V1 and V2, respectively) H and V L They typically have similar structures, with each domain containing four conserved frame regions (FRs) and three highly variable regions (HVRs) (see, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91). A single V H or V L The structural domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies binding to a specific antigen can utilize the V domain derived from the antibody binding to that antigen. H Domain or V L The structural domains are selected to screen complementary V. L Domain or V H The structural domain library is thus separated (see, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0070] As used herein, a “hypervariant region” or “HVR” refers to each of the regions of an antibody’s variable domain that is hypervariable in sequence and / or forms a structurally defined loop (“hypervariant loop”). Generally, natural antibodies contain four chains and six HVRs: three in the heavy chain variable domain, three in the V… H (HVR-H1, HVR-H2, HVR-H3), and three in the variable structural domain of the light chain, V L(HVR-L1, HVR-L2, HVR-L3). HVRs generally contain amino acid residues from hypervariable loops and / or from “complementarity-determining regions” (CDRs). Various hypervariable region classifications are in use and are covered in this paper. The Kabat CDR is based on sequence variability and is 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 to the location of the structural loop (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). AbM hypervariable regions represent a compromise between the Kabat CDR and the Chothia structural loop and are used by the OxfordMolecular AbM antibody modeling software. “Contact” hypervariable regions are based on analysis of available complex crystal structures. Residues from each of these hypervariable regions are indicated in Table 1 below.

[0071] Table 1

[0072] Unless otherwise stated, HVR residues and other residues (e.g., FR residues) in the variable domain are numbered in accordance with Kabat et al., ibid.

[0073] As used herein, hypervariable regions may include the following extended or alternative hypervariable regions: V H The domains 26-35 or 30-35 (H1), 50-61, 50-65 or 49-65 (H2), and 93-102, 94-102 or 95-102 (H3); and V L The domains 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3). For each of these definitions, the variable domain residues are numbered as above, according to Kabat et al.

[0074] As used in this article, the "complementarity-determining region" or "CDR" refers to the region within the variable domain HVR that has the highest sequence variability and / or participates in antigen recognition. Generally, natural antibodies contain four chains and six CDRs: three in the heavy chain variable domain, three in the V... H (H1, H2, H3), and three in the variable structural domain of the light chain, V L(L1, L2, L3). Typically, VHH antibodies contain only the CDR (V...) of the heavy chain (VH). H (H1, H2, H3). Exemplary CDRs (according to Kabat et al., ibid.) are present at the following amino acid residue positions in 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.

[0075] "Frame" or "FR" refers to variable domain residues other than the hypervariable region (HVR) residues. A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences in V H (or V) L In FR1, H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0076] "Natural antibodies" refer to naturally occurring immunoglobulin molecules. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. Each heavy chain has a variable region (VT) from the N-terminus to the C-terminus. H (Also known as the variable heavy chain domain or heavy chain variable domain), followed by three constant domains (CH1, CH2, and CH3). Similarly, each light chain has a variable region (V) from the N-terminus to the C-terminus. L The light chain (also known as the variable light chain domain or light chain variable domain) is followed by the constant light chain (CL) domain. Based on the amino acid sequence of its constant domain, the antibody light chain can be assigned to one of two types (called kappa (κ) and lambda (λ)).

[0077] As used herein, a “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising that population are identical and / or bind to the same epitopes, except for possible variant antibodies (e.g., those containing naturally occurring or acquired mutations during monoclonal antibody production and generally present in smaller quantities). In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Therefore, the term “monoclonal” indicates the characteristic of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies to be used can be prepared using a variety of techniques, including but not limited to hybridoma methods, 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 preparing monoclonal antibodies are described herein.

[0078] "Chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remainder of the heavy chain and / or light chain originates from different sources or species.

[0079] "Humanized antibody" refers to a chimeric antibody comprising an amino acid sequence from a non-human HVR and an amino acid sequence from a human FR. In some embodiments, the humanized antibody will comprise at least one, and typically substantially all, of two variable domains, wherein all or substantially all of the HVR corresponds to those of the non-human antibody, and all or substantially all of the FR corresponds to those of the human antibody. Optionally, the humanized antibody may comprise at least a portion of the antibody constant region derived from a human antibody. The "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized.

[0080] "Human antibody" refers to an antibody having an amino acid sequence corresponding to the following antibodies: antibodies produced by humans or human cells, or antibodies derived from non-human sources using sequences encoded by human antibody libraries or other human antibodies. This definition of human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues.

[0081] The "human common framework" represents human immunoglobulin V. L or H The selection of the framework sequence is based on the most frequently occurring amino acid residues. Typically, human immunoglobulin V... L or V HThe sequence selection is based on a subgroup of variable domain sequences. Generally, the sequence subgroups are those shown in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In some implementations, for V... L The subgroup is like that of Kabat et al., the same as subgroup κ I mentioned above. In some implementations, for V H Subgroups include those like Kabat et al., which are subgroups III mentioned above.

[0082] As used in this article, "receptor human framework" refers to a light chain variable domain (V) containing a light chain derived from the human immunoglobulin framework or the human common framework. L )Frame or heavy-chain variable structural domain (V H The frame of amino acid sequences of the frame. Receptor human frames “derived from” the human immunoglobulin frame or the human common frame may contain the same amino acid sequence or may contain amino acid sequence variations. In some embodiments, the number of amino acid variations is 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, V L Receiver human frame sequence and V L The human immunoglobulin framework sequence or the human common framework sequence is the same.

[0083] The “Fc region” or “crystallizable fragment region” refers to a dimeric complex containing the C-terminal polypeptide sequence of the immunoglobulin heavy chain, where the C-terminal polypeptide sequence is a sequence obtainable by digesting an intact antibody with papain. The Fc region of an immunoglobulin typically includes the CH2 and CH3 domains of the heavy chain, and optionally the CH4 domain. The Fc region may contain native or variant Fc sequences. Although the boundaries of the Fc sequence of the immunoglobulin heavy chain can vary, the human IgG heavy chain Fc sequence is generally defined as an extension from approximately position Cys226 or approximately position Pro230 to the C-terminus of the Fc sequence.

[0084] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a natural human FcR. In some embodiments, the FcR is a receptor that binds to IgG antibodies (γ receptors) and includes the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splicing forms of those receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an activation motif (ITAM) based on the immunoreceptor tyrosine residue in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an inhibitory motif (ITIM) based on the immunoreceptor tyrosine residue in its cytoplasmic domain (see, for example, Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). As used in this article, FcR also includes the neonatal receptor FcRn, which is responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). FcR is reviewed in, for example, 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).

[0085] As used herein, a "multivalent antibody" is an antibody containing three or more antigen-binding sites. Multivalent antibodies are preferably engineered to have three or more antigen-binding sites and are typically not naturally occurring IgM or IgA antibodies.

[0086] A "multispecific antibody" is an antibody with at least two different binding sites, each with different binding specificities. Multispecific antibodies can be full-length antibodies or antibody fragments, and the different binding sites can bind to different antigens, or the different binding sites can bind to two different epitopes of the same antigen.

[0087] "Fv fragment" refers to an antibody fragment containing complete antigen recognition and binding sites. This region consists of a dimer of a tightly associated heavy-chain variable domain and a light-chain variable domain; this association can be covalent, as in scFv. It is in this configuration that the three HVRs of each variable domain interact, thereby achieving V... H -V LAntigen-binding sites are defined on the surface of the dimer. In general, the six HVRs or a subset thereof confer antigen-binding specificity to the antibody. However, even a single variable domain (or half an Fv containing only three HVRs that are specific to the antigen) has the ability to recognize and bind to the antigen, although its affinity is generally lower than that of the complete binding site.

[0088] "Fab fragment" refers to an antibody fragment containing a variable and constant domain of the light chain and a variable and first constant domain (CH1) of the heavy chain. "F(ab')2 fragment" comprises a pair of Fab fragments, typically covalently linked near the carboxyl terminus via a hinge cysteine ​​residue between them. Other chemical conjugations of antibody fragments are also known in the art.

[0089] As used herein, an "antigen-binding arm" refers to a component of an antibody that has the ability to specifically bind to a target molecule. Typically, an antigen-binding arm is a complex of immunoglobulin polypeptide sequences, such as the HVR and / or variable domain sequences of the immunoglobulin light and heavy chains.

[0090] "Single-chain Fv" or "scFv" refers to a V containing an antibody. H and V L Antibody fragments containing domains, wherein these domains are present within a single polypeptide chain. Typically, Fv polypeptides also contain V... H and V L The polypeptide linkers between the domains enable scFv to form the desired antigen-binding structure.

[0091] "Dual antibody" refers to a small antibody fragment with two antigen-binding sites contained within the same polypeptide chain (V). H and V L In the light chain variable structural domain (V) L ) connected heavy chain variable structural domain (V H By using excessively short linkers, pairing between two domains on the same strand is allowed, forcing that domain to pair with a complementary domain on another strand and creating two antigen-binding sites.

[0092] "Linear antibodies" refer to the antibodies described in Zapata et al., Protein Eng., 8(10): 1057-1062 (1995). In short, these antibodies contain a pair of tandem Fd regions (VH-CH1-VH-CH1) that, together with a complementary light chain polypeptide, form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

[0093] "Naked antibody" refers to an antibody that is not conjugated with a heterologous part (e.g., a cytotoxic part) or a radioactive label.

[0094] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an IL-2 peptide) and its binding partner (e.g., an IL-2 receptor). "Binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair. The affinity of molecule X for its partner Y can generally be determined by the equilibrium dissociation constant (K0). D Affinity can be measured using methods commonly known in the art, including those described herein. "Specific binding" or "specific binding" refers to the binding affinity between the IL-2 peptide and its receptor at a rate not exceeding about 1 × 10⁻⁶. -7 The affinity value of M is used for binding. Illustrative and exemplary embodiments for measuring binding affinity and / or specific binding are described elsewhere herein (including examples).

[0095] "Specific binding" or "specific binding" refers to the interaction between an antibody and an antigen at a rate not exceeding approximately 1 × 10⁻⁶. -7 The affinity value of M is used for binding. In some embodiments, the antibody may have secondary affinity for antigens other than the antigen it specifically binds to, where "secondary affinity" generally refers to the antibody binding to a secondary antigen with an affinity value exceeding about 10 nM, as described elsewhere herein. When an antibody may have secondary affinity for a secondary antigen, it will still specifically bind to the primary antigen.

[0096] "Affinity maturation" antibodies are antibodies that have one or more alterations in one or more HVRs, which result in improved affinity of the antibody for the antigen compared to parental antibodies that do not have such alterations.

[0097] An antibody's "functional antigen-binding site" is a site that can bind to a target antigen. The antigen-binding affinity of an antigen-binding site may not be as strong as that of the parent antibody from which the antigen-binding site originates, but the ability to bind to the antigen must be measurable using any of the various methods known for evaluating antibody-antigen binding.

[0098] "Isolated antibody" is an antibody that has been separated from components of its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87.

[0099] As used herein, “substantially similar” or “substantially identical” means that two values ​​(e.g., one related to a test antibody and the other to a reference antibody) are similar enough that a person skilled in the art would consider the difference between the two values ​​to be in terms of biological characteristics (e.g., K) as measured by said values. D In cases where the value is very small or statistically significant, or not significant at all, the biological and / or statistical significance is very small.

[0100] As used herein, “substantially different” means a sufficiently high degree of difference between two values ​​(typically one related to a molecule and the other to a reference molecule) such that a person skilled in the art would consider the difference between the two values ​​to be significant in terms of biological properties (e.g., K) as measured by said values. D It is statistically significant when the value is less than or equal to 1.

[0101] "Effective functions" refer to the biological activities induced by the Fc region of an antibody, which vary with antibody isotypes. Examples of antibody effector functions include: Clq binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0102] As used in this article, “host cell” refers to a cell that can be functionally modified with recombinant nucleic acids and function to express recombinant products (including peptides and compounds generated from the activity of peptides).

[0103] As used interchangeably herein, “nucleic acid” or “polynucleotide” refers to two or more nucleosides covalently linked together. Nucleic acids may consist entirely of ribonucleosides (e.g., RNA), entirely of 2'-deoxyribonucleotides (e.g., DNA), or a mixture of ribonucleosides and 2'-deoxyribonucleosides. The nucleoside units of a nucleic acid may be linked together via phosphodiester bonds (e.g., as in naturally occurring nucleic acids), or the nucleic acid may contain one or more non-natural bonds (e.g., phosphate thioester bonds). Nucleic acids or polynucleotides are intended to include single-stranded or double-stranded molecules, or molecules having both single-stranded and double-stranded regions. Nucleic acids or polynucleotides are intended to include molecules composed of naturally occurring nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), or molecules containing one or more modified and / or synthetic nucleobases (such as inosine, xanthine, hypoxanthine, etc.).

[0104] The terms “protein,” “polypeptide,” and “peptide” are used interchangeably herein to refer to a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modifications (e.g., glycosylation, phosphorylation, esterification, myristylation, ubiquitination, etc.). As used herein, a “protein,” “polypeptide,” or “peptide” polymer may contain D-amino acids and L-amino acids, as well as mixtures of D-amino acids and L-amino acids.

[0105] As used in this article, “naturally occurring” or “wild-type” refers to a form that exists in nature. For example, a naturally occurring nucleic acid sequence is a sequence that exists in an organism, can be isolated from natural sources, and has not been intentionally modified by human intervention.

[0106] When used herein with reference to, for example, cells, nucleic acids, or peptides, "recombinant," "engineered," or "non-natural" means a material or a material in its natural or native form that is modified in a manner not originally present in nature, or is identical to such a material but produced or derived from synthetic materials and / or through manipulation using recombinant techniques. Non-limiting examples include, in particular, recombinant cells expressing genes not present in naturally (non-recombinant) cells or expressing natural genes originally expressed at different levels.

[0107] As used herein, “nucleic acid derived from” means a nucleic acid having a sequence that is at least substantially identical to a sequence naturally present in an organism. For example, a cDNA molecule prepared from mRNA isolated from an organism by reverse transcription or a nucleic acid molecule prepared synthetically having a sequence that is at least substantially identical to or hybridizes with a nucleic acid sequence present in an organism.

[0108] "Coding sequence" refers to the nucleic acid portion (e.g., gene) that encodes the amino acid sequence of a protein.

[0109] As used in this article, “heterologous nucleic acid” means any polynucleotide introduced into a host cell through laboratory techniques, including polynucleotides that are removed from a host cell, manipulated in the laboratory, and then reintroduced into the host cell.

[0110] "Codon-optimized" refers to the alteration of codons in polynucleotides encoding proteins to codons preferred by a particular organism, resulting in efficient protein expression in the target organism. Although the genetic code is degenerate, as most amino acids are represented by several codons (called "synonymous codons"), codon usage in a particular organism is known to be non-random and biased towards specific codon triples. This codon preference may be even greater for a given gene, genes with a common function or ancestral origin, highly expressed proteins versus low-copy-number proteins, and protein-coding regions that aggregate in an organism's genome. In some implementations, polynucleotides encoding imine reductases may be codon-optimized for optimal production from selected host organisms for expression.

[0111] "Preferred, optimal, high-codon-use-preference codons" refer to codons used in protein-coding regions at a higher frequency than other codons encoding the same amino acid. Preferred codons can be determined based on factors such as: codon usage in a single gene, a group of genes with a common function or origin, codon usage in highly expressed genes, codon frequency in aggregated protein-coding regions of the entire organism, codon frequency in aggregated protein-coding regions of related organisms, or combinations thereof. Codons whose frequency increases with gene expression levels are generally the optimal codons for expression. Several methods are known for determining codon frequencies (e.g., codon usage, relative synonymous codon usage) and codon preferences in a particular organism, including multivariate analysis, such as using cluster analysis or correspondence analysis, and the number of effective codons used in a gene (see GCG CodonPreference, Genetics Computer Group Wisconsin Package; Codon W, 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). The list of organisms covered by available codon usage tables is constantly growing (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., ibid.; Henaut and Danchin, "Escherichia coli and Salmonella," 1996, eds. Neidhardt et al., ASM Press, Washington DC, pp. 2047-2066). Data sources for obtaining codon usage can rely on any available nucleotide sequence capable of encoding a protein.These datasets include nucleic acid sequences that are actually known to encode the expressed protein (e.g., complete protein-coding sequences - CDS), expressed sequence tags (ESTS), or predicted coding regions of genome sequences (see, for example, Mount, D., Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001; Uberbacher, EC, 1996, Methods Enzymol. 266:259-281; ​​Tiwari et al., 1997, Comput. Appl. Biosci. 13:263-270).

[0112] As used herein, a “control sequence” refers to any sequence that is essential or advantageous for the expression of polynucleotides and / or polypeptides as used in this disclosure. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, leader sequences, promoters, polyadenylated sequences, propeptide sequences, signal peptide sequences, and transcription terminators. At a minimum, control sequences typically include promoters, as well as transcription and translation termination signals. Control sequences may be provided with linkers to introduce specific restriction sites that facilitate the connection of the control sequence to the coding region of the nucleic acid sequence encoding the polypeptide.

[0113] As used herein, “operationally linked” refers to a configuration in which a control sequence is appropriately placed (e.g., in a functional relationship) at a position relative to a target polynucleotide or polypeptide sequence such that the control sequence directs or regulates the expression of the target sequence.

[0114] A promoter sequence is a nucleic acid sequence that is recognized by the host cell for the expression of a target polynucleotide (such as a coding sequence). A promoter sequence contains a transcriptional control sequence that mediates the expression of the target polynucleotide. A promoter can be any nucleic acid sequence that exhibits transcriptional activity in a selected host cell, including mutant, truncated, and heterozygous promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to those of the host cell.

[0115] The terms "percentage of sequence identity," "percentage of sequence identity," "percentage homology," or "percentage of homology" are used interchangeably herein and refer to a value quantifying the comparison of polynucleotide or polypeptide sequences. This value is determined by comparing two optimally aligned sequences within a comparison window, where the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (or vacancies) compared to a reference sequence used for optimal alignment of the two sequences. The percentage value can be calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue appears to obtain the number of matching positions, dividing this number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Alternatively, the percentage can be calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue appears or where a nucleic acid base or amino acid residue is aligned with a vacancy to obtain the number of matching positions, dividing this number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Those skilled in the art will understand that many established algorithms are available for aligning two sequences. The best alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, and the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin software package), or by visual inspection (generally see Current Protocols in Molecular Biology, FM 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 suitable for determining sequence identity and sequence similarity percentages are the BLAST and BLAST 2.0 algorithms, 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 analysis is publicly available from the website of the National Center for Biotechnology Information (NCBI).The algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match when compared to words of the same length in a database sequence, or that satisfy a positive threshold score T. T is called the neighboring word score threshold (Altschul et al., ibid.). These initial neighboring word hits act as seeds to initiate a search for longer HSPs containing them. Then, word hits are extended in both directions along each sequence until the cumulative alignment score can increase. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction stops when: the cumulative alignment score decreases by an amount X from its maximum value; the cumulative score drops to zero or below due to the accumulation of one or more negative 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) defaults to a word length (W) of 11, an expected value (E) of 10, M=5, and N=-4, and compares two strands. For amino acid sequences, the BLASTP program defaults to a word length (W) of 3, an expected value (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:10915). Exemplary determination of sequence alignment and sequence identity percentage can be performed using the BESTFIT or GAP programs in the GCG Wisconsin software package (Accelrys, Madison Wis.) with the provided default parameters.

[0116] A “reference sequence” is a defined sequence used as a benchmark for sequence comparison. A reference sequence can be a subset of a larger sequence, such as a segment of a full-length nucleic acid or polypeptide sequence. The length of a reference sequence is typically at least 20 nucleotide or amino acid residues, but can also be the full length of a nucleic acid or polypeptide. Since two polynucleotides or polypeptides can each (1) be contained within a similar sequence (i.e., a portion of the complete sequence) and (2) also be contained within a different sequence, sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing the sequences of the two polynucleotides or polypeptides within a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” is a conceptual segment of at least about 20 consecutive nucleotide positions or amino acid residues, in which the sequence can be compared to a reference sequence of at least 20 consecutive nucleotides or amino acids, and in which the portion of the sequence in the comparison window may contain 20% or less of additions or deletions (or vacancies) compared to the reference sequence (which does not contain additions or deletions) to achieve optimal alignment of the two sequences.

[0117] "Substantially identical" or "substantially the same" means that, within a comparison window of at least 20 nucleotide or amino acid residue positions, typically at least 30-50 positions, the polynucleotide or polypeptide sequence has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity compared to a reference sequence, wherein the percentage of sequence identity is calculated by comparing the reference sequence with a sequence containing deletions or additions that total 20% or less of the reference sequence within the comparison window.

[0118] When used in the context of numbering a given amino acid or polynucleotide sequence, "corresponding to," "reference," or "relative to" means that the residue number of the reference sequence is specified when the given amino acid or polynucleotide sequence is compared to a reference sequence. In other words, the residue number or position of a given polymer is specified relative to the reference sequence, not by the actual numerical position of the residues within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence (such as the amino acid sequence of an engineered imine reductase) can be aligned to a reference sequence by introducing vacancies to optimize residue matching between the two sequences. In these cases, the numbering of residues in the given amino acid or polynucleotide sequence is relative to the reference sequence with which it is aligned, despite the presence of vacancies.

[0119] As used herein, “isolated” in relation to a molecule means that the molecule (e.g., cannabinoids, polynucleotides, polypeptides) is substantially isolated from other compounds that naturally accompany it (e.g., proteins, lipids, and polynucleotides). The term covers nucleic acids that have been removed or purified from their natural environment or expression system (e.g., host cells or in vitro synthesis).

[0120] "Substantially pure" means a composition in which the desired molecule is the predominant species (i.e., more abundant than any other single macromolecular species in the composition, based on mole or weight), and is typically substantially purified when the target species accounts for at least about 50% of the present macromolecular species in mole or weight %.

[0121] "Treatment" refers to a clinical intervention that attempts to alter the natural course of a disease in an individual receiving treatment, and may be used for prevention or in the clinicopathological process. Desired treatment outcomes may include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of progression, improving or slowing the disease state, and alleviating or improving prognosis. For example, treatment may include administering to a subject a therapeutically effective amount of a pharmaceutical preparation containing an IL-2 mutant peptide to delay or slow the development of a disease or symptom mediated by IL-2R, or in which IL-2R may play a role in the pathogenesis and / or progression.

[0122] A "pharmaceutical formulation" is a preparation whose form allows the bioactivity of the active ingredient to be effectively exerted and does not contain any additional components that are toxic to a subject administering the formulation. A pharmaceutical formulation may contain one or more active agents. For example, a pharmaceutical formulation may contain a mutant IL-2 peptide as the sole active agent of the formulation, or it may contain a mutant IL-2 peptide and one or more other active agents (such as immune checkpoint inhibitors).

[0123] "Pharmaceutically acceptable carriers" refer to components in a pharmaceutical preparation other than the active ingredient that are non-toxic to the subject administering the drug. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0124] "Therapeutic effective amount" refers to the amount of an active ingredient or agent (e.g., a pharmaceutical preparation) that achieves the desired therapeutic or preventative outcome (e.g., treatment or prevention of a subject's disease, condition, or symptom). In the case of IL-2-mediated diseases or conditions, a therapeutically effective amount of a therapeutic agent is the amount that reduces, prevents, inhibits, and / or alleviates one or more of the symptoms associated with the disease, condition, or symptom to a certain extent. For cancer therapies, in vivo efficacy can be measured, for example, by assessing the following parameters: growth of the primary tumor, occurrence and / or growth of secondary tumors, occurrence and / or number of metastases, duration, severity, and / or recurrence of symptoms, response rate (RR), duration of response, and / or quality of life.

[0125] "Individual" or "subject" refers to mammals, including but not limited to domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0126] IL-2 receptor IL-2 signaling is mediated by binding to three distinct IL-2 receptor protein subunits: IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132). Immune cells express either dimeric or trimeric IL-2 receptors. The dimeric receptor is expressed on cytotoxic CD8+ T cells and natural killer (NK) cells, while the trimeric receptor is primarily expressed on activated lymphocytes and CD4+CD25+ FoxP3+ inhibitory regulatory T cells (Tregs) (see Byman et al., J. Nat. Rev. Immunol. 12, 180-190 (2012)). Resting effector T cells and NK cells lack CD25 on their cell surface and are therefore relatively insensitive to IL-2. However, Treg cells express high levels of CD25, and thus Treg proliferation is stimulated by IL-2.

[0127] The trimeric receptor IL-2Rαβγ, formed by the combination of IL-2Ra, IL-2Rβ, and IL-2Rγ, is a high-affinity receptor for IL-2. D It is approximately 10 pM. The dimer receptor (IL-2Rβγ) is a medium-affinity receptor, K DIt is approximately 1 nM. The monomeric IL-2Rα receptor is a low-affinity IL-2 receptor. The IL-2 signaling activities mediated by the receptor and its complex also differ significantly. Generally, IL-2Rβ and IL-2Rγ have been found to be crucial for IL-2 signaling, while IL-2Rα (CD25) is not essential for signaling, but the presence of IL-2Rα enables high-affinity binding of IL-2 to the receptor complex (see, for example, Krieg et al., Proc Natl Acad Sci 107, 11906-11 (2010)).

[0128] The amino acid sequence of the IL-2Rα subunit can be found in UniProt P01589, and herein it is shown as SEQ ID NO: 2, having the encoding polynucleotide sequence as shown in SEQ ID NO: 1. The amino acid sequence of the IL-2Rβ subunit can be found in UniProt P14784, and herein it is shown as SEQ ID NO: 4, having the encoding polynucleotide sequence as shown in SEQ ID NO: 3. The amino acid sequence of the IL-2Rγ subunit can be found in UniProt 31785, and herein it is shown as SEQ ID NO: 6, having the encoding polynucleotide sequence as shown in SEQ ID NO: 5. Table 2 below provides an overview of the sequences and sequence identifiers of the various IL-2R peptides of this disclosure. Sequences are also included in the accompanying sequence listing.

[0129] Table 2: IL-2R protein subunits

[0130] mutant IL-2 peptide This disclosure provides mutant IL2 fusion proteins comprising polypeptide subunits, wherein the polypeptide subunits are mutant IL-2 polypeptides (or IL-2 mutant proteins). Typically, the mutant IL2 subunit polypeptides used in the fusion proteins of this disclosure contain mutations that alter the glycosylation of the polypeptide and affect a variety of physicochemical and functional properties of IL-2, including recombinant expression titers, solubility, and binding affinity to the monomeric, dimer, and trimeric forms of the IL-2R chains IL-2Rα, IL-2Rβ, and IL-2Rγ. The altered binding properties of the mutant IL-2 polypeptides to different IL-2R chains allow for inhibition, reduction, and / or complete blockade of the function of the IL-2R receptor, particularly its function as a cell surface receptor in mediating immune regulation. Therefore, any of the mutant IL2 fusion protein compositions or formulations of this disclosure are contemplated for use as therapeutic agents for diseases mediated by the function of IL-2R or its homologous ligand IL-2, such as cancer and autoimmune diseases. Furthermore, as described elsewhere herein, the mutant IL-2 fusion protein of this disclosure is anticipated to be used as a therapeutic agent in combination with other therapeutic agents, such as antibodies that target immune checkpoint molecules.

[0131] Naturally occurring human IL-2 is a 153-amino acid polypeptide sequence (Uniprot: P60568; disclosed herein as SEQ ID NO: 8, having a coding polynucleotide sequence as shown in SEQ ID NO: 7), which includes a 20-amino acid N-terminal signal peptide: MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 8) The structure of the IL-2 polypeptide comprises four antiparallel and amphiphilic α-helices, which form a quaternary structure crucial to its function (see, for example, Smith, Science 240, 1169-76 (1988); Bazan, Science 257, 410-413 (1992)). The 153-amino acid precursor IL-2 of SEQ ID NO: 8 was processed to remove the signal peptide, yielding the mature secreted IL-2 polypeptide of SEQ ID NO: 9, consisting of 133 amino acids, as shown below: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:9) The mature IL-2 peptide of SEQ ID NO: 9 has been engineered for human pharmaceutical use by removing a cysteine ​​residue at position C125, thereby reducing peptide aggregation. The C125S IL-2 mutant peptide (disclosed herein as SEQ ID NO: 10) is the active ingredient in adefolecin, an approved drug for the treatment of cancer in humans.

[0132] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO:10) The mutations in the IL-2 polypeptide subunits of the fusion proteins disclosed herein are mutations relative to the amino acid sequence of wild-type IL-2 or C125S IL-2. These mutations alter glycosylation, thereby affecting the functional properties of fusion proteins comprising these IL-2 polypeptides. These altered functional properties include increased expression titers, increased solubility, and / or altered binding affinity to the IL-2 receptor complex. The alterations in glycosylation and functional properties resulting from mutations in the IL-2 amino acid sequence relative to the parent IL-2 polypeptide (also known as amino acid substitutions or differences) result in IL-2 polypeptides with improved properties, making them suitable for use in pharmaceutical compositions for treating IL-2-mediated diseases such as cancer and autoimmune diseases.

[0133] The mutant IL-2 peptide used in the fusion protein disclosed herein is derived from the parental C125S IL-2 peptide of SEQ ID NO: 10. The mutant IL-2 peptide has amino acid substitutions that create novel N-glycosylation sites on the expressed mutant IL-2 peptide. The engineering of N-glycosylation motifs in proteins is well known in the art. Typically, a potential N-glycosylation site on a peptide is obtained by the three-amino acid sequence motifs asparagine-X-serine (NXS) and asparagine-X-threonine (NXT) (where X can be any amino acid except proline). Depending on the peptide sequence, N-glycosylation sites can be introduced into the peptide by engineering at one, two, or three positions in the amino acid sequence using amino acid substitutions.

[0134] In at least one embodiment of this disclosure, the fusion protein may comprise a mutant IL-2 polypeptide subunit having at least two mutations relative to SEQ ID NO: 10, wherein the group of mutations may 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 comprising one of these groups of mutations relative to SEQ ID NO: 10.

[0135] Table 3: Exemplary mutant IL2 peptides

[0136] The mutant IL-2 peptides of SEQ ID NO: 11, 12, 13, 14, 15, 16 and 17, as well as a series of other exemplary mutant IL-2 peptides that can be used in the fusion proteins of this disclosure, are described in U.S. Provisional Patent Application No. 63 / 385,610 (filed November 30, 2022), which is hereby incorporated by reference for all purposes. Table 4 (below) provides a summary of additional groups of amino acid differences of the mutant IL-2 peptide subunits of SEQ ID NO: 10 of the exemplary mutant IL-2 peptide disclosed in U.S. Provisional Patent Application No. 63 / 385,610 that can be used in the fusion proteins of this disclosure.

[0137] Table 4: Exemplary IL-2 peptide subunit mutations relative to SEQ ID NO: 10

[0138] As described elsewhere herein, the use of mutant IL-2 peptides in the fusion proteins of this disclosure can produce technical effects such as increased expression titers and solubility of the fusion protein during preparation in mammalian cell cultures, and altered binding affinity to trimer and dimer IL-2R complexes formed by the IL-2Rα subunit of SEQ ID NO: 2, the IL-2Rβ subunit of SEQ ID NO: 4, and the IL-2Rγ subunit of SEQ ID NO: 6. For example, engineered N-glycosylation mutations in recombinant genes encoding IL-2 peptides have been observed to produce mutant IL-2 peptides exhibiting altered binding affinity to different IL-2R receptor subunits IL-2Rα, IL-2Rβ, and IL-2Rγ in monomeric, dimer, and trimeric complex forms. Such mutant IL-2 peptides exhibiting reduced binding affinity to IL-2Rα, for example due to reduced or no stimulation of immunosuppressive CD25+ cells, are believed to provide improved therapeutic compounds for the treatment of cancer. In addition, mutant IL-2 peptides, which exhibit reduced binding affinity for IL-2Rβγ and little or no reduction in binding affinity for IL-2Rα, are believed to provide improved therapeutic compounds for the treatment of autoimmune diseases, for example, due to preferential stimulation of immunosuppressive CD25+ cells.

[0139] As described elsewhere herein, in at least one embodiment, the fusion protein of this disclosure comprises a mutant IL-2 polypeptide subunit (e.g., IL-2 mutants of SEQ ID NO: 11, 12, 13, 14, 15, 16, 17), which results in a reduced affinity of the fusion protein for IL-2Rα, while the loss of binding affinity for IL-2β, IL-2γ, or IL-2βγ dimer complex is minimal or nonexistent.

[0140] Specific antigen-binding subunit The first and second antigen-binding subunits (e.g., A1B and A2B) of the mutant IL2 fusion protein are able to bind with high affinity to target antigens expressed on the surface of immune cells. This affinity for its target antigen allows the fused IL2 subunits to bind to the IL-2 receptor, providing a combinatorial effect on targeted immune cells, such as tumor-reactive T cells expressing the target antigen.

[0141] Typically, for binding to PD-1, the antigen-binding subunit in the fusion protein should possess an equilibrium dissociation constant (K0). D <100nM, <10nM, <1nM, <0.1nM, <0.01nM, or <0.001nM (e.g., 10 -8 M or lower, 10-8 M to 10 -13 M, for example, 10 -9 M to 10 -13 M). The binding affinity of an antigen-binding subunit to its target antigen can be determined using any of a variety of assays and expressed in a variety of quantitative values. Specific binding affinity assays for determining the affinity of antigen-binding subunit peptides are disclosed in the embodiments herein. Additionally, antigen binding assays are known in the art and can be used herein, including but not limited to any direct or competitive binding assays using techniques such as: Western blotting, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), sandwich immunoassay, surface plasmon resonance-based assays (such as the BIAcore assay described in WO2005 / 012359), immunoprecipitation assay, fluorescence immunoassay, protein A immunoassay, flow cytometry, and fluorescence activated cell sorting (FACS) assays, etc. Therefore, in some embodiments, binding affinity is expressed as K. D The value reflects the intrinsic binding affinity (e.g., having a minimized affinity effect).

[0142] Antigen-binding subunits targeting PD-1 It is anticipated that the antigen-binding subunit (e.g., A1B or A2B) of the mutant IL2 fusion protein of this disclosure may comprise a polypeptide that specifically binds to the target antigen PD-1. Therefore, in at least one embodiment, the antigen-binding subunit may comprise an anti-PD-1 antibody. Anti-PD-1 antibodies that can be used as antigen-binding subunits in the fusion protein may 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, (only) heavy-chain antibodies (e.g., sdAb or VHH, IgNAR, nanobodies (or nanoAbs)), antigen-binding fragments (e.g., Fab′, F(ab′)2, Fab, Fv, rIgG, and scFv fragments), antibody fusions, and synthetic antibodies (or antibody mimics).

[0143] Typically, for binding to PD-1, the anti-PD-1 antibody used in the fusion protein should have an equilibrium dissociation constant (K0). D <100nM, <10nM, <1nM, <0.1nM, <0.01nM, or <0.001nM (e.g., 10 -8 M or lower, 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 M). Binding affinity can be determined by the equilibrium dissociation constant (K) of the hu-PD-1 ECD peptide. DThe binding affinity is measured using [the method described herein]. In some embodiments, the anti-PD-1 antibody used in the fusion protein as disclosed herein is capable of binding cyno-PD-1 with high affinity, and / or binding both hu-PD-1 and cyno-PD-1. In some embodiments, the binding affinity of the anti-PD-1 antibody of this disclosure to cyno-PD-1 is 1 × 10⁻⁶. -8 M or lower, 1×10 -9 M or lower, 1×10 -10 M or lower, or 1×10 -11 M or lower.

[0144] In at least one embodiment, the anti-PD1 antibody, which can be used as an antigen-binding subunit in a fusion protein, may have a single heavy chain variable domain (VH), the amino acid and encoding nucleotide sequence of which are defined according to various well-known immunoglobulin features (e.g., CDR, VH domain). Table 5 below provides an overview of the anti-PD-1 antibody sequences and their sequence identifiers of the present disclosure. The sequences are included in the accompanying sequence listing.

[0145] Table 5 Anti-PD-1 VHH antibody sequence

[0146] A number of additional exemplary anti-PD1 antibodies (including anti-PD1 VHH antibodies) that can be used in the fusion proteins of this disclosure are described in U.S. Provisional Patent Application No. 63 / 488,176 (filed March 3, 2023), which is hereby incorporated herein by reference for all purposes.

[0147] The anti-PD-1 antibody provided herein, which serves as an antigen-binding subunit in a mutant IL2 fusion protein, specifically targets and binds to PD-1 expressed on T cells without diminishing, inhibiting, or blocking T cell-mediated immune regulatory effects, including T cell activation in response to specific MHC antigen binding. In some embodiments, the anti-PD-1 antibody provided herein specifically targets and binds to PD-1 expressed on T cells and inhibits immune regulation and / or immune signaling pathways mediated by activated T cell binding. The effect of antibody binding on PD-1-mediated immune responses can be measured in vitro using known cell-based assays (including those described in the embodiments of this disclosure). Therefore, in some embodiments, the anti-PD-1 antibody of this disclosure is characterized by one or more of the following functional properties based on its ability to alter T cell activation and associated immune responses.

[0148] In at least one embodiment, the anti-PD-1 antibody, used as an antigen-binding subunit in the fusion protein of this disclosure, is capable of blocking binding to hu-PD-1 ECD, as measured by ELISA.50 The concentrations are 10 nM or lower, 7 nM or lower, 5 nM or lower, 2 nM or lower, 1 nM or lower, 0.5 nM or lower, or 0.25 nM or lower. In at least one embodiment, the anti-PD-1 antibody blocks the binding to hu-PD-1 ECD expressed on cells; its IC50 value is 10 nM or lower, 7 nM or lower, 5 nM or lower, 2 nM or lower, 1 nM or lower, 0.5 nM or lower, or 0.25 nM or lower. 50 The concentration is 2.5 nM or lower, 1 nM or lower, 0.5 nM or lower, or 0.25 nM or lower; optionally, 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 the binding to hu-PD-1 ECD expressed on human T cells, with an IC50 value of 2.5 nM or lower, 1 nM or lower, 0.5 nM or lower, or 0.25 nM or lower; optionally, the cell is a HEKBlue cell that stably expresses PD-1 ECD. 50 The concentrations are 5 nM or lower, 2.5 nM or lower, 1 nM or lower, 0.5 nM or lower, or 0.25 nM or lower; optionally, the cells are human CD8+. + T cells.

[0149] Antigen-binding subunit targeting CD8 It is anticipated that the antigen-binding subunit (e.g., A1B or A2B) of the mutant IL2 fusion protein of this disclosure may comprise a polypeptide that specifically binds to the target antigen CD8. Therefore, in at least one embodiment, the antigen-binding subunit may comprise an anti-CD8 antibody. Anti-CD8 antibodies that can be used as antigen-binding subunits in fusion proteins may 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, (only) heavy-chain antibodies (e.g., sdAb or VHH, IgNAR, nanobodies (or nanoAbs)), antigen-binding fragments (e.g., Fab′, F(ab′)2, Fab, Fv, rIgG, and scFv fragments), antibody fusions, and synthetic antibodies (or antibody mimics).

[0150] Typically, for binding to CD8, the anti-CD8 antibody used in the fusion protein should have an equilibrium dissociation constant (K0). D <100nM, <10nM, <1nM, <0.1nM, <0.01nM, or <0.001nM (e.g., 10 -8 M or lower, 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 M). Binding affinity can be determined by the equilibrium dissociation constant (K) of the hu-CD8 ECD peptide. DThe binding affinity is measured using [the method described herein]. In some embodiments, the anti-CD8 antibody used in the fusion protein as disclosed herein is capable of binding cyno-CD8 with high affinity, and / or binding both hu-CD8 and cyno-CD8. In some embodiments, the binding affinity of the anti-CD8 antibody of this disclosure for cyno-CD8 is 1 × 10⁻⁶. -8 M or lower, 1×10 -9 M or lower, 1×10 -10 M or lower, or 1×10 -11 M or lower.

[0151] In at least one embodiment, the anti-CD8 antibody, which can be used as an antigen-binding subunit in a fusion protein, may have a single heavy chain variable domain (VH), the amino acid and encoding nucleotide sequence of which are defined according to various well-known immunoglobulin features (e.g., CDR, VH domain). Table 6 below provides an overview of the anti-CD8 antibody sequences and their sequence identifiers of the present disclosure. The sequences are included in the accompanying sequence listing.

[0152] Table 6 Anti-CD8 VHH antibody sequence

[0153] A number of additional exemplary anti-CD8 antibodies (including anti-CD8 VHH antibodies) that can be used in the fusion proteins of this disclosure are described in U.S. Provisional Patent Application No. 63 / 477,529 (filed December 28, 2022), which is hereby incorporated herein by reference for all purposes.

[0154] The anti-CD8 antibody provided herein, used as an antigen-binding subunit in a mutant IL2 fusion protein, specifically targets and binds to CD8 expressed on T cells without reducing, inhibiting, or blocking T cell-mediated immune regulatory effects, including T cell activation in response to specific MHC antigen binding. In some embodiments, the anti-CD8 antibody provided herein specifically targets and binds to CD8 expressed on T cells and inhibits immune regulation and / or immune signaling pathways mediated by activated T cell binding. The effect of antibody binding on CD8-mediated immune responses can be measured in vitro using known cell-based assays (including those described in the embodiments of this disclosure). Therefore, in some embodiments, the anti-CD8 antibody of this disclosure is characterized by one or more of the following functional properties based on its ability to alter T cell activation and associated immune responses.

[0155] In at least one embodiment, the anti-CD8 antibody, which serves as the antigen-binding subunit in the fusion protein of this disclosure, is capable of blocking binding to hu-CD8 ECD, as measured by ELISA. 50 The concentrations are 10 nM or lower, 7 nM or lower, 5 nM or lower, 2 nM or lower, 1 nM or lower, 0.5 nM or lower, or 0.25 nM or lower. In at least one embodiment, the anti-CD8 antibody blocks the binding to hu-CD8 ECD expressed on cells, with an IC50 value of 10 nM or lower, 7 nM or lower, 5 nM or lower, 2 nM or lower, 1 nM or lower, 0.5 nM or lower, or 0.25 nM or lower. 50 The concentration is 2.5 nM or lower, 1 nM or lower, 0.5 nM or lower, or 0.25 nM or lower; optionally, 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 the binding to hu-CD8 ECD expressed on human T cells, with an IC50 value of 2.5 nM or lower, 1 nM or lower, 0.5 nM or lower, or 0.25 nM or lower; optionally, the cell is a HEKBlue cell that stably expresses CD8 ECD. 50 The concentrations are 5 nM or lower, 2.5 nM or lower, 1 nM or lower, 0.5 nM or lower, or 0.25 nM or lower; optionally, the cells are human CD8+. + T cells.

[0156] mutant IL-2 fusion with antigen-binding polypeptide subunit Figures 1A-1J A schematic diagram illustrating the fusion single-chain polypeptide structure of an exemplary mutant IL2 fusion protein of the present disclosure is shown. Figure 1A , Figure 1B , Figure 1C , Figure 1D and Figure 1E Exemplary trispecific fusion proteins are depicted. These fusion proteins can bind IL-2R, as well as a first and second antigen expressed on the surface of immune cells. These fusion proteins comprise a single polypeptide chain containing a mutant IL2 subunit, a first antigen-binding subunit A1B with different antigen-binding specificities and a second antigen-binding subunit A2B (e.g., AB1 binds PD1 and AB2 binds CD8), and a polypeptide subunit HLE with an extended half-life (e.g., the Fc region of IgG1). Additionally, polypeptide linkers (L1, L2, and L3) connect the C-terminus to the N-terminus of adjacent polypeptide subunits.

[0157] Figure 1F , Figure 1G , Figure 1H , Figure 1I and Figure 1J An exemplary bispecific fusion protein comprising a single polypeptide chain containing a mutant IL2 polypeptide subunit that binds IL-2R and a single antigen-binding subunit (e.g., A1B or A2B). The bispecific fusion protein also includes a polypeptide subunit HLE with an extended half-life and a polypeptide linker connecting the C-terminus to the N-terminus of an adjacent polypeptide subunit.

[0158] This disclosure also provides Figures 1A-1J The dimer complex of the mutant IL2 fusion protein described in the article. Figures 2A-2I Schematic diagrams depict exemplary mutant IL2 fusion proteins having homodimeric or heterodimeric structures comprising a pair of single-stranded mutant IL2 fusion polypeptides. These exemplary structures include bispecific or trispecific single polypeptide chains (such as...) Figures 1A-1J Homodimers or heterodimers (those depicted herein). In at least one embodiment, it is contemplated that the formation of a dimeric complex can be facilitated by including an HLE subunit containing the amino acid sequence features of a "palm" (e.g., Fc-Kb) and a "mortar" (e.g., Fc-Ho). Such palm and mortar features are well known and commonly used in the formation of bispecific antibodies. As described elsewhere herein, these same palm and mortar features can be used to form dimeric complexes of mutant IL2 protein fusions of this disclosure, such as... Figures 2A-2I Those shown in the image.

[0159] Table 7 (below) provides a series of exemplary peptides that can be used for the mutant IL2 fusion protein of this disclosure.

[0160] Table 7: Exemplary fusion protein peptides

[0161] The exemplary fusion protein peptides in Table 7 are single-chain peptides that can be used to prepare the exemplary mutant IL2 fusion protein of this disclosure, such as... Figures 1A-1J The single-chain structures shown, and Figures 2A-2I The dimer complex shown is illustrated in Table 7. Schematic fusion structures of the peptides from Table 7 are summarized in Table 8 (below).

[0162] Table 8: Exemplary fusion protein structures

[0163] The component subunits of the fusion protein in Table 8 are summarized in Table 9 (below).

[0164] Table 9: Exemplary fusion protein subunits

[0165] HLE subunit polypeptide As illustrated by the exemplary structure of the mutant IL-2 fusion protein of this disclosure, the mutant IL-2 fusion protein includes an HLE (extended half-life) subunit, which may contain an Fc polypeptide, such as the wild-type monomeric human IgG1 Fc lower hinge region polypeptide of SEQ ID NO: 72 shown below: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 72).

[0166] In at least one embodiment, the HLE subunit used in the mutant IL-2 fusion protein comprises a variant of the wild-type human IgG1 Fc polypeptide of SEQ ID NO:72, which includes the “KK” variant of SEQ ID NO:73: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSPGK(SEQ ID NO: 73) In at least one embodiment, the HLE subunit used in the mutant IL-2 fusion comprises a variant of the wild-type human IgG1 Fc polypeptide of SEQ ID NO: 72, which includes the “DSDL” variant of SEQ ID NO: 74, both as follows: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLSSDLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSPGK(SEQ ID NO: 74).

[0167] In addition to the three human IgG1 Fc lower hinge region peptides described above, the mutant IL-2 fusion protein of this disclosure is expected to be prepared using HLE subunits containing other IgG1 Fc region fragments and variants known to provide improved properties when conjugated with peptides such as cytokines for therapeutic purposes. For example, Fc peptide variants with the effector function removed, such as Fc regions with amino acid substitutions L234A / L235A (“LALA”) (Woodle, E. Steve et al., Transplantation, 68 (5): 608-616 (1999)), may be used. Other effectless Fc region mutations are well known in the art, such as L234A / L235A / P329G (“LALAPG”) (see, for example, Schlothauer, T. et al., “Novelhuman IgG1 and IgG4 Fc-engineered antibodies with completely abolished immuneeffector functions”, Protein Eng. Des. Sel., 29 (10): 457–466 (2016)), or, when the Fc is isotype IgG2 or IgG4, amino acid substitutions of S228P and / or L235E.

[0168] Additionally, the HLE subunits that can be used in mutant IL-2 fusion proteins may include IgG1 hinge and Fc region amino acid sequences that provide "pestle" or "mortar" functionality, thereby allowing fusions containing both "pestle" and "mortar" HLE subunits to be used to form a dimer complex of the mutant IL-2 fusion protein of this disclosure. Exemplary IgG1 hinge and Fc region amino acid sequences that can be used as HLE subunits in the fusion proteins of this disclosure are provided in Table 10 (below).

[0169] Table 10: Exemplary IgG1 hinge and Fc region sequences

[0170] connector In at least one embodiment, the mutant IL-2 fusion protein of this disclosure is conjugated between subunit polypeptides via linkers (e.g., L1, L2, L3). Any of a variety of synthetic chain molecules known in the art that can be used as linkers between biomolecules can be used to fuse the subunits of the mutant IL-2 fusion protein. In at least one embodiment, a polypeptide linker may be used. Such a polypeptide linker comprises an amino acid chain, each end of which is covalently attached to one of two different polypeptides, thereby performing the function of conjugation or fusion with them. Typically, such a polypeptide linker comprises a chain of 5 to 30 amino acids.

[0171] A wide variety of peptide linkers are known in the art and can be used in the mutant IL-2 peptide fusions disclosed herein. Peptides known in the art may 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 peptide linkers that can be used in IL-2 fusions of this disclosure include, but are not limited to, the linkers provided in Table 11 (below).

[0172] Table 11: Exemplary Connectors

[0173] Recombination Method and Composition The fusion protein of this disclosure can be produced using recombinant methods and materials well known in the field of peptide and protein production. In some embodiments, this disclosure provides an isolated nucleic acid encoding a mutant IL-2 peptide. The nucleic acid may encode an amino acid sequence containing only the IL-2 peptide or a fusion of itself with another peptide, such as a monomeric Fc peptide. In some embodiments, one or more vectors (e.g., expression vectors) are provided containing a nucleic acid sequence encoding the mutant IL-2 peptide of this disclosure. In some embodiments, a host cell is provided containing a nucleic acid sequence encoding the mutant IL-2 peptide of this disclosure. In one embodiment, the host cell has been transformed with a vector containing nucleic acid encoding an amino acid sequence containing the mutant IL-2 peptide. In some embodiments, the host cell used is a eukaryotic cell, such as Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NSO, Sp20).

[0174] In at least one embodiment, a method for preparing a mutant IL-2 polypeptide is provided, wherein the method includes culturing a host cell containing a nucleic acid encoding the polypeptide, as described above, under conditions suitable for polypeptide expression, and optionally recovering the polypeptide from the host cell (or host cell culture medium). In short, the recombinant production of the mutant IL-2 polypeptide is carried out by the following steps: synthesizing or isolating a nucleic acid encoding the mutant IL-2 polypeptide (e.g., as described herein), and inserting the nucleic acid into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids are easily isolated and sequenced using conventional procedures well known in the art (e.g., by using oligonucleotide probes capable of specifically binding to a gene encoding IL-2). Suitable host cells and culture methods for vectors encoding IL-2 polypeptides are well known in the art and include prokaryotic or eukaryotic cells. Typically, after expression, the mutant IL-2 polypeptide can be isolated from the cell paste in the soluble fraction and further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for the vector.

[0175] Examples of suitable mammalian host cell lines that can be used to produce the mutant IL-2 fusion protein of this disclosure include: Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (see, for example, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); myeloma cell lines such as Y0, NSO, and Sp2 / 0; monkey kidney CV1 cell lines transformed with SV40 (COS-7); human embryonic kidney cell lines (293 or 293 cells, as described, for example, in Graham et al., J. Gen Virol. 36:59 (1977)); juvenile hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, for example, Mather, Biol. Reprod. 23:243-251). (described in 1980); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor cells (MMT 060562); TR1 cells (see, for example, Mather et al., Annals NY. Acad. Sci. 383:44-68 (1982) and US 6,235,498); Medical Research Council 5 (MRC 5) cells (such as cells available from ATCC, also known as CCL-171); and foreskin 4 (FS-4) cells (see, for example, Vilcek et al. Ann. NY Acad. Sci. 284:703-710 (1977); Gardner and Vilcek. J. Gen. Virol. 44:161-168 (1979); and Pang et al. Proc. Natl. Acad. Sci. USA 77:5341-5345 (1980)).

[0176] Pharmaceutical compositions and formulations of mutant IL-2 fusion protein This disclosure also provides pharmaceutical compositions and formulations comprising a mutant IL-2 peptide. In some embodiments, this disclosure provides a pharmaceutical formulation comprising a mutant IL-2 peptide as described herein and a pharmaceutically acceptable carrier. In some embodiments, the mutant IL-2 peptide is the sole active agent in the pharmaceutical composition. Such pharmaceutical formulations can be prepared by mixing a mutant IL-2 peptide of desired purity with one or more pharmaceutically acceptable carriers. Typically, such mutant IL-2 peptide formulations are prepared as aqueous solutions or lyophilized formulations.

[0177] Pharmaceutically acceptable carriers are generally non-toxic to recipients at the doses and concentrations used. A variety of such pharmaceutically acceptable carriers are well known in the art (see, for example, Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). Exemplary pharmaceutically acceptable carriers that may be used in formulations of this disclosure may include, but are not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride, hexamethyl ammonium chloride, benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) peptides; proteins 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 dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG).

[0178] Pharmaceutically acceptable carriers that can be used in formulations disclosed herein may also include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP) (see, for example, U.S. Patent Publications 2005 / 0260186 and 2006 / 0104968), such as human soluble PH-20 hyaluronidase glycoprotein (e.g., rHuPH20 or HYLENEX). ® , Baxter International, Inc.).

[0179] It is also conceivable that the formulations disclosed herein may contain active ingredients other than the mutant IL-2 peptide, such as those required for a specific indication in which the subject administering the formulation is receiving treatment. Preferably, any additional active ingredient has an activity complementary to the IL-2 activity, and such activity does not produce an adverse effect on each other.

[0180] As disclosed elsewhere herein (including examples), the mutant IL-2 peptide of this disclosure has been demonstrated to be usable as a fusion with an Fc peptide to provide improved therapeutic effects in the treatment of autoimmune diseases and / or cancer.

[0181] As described elsewhere herein, in some embodiments, this disclosure provides a pharmaceutical composition or formulation for use in a treatment method, wherein the composition comprises a mutant IL-2 polypeptide fused to an antigen-binding subunit polypeptide and an extended-half-life subunit polypeptide. In some embodiments, such a pharmaceutical composition or formulation may comprise a mutant IL-2 protein fusion containing a linker polypeptide, such as a polypeptide linker with the amino acid sequence SEQ ID NO: 82-89, between the subunit polypeptides. Examples demonstrating such mutant IL-2 protein fusions and their use in pharmaceutical compositions for the treatment of cancer or autoimmune diseases are described elsewhere herein.

[0182] In some embodiments, the pharmaceutical composition may comprise a mutant IL-2 protein fusion of the present disclosure and additional active agents (such as immune checkpoint inhibitors) for the treatment of cancer. Checkpoint inhibitors that can be used in such embodiments include, but are not limited to, antibodies specific to an antigen, such as 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.

[0183] Typically, the active ingredient in a pharmaceutical composition can be encapsulated in microcapsules, for example, prepared by coagulation techniques or by interfacial polymerization, such as in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in crude emulsions using hydroxymethyl cellulose or gelatin microcapsules and polymethyl methacrylate microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (1980).

[0184] In some embodiments, the formulation of the mutant IL-2 protein fusion may be a sustained-release formulation of a peptide and / or other active ingredient. Suitable examples of sustained-release formulations include a semi-permeable matrix containing a solid hydrophobic polymer of the mutant IL-2 protein fusion, the matrix being in the form of a molded article, such as a film or microcapsule.

[0185] Typically, the formulations of this disclosure intended for administration to a subject are sterile. Sterile formulations can be readily prepared using well-known techniques, such as filtration through a sterile filter membrane.

[0186] Uses and treatments Any composition or formulation comprising the mutant IL-2 fusion protein of this disclosure is intended for use in any method or application utilizing the ability of a peptide to specifically bind to an IL-2 receptor protein, such as therapeutic methods. The binding of IL-2 to IL-2 receptor proteins expressed on different cells mediates different immune responses. IL-2 binding can stimulate immune responses such as T cell proliferation and differentiation, cytotoxic T lymphocyte (CTL) production, B cell proliferation and differentiation, immunoglobulin synthesis and production, and NK cell proliferation and activation. IL-2 peptides such as C125S IL-2 (Proleukin) have been approved as immunotherapeutic agents for the treatment of cancer and chronic viral infections. However, IL-2 peptides may also promote the activation and proliferation of immunosuppressive CD4+CD25+Treg cells, leading to immunosuppression. Therefore, there is a range of diseases, conditions, and symptoms that could potentially be treated by altering the immunomodulatory and / or immune signaling activity of IL-2 binding to IL-2 receptor proteins, particularly the effect of IL-2 on tumor progression. Diseases, conditions, and symptoms include, but are not limited to, cancers, including but not limited to, colon cancer, pancreatic cancer, ovarian cancer, liver cancer, kidney cancer, breast cancer, lung cancer, stomach cancer, head and neck cancer, or oral cancer. Any composition or formulation comprising the mutant IL-2 fusion protein of this disclosure is intended for use in the treatment of any of the cancers listed above. Therefore, in at least one embodiment, this disclosure provides a method of treating a subject with cancer, wherein the method comprises administering to a subject in need a therapeutically effective amount of the mutant IL-2 fusion protein of this disclosure, or administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising the mutant IL-2 fusion protein of this disclosure and a pharmaceutically acceptable carrier.

[0187] As disclosed herein, including in the embodiments described below, the mutant IL-2 fusion protein of this disclosure has the ability to differentially and specifically bind to IL-2 receptor proteins, thereby differentially altering immune signaling pathways mediated by the binding of IL-2 to IL-2 receptor proteins expressed on different cells. Therefore, in some embodiments, this disclosure provides a method of treating a subject for an IL-2-mediated disease or condition, the method comprising administering to the subject a therapeutically effective amount of the mutant IL-2 fusion protein of this disclosure, or administering to a subject in need a therapeutically effective amount of a pharmaceutical composition comprising the mutant IL-2 fusion protein of this disclosure and a pharmaceutically acceptable carrier. Similarly, in some embodiments, this disclosure provides a method of treating a subject for a disease mediated by the binding of IL-2 to IL-2 receptor proteins expressed on cells, the method comprising administering to the subject a therapeutically effective amount of the mutant IL-2 fusion protein of this disclosure, or administering to a subject in need a therapeutically effective amount of a pharmaceutical composition comprising the mutant IL-2 fusion protein of this disclosure and a pharmaceutically acceptable carrier.

[0188] The administration of a mutant IL-2 fusion protein, composition, or pharmaceutical formulation, according to the treatment method, provides an antibody-induced therapeutic effect that prevents the progression of IL-2-mediated disease in a subject and / or treats the progression of that disease. In some embodiments, the treatment method may also include the administration of one or more additional therapeutic agents or therapies known to those skilled in the art to prevent and / or treat IL-2-mediated disease or symptoms. Such methods involving the administration of one or more additional agents may cover combined administration (where two or more therapeutic agents are contained in the same formulation or separate formulations) and separate administration, in the latter case, where the administration of the mutant IL-2 fusion protein composition or formulation may occur before, simultaneously with, and / or after the administration of the additional therapeutic agent.

[0189] In some embodiments of the treatment methods disclosed herein, the mutant IL-2 fusion protein or a pharmaceutical preparation containing the mutant IL-2 fusion protein is administered to the subject via any administration modality, either through systemic delivery or delivery to the desired target tissue. Systemic administration generally refers to any modality that applies the antibody to a site on the subject rather than directly to the desired target site, tissue, or organ, allowing the antibody or its preparation to enter the subject's circulatory system and thus undergo metabolism and other similar processes. Therefore, administration modalities that can be used in the treatment methods of this disclosure may include, but are not limited to, injection, infusion, drip, and inhalation. Injection administration may include intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, intracerebrospinal fluid system, and intrasternal injection and infusion.

[0190] In at least one embodiment, a pharmaceutical formulation of the mutant IL-2 fusion protein is formulated such that IL-2 is protected from inactivation in the intestine. Therefore, treatment methods may include oral administration of the formulation.

[0191] In at least one embodiment, the use of a composition or formulation comprising the mutant IL-2 fusion protein of this disclosure as a medicament is also provided. Additionally, in some embodiments, the disclosure also provides the use of a composition or formulation comprising the mutant IL-2 fusion protein in the manufacture or preparation of a medicament, particularly a medicament for treating IL-2-mediated diseases. In another embodiment, in a method of using the medicament to treat a disease, the method includes administering an effective amount of the medicament to an individual suffering from the disease. In some embodiments, the medicament further comprises an effective amount of at least one other therapeutic agent or therapy.

[0192] As disclosed elsewhere herein, other therapeutic agents or therapies are also contemplated for use in such pharmaceuticals in conjunction with the mutant IL-2 fusion protein of this disclosure. Generally, the mutant IL-2 fusion protein of this disclosure is intended to be used in conjunction with any therapeutic agent or therapy (such as a therapeutic antibody) that specifically targets cell surface receptors on immune cells, tumor cells, or myeloid cells. In at least one embodiment, the additional therapeutic agent may include, but is not limited to, therapeutic antibodies that specifically bind to immune checkpoint molecules such as PD1, PD-L1, LAG3, CTLA-4, A2AR, TIM-3, BTLA, CD276, CD328, VTCN1, IDO, KIR, NOX2, VISTA, OX40, CD27, CD28, CD40, CD122, CD137, GITR, and ICOS.

[0193] In another embodiment, the drug is used to treat a subject with an IL-2-mediated disease, such as cancer, including administering an effective amount of the drug to the subject to treat, inhibit, or prevent the IL-2-mediated disease. The appropriate dose of the mutant IL-2 fusion protein contained in the compositions and formulations disclosed herein (when used alone or in combination with one or more other additional therapeutic agents) will depend on factors such as: the specific disease or symptom being treated, the severity and duration of the disease, whether the dose is administered for prophylactic or therapeutic purposes, previous therapies administered to the patient, the patient's clinical history and response to the mutant IL-2 fusion protein composition, and the judgment of the attending physician. It is contemplated that the mutant IL-2 fusion protein contained in the compositions and formulations described herein can be appropriately administered to a patient in a single dose or in a series of treatments. Various dosing regimens are envisioned herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulse infusion.

[0194] Depending on the type and severity of the disease, a mutant IL-2 fusion protein of about 1 µg / kg to 30 mg / kg in the formulation disclosed herein is the initial candidate dose for administration to human subjects, whether, for example, by single or multiple administrations alone, or by continuous infusion. Typically, the antibody administration dose will be in the range of about 0.05 mg / kg to about 10 mg / kg. In some embodiments, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the patient.

[0195] Depending on the subject's condition, dosing may be maintained for several days or longer; for example, dosing may continue until the IL-2-mediated disease is adequately treated, as determined by methods known in the art. In some embodiments, an initial higher loading dose may be administered, followed by one or more lower doses. However, other dosing regimens may be useful. The progression of the therapeutic effect of the dosing administration can be monitored using conventional techniques and assays. Thus, in some embodiments of the methods disclosed herein, administration of the mutant IL-2 fusion protein comprises a daily dose of about 0.05 mg / kg to about 50 mg / kg, at least about 0.5 mg / kg to about 30 mg / kg, or at least about 1 mg / kg to about 25 mg / kg. In some embodiments, the dose of the mutant IL-2 fusion protein comprises a daily dose of at least about 0.1 mg / kg, at least about 1 mg / kg, at least about 5 mg / kg, at least about 10 mg / kg, at least about 20 mg / kg, at least about 30 mg / kg, at least about 40 mg / kg, or at least about 50 mg / kg.

[0196] Example Various features and embodiments of this disclosure are illustrated in the following representative examples, which are intended to be illustrative and not restrictive. Those skilled in the art will readily recognize that the specific embodiments are for illustrative purposes only, as more fully described in the appended claims. Each embodiment and feature described in this application should be understood to be interchangeable and combined with each embodiment contained therein.

[0197] Example 1: Cloning, expression, and purification of mutant IL-2 fusions containing anti-PD1 VHH and / or anti-CD8 VHH antibodies This embodiment illustrates the design, cloning, expression, and purification of exemplary mutant IL2 fusion proteins of this disclosure.

[0198] A. Design and cloning of fusion protein constructs 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, are designed to have... Figures 1A-1J The schematic depiction shows the general form of the polypeptide chain structure. This protein fuses VHH to the hinge and N-terminus of the Fc region of human IgG1, including the human constant heavy chain 2 (CH2) and constant heavy chain 3 (CH3) domains of SEQ ID NO: 57 (Uniprot ID: P01857). Polynucleotides encoding the structures of these bispecific and trispecific fusion proteins were cloned into the pcDNA3.1(+) vector using standard molecular biology techniques. Figures 2A-2I The heterodimeric form of the exemplary fusion protein, schematically depicted in the diagram, is prepared using a single-chain fusion polypeptide. Table 12 below provides schematic structures and polypeptide sequences used in the exemplary IL2 fusion protein prepared in this embodiment.

[0199] B. Expression of the fusion protein in CHO cells The cloned mutant IL2 fusion protein construct was expressed in Chinese hamster ovary (CHO) cells using the ExpiCHO expression system (Thermo Fisher Scientific). ExpiCHO cells (Thermo Fisher Scientific) were maintained in 60 mL of ExpiCHO expression medium in Erlenmeyer flasks at 37°C, 5% CO2, and 130 rpm. Transfection was performed according to the instructions provided with the ExpiCHO™ Expression System Kit (ThermoFisher – Catalog No. A29133). In short, when the cell count reached 6 × 10⁶ cells / year... 6 At a viable cell density (VCD) of 10 cells / mL and a doubling time of 18-20 hours, each 10 6 1 μg of plasmid DNA and ExpiFectamine CHO per cell were mixed by repeated inversion, diluted with cold OptiPRO serum-free medium, and finally complexed with the diluted plasmid DNA at room temperature. After 5 minutes, the mixture was added dropwise to the cell culture at room temperature. After transfection, cells were cultured in 25 mL or larger flasks at 130 RPM. 7–12 days after transfection, the culture medium was centrifuged at 25 °C and 500 × g for 5 minutes to pellet the cells, and the supernatant was further centrifuged at 4 °C and 4500 × g for 30 minutes. The clarified supernatant was filtered through a 0.45 μm filter and purified with protein A affinity column chromatography to obtain VHH antibody.

[0200] C. Purification of IL-2 fusion protein by protein A affinity chromatography CHO cells expressing various IL-2 fusion constructs were purified using affinity chromatography with protein-A resin (Atmosphere A3, JSR Life Sciences). Droplets of protein-A resin (column volume = 0.2 mL) were equilibrated with 5 column volumes (CV) of 1X PBS buffer, and 4 mL of supernatant was loaded directly. Unbound proteins were removed by washing with 10 CV of 1X PBS buffer, and the target protein was eluted with either 100 mM triethanolamine (pH 11.5) or 100 mM Na₂HPO₄-NaOH buffer (pH 11.5). The eluted fractions were collected, and the pH was adjusted to 9.5 using 1 M Tris buffer (pH 8.0).

[0201] The expression titer of each sample was quantified in HPLC using a POROS™ A column, as per the manufacturer's protocol (ThermoFisher, catalog number 2100100). Exemplary IL-2 fusion protein expression levels are shown in Table 12 (below).

[0202] Table 12

[0203] D. SEC-HPLC characterization of the IL-2 fusion protein Size exclusion-high performance liquid chromatography (SEC-HPLC) was performed on an Agilent 1100 HPLC system (Santa Clara, California), with 1X PBS used as the run buffer. The purified IL-2 fusion sample was injected into a pre-packed Superose 12-300 column (Cytiva) equilibrated with 1X PBS buffer. The flow rate was 0.65 mL / min, and the total run time was 40 minutes.

[0204] An exemplary SEC-HPLC spectrum showing the protein conformation of the purified fusion protein is shown in [image / image / etc.]. Figure 3A middle.

[0205] E. AEX ion exchange chromatography The IL-2 fusion purified from protein A was diluted with 20 mM Tris propane buffer (buffer A, pH 9.5) and then loaded onto an AEX column packed with Q-FF resin (column volume = 5 mL). After washing with 5 column volumes of buffer A, the target protein was eluted with 20 column volumes of buffer B (20 mM Tris propane + 1 M NaCl, pH 9.5) at a linear gradient from 0% to 60%.

[0206] Comparative SEC-HPLC chromatograms of the IL2 fusion protein P703 further purified by AEX chromatography are shown in [image / image / etc.]. Figure 3B(No AEX steps) and Figure 3C (In the AEX step)

[0207] Example 2: Generation of stable cell lines and IL-2 reporter gene assay for IL-2 fusion protein This example illustrates the study of IL-2 stimulatory activity of mutant IL2 fusion protein using the HEKBlue cell line and the IL-2-inducible STAT5 reporter gene assay.

[0208] Materials and methods A. Stable cell line generation 1) HEKBlue-PD1+ and HEKBlue CD122 / CD132+ cell lines: HEKBlue CD122 / CD132 cells (Invivogen catalog number hkb-il2bg) were infected with human-tagged ORF lentiviral particles of PD1 (PDCD1) (NM_005018) (Origene catalog number RC210364L4V) at an MOI of 20. Approximately 60% of the transduced cells expressed PD1 (captured by flow cytometry using a GFP tag). After two rounds of single-cell cloning (limited dilution at 0.3 cells / well), cell clones with a PD-1 positivity rate higher than 95% were selected for amplification and subsequent cell-based assays.

[0209] 2) HEKBlue-CD8+ HEKBlue CD122 / CD132 cell lines: HEKBlue-IL2 cells (Invivogen catalog number hkb-il2) were infected with CD8A (NM_001768) human-tagged ORF clone lentiviral particles (Origene catalog number RC206608L3V) at an MOI of 20. Transduced cells with approximately 60% CD8 positivity (measured by flow cytometry using BV421-CD8 antibody) underwent two rounds of single-cell cloning (limited dilution at 0.3 cells / well). Cell clones with a CD8 positivity rate higher than 95% were selected for amplification and subsequent cell-based assays.

[0210] 3) HEKBlue-PD1+CD8+ cell line: HEKBlue-PD1+ cells (in-house prepared) were infected with CD8A (NM_001768) human-tagged ORF clone lentiviral particles (Origene catalog number RC206608L3V) at an MOI of 20. Transduced cells with approximately 40% CD8 positivity (measured by flow cytometry using BV421-CD8 antibody) underwent two rounds of single-cell cloning (limited dilution at 0.3 cells / well). Cell clones with CD8 and PD-1 positivity rates higher than 95% were selected for amplification and subsequent cell-based assays.

[0211] B. Reporter gene assay of IL-2-induced STAT5 activity HEK293 stable cell lines expressing IL-2 receptor subunits (CD122 / CD132) and STAT5-induced SEAP reporter genes (prepared as above) were transfected with PD-1, CD8A, or PD-1+CD8A genes, respectively, and used for cell-based functional assays of the IL-2 fusion protein. In short, cell suspensions with viability greater than 90% were prepared. The specified concentrations of the test sample for each experiment were prepared by serial dilution in DMEM + 10% heat-inactivated FBS, and the cell suspensions were added to 96-well flat-bottom plates at 50 K / well and incubated at 37°C for 20–24 h. The next day, QuantI-Blue solution was prepared according to Invivogen's instructions. 20 μl of induced HEKBlue cell supernatant was added to each well of a 96-well flat-bottom plate, followed by 100 μl of resuspended QuantI-Blue solution, and the plate was incubated at 37°C for 1–3 h. SEAP level was measured using a spectrophotometer at 630 nm (OD). 630 ) to be measured.

[0212] result Figure 4A Exemplary results demonstrate that, in the absence of A1B and / or A2B antigens, the A2B antigen-binding peptide is more effective than the A1B antigen-binding peptide in reducing the IL-2-inducible STAT5 activity of the fusion protein. However, when using PD-1 ( Figure 4C and Figure 4D ) or CD8A ( Figure 4B and Figure 4D In cell lines expressing IL2, the presence of A1B and A2B polypeptide subunits in the fusion protein can completely or partially salvage the lost IL2 activity of the fusion protein.

[0213] Example 3: IL-2 stimulation of IL-2 fusion protein in PBMCs This example illustrates the study of the IL-2 stimulatory activity of a mutant IL2 fusion protein in PBMCs using phosphorylated STAT5 and isotype control staining flow cytometry.

[0214] Materials and methods A. Preparation of PBMCs Peripheral blood mononuclear cells (PBMCs) were isolated from fresh erythrocyte sedimentation rate (ESR) amber layers ordered from a blood center by density centrifugation using a Ficoll-Histopaque 1077 (Sigma H8889). In short, the ESR amber layer was diluted 1:1 with PBS, and then 15 ml of Ficoll was applied to each PBMC layer with 30 ml of the diluted ESR amber layer. The mixture was then centrifuged at 600 x g for 30 minutes with the centrifuge brake off. The PBMC layer was harvested and washed twice with RPMI 1640 medium (Sigma R0883) at 500 x g for 10 minutes each time. Cell count and viability were then determined. Cells with 99% viability were used for subsequent experiments.

[0215] B. PBMC stimulation and phosphorylation of STAT5 and isotype control staining PBMC was used at 100ul, 10 6 Cells were seeded per well in 96-well cell culture plates and stimulated at 37°C for 30 min with the specified concentration of IL2 bispecific antibody, followed immediately by fixation on ice for 30 min with 2% PFA (paraformaldehyde, Electron Microscopy Science catalog number: 15710). The fixed cells were washed twice with HBSS at 500 x g for 7 min each time at 10°C, and then treated on ice for 30 min with pre-chilled 90% methanol (at -80°C). Cells were then washed sequentially with HBSS + 10% FBS and FACs buffer. Cells were divided into two replicates for isotype control and experimental staining, respectively. Cells were then pre-incubated at 2–25°C with 20 μL of anti-human Fc receptor binding inhibitor antibody / 100 μL for 10–20 min, followed by staining. Cells were resuspended at room temperature in 50 μL of eBioscience flow cytometry staining buffer for 2 h. (Details of the antibodies used are provided in Table 13 below).

[0216] Table 13

[0217] The stained cells were washed twice with PBS and read in a Beckman Cytoflex flow cytometer.

[0218] C. Data Collection and Analysis Data were collected on Cytoflex and analyzed using CytExpert software (Beckman). Treg cells were gated as CD3+CD4+CD25- hypercellular cells, CD8+ T cells as CD3+CD8+ cells, and TConv cells as CD3+CD4+CD25- cells. pSTAT5 dose-response curves were fitted using a logistic model, and EMax and EC50 values ​​were calculated using GraphPad Prism data analysis software after subtracting the MFI of unstimulated cells and normalizing to the maximum signal intensity.

[0219] result The assay was performed using an exemplary trispecific mutant IL-2 fusion protein, P402, with 3H9 anti-PD1 VHH as A1B, 3A8 anti-CD8 VHH as A2B, and a mutant IL-2 subunit polypeptide. As shown in the results of PBMC assays performed on samples collected from two blood donors (donor A and donor B) after IL-2 stimulation, the results were plotted on… Figure 5A and Figure 5B As the results depicted show, p402 exhibited significantly lower stimulatory activity against Tregs (Kd=620 nM), CD4+ (Kd<1000 nM), and NK cells (Kd=1 nM) than wild-type IL-2 and non-aIL-2 p132. This suggests that p402 would have much lower immunosuppressive and toxic effects. However, in the presence of A1B and A2B VHH binding targets on CD8+ T cells, p402 showed much stronger stimulatory activity than wild-type IL-2 (Kd=0.31 nM), which is required for tumor killing. It is believed that the binding of p402 to its A1B and A2B VHH targets on CD8+ T cells causes a conformational change in the protein, thereby reopening the IL-2 binding site to the IL-2bg receptor. Compared to wild-type IL-2, the affinity of both A1B / A2B VHH for their targets and the IL2-ILRbg interaction further increased CD8+ stimulation. The CD8+ to Treg stimulation ratio is considered the therapeutic window of candidate drugs. For p402, the therapeutic window can reach 2000-fold. However, non-α (p132) and wild-type IL-2 have therapeutic windows of approximately 2-fold and <0.05-fold, respectively.

[0220] Example 4: Differential Scanning Fluorescence (DSF) This example illustrates the thermostability studies of mutant IL-2 fusion proteins P703 and P709, the latter including the nivolumab antigen-binding subunit.

[0221] Materials and methods The thermostability of mutant IL2 fusion protein samples was analyzed using differential scanning fluorometry (DSF) with a StepOne™ real-time PCR system (ThermoFisher) and Sypro Orange as the fluorescent dye. Samples were exchanged for 1X PBS buffer containing 1 mg / mL. An appropriate amount of 5000x Sypro Orange stock solution was added to achieve a dye concentration of 20X. The sample containing Sypro Orange dye was added to a 0.1 mL PCR tube and heated from 25°C to 95°C over 90 minutes. Temperature-dependent fluorescence signal curves were recorded, and the melting temperature (Tm) was determined using the first derivative curve method.

[0222] result Exemplary fusion proteins P703 and P709, as well as individual nivolumab antibodies, are illustrated using differential scanning fluorescence (DSF) spectra. Figure 6 P709 comprises a mutant IL2 polypeptide with mutant T3A, F42A, Y45A, and L72G fused to nivolumab antibody. As shown in the Tm values ​​summarized in Table 14 (below), the P703 fusion protein exhibits surprising thermostability comparable to the nivolumab monoclonal antibody.

[0223] Table 14: Results of Differential Scanning Fluorescence (DSF)

[0224] Example 5: Pharmacokinetics of IL-2 mutant fusion protein in mice This example illustrates the pharmacokinetic study of the mutant IL2 fusion protein P610 in mice.

[0225] Materials and methods Six female C57BL6 mice were selected for the study group to test the pharmacokinetics (PK) of the mutant IL-2 fusion protein P610 at a specific dose (mg / kg). The test sample was injected intravenously into the tail of the mice, and approximately 0.150 mL of blood samples were collected from two mice in each group at various time points (5 min, 1 h, 4 h, 8 h, 24 h, 48 h, 4 days, 7 days, 10 days, and 14 days). Serum was separated from blood cells and stored in new tubes. Samples from each mouse provided data at 3–4 time points. The concentration of the IL-2 fusion protein test sample was detected using ELISA according to the manufacturer's protocol.

[0226] result The pharmacokinetic profile of the exemplary IL-2 fusion protein p610 is shown in the figure. Figure 7The results are summarized in Table 15 (below).

[0227] Table 15

[0228] Example 6: The efficacy of mutant IL2 fusion protein in inhibiting tumor growth in a syngeneic mouse tumor model This example illustrates the in vivo antitumor efficacy of the mutant IL2 fusion protein P701 in a syngeneic mouse tumor model.

[0229] Materials and methods According to the IACUC guidelines, 5×10 5 One mouse tumor cell line, MC-38, was implanted into the left ventral region of C57BL / 6 mice. Tumor volume was measured using calipers and calculated as L × (W / 2). 2 Calculate the tumor volume. Wait until the tumor volume reaches 80-120 mm. 3 Mice were then randomly assigned to treatment groups. The mutant IL2 fusion protein substitute P701 was administered intraperitoneally at doses of 0.3 mg / kg, 1.0 mg / kg, and 30 mg / kg on day 1 and weekly. Mouse body weight was also measured on specified days.

[0230] result Figure 8A The results were plotted to show that tumor growth was significantly inhibited in mice treated with the mutant IL2 fusion protein P701 at all three doses. Figure 8B A graph of mouse weight was plotted, showing no significant weight loss in the treated mice.

[0231] While the foregoing disclosure of the present invention has been described in considerable detail by way of examples and illustrations for clarity and understanding, this disclosure, including the examples, descriptions, and embodiments described herein, is for illustrative purposes and is intended to be exemplary, and should not be construed as limiting the scope of this disclosure. It will be apparent to those skilled in the art that various modifications or alterations can be made to the examples, descriptions, and embodiments described herein, and that such modifications or alterations are included within the spirit and scope of this disclosure and the appended claims. Furthermore, those skilled in the art will recognize many methods and procedures equivalent to those described herein. All such equivalents should be understood to be within the scope of this disclosure and covered by the appended claims.

[0232] Further embodiments of the invention are set forth in the following claims.

[0233] All publications, patent applications, patents or other documents mentioned herein are expressly incorporated in their entirety for all purposes, as if each such individual publication, patent, patent application or other document were individually and specifically indicated to be incorporated herein in its entirety for all purposes and is set forth herein in its whole. In the event of any conflict, this specification (including the specified terminology) shall prevail.

Claims

1. A fusion protein comprising a first polypeptide chain, wherein the first polypeptide chain comprises: (a) The A1B subunit contains a polypeptide that specifically binds to the first antigen expressed on the surface of immune cells; (b) HLE subunit, which contains a polypeptide with half-life extension activity; (c) An IL2 subunit comprising an amino acid sequence having at least 90% identity with SEQ ID NO: 10 and a group of polypeptides differing from SEQ ID NO: 10 from the following: 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 optional location, (d) The A2B subunit contains a polypeptide that specifically binds to a second antigen expressed on the surface of the immune cells.

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 VHH antibodies, antibodies, Fab, scFv and nanobodies.

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

5. The fusion protein of claim 4, wherein the A1B subunit and / or the A2B subunit VHH antibody: (a) Specifically binds to CD8 and includes CDR1 of SEQ ID NO: 28, CDR2 of SEQ ID NO: 29 and CDR3 of SEQ ID NO: 30; (b) Specifically binds to CD8 and contains 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 to PD1 and includes CDR1 of SEQ ID NO: 19, CDR2 of SEQ ID NO: 20, and CDR3 of SEQ ID NO: 21; and / or (d) Specifically binds to PD1 and contains 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 comprises an amino acid sequence characteristic selected from the following: 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 polypeptide chain and the second polypeptide chain 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 polypeptide chain and the second polypeptide chain 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 to at least one of the other subunits via a linker 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 according to any one of claims 1-13, wherein: (a) The immune cells are tumor-reactive T cells or Treg cells; (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 antigen and the second antigen are different; optionally, the first antigen and the second antigen are CD8 and PD-1.

15. A polynucleotide encoding the fusion protein as described in claim 1.

16. An expression vector comprising the polynucleotide as described in 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 selected from mammalian cells: Chinese hamster ovary (CHO) cells, myeloma cells (e.g., Y0, NSO, Sp2 / O), monkey kidney cells (COS-7), human embryonic kidney cell line (293), young hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells, human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor cells, TR1 cells, Medical Research Council 5 (MRC 5) cells, and FS4 cells.

19. A method for producing the fusion protein of claim 1, comprising culturing a host cell as described in any one of claims 17 or 18 under conditions suitable for expressing the polypeptide.

20. A pharmaceutical composition comprising the fusion protein as described in claim 1 and a pharmaceutically acceptable carrier.

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

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

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

24. The method of claim 21, wherein the disease or condition 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, Sjögren's syndrome, type 1 diabetes, atopic dermatitis, psoriasis, and multiple sclerosis.

26. The method of claim 21, wherein the disease or condition 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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