Mutant IL-2 Polypeptides and IL-2 Prodrugs

Abstract

Provided herein are IL-2 mutants, IL-2 prodrugs, and IL-2 antibody fusion molecules, and methods of using them to modulate the immune system in a subject.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 429,940, filed December 2, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format. Said XML copy, created on November 30, 2023, is named "025471.WO015.xml" and is 525,382 bytes in size. The Sequence Listing contained in this XML file is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0003] Interleukin-2 (IL-2) plays a central role in lymphocyte development, survival, and homeostasis. IL-2 has 133 amino acids and consists of four antiparallel amphipathic alpha-helices that form a quaternary structure essential for its function [Smith, Science (1988) 240:1169-76; Bazan, Science (1992) 257:410-13].

[0004] IL-2 exerts its activity by binding to the IL-2 receptor (IL-2R), which consists of three individual subunits. The association of the α (CD25 or Tac antigen), β (CD122), and γ (γc, common γ chain, or CD132) subunits results in a trimeric, high-affinity receptor for IL-2 (KD ∼0.01 nM). The dimeric IL-2 receptor, consisting of the β and γ subunits, is called the intermediate-affinity IL-2R (KD ∼1 nM). The α subunit alone forms a monomeric, low-affinity IL-2 receptor (KD ∼10 nM). See, e.g., Kim et al., Cytokine Growth Factor Rev. (2006) 17:349-66. The dimeric intermediate-affinity IL-2 receptor binds IL-2 with approximately 100-fold lower affinity than the trimeric high-affinity receptor, but both the dimeric and trimeric IL-2 receptors can transduce signals upon IL-2 binding [Minami et al., Annu Rev Immunol. (1993) 11:245-68]. Thus, although the α subunit mediates high-affinity binding of the receptor to IL-2, it appears not to be essential for IL-2 signaling. However, the β and γ subunits are essential for IL-2 signaling [Krieg et al., Proc Natl Acad Sci. (2010) 107:11906-11]. The trimeric IL-2 receptor is expressed by CD4+FoxP3+ regulatory T (Treg) cells. Treg cells consistently express the highest levels of IL-2Rα (CD25) in vivo [Fontenot et al., Nature Immunol. (2005) 6:1142-51]. Trimeric IL-2 receptors are also transiently induced in conventionally activated T cells, but in the resting state, these cells express only dimeric IL-2 receptors. Summary of the Invention [Problem to be solved by the invention]

[0005] Mutated versions of IL-2 have been developed to optimize the treatment of cancer and autoimmune diseases. However, the immunogenicity of mutated IL-2 molecules is a potential risk in the clinical development of these molecules. Therefore, there is a need to develop IL-2-based therapeutics with reduced immunogenicity. [Means for solving the problem]

[0006] The present disclosure provides mutant human IL-2 polypeptides comprising a mutation at position L36 (e.g., L36I) according to SEQ ID NO: 1. In one aspect, the present disclosure provides mutant human IL-2 polypeptides comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 and an L36I mutation relative to SEQ ID NO: 1. In some embodiments, the mutant IL-2 polypeptide comprises additional mutations as further described herein.

[0007] In some embodiments, the additional one or more IL-2 polypeptide mutations reduce the binding affinity of the polypeptide to CD25. In some embodiments, the IL-2 polypeptide mutation is a C125A mutation relative to SEQ ID NO:1. In some embodiments, the additional one or more mutations are: T3, wherein the mutation is an N3A mutation; D20, wherein the mutation is a D20H, D20K, D20L, D20M, D20N, D20Q, D20R, D20S, D20V, or D20Y mutation; R38, wherein the mutation is an R38A, R38K, or R38S; F42, wherein the mutation is F42A, F42G, F42I, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, or F42K; Y45, wherein the mutation is Y45A, Y45G, Y45I, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, or Y45K; E6, wherein the mutation is E62L, E62A, or E62I. 2; E68, in which the mutation is E68V; L72, in which the mutation is L72G; A73, in which the mutation is A73T; N88, in which the mutation is N88A, N88E, N88F, N88H, N88K, N88T, N88L, N88M, N88S, N88V, N88W, or N88Y; N90, in which the mutation is N90T; V91, which is V91A, V91H, or V91R; I92; the mutation is at a position selected from Q126A, Q126D, Q126F, Q126G, Q126H, Q126I, Q126K, Q126L, Q126P, Q126S, Q126T, Q126W, or Q126Y (numbering according to SEQ ID NO: 1).

[0008] In some embodiments, the mutant IL-2 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 3 and 5-33, or an amino acid sequence at least 95% identical thereto. In other embodiments, the mutant IL-2 polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 2 or 4.

[0009] In another aspect, the present disclosure provides an anti-IL-2 antibody or antigen-binding fragment thereof comprising heavy chain CDR1-3 of SEQ ID NOs: 170-172, respectively, and light chain CDR1-3 of SEQ ID NOs: 173-175, respectively. In some embodiments, the anti-IL-2 antibody or antigen-binding fragment thereof herein comprises a light chain variable domain (V) comprising SEQ ID NO: 190 or an amino acid sequence at least 95% identical thereto. L ), and a heavy chain variable domain (V) comprising an amino acid sequence of SEQ ID NO: 191 or at least 95% identical thereto. H ); V comprising an amino acid sequence of SEQ ID NO: 192 or at least 95% identical thereto L and V comprising an amino acid sequence of SEQ ID NO: 193 or at least 95% identical thereto. H or V comprising an amino acid sequence of SEQ ID NO: 194 or at least 95% identical thereto. L and V comprising an amino acid sequence of SEQ ID NO: 195 or at least 95% identical thereto. H In some embodiments, the anti-IL-2 antigen-binding fragment herein comprises an amino acid sequence selected from SEQ ID NOs: 34, 35, 36, and 250-258, or an amino acid sequence at least 90% identical thereto. In some embodiments, the antibody or antigen-binding fragment, when bound to an IL-2 polypeptide, reduces binding of the IL-2 polypeptide to IL-2Rβ (CD122) or the complex of IL-2β and IL-2Rγ (CD132). In some embodiments, the antibody or antigen-binding fragment enhances the thermal stability of an IL-2 polypeptide when in a complex with a human IL-2 polypeptide. In some embodiments, the complex has a higher thermal stability or Tag temperature than the complex formed by the IL-2 polypeptide and IL-2Rβ; and / or the IL-2 polypeptide in the complex has an increased Tag temperature. In some embodiments, Tag is increased by more than 2°C, more than 5°C, more than about 10°C, or more than 10°C.

[0010] Also provided are anti-IL-2 antibodies or antigen-binding fragments thereof that compete for binding to human IL-2 with or bind to the same epitope as the antibodies or antigen-binding fragments specifically exemplified herein.

[0011] In another aspect, the disclosure provides a prodrug comprising an IL-2 cytokine portion, a masking portion, and optionally a carrier portion, wherein the masking portion comprises an antibody or antigen-binding fragment thereof, and the cytokine portion comprises SEQ ID NO: 1, or an amino acid sequence at least 90% identical thereto (e.g., a mutant IL-2 polypeptide herein). In some embodiments, the cytokine portion comprises a mutant IL-2 polypeptide described herein. In some embodiments, the IL-2 cytokine portion comprises an amino acid sequence selected from SEQ ID NOs: 1-33. In some embodiments, the masking portion is an antibody comprising a VH of SEQ ID NO: 191, or an amino acid sequence at least 95% identical thereto, and a VL of SEQ ID NO: 190, or an amino acid sequence at least 95% identical thereto. In some embodiments, the IL-2 cytokine portion comprises SEQ ID NO: 1, and in other embodiments, the one or more mutations are selected from T3A, L36I, V69A, Q74P, and C125A.

[0012] In a related aspect, the present disclosure also provides a prodrug comprising an IL-2 cytokine portion, a masking portion, and an optional carrier portion, wherein the masking portion binds to and inhibits the biological activity of the cytokine portion, and wherein the IL-2 cytokine portion comprises a mutant IL-2 polypeptide described herein. In some embodiments, the prodrug comprises an IL-2 cytokine portion, a masking portion, and an optional carrier portion, wherein the masking portion binds to and inhibits the biological activity of the cytokine portion, and wherein the IL-2 cytokine portion comprises a mutant IL-2 polypeptide. In some embodiments, the masking portion comprises the extracellular domain (ECD) of IL-2Rβ or a functional fragment thereof, or a single-chain variable fragment (scFv) or Fab. In some embodiments, the masking portion comprises an IL-2Rβ ECD comprising SEQ ID NO: 37, or an amino acid sequence at least 90% identical thereto. In some embodiments, the mutant IL-2 polypeptide comprises an L36I mutation and may further comprise a C125A mutation, and the masking portion may comprise an amino acid sequence at least 90% identical thereto. In some embodiments, the mutant IL-2 polypeptide comprises an L36I mutation, a C125A mutation, and the masking moiety may comprise SEQ ID NO: 34, or an amino acid sequence at least 90% identical thereto. In some embodiments, the masking moiety comprises an anti-IL-2 antibody or antigen-binding fragment thereof. In some embodiments, the prodrug comprises a carrier moiety selected from an antigen-binding moiety, an Fc domain, albumin or a fragment thereof, and PEG.

[0013] In some embodiments, the carrier moiety comprises an antigen-binding moiety that targets an antigen presented on an immune cell or cancer cell, and the antigen-binding moiety may be a bispecific antibody, a single-domain antibody, a Fab, or an scFv. In some embodiments, the antigen-binding moiety targets an antigen presented on a T cell, an NK cell, a macrophage, or a cell within the tumor microenvironment (TME), and the antigen may be PD-1, CD3, CD4, CD8, Tim-3, LAG-3, TIGIT, HER2, signal regulatory protein alpha (SIRPα), CTLA-4, CSF1R, NKG2A, NKG2D, CD16A, NKp30, NKp46, ILT2, ILT4, CD40, CD163, LRRC15, fibroblast activation protein (FAP), the A1 domain of tenascin-C (TNC A1), the A2 domain of tenascin-C (TNC A2), fibronectin extra domain B (EDB), fibronectin (α5β1), vitronectin (αvβ3 integrin and αvβ5 integrin), carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), 5T4, BCMA, PD-L1, CD47, epidermal growth factor receptor (EGFR), c-MET, claudin 18.2, claudin 6, CD20, CD24, CD38, CD47, GPC3, mesothelin, ROR1, and melanoma-associated chondroitin sulfate proteoglycan (MCSP). In some embodiments, the antigen-binding moiety comprises an anti-PD-1 antibody or antigen-binding fragment thereof, and the anti-PD-1 antibody may be selected from nivolumab and pembrolizumab. In some embodiments, the antigen-binding portion comprises an anti-CD8 antibody or antigen-binding fragment thereof, and the antigen-binding portion may comprise OKT8 or humanized OKT8, heavy and light chain CDRs 1-3 from OKT8, and / or a light chain variable domain comprising SEQ ID NO: 52 or an amino acid sequence at least 90% identical thereto, and a heavy chain variable domain comprising SEQ ID NO: 53 or 54 or an amino acid sequence at least 90% identical thereto.In some embodiments, the carrier moiety comprises an IgG Fc domain or an IgG antibody comprising L234A and L235A ("LALA") mutations (Eu numbering) and / or knobs-into-holes mutations, and the IL-2 cytokine moiety and the masking moiety are fused to different polypeptide chains of the Fc domain, different heavy chains of the IgG antibody, or the light and heavy chains, respectively, of the IgG antibody.

[0014] In some embodiments, the prodrug comprises one or more cleavable and / or non-cleavable peptide linkers. In some embodiments, the masking moiety is fused to the carrier moiety via a cleavable or non-cleavable peptide linker. In some embodiments, the cytokine moiety is fused to the carrier moiety via a cleavable or non-cleavable peptide linker or to the masking moiety via a cleavable peptide linker. In some embodiments, the cleavable peptide linker is cleavable by one or more proteases located in the tumor microenvironment (TME), and cleavage results in activation of the prodrug in the TME. The cleavable peptide linker may include a substrate sequence for urokinase-type plasminogen activator (uPA), matrix metallopeptidase 2 (MMP2), MMP7, MMP9, MMP14, legumain, or matriptase, and / or substrate sequences for two, three, four, or more proteases preferentially expressed in the TME. In some embodiments, the cleavable peptide linker comprises an amino acid sequence selected from SEQ ID NOs: 55-124, 268, and 269.

[0015] In some embodiments, the disclosure provides a prodrug comprising a mutant IL-2 polypeptide described herein, wherein the prodrug comprises a first heavy chain polypeptide chain, a second heavy chain polypeptide chain, and one or two light chains, wherein: a. the first heavy chain polypeptide chain comprises SEQ ID NO: 275 or an amino acid sequence at least 95% identical thereto; and the second heavy chain polypeptide chain comprises SEQ ID NO: 277 or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto; the two identical light chains comprise an amino acid sequence set forth in SEQ ID NO:276 or at least 95% identical thereto; b. the first heavy chain polypeptide chain comprises an amino acid sequence set forth in SEQ ID NO:278 or at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto, and the second heavy chain polypeptide chain comprises an amino acid sequence set forth in SEQ ID NO:279 or at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto, and the two identical light chains comprise an amino acid sequence set forth in SEQ ID NO:189 or at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto; c. the first and second heavy chain polypeptide chains comprise an amino acid sequence of SEQ ID NO:286 or at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto, and the two identical light chains comprise an amino acid sequence of SEQ ID NO:276 or at least 95% identical thereto; d. the first and second heavy chain polypeptide chains comprise an amino acid sequence of SEQ ID NO:187 or at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto, and the two identical light chains comprise an amino acid sequence of SEQ ID NO:276 or at least 95% identical thereto. the light chain comprises SEQ ID NO: 189 or an amino acid sequence at least 95% identical thereto; or e. a first heavy chain polypeptide chain comprises SEQ ID NO: 283 or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto, a second heavy chain polypeptide chain comprises SEQ ID NO: 284 or an amino acid sequence at least 95% (e.g., at least 96, 97, 98, or 99%) identical thereto, and one light chain comprises SEQ ID NO: 189 or an amino acid sequence at least 95% identical thereto.

[0016] In another aspect, the disclosure provides an IL-2 antibody fusion molecule comprising two identical antibody light chains, a first antibody heavy chain, and a second antibody heavy chain, wherein: a. each of the light chains comprises an amino acid sequence set forth in SEQ ID NO: 38 or at least 90% identical thereto, the first heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 40, 41, 45, 46 or at least 95% identical thereto, and the second heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 42, 43, 44, or 47 or at least 95% identical thereto; or b. each of the light chains comprises an amino acid sequence set forth in SEQ ID NO: 38 or at least 90% identical thereto. or c. the first and second heavy chains each comprise an amino acid sequence set forth in SEQ ID NO: 189 or an amino acid sequence at least 90% identical thereto, and the first and second heavy chains each comprise an amino acid sequence set forth in SEQ ID NO: 185 or an amino acid sequence at least 95% identical thereto; or b. the light chains each comprise an amino acid sequence set forth in SEQ ID NO: 189 or an amino acid sequence at least 90% identical thereto, and the second heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 186 or an amino acid sequence at least 95% identical thereto.

[0017] In yet another aspect, the disclosure provides a polypeptide comprising: a. two identical light chains and two identical heavy chains, wherein the light chains and heavy chains, respectively, comprise: (i) SEQ ID NO: 207, or an amino acid sequence at least 95% identical thereto, and SEQ ID NO: 205, 206, 211, 212, 213, or 214, or an amino acid sequence at least 95% identical thereto, or (ii) SEQ ID NO: 208, or an amino acid sequence at least 95% identical thereto, and SEQ ID NO: 215, or an amino acid sequence at least 95% identical thereto; or b. a first polypeptide chain and Provided is an IL-2 antibody fusion molecule comprising a second polypeptide chain, wherein the first and second polypeptide chains comprise, respectively: (i) an amino acid sequence set forth in SEQ ID NO: 196 or 197, or at least 95% identical thereto, and an amino acid sequence set forth in SEQ ID NO: 209 or 210, or at least 95% identical thereto; or (ii) an amino acid sequence set forth in SEQ ID NO: 198, or at least 95% identical thereto, and an amino acid sequence set forth in SEQ ID NO: 199, 202, 203, or 204, or at least 95% identical thereto.

[0018] Also provided herein are pharmaceutical compositions comprising a mutant human IL-2 polypeptide, prodrug, or IL-2 antibody fusion molecule and a pharmaceutically acceptable excipient.

[0019] Also provided herein are methods for producing novel proteins by culturing one or more polynucleotides encoding the novel proteins (i.e., mutant human IL-2 polypeptides, prodrugs, antibodies or antigen-binding fragments thereof, or IL-2 antibody fusion molecules); expression vectors comprising the polynucleotides; host cells comprising the expression vectors; and mammalian host cells capable of expressing the novel proteins, and isolating the expressed novel proteins from the culture.

[0020] In yet another aspect, the disclosure provides methods of treating cancer or an infectious disease or modulating (e.g., stimulating) the immune system in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a mutant IL-2 polypeptide, prodrug, IL-2 antibody fusion molecule, or pharmaceutical composition of the invention. Also provided are mutant IL-2 polypeptides, prodrugs, IL-2 antibody fusion molecules, and pharmaceutical compositions for use in treating cancer or an infectious disease or modulating (e.g., stimulating) the immune system in a patient (e.g., a human patient) in need thereof; and the use of these mutant IL-2 polypeptides, prodrugs, and IL-2 antibody fusion molecules for the manufacture of a medicament for treating cancer or an infectious disease or modulating (e.g., stimulating) the immune system in a patient in need thereof. In some embodiments, the patient has a viral infection (e.g., HIV infection); a cancer selected from the group consisting of leukemia, lymphoma, kidney cancer, bladder cancer, urinary tract cancer, cervical cancer, brain cancer, head and neck cancer, skin cancer, uterine cancer, testicular cancer, esophageal cancer, liver cancer, colorectal cancer, gastric cancer, squamous cell carcinoma, prostate cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer, bile duct cancer, breast cancer, and ovarian cancer, and medullary thyroid cancer; or an inflammatory disease or autoimmune disease, such as asthma, type I diabetes, rheumatoid arthritis, allergy, systemic lupus erythematosus, organ transplant rejection, and graft-versus-host disease. Articles of manufacture (e.g., kits) containing one or more dosage units of the novel proteins of the present invention are also provided.

[0021] Other features, objects, and advantages of the present invention will be apparent in the following detailed description. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the present invention, is given by way of illustration only, and not of limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description. [Brief explanation of the drawings]

[0022] [Figure 1A]Figures 1A-D show the results of blocking assays. Supernatants from candidate scFv clones were tested for their ability to block Fc-IL-2 binding to Fc-IL-2Rβγ. Blocking was tested using an Octet Red96e Kinetics binding assay. Figure 1A is a graph showing the binding affinity of IL-2 (1 μg / ml, 0.3 μg / ml, and 0.1 μg / ml) to IL-2Rβγ. Figure 1B is a graph showing blocking of IL-2 binding to IL-2Rβγ. Figure 1C is a graph showing partial inhibition of IL-2 binding to IL-2Rβγ. Figure 1D shows a blocking assay with 100 μl of supernatant from clone K3-23A2, which did not block IL-2 binding to IL-2Rβγ. [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 1D] Same as above. [Figure 2] FIG. 2 shows the results of a CTLL2 cell-based IL-2 activity inhibition assay using different fusion (scFv-Fc) proteins. [Figure 3A] Figure 3A is a table showing the heavy chain and expression titers of scFv clones, and Figure 3B is a graph showing a CTLL2 cell-based IL-2 activity inhibition assay using various scFv clones. [Figure 3B] Same as above. [Figure 4A] 4A and 4B show the binding kinetics of selected scFv-Fc fusion molecules. The name, binding affinity, and response of each clone are shown. [Figure 4B] Same as above. [Figure 5A]Figures 5A-C show PD-1 antibody-IL-2 prodrug information and screening data for the prodrugs after CTLL2-based activity assays. Figure 5A shows a schematic diagram of the anti-PD-1 antibody Keytruda® (JR11.20.1), the PD-1 antibody-IL-2 fusion molecule (JR11.20.2), and the PD-1 antibody-IL-2 prodrug fusion molecule with a mask in which the IL-2 portion of each prodrug contains an scFv against IL-2 (JR11.20.3-JR11.20.6) or IL-2Rβ-ECD (JR11.20.7). Figure 5B is a table showing the sample / molecule name, plasmid code, and sequence number of each molecule. Expression titers and a brief description of each molecule are also included. Figure 5C shows the results from a CTLL2 cell-based activity assay of the prodrug samples before and after activation. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 6A] Figures 6A-D show information and data from the screening of PD-1 antibody-IL-2 prodrugs based on the NK92-based activity assay. Figure 6A is a table showing the name, plasmid code, sequence number, and transient expression titer of additional PD-1 antibody-IL-2 prodrug molecules masked by scFvs screened from the yeast library. Figure 6B shows a schematic diagram of anti-PD-1 antibody-IL-2 prodrug fusion molecules (JR11.29.1, JR11.31.1-8) in which the IL-2 portion of each prodrug is masked with a mask containing an scFv against IL-2. Figure 6C shows SDS-PAGE analysis of prodrug samples JR11.29.1 and JR11.31.1 before activation and all prodrug samples after activation based on the protease MMP-2. All samples were digestible by MMP-2. Figure 6D shows the results of an NK92 cell-based activity assay (left panel) measuring the activity of prodrug samples before and after activation with MMP-2. Activity is shown in the right panel. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 6D]Same as above. [Figure 7A] Figures 7A-C show information and results from peptide linker optimization experiments. Figure 7A shows the plasmid and SEQ ID NO for each prodrug sample. Figure 7B shows a schematic diagram of anti-PD-1 antibody-IL-2 prodrug fusion molecules containing an IL-2 moiety (IL-2v) linked to the heavy chain using different linkers (JR11.101.1-3) or no linker (JR11.101.4). Figure 7C shows results from SEC-HPLC analysis. [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 8A] Figures 8A-E show the results of an ex vivo assay of PD-1 antibody-IL-2 prodrugs using human PBMCs. Figure 8A is a table showing the plasmids and SEQ ID NOs for two prodrug molecules, ASKG812K-C7 and ASKG812K-G3, and a control molecule, i.e., an unmasked PD-1 antibody-IL-2v fusion molecule (PD1-mab-IL-2vRef). The prodrug ASKG812K-G3 does not have a cleavable linker. Figure 8B shows a schematic diagram of the prodrugs. IL-2 induces STAT5 phosphorylation (pSTAT5) in immune cells. Induction of pSTAT5 is shown in Figure 8C for CD4+ T cells, Figure 8D for CD8+ T cells, and Figure 8E for NK cells. [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 8D] Same as above. [Figure 8E] Same as above. [Figure 9A]Figures 9A-D show sample information and the improved stability and thermal stability achieved by scFv masking. Figure 9A is a table showing the plasmids and SEQ ID NOs for three prodrug molecules, ASKG812K-C7, ASKG812K-F7, and ASKG812K-G3, as well as a control molecule, the ASKG812-β-ECD prodrug masked with the IL-2Rβ extracellular domain (ECD). Figure 9B is a table showing the thermal stability of the prodrugs. Figure 9C shows a schematic diagram of the prodrugs. Figure 9D is a graph showing accelerated stability studies performed on the prodrugs. [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 9D] Same as above. [Figure 10A] Figures 10A-C show sample information and results of CTLL2 assays of several prodrug molecules. Figure 10A is a table showing the plasmids and SEQ ID NOs for the following molecules: PD-1 antibody-IL-2v reference molecule (EB01-08; PD1-mab-IL-2v Ref), PD-1 antibody-IL-2v (LL24-68; PD1-mab-IL-2v), PD-1 antibody-IL-2v / L36I (JR11.145.2; PD1-mab-IL-2v / L36I), and F7-masked PD-1 antibody-IL-2v / L36I (Masked JR11.145.4; Masked PD-1 mab-IL-2v / L36I_C7). All prodrugs were tested by CTLL2 assay. Assay results are shown in Figure 10B (left and right panels). Figure 10C shows a schematic diagram of the prodrugs. [Figure 10B] Same as above. [Figure 10C] Same as above. [Figure 11A]Figures 11A-F show schematic diagrams of the structures of antibody-cytokine fusion molecules. Figure 11A shows the structure of ASKG222C7-C, in which the cytokine IL-2 and scFv mask K1-69C7, i.e., "C7," are fused to the C-terminus of the Fc domain. Figure 11B shows the structure of ASKG222C7-N, in which the cytokine IL-2 and mask C7 are fused to the N-terminus of the Fc domain. Figure 11C shows the structure of ASKG222A, in which the antibody contains the same VL and VH as C7; the IL-2 cytokine is fused to the N-terminus of each heavy chain. Figure 11D shows the structure of ASKG222B, in which the antibody contains the same VL and VH as C7; the IL-2 cytokine is fused to the N-terminus of each light chain. Figure 11E shows the structure of ASKG222G, which is essentially the same as the structure of ASKG222A (Figure 11C), except that it has only a single Fab. FIG. 11F shows the structure of ASKG222H, which is essentially the same as the structure of ASKG222B (FIG. 11D) except that it has only a single Fab. [Figure 11B] Same as above. [Figure 11C] Same as above. [Figure 11D] Same as above. [Figure 11E] Same as above. [Figure 11F] Same as above. [Figure 12A] Figure 12A shows the plasmid and sequence information for ASKG222C7-C and ASKG222C7-D. Figure 12B shows the DRC-HPLC purity of the molecules expressed in CHO cells and purified by Protein A affinity chromatography. [Figure 12B] Same as above. [Figure 13A] Figure 13A shows the plasmid and sequence information for ASKG222A-C7-Ab and ASKG222B-C7-Ab, and Figure 13B shows the DRC-HPLC purity of the molecules expressed in CHO cells and purified by Protein A affinity chromatography. [Figure 13B] Same as above. [Figure 14A]Figures 14A and 14B show the results of ex vivo assays using antibody-cytokine fusion molecules. Figure 14A is a graph showing the activity of antibody fusion molecules to stimulate the proliferation of Ki67+ regulatory T cells (Tregs). Figure 14B is a graph showing the activity of antibody fusion molecules to stimulate the proliferation of Ki67+ effector T cells (Teffs). [Figure 14B] Same as above. [Figure 15] 15 is a graph showing the PK data of ASKG222A-C7-Ab in rats. As a result, the half-life of ASKG222A-C7-Ab was shown to be 66 to 88 hours. [Figure 16-1] 16A-16E show schematic diagrams of the structure of antibody-cytokine fusion molecules and the sequences of the polypeptide chains that form the fusion molecules. [Figure 16-2] Same as above. [Figure 16-3] Same as above. [Figure 16-4] Same as above. [Figure 17A] Figures 17A and 17B show the in vivo efficacy and safety results of two fusion molecules (812mN-mut4 and 812mW5-mut4) compared with the reference molecule Ref3 and an anti-mouse PD-1 antibody. [Figure 17B] Same as above. [Figure 18] FIG. 18 shows the sequence information of the antibody-cytokine fusion molecules. [Figure 19A] Figures 19A and 19B show the in vivo efficacy and safety results of the fusion molecule (678F3-Fab-B) at two different doses. [Figure 19B] Same as above. [Figure 20] FIG. 20 shows the results of an in vitro cell-based activity assay (HEK Blue Reporter Assay). [Figure 21] FIG. 21 shows a schematic diagram of the structure of the fusion molecule 678F3-Fab-B. DETAILED DESCRIPTION OF THE INVENTION

[0023] As used in this specification and the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0024] As used herein, a reference to "about" a value or parameter includes (and describes) the variable that refers to the value or parameter itself. For example, a description that refers to "about X" includes a description of "X." Furthermore, the use of "about" before a series of numbers includes "about" for each description of the numbers in the series. For example, a description that refers to "about X, Y, or Z" is intended to describe "about X, about Y, or about Z."

[0025] The term "antigen-binding portion" refers to a polypeptide or set of interacting polypeptides that specifically binds to an antigen, and includes, but is not limited to, antibodies (e.g., monoclonal antibodies, polyclonal antibodies, multispecific antibodies, bispecific or diaspecific antibodies, anti-idiotypic antibodies, or bifunctional hybrid antibodies) or antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv, disulfide-linked Fv, scFv, single-domain antibodies (dAbs), or diabodies), single-chain antibodies, and Fc-containing polypeptides such as immunoadhesins or scFv-Fc. In some embodiments, the antibody may be of any heavy chain isotype (e.g., IgG, IgA, IgM, IgE, or IgD) or subtype (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the antibody may be of any light chain isotype (e.g., kappa or lambda). Antibodies may be human, non-human (e.g., from mouse, rat, rabbit, goat, or other non-human animal), chimeric (e.g., comprising non-human variable regions and human constant regions), or humanized (e.g., comprising non-human CDRs and human framework and constant regions). In some embodiments, the antibody is a derivatized antibody.

[0026] The term "cytokine agonist polypeptide" refers to a wild-type cytokine or an analog thereof. An analog of a wild-type cytokine has the same biological specificity (e.g., binding to the same receptor and activating the same target cells) as the wild-type cytokine, but the activity level of the analog may differ from that of the wild-type cytokine. An analog may be, for example, a mutein (i.e., a mutated polypeptide) of a wild-type cytokine and may contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten mutations relative to the wild-type cytokine.

[0027] As used herein, the term "prodrug" refers to a cytokine fusion protein that contains a cytokine moiety attached by a masking moiety and that has not yet been activated to remove the mask from the cytokine moiety. Once the cytokine moiety is unattached, the fusion molecule is "activated."

[0028] The terms "cytokine antagonist," "cytokine mask," and "masking moiety" refer to a moiety (e.g., a polypeptide) that binds to a cytokine and thereby inhibits the cytokine from binding to a receptor on the surface of a target cell and / or inhibits the cytokine from exerting its biological function while bound by the antagonist or mask. Examples of cytokine antagonists or masks include, but are not limited to, polypeptides derived from the extracellular domain of the cytokine's natural receptor that contacts the cytokine, and antibodies that bind to the cytokine or its antigen-binding fragment (e.g., scFv).

[0029] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or composition sufficient to treat a particular disorder, condition, or disease, e.g., ameliorate, alleviate, relieve, and / or delay one or more of its symptoms. With respect to diseases such as cancer, an effective amount can be an amount sufficient to delay the onset or progression of cancer (e.g., reduce the rate of tumor growth and / or delay or prevent tumor angiogenesis, metastasis, or cancer cell invasion into peripheral organs), reduce the number of epithelial cells, cause cancer regression (e.g., shrink or eradicate tumors), and / or prevent or delay the onset or recurrence of cancer. An effective amount can be administered in one or more doses.

[0030] The term "functional analog" refers to a molecule that has the same biological specificity (e.g., binding to the same ligand) and / or activity (e.g., activating or inhibiting target cells) as a reference molecule.

[0031] The term "fused" or "fusion" in reference to two polypeptide sequences refers to the joining of the two polypeptide sequences via a backbone peptide bond. The two polypeptides may be fused directly or via a peptide linker that is one or more amino acids in length. A fusion polypeptide is produced recombinantly from a coding sequence containing the coding sequences for each of the two fusion partners, with or without the coding sequence for a peptide linker between them. In some embodiments, fusion encompasses chemical conjugation.

[0032] The term "pharmaceutically acceptable excipient," when used to refer to an ingredient in a composition, means that the excipient is suitable for administration to a subject to be treated, including a human subject, without undue adverse side effects to the subject and without affecting the biological activity of the active ingredient (API).

[0033] The term "subject" refers to a mammal, including, but not limited to, a human, a pet (e.g., a dog or cat), a livestock (e.g., a cow or horse), a rodent, or a primate.

[0034] As used herein, "treatment" or "treating" is an approach for obtaining beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms attributable to the disease, diminishing the severity of the disease, improving the disease state, stabilizing the disease (e.g., preventing or slowing the worsening or progression of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, providing partial or complete remission of the disease, reducing the dose of one or more other medications required to treat the disease, improving the patient's quality of life, and / or prolonging survival. The methods of the present disclosure contemplate any one or more of these treatment aspects.

[0035] It is understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present invention. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described thereunder.

[0036] I. Mutant IL-2 Prodrugs The present disclosure further provides cytokine prodrugs that are metabolized in vivo at the target site to become active cytokine therapeutics. Cytokine prodrugs have fewer side effects (including reduced risk of cytokine immunogenicity compared to wild-type cytokines), a better in vivo PK profile (e.g., longer half-life), and better target specificity, making them more effective than prior cytokine therapeutics. The prodrugs of the present invention comprise a cytokine agonist polypeptide linked to a carrier moiety, and a cytokine moiety masked (bound) by a cytokine antagonist (masking moiety). The cytokine antagonist may be, for example, the extracellular domain of a cytokine receptor, linked to the cytokine moiety or carrier moiety via a peptide linker (e.g., a cleavable or non-cleavable peptide linker). While bound to the cytokine, the mask inhibits the biological function of the cytokine moiety. The prodrug is activated at the target site (e.g., a tumor site or the surrounding environment) in a patient by cleavage of the linker and subsequent release of the cytokine mask from the prodrug, exposing the previously masked cytokine moiety and allowing the cytokine moiety to bind to a receptor on the target cell and exert its biological function on the target cell. In other embodiments, when the masking moiety is linked to the carrier moiety via a non-cleavable peptide linker, the prodrugs disclosed herein can engage target cells via "cis-binding" of cytokine receptors and antigens expressed on the cell surface, resulting in increased activity of the prodrug without cleavage and removal of the masking moiety. The cytokine portion of the prodrug may increase activity at target sites (e.g., tumor sites or surrounding environments) where both the antigen targeted by the carrier and the cytokine receptor are expressed on the same cells.

[0037] In some embodiments, the carrier for the prodrug is an antigen-binding moiety, such as an antibody or antigen-binding fragment thereof, that binds to an antigen at a target site.

[0038] In some embodiments, the prodrugs of the present invention are proinflammatory cytokine prodrugs that are metabolized to a proinflammatory cytokine at a target site in the body targeted by the carrier moiety. In further embodiments, the carrier moiety in the prodrug is an antibody or antigen-binding fragment thereof that targets a tumor antigen such that the prodrug is delivered to a tumor site in a patient and metabolized locally (e.g., within or near the tumor microenvironment) upon cleavage of a linker connecting the cytokine mask to the carrier moiety or cytokine moiety, allowing the proinflammatory cytokine moiety to interact with a receptor on the target cell and locally stimulate the target immune cell.

[0039] An IL-2 prodrug may comprise a cytokine moiety comprising an IL-2 agonist polypeptide, a carrier moiety, and a masking moiety (IL-2 antagonist), where the cytokine moiety is fused to the carrier moiety directly or via a linker (e.g., a cleavable or non-cleavable peptide linker), and the IL-2 antagonist is linked to the IL-2 agonist polypeptide or the carrier moiety via a cleavable peptide linker. In the IL-2 prodrug of the present invention, the IL-2 agonist polypeptide may be an IL-2 mutein, such as the IL-2 muteins described herein, derived from human IL-2. The IL-2 mutein may have significantly reduced affinity for CD25 or trimeric high-affinity IL-2R compared to wild-type IL-2. In some embodiments, the IL-2 mutein has 100-, 300-, 500-, 1,000-, or 10,000-fold reduced binding affinity for the high-affinity IL-2R compared to wild-type IL-2. Unless otherwise indicated, all residue numbers in IL-2 and IL-2 muteins described herein follow the numbering of SEQ ID NO:1.

[0040] The present disclosure further provides a prodrug comprising the above-described mutant IL-2 polypeptide and a carrier moiety. In some embodiments, the carrier moiety comprises an antigen-binding moiety, which binds to an antigen expressed on an immune cell. In some embodiments, the prodrug comprises an antigen-binding moiety, which binds to an antigen expressed on a tumor cell or in the tumor microenvironment. In some embodiments, the carrier moiety comprises two or more antigen-binding moieties, which bind to two different antigens, one of which is expressed on an immune cell and the other of which is expressed on a tumor cell or in the tumor microenvironment.

[0041] In some embodiments, the prodrug further comprises a masking moiety, which binds to the mutant IL-2 polypeptide and inhibits the biological activity of the mutant IL-2 polypeptide. In some embodiments, the masking moiety comprises the extracellular domain of an IL-2 receptor subunit. In some embodiments, the masking moiety comprises the extracellular domain (ECD) of IL-2 receptor beta (IL-2Rβ) or a functional analog thereof. In some embodiments, the IL-2Rβ-ECD comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the masking moiety comprises an antibody or antigen-binding fragment thereof, wherein the antibody binds to IL-2. In some embodiments, the masking moiety is an scFv. In some embodiments, the scFv comprises an amino acid sequence selected from SEQ ID NOs: 34, 35, and 36. In some embodiments, the IL-2 prodrug further comprises a cleavable peptide linker. In some embodiments, the cleavable linker links the masking moiety to the carrier. In some embodiments, the cleavable linker links the mutant IL-2 polypeptide to the carrier.

[0042] A. Mutant IL-2 Polypeptides In the IL-2 prodrug of the present invention, the IL-2 agonist polypeptide is an IL-2 mutein derived from human IL-2. IL-2 with mutations that reduce or eliminate the interaction of IL-2 with CD25 has previously been disclosed. For example, WO2008 / 0034473 mentions the R38W and F42K mutations, while WO2012 / 107417 mentions a mutation at position 72. U.S. Patent Application Publication No. 2003 / 0124678 mentions the introduction of the R38W mutation to eliminate the vascular permeability activity of IL-2. Heaton et al. [Cancer Res. (1993) 53:2597-2602; U.S. Patent No. 5,229,109] describe the introduction of two mutations, R38A and F42K, to obtain an IL-2 mutein with reduced ability to induce the secretion of inflammatory cytokines from natural killer (NK) cells. EP2639241 is T regReference is made to IL-2 muteins that are at least 1,000-fold less effective than native IL-2 in stimulating cells, including IL-2 muteins having mutations selected from: 1) R38K, F42I, Y45N, E62L, and E68V; 2) R38A, F42I, Y45N, E62L, and E68V; 3) R38K, F42K, Y45R, E62L, and E68V; or 4) R38A, F42A, Y45A, and E62A. U.S. Patent Application Publication No. 2014 / 0328791 refers to pegylated IL-2 with reduced affinity for CD25. These IL-2 mutants, referred to as "non-alpha" IL-2 agonist polypeptides, have been shown to preferentially activate effector T cells and natural killer (NK) cells over regulatory T cells. Such IL-2 mutants may contain one or more mutations selected from T3A, R38A, R38K, R38S, F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, E62A, E62L, E62I, E68A, E68V, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, L72K, and C125S (numbering according to SEQ ID NO: 1). Unfortunately, such mutations may pose a risk of immunogenicity. For example, an IL-2 mutant containing the mutations T3A / R38S / F42A / Y45A / E62A / C125S (numbering according to SEQ ID NO: 1) showed additional hotspots based on MHC class II binding prediction using the T Cell Epitope Prediction Tool. Table 1 shows the prediction results for wild-type IL-2. Table 2 shows the results for an IL-2 mutant with T3A / R38S / F42A / Y45A / E62A / C125S (numbering according to SEQ ID NO: 1). This mutant was named IL-2V1 and contains the amino acid sequence of SEQ ID NO: 176.

[0043] [Table 1]

[0044] [Table 2]

[0045] In some embodiments, the present invention provides novel IL-2 muteins (mutant IL-2 polypeptides) with reduced immunogenicity risk. In some embodiments, the mutant IL-2 polypeptide comprises a mutation at position L36 (numbering according to SEQ ID NO: 1). In particular embodiments, the mutant IL-2 polypeptide comprises an L36I mutation (numbering according to SEQ ID NO: 1). In some embodiments, the mutant IL-2 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 2-33, or at least 95%, at least 98%, or at least 99% of an amino acid sequence selected from SEQ ID NOs: 2-33.

[0046] In some embodiments, the mutant IL-2 polypeptides disclosed herein may further include a mutation at A73 (e.g., to T or another amino acid residue) and / or a K35N mutation. Without being bound by theory, the inventors believe that A73 and K35 are potential glycosylation sites on IL-2, and that mutation of these glycosylation sites modulates the affinity of the IL-2 mutein for the IL-2 receptor. The mutant IL-2 polypeptides have a safer clinical profile and can be used in patients requiring IL-2 activity, for example, in patients requiring a stimulated immune system (e.g., cancer and AIDS patients). The mutant IL-2 polypeptides can be used as separate entities or as conjugates (e.g., fused to a carrier, such as in the prodrugs of the present invention).

[0047] In some embodiments, mutant IL-2 polypeptides of the invention may comprise a mutation at L36 (e.g., L36I) and one or more mutations at positions selected from T3, D20, K35, R38, F42, F44, Y45, E62, E68, L72, A73, N88, N90, C125, and Q126 (numbering according to SEQ ID NO: 1). In certain embodiments, mutant IL-2 polypeptides comprise mutations at L36, R38, F42, Y45, and A73 (numbering according to SEQ ID NO: 1).

[0048] In some embodiments, mutant human IL-2 polypeptides of the invention may comprise a K35N mutation and one or more mutations at positions selected from T3, D20, R38, F42, F44, Y45, E62, E68, L72, A73, N88, N90, C125, and Q126 (numbering according to SEQ ID NO: 1). In certain embodiments, mutant human IL-2 polypeptides comprise the mutation K35N and additional mutations at R38, F42, and Y45, with or without a mutation at A73. In some embodiments, in addition to a mutation at L36, mutant human IL-2 polypeptides of the invention may comprise a K35N mutation and one or more mutations at positions selected from T3, D20, R38, F42, F44, Y45, E62, E68, L72, A73, N88, N90, C125, and Q126 (numbering according to SEQ ID NO: 1).

[0049] In some embodiments, the mutant human IL-2 polypeptide may comprise one or more mutations at K35, R38, F42, F44, Y45, E62, E68, L72, and A73 (numbering according to SEQ ID NO: 1). In some embodiments, the mutant human IL-2 polypeptide further comprises one or more mutations at D20, N88, N90, and Q126 (numbering according to SEQ ID NO: 1). Additional mutations at T3 and / or C125 may be included.

[0050] B. Masking moiety of the prodrug The cytokine antagonist, i.e., masking moiety, in the immunoconjugates of the invention may comprise a peptide, antibody, or antibody fragment that binds to the cytokine moiety in the prodrug, thereby masking the cytokine moiety and inhibiting its biological function. In some embodiments, the prodrug comprises a masking moiety that binds to a mutant IL-2 polypeptide disclosed herein and inhibits the biological activity of the mutant IL-2 polypeptide.

[0051] By way of example, IL-2 antagonists may include peptides and antibodies that bind to IL-2 and prevent the IL-2 moiety from binding to the receptor, resulting in reduced biological activity of the IL-2 moiety when masked. In some embodiments, the IL-2 antagonist comprises the IL-2Rβ or IL-2Rγ extracellular domain, or a functional analog thereof, such as that derived from human IL-2Rβ or IL-2Rγ. In some embodiments, the IL-2Rβ-ECD comprises the amino acid sequence of SEQ ID NO: 37 or an amino acid sequence at least 90% identical thereto. In some embodiments, the IL-2 antagonist comprises a peptide identified from screening a peptide library. In some embodiments, the masking moiety comprises an antibody or antigen-binding fragment thereof. In certain embodiments, the IL-2 antagonist comprises an antibody or fragment thereof that blocks binding of IL-2 or an IL-2 mutein to the IL-2 receptor. In certain embodiments, the IL-2 antagonist comprises an scFv, Fab, or single-chain Fab having the same CDR sequences as an scFv comprising an amino acid sequence selected from SEQ ID NOs: 34, 35, and 36.

[0052] In some other embodiments, the prodrug further comprises a peptide linker (e.g., cleavable or non-cleavable), which links the masking moiety to the carrier. In some other embodiments, the peptide linker links the mutant IL-2 polypeptide to the carrier moiety.

[0053] C. Carrier Moieties of Prodrugs The carrier moiety of the prodrug of the present invention may be an antigen-binding moiety or a moiety that does not bind to an antigen. The carrier moiety may improve the PK profile, such as serum half-life, of the cytokine agonist polypeptide and may target the cytokine agonist polypeptide to a target site in the body, such as a tumor site.

[0054] 1. Antigen-binding carrier moiety The carrier moiety may be an antibody or antigen-binding fragment thereof, or an immunoadhesin. In some embodiments, the antigen-binding moiety is a full-length antibody having two heavy chains and two light chains, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a disulfide-linked Fv fragment, a single-domain antibody, a nanobody, or a single-chain variable fragment (scFv). In some embodiments, the antigen-binding moiety is a bispecific antigen-binding moiety, capable of binding to two different antigens or two different epitopes on the same antigen. The antigen-binding moiety may provide additional, potentially synergistic, therapeutic efficacy to the cytokine agonist polypeptide.

[0055] The cytokine agonist polypeptide and its mask may be fused to the N-terminus or C-terminus of the light chain and / or heavy chain of the antigen-binding portion. For example, the cytokine agonist polypeptide and its mask may be fused to the heavy chain of an antibody or its antigen-binding fragment. In some embodiments, the cytokine agonist polypeptide and its mask may be fused to the light chain of an antibody or its antigen-binding fragment. In some embodiments, the cytokine agonist polypeptide is fused to the C-terminus of one or both heavy chains of the antibody, and the cytokine mask (masking moiety) is fused to the C-terminus of the cytokine agonist polypeptide via a cleavable or non-cleavable peptide linker. In some embodiments, the cytokine agonist polypeptide is fused to the C-terminus of one heavy chain of the antibody, and the cytokine mask is fused to the C-terminus of the other heavy chain of the antibody via a cleavable or non-cleavable peptide linker, and the two heavy chains contain mutations that allow them to specifically pair.

[0056] Strategies for forming heterodimers are well known (see, for example, Spies et al., Mol Imm. (2015) 67(2)(A):95-106). For example, the two heavy chain polypeptides in a prodrug can form stable heterodimers by "knob-into-hole" mutations. "Knob-into-hole" mutations are made to promote heterodimer formation of antibody heavy chains and are commonly used to create bispecific antibodies (see, for example, U.S. Patent No. 8,642,745). For example, the Fc domain of an antibody can contain a T366W mutation in the CH3 domain of the "knob chain" and a T366S, L368A, and / or Y407V mutation in the CH3 domain of the "hole chain." Additional interchain disulfide bridges between CH3 domains can also be used, for example, by introducing a Y349C mutation in the CH3 domain of the "knob chain" and an E356C or S354C mutation in the CH3 domain of the "hole chain" (see, e.g., Merchant et al., Nature Biotech. (1998) 16:677-81). In other embodiments, the antibody portion can include a Y349C and / or T366W mutation in one of the two CH3 domains and an E356C, T366S, L368A, and / or Y407V mutation in the other CH3 domain. In certain embodiments, the antibody portion may comprise a Y349C and / or T366W mutation in one of the two CH3 domains and an S354C (or E356C), T366S, L368A, and / or Y407V mutation in the other CH3 domain, wherein the additional Y349C mutation in one CH3 domain and the additional E356C or S354C mutation in the other CH3 domain form an interchain disulfide bridge (numbering always according to the EU index of Kabat; Kabat et al., "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).Other knobs-into-hole technologies may alternatively or additionally be used, such as those described in EP 1870459. Thus, another example of a knobs-into-hole mutation for an antibody moiety is to have R409D / K370E mutations in the CH3 domain of the "knob chain" and D399K / E357K mutations in the CH3 domain of the "hole chain" (Eu numbering).

[0057] In some embodiments, the antibody portion of the prodrug contains L234A and L235A ("LALA") mutations in its Fc domain. The LALA mutations eliminate complement binding and fixation, as well as Fcγ-dependent ADCC (see, e.g., Hezareh et al., J. Virol. (2001) 75(24):12161-8). In further embodiments, the LALA mutations are present in the antibody portion in addition to the knobs-into-holes mutations.

[0058] In some embodiments, the antibody portion comprises M252Y / S254T / T256E ("YTE") mutations in the Fc domain. The YTE mutation can simultaneously modulate the serum half-life, tissue distribution, and activity of IgG1 (see Dall'Acqua et al., J Biol Chem. (2006) 281:23514-24; and Robbie et al., Antimicrob Agents Chemother. (2013) 57(12):6147-53). In further embodiments, the YTE mutation is present in the antibody portion in addition to knob-into-hole mutations. In certain embodiments, the antibody portion has YTE, LALA, knob-into-hole mutations, or any combination thereof.

[0059] The antigen-binding moiety may bind to antigens on the cell surface of immune cells, such as T cells, NK cells, and macrophages. In other cases, the antigen-binding moiety may bind to cytokines. For example, the antigen-binding moiety may be an antibody or antigen-binding fragment thereof that binds to PD-1, LAG-3, TIM-3, TIGIT, SIRP alpha, CTLA-4, CSF1R, NKG2A, NKG2D, CD16A, NKp30, NKp46, fibroblast activation protein (FAP), the A1 domain of tenascin-C (TNC A1), the A2 domain of tenascin-C (TNC A2), fibronectin extradomain B (EDB), PSA, 5T4, CD47, CMET, claudin 6, CD24, melanoma-associated chondroitin sulfate proteoglycan (MCSP), or TGF-beta. The antibody may have the ability to activate immune cells and enhance anti-cancer activity.

[0060] The antigen-binding portion can be an antibody or its antigen-binding fragment that binds to an antigen on the surface of tumor cells.For example, the antigen-binding portion can bind to FAP alpha, 5T4, Trop-2, PD-L1, HER-2, EGFR, claudin 18.2, or carcinoembryonic antigen (CEA).The antibody may or may not have antibody-dependent cellular cytotoxicity (ADCC) activity.The antibody can also be conjugated with a cytotoxic drug.

[0061] In some embodiments, the antigen-binding moiety is an anti-PD1 antibody. In some embodiments, the anti-PD-1 antibody is pembrolizumab or nivolumab. In some embodiments, the antigen-binding moiety is an anti-CD8 antibody or a CD8-binding fragment thereof. In some embodiments, the antigen-binding moiety is an anti-NKG2A, NKG2D, CD16A, NKp30, NKP44, or NKP46 antibody or a binding fragment thereof.

[0062] In some embodiments, the PD-1 binding moiety comprises an antibody or fragment thereof known in the art that binds to PD-1 and disrupts the interaction between PD-1 and its ligand (PD-L1) to stimulate an anti-tumor immune response. In some embodiments, the antibody or antigen-binding portion thereof specifically binds to PD-1. For example, antibodies that target PD-1 and may find use in the present invention include nivolumab (BMS-936558, Bristol-Myers Squibb), pembrolizumab (lambrolizumab, MK03475 or MK-3475, Merck), humanized anti-PD-1 antibody JS001 (ShangHai JunShi), monoclonal anti-PD-1 antibody TSR-042 (Tesaro, Inc.), pidilizumab (anti-PD-1 mAb CT-011, Medivation), anti-PD-1 monoclonal antibody BGB-A317 (BeiGene), and / or anti-PD-1 antibody SHR-1210 (ShangHai HengRui), human monoclonal antibody REGN2810 (Regeneron), human monoclonal antibody MDX-1106 (Bristol-Myers Squibb), and / or human monoclonal antibody PD-1 (Bristol-Myers Squibb). Examples of suitable PD-1 antibodies include, but are not limited to, the humanized anti-PD-1 IgG4 antibody PDR001 (Novartis), from BioXcell (Squibb), and / or the humanized anti-PD-1 IgG4 antibody PDR001 (Novartis). In some embodiments, the PD-1 antibody is derived from clone: ​​RMP1-14 (rat IgG) - BioXcell catalog number BP0146. Other suitable anti-PD-1 antibodies include those disclosed in U.S. Patent No. 8,008,449. In some embodiments, the PD-1 binding moiety comprises a single domain antibody or nanobody. In some embodiments, the single domain antibody includes those disclosed in WO2019 / 137541.

[0063] In some embodiments, the antigen binding moiety is selected from the group consisting of guanyl cyclase C (GCC), carbohydrate antigen 19-9 (CA19-9), glycoprotein A33 (gpA33), mucin 1 (MUC1), insulin-like growth factor 1 receptor (IGF1-R), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), delta-like protein 3 (DLL3), delta-like protein 4 (DLL4), epidermal growth factor receptor (EGFR), glypican-3 (GPC3), c-MET, vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), Nectin-4, Liv-1, glycoprotein NMB (GPNMB), prostate-specific membrane antigen (PSMA), Trop-2, carbonic anhydrase IX ( CA9), endothelin B receptor (ETBR), prostate six-transmembrane epithelial antigen 1 (STEAP1), folate receptor alpha (FR-α), SLIT and NTRK-like protein 6 (SLITRK6), carbonic anhydrase VI (CA6), ectonucleotide pyrophosphatase / phosphodiesterase family member 3 (ENPP3), mesothelin, trophoblast glycoprotein (TPBG), CD19, CD20, CD22, CD33, CD40, CD56, CD66e, CD70, CD74, CD79b, CD98, CD123, CD138, CD352, CD47, signal regulatory protein alpha (SIRPα), claudin 18.2, claudin 6, BCMA, or EPCAM. In some embodiments, the antigen-binding moiety binds to the epidermal growth factor (EGF)-like domain of DLL3. In some embodiments, the antigen-binding moiety binds to the Delta / Serrate / Lag2 (DSL)-like domain of DLL3. In some embodiments, the antigen-binding moiety binds to an epitope located after amino acid 374 of GPC3. In some embodiments, the antigen-binding moiety binds to a heparin sulfate glycan of GPC3. In some embodiments, the antigen-binding moiety binds to claudin 18.2 but not to claudin 18.1. In some embodiments, the antigen-binding moiety binds to claudin 18.1 with at least 10-fold weaker binding affinity than to claudin 18.2.

[0064] Exemplary antigen-binding moieties include trastuzumab, rituximab, brentuximab, cetuximab, panitumumab, GC33 (or a humanized version thereof), anti-EGFR antibody mAb806 (or a humanized version thereof), anti-dPNAG antibody F598, and antigen-binding fragments thereof. In some embodiments, the antigen-binding moiety has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with trastuzumab, rituximab, brentuximab, cetuximab, or panitumumab, GC33 (or a humanized version thereof), anti-EGFR antibody mAb806 (or a humanized version thereof), anti-dPNAG antibody F598, or a fragment thereof. In some embodiments, the antigen-binding portion has an antibody heavy chain that has at least 90%, 91%, 92%, 93%, 95%, 96%, 97%, 98%, or 99% identity to an antibody heavy chain of trastuzumab, rituximab, brentuximab, cetuximab, panitumumab, GC33 (or a humanized version thereof), anti-EGFR antibody mAb806 (or a humanized version thereof), anti-dPNAG antibody F598, or a fragment thereof. In some embodiments, the antigen-binding portion has an antibody light chain that is at least 90%, 91%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the antibody light chain of trastuzumab, rituximab, brentuximab, cetuximab, panitumumab, GC33 (or a humanized version thereof), anti-EGFR antibody mAb806 (or a humanized version thereof), anti-dPNAG antibody F598, or a fragment thereof. In some embodiments, the antigen-binding portion is fused to an IL-2 agonist polypeptide. In some embodiments, the antigen-binding portion comprises the six complementarity-determining regions (CDRs) of trastuzumab, rituximab, brentuximab, cetuximab, panitumumab, GC33, anti-EGFR antibody mAb806, or anti-dPNAG antibody F598.

[0065] Several CDR delineations are known in the art and are encompassed herein. One skilled in the art can easily determine the CDRs of a given delineation based on the sequence of the variable region of the heavy or light chain. The "Kabat" CDRs are based on sequence variability and are the most commonly used [Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)]. "Chothia" CDRs refer to the location of structural loops [Chothia & Lesk, Canonical structures for the hypervariable regions of immunoglobulins, J. Mol. Biol., vol. 196, pp. 901-917 (1987)]. "AbM" CDRs are a compromise between Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. "Contact" CDRs are based on analysis of available complex crystal structures. The residues of each of these CDRs are listed in Table 3 with reference to a common antibody numbering scheme. Unless otherwise specified herein, amino acid numbers in antibodies refer to the Kabat numbering scheme described in Kabat et al., supra, including when CDRs are delineated with reference to the Kabat, Chothia, AbM, or Contact schemes. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the framework regions (FRs) or CDRs of the variable domain. For example, a heavy chain variable domain may contain a single amino acid inserted after residue 52 of H2 (residue 52a according to Kabat) and residues inserted after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat, etc.). The Kabat numbering of residues can be determined for a given antibody by alignment of the antibody's sequence with the "standard" Kabat numbering sequence at the regions of homology.

[0066] [Table 3]

[0067] In some embodiments, the CDRs are "extended CDRs" and encompass regions that start or end according to different schemes. For example, the extended CDRs can be: L24 to L36, L26 to L34, or L26 to L36 (VL-CDR1); L46 to L52, L46 to L56, or L50 to L55 (VL-CDR2); L91 to L97 (VL-CDR3); H47 to H55, H47 to H65, H50 to H55, H53 to H58, or H53 to H65 (VH-CDR2); and / or H93 to H102 (VH-CDR3).

[0068] In some embodiments, the antigen-binding portion binds to HER2 and comprises a light chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 126, or a fragment thereof, and a heavy chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 127, or a fragment thereof. In some embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO: 126 and CDR1, CDR2, and CDR3 from SEQ ID NO: 127.

[0069] In some embodiments, the antigen-binding portion binds to CD20 and comprises a light chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 128, or a fragment thereof, and a heavy chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 129, or a fragment thereof. In some embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO: 128 and CDR1, CDR2, and CDR3 from SEQ ID NO: 129.

[0070] In some embodiments, the antigen-binding portion binds to CD30 and comprises a light chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 130, or a fragment thereof, and a heavy chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 131, or a fragment thereof. In some embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO: 130 and CDR1, CDR2, and CDR3 from SEQ ID NO: 131.

[0071] In some embodiments, the antigen-binding portion binds to EGFR and comprises a light chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 132, or a fragment thereof, and a heavy chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 133, or a fragment thereof. In some embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO: 132 and CDR1, CDR2, and CDR3 from SEQ ID NO: 133.

[0072] In some embodiments, the antigen-binding portion binds to EGFR and comprises a light chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 134, or a fragment thereof, and a heavy chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 135, or a fragment thereof. In some embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO: 134 and CDR1, CDR2, and CDR3 from SEQ ID NO: 135.

[0073] In some embodiments, the antigen-binding portion binds to c-MET and comprises a light chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 136, or a fragment thereof, and a heavy chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 137, or a fragment thereof. In some embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO: 136 and CDR1, CDR2, and CDR3 from SEQ ID NO: 137.

[0074] In some embodiments, the antigen-binding portion binds to GPC3 and comprises a light chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 138, or a fragment thereof, and a heavy chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 139, or a fragment thereof. In some embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO: 138, and CDR1, CDR2, and CDR3 from SEQ ID NO: 139.

[0075] In some embodiments, the antigen-binding portion binds to claudin 18.2 and comprises a light chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 140, or a fragment thereof, and a heavy chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 141, or a fragment thereof. In some embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO: 140 and CDR1, CDR2, and CDR3 from SEQ ID NO: 141.

[0076] In some embodiments, the antigen-binding portion binds to FAP alpha and comprises a light chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 158 or 159, or a fragment thereof, and a heavy chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 160, or a fragment thereof. In some embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO: 158 or 159, and CDR1, CDR2, and CDR3 from SEQ ID NO: 160. In some embodiments, the antigen-binding portion binds to FAP alpha and comprises a light chain variable domain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 161, and a heavy chain variable domain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 162. In some embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO: 161 and CDR1, CDR2, and CDR3 from SEQ ID NO: 162. In certain embodiments, the humanized FAP antibody comprises the light chain amino acid sequence set forth in SEQ ID NO: 158 or 159 and the heavy chain amino acid sequence set forth in SEQ ID NO: 160.

[0077] In some embodiments, the antigen-binding portion binds to carcinoembryonic antigen (CEA) and may be derived from antibody PR1A3 (U.S. Patent No. 8,642,742). The anti-CEA antibody comprises a light chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 156, or a fragment thereof, and a heavy chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 157, or a fragment thereof. In some embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO: 154 and CDR1, CDR2, and CDR3 from SEQ ID NO: 155. In certain embodiments, the PR1A3 antibody is a humanized antibody comprising the light chain variable domain amino acid sequence set forth in SEQ ID NO:156 and the heavy chain variable domain amino acid sequence set forth in SEQ ID NO:157.

[0078] In some embodiments, the antigen-binding portion binds to PDL1 and comprises a light chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 167, or a fragment thereof, and a heavy chain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 168, or a fragment thereof. In some embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO: 167, and CDR1, CDR2, and CDR3 from SEQ ID NO: 168.

[0079] In some embodiments, the antigen-binding portion binds to 5T4 and comprises a light chain variable domain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 165 or 166, and a heavy chain variable domain having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 163 or 164. In some embodiments, the antigen-binding domain comprises CDR1, CDR2, and CDR3 from SEQ ID NO: 165 or 166, and CDR1, CDR2, and CDR3 from SEQ ID NO: 163 or 164.

[0080] In some embodiments, the antigen-binding portion binds to Trop-2 and comprises a light chain variable region comprising CDR1 comprising the amino acid sequence of KASQDVSIAVA (SEQ ID NO: 142), CDR2 comprising the amino acid sequence of SASYRYT (SEQ ID NO: 143), and CDR3 comprising the amino acid sequence of QQHYITPLT (SEQ ID NO: 144); and a heavy chain variable region comprising CDR1 comprising the amino acid sequence of NYGMN (SEQ ID NO: 145), CDR2 comprising the amino acid sequence of WINTYTGEPTYTDDFKG (SEQ ID NO: 146), and CDR3 comprising the amino acid sequence of GGFGSSYWYFDV (SEQ ID NO: 147).

[0081] In some embodiments, the antigen-binding portion binds to mesothelin and comprises a light chain variable region comprising CDR1 comprising the amino acid sequence of SASSSVSYMH (SEQ ID NO: 148), CDR2 comprising the amino acid sequence of DTSKLAS (SEQ ID NO: 149), and CDR3 comprising the amino acid sequence of QQWSGYPLT (SEQ ID NO: 150); and a heavy chain variable region comprising CDR1 comprising the amino acid sequence of GYTMN (SEQ ID NO: 151), CDR2 comprising the amino acid sequence of LITPYNGASSYNQKFRG (SEQ ID NO: 152), and CDR3 comprising the amino acid sequence of GGYDGRGFDY (SEQ ID NO: 153).

[0082] In some embodiments, the antigen-binding moiety comprises one, two, or three antigen-binding domains. For example, the antigen-binding moiety is bispecific and binds to two different antigens selected from the group consisting of HER2, HER3, EGFR, 5T4, FAP alpha, Trop-2, GPC3, VEGFR2, claudin 18.2, and PD-L1. In some embodiments, the bispecific antigen-binding moiety binds to two different epitopes of HER2.

[0083] 2. Other carrier parts Other non-antigen-binding carrier moieties may be used in the prodrugs of the present invention, such as antibody Fc domains (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc), polymers (e.g., PEG), albumin (e.g., human albumin) or fragments thereof, or nanoparticles.

[0084] For example, a cytokine agonist polypeptide and its antagonist can be fused to an antibody Fc domain to form an Fc fusion protein. In some embodiments, the cytokine agonist polypeptide is fused (directly or via a peptide linker) to the C-terminus or N-terminus of one of the Fc domain polypeptide chains, and the cytokine mask is fused to the C-terminus or N-terminus of the other Fc domain polypeptide chain via a cleavable or non-cleavable peptide linker, and the two Fc domain polypeptide chains contain mutations that enable their specific pairing. In some embodiments, the Fc domain contains the knobs-into-holes mutation described above. In further embodiments, the Fc domain can contain the YTE and / or LALA mutation described above.

[0085] The carrier moiety of the prodrug may comprise albumin (e.g., human serum albumin) or a fragment thereof. An exemplary sequence of albumin is set forth in SEQ ID NO: 124. In some embodiments, the albumin or albumin fragment is about 85% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.5% or more, or about 99.8% or more identical to human serum albumin or a fragment thereof.

[0086] In some embodiments, the carrier moiety comprises an albumin fragment (e.g., a human serum albumin fragment) that is about 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 120 or more, 140 or more, 160 or more, 180 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, or 550 or more amino acids in length. In some embodiments, the albumin fragment is about 10 amino acids to about 584 amino acids in length (e.g., about 10 to about 20, about 20 to about 40, about 40 to about 80, about 80 to about 160, about 160 to about 250, about 250 to about 350, about 350 to about 450, or about 450 to about 550 amino acids in length). In some embodiments, the albumin fragment comprises the Sudlow I domain or a fragment thereof, or the Sudlow II domain or a fragment thereof.

[0087] D. Linker Moieties of Prodrugs The IL-2 agonist polypeptide may be fused to a carrier moiety with or without a peptide linker. The peptide linker may be non-cleavable. In certain embodiments, the peptide linker comprises the amino acid sequence GGS (SEQ ID NO: 177), GGGGS (SEQ ID NO: 178), GGSGGS (SEQ ID NO: 179), GGGSGGGGS (SEQ ID NO: 180), GGGGSGGGSGGGGGS (SEQ ID NO: 181), GGGGSAAGGGGAGGGGA (SEQ ID NO: 182), or GGGGSGGGGSAAGGGGSGGGGS (SEQ ID NO: 183).

[0088] The IL-2 mask may be fused to the cytokine moiety or carrier via a cleavable linker. The cleavable linker may contain one or more (e.g., two or three) cleavable moieties (CMs). Each CM may be a substrate for an enzyme or protease selected from legumain, plasmin, TMPRSS-3 / 4, MMP-2, MMP-9, MT1-MMP, cathepsin, caspase, human neutrophil elastase, beta-secretase, uPA, and PSA. Examples of cleavable linkers include, but are not limited to, those comprising an amino acid sequence selected from SEQ ID NOs: 55-124.

[0089] Specific, non-limiting examples of cytokine agonist polypeptides, cytokine masks, carriers, peptide linkers, and prodrugs are provided in the sequence section below. Furthermore, the prodrugs and novel IL-2 muteins of the present disclosure can be produced by well-known recombinant techniques. For example, one or more expression vectors containing the coding sequences for the polypeptide chains of the prodrugs can be transfected into mammalian host cells (e.g., CHO cells), and the cells are cultured under conditions that allow expression of the coding sequences and assembly of the expressed polypeptides into prodrug complexes. To ensure that the prodrugs are inactive, host cells that do not express or barely express uPA, MMP-2, and / or MMP-9 can be used. In some embodiments, the host cells can contain null mutations (knockouts) of the genes for these proteases.

[0090] II. Examples of Prodrugs In some embodiments, the prodrugs provided herein comprise an antibody fused to one or two of the above mutant IL-2 polypeptides.

[0091] In some embodiments, the prodrug comprises one mutant IL-2 polypeptide. By way of example, the prodrug comprises two identical light chains having the amino acid sequence of SEQ ID NO: 38 or an amino acid sequence at least 90% identical thereto, a first heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 39 or an amino acid sequence at least 90% identical thereto, and a second heavy chain polypeptide chain comprising the amino acid sequence of SEQ ID NO: 40 or 41 or an amino acid sequence at least 90% identical thereto.

[0092] In some embodiments, the prodrug comprises one mutant IL-2 polypeptide. By way of example, the prodrug comprises two identical light chains having the amino acid sequence of SEQ ID NO: 38, or an amino acid sequence at least 90% identical thereto, a first heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 47, or an amino acid sequence at least 90% identical thereto, and a second heavy chain polypeptide chain comprising the amino acid sequence of SEQ ID NO: 45 or 46, or an amino acid sequence at least 90% identical thereto.

[0093] In some embodiments, the prodrug comprises two mutant IL-2 polypeptides. By way of example, the prodrug comprises two identical light chains having the amino acid sequence of SEQ ID NO: 38, or an amino acid sequence at least 90% identical thereto, and two identical heavy chain polypeptide chains having the amino acid sequence of SEQ ID NO: 40 or 41, or an amino acid sequence at least 90% identical thereto.

[0094] By way of example, the prodrug comprises two identical light chains having the amino acid sequence of SEQ ID NO: 38, or an amino acid sequence at least 90% identical thereto, a first heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 42, 43 or 44, or an amino acid sequence at least 90% identical thereto, and a second heavy chain polypeptide chain of the amino acid sequence of SEQ ID NO: 40, 41 or 125, or an amino acid sequence at least 90% identical thereto.

[0095] By way of example, the prodrug comprises two identical light chains having the amino acid sequence of SEQ ID NO: 38, or an amino acid sequence at least 90% identical thereto, and two identical heavy chain polypeptide chains having the amino acid sequence of SEQ ID NO: 48, 49, 50, or 51, or an amino acid sequence at least 90% identical thereto.

[0096] III. Pharmaceutical Compositions Pharmaceutical compositions comprising the prodrugs and muteins (i.e., active pharmaceutical ingredients or API) of the present disclosure can be prepared by mixing the API having the desired purity with one or more optional pharmaceutically acceptable excipients (see, e.g., Remington's Pharmaceutical Sciences, 16th Edition., Osol, A. Ed. (1980)) in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable excipients (or carriers) are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers containing, for example, phosphate, citrate, succinate, histidine, acetate, or another inorganic or organic acid or its salt; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including sucrose, glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes [e.g., Zn-protein complexes]; and / or non-ionic surfactants, such as polyethylene glycol (PEG)].

[0097] Buffers are used to control the pH within a range that optimizes therapeutic efficacy, particularly when stability is pH-dependent. The buffer is preferably present at a concentration ranging from about 50 mM to about 250 mM. Buffers suitable for use in the present invention include organic and inorganic acids and their salts, such as citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, and acetate. Additionally, buffers may include histidine salts and trimethylamine salts, such as Tris.

[0098] Preservatives are added to inhibit microbial growth and are typically present in the range of 0.2% to 1.0% (w / v). Suitable preservatives for use in the present invention include octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

[0099] Isotonicity agents, sometimes known as "stabilizers," are present to adjust or maintain the tonicity of the liquid in the composition. When used with highly charged biomolecules such as proteins and antibodies, they are often called "stabilizers" because they can interact with the charged groups on amino acid side chains, reducing the likelihood of inter- and intra-molecular interactions. Taking into account the relative amounts of other components, isotonicity agents can be present in any amount between 0.1% and 25% by weight, or more preferably between 1% and 5% by weight. Preferred isotonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0100] The non-ionic surfactant or detergent (also known as a "wetting agent") is present not only to aid in solubilizing the therapeutic agent, but also to protect the therapeutic protein from agitation-induced aggregation, allowing the formulation to be subjected to shear surface stresses without causing denaturation of the active therapeutic protein or antibody. The non-ionic surfactant is present in a range of about 0.05 mg / ml to about 1.0 mg / ml, preferably about 0.07 mg / ml to about 0.2 mg / ml.

[0101] Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (184, 188, etc.), PLURONIC® polyol, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glycerol monostearate, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0102] The choice of pharmaceutical carrier, excipient, or diluent can be selected having regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition can further comprise any suitable binder, lubricant, suspending agent, coating agent, or solubilizing agent.

[0103] There may be different composition / formulation requirements depending on different delivery systems. For example, the pharmaceutical compositions useful in the present invention can be formulated to be administered using a minipump, or by mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestible solution, or parenterally (for example, for delivery by intravenous, intramuscular or subcutaneous route, the composition is formulated in an injectable form).

[0104] In some embodiments, the pharmaceutical composition of the present disclosure is a lyophilized protein formulation, hi other embodiments, the pharmaceutical composition may be an aqueous liquid formulation.

[0105] IV. Treatment Methods The prodrugs and novel IL-2 muteins (mutant IL-2 polypeptides) of the present invention can be used to treat diseases. In some embodiments, the prodrugs or mutant IL-2 polypeptides are used to treat cancer. In some embodiments, the prodrugs or mutant IL-2 polypeptides are used to treat infectious diseases, for example, when the drug molecule is an antibacterial or antiviral agent.

[0106] In some embodiments, a method of treating a disease (e.g., cancer, viral infection, or bacterial infection) in a subject comprises administering to the subject an effective amount of a prodrug or mutant IL-2 polypeptide disclosed herein.

[0107] In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a blood cancer or a solid tumor. Exemplary cancers that can be treated include, but are not limited to, leukemia, lymphoma, kidney cancer, bladder cancer, urinary tract cancer, cervical cancer, brain cancer, head and neck cancer, skin cancer, uterine cancer, testicular cancer, esophageal cancer, liver cancer, colorectal cancer, gastric cancer, squamous cell carcinoma, prostate cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer, cholangiocarcinoma, breast cancer, and ovarian cancer, and medullary thyroid cancer.

[0108] In some embodiments, the prodrug or mutant IL-2 polypeptide is used to treat a bacterial infection, such as sepsis. In some embodiments, the bacteria causing the bacterial infection are drug-resistant bacteria. In some embodiments, the antigen-binding moiety (carrier moiety) disclosed herein binds to a bacterial antigen.

[0109] In some embodiments, the prodrug or mutant IL-2 polypeptide is used to treat a viral infection. In some embodiments, the virus causing the viral infection is hepatitis C virus (HCV), hepatitis B virus (HBV), human immunodeficiency virus (HIV), or human papillomavirus (HPV). In some embodiments, the antigen-binding moieties disclosed herein bind to a viral antigen.

[0110] Generally, the dosage and administration route of the pharmaceutical composition of the present invention are determined according to standard pharmaceutical practice according to the weight and condition of the subject.In some embodiments, the pharmaceutical composition is administered to the subject by any route, including oral, transdermal, inhalation, intravenous, intraarterial, intramuscular, direct application to wound site, application to surgical site, intraperitoneal, suppository, subcutaneous, intradermal, transdermal, nebulization, intrathoracic, intraventricular, intraarticular, intraocular, intracranial or intraspinal.In some embodiments, the composition is administered to the subject intravenously.

[0111] In some embodiments, the dosage of the pharmaceutical composition is a single dose or multiple doses. In some embodiments, the dose is given to the subject once a day, twice a day, three times a day, or four or more times a day. In some embodiments, the dose is given about once a week or more (such as about 2, 3, 4, 5, 6, or 7 or more times). In some embodiments, the pharmaceutical composition is administered weekly, once every two weeks, once every three weeks, once every four weeks, every two weeks out of three weeks, or every three weeks out of four weeks. In some embodiments, multiple doses are given over the course of several days, weeks, months, or years. In some embodiments, the course of treatment is about one or more doses (such as about 2, 3, 4, 5, 7, 10, 15, or 20 or more times).

[0112] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this disclosure. In the case of conflict, the present specification, including definitions, will control. Furthermore, unless the context dictates otherwise, singular terms shall include the plural, and plural terms shall include the singular. Throughout this specification and the embodiments, the words "have" and "comprise," or variations such as "has," "having," "comprises," or "comprising," are understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although several documents are cited herein, this citation is not an admission that any of these documents form part of the general knowledge in the art. As used herein, the term "approximately" or "about" as applied to one or more values ​​of interest refers to a value similar to the stated reference value. In certain embodiments, the term refers to a range of values ​​that falls within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less) of the stated reference value, unless otherwise stated or apparent from the context.

[0113] According to this disclosure, backward references in dependent claims are intended as shorthand for directly and unambiguously disclosing each combination of claims indicated by the backward reference. Furthermore, the headings herein are created for ease of organization and are not intended to limit the scope of the claimed invention in any way.

[0114] V. Illustrative Embodiments Further specific embodiments of the present disclosure are described below, which are intended to illustrate the compositions and methods described in this disclosure and are not intended to limit the scope of the disclosure. 1. A mutant human interleukin-2 (IL-2) polypeptide comprising the mutation L36I (numbering according to SEQ ID NO:1), wherein the IL-2 polypeptide comprises an amino acid sequence that is at least 90% identical to the sequence set forth in SEQ ID NO:1. 2. The mutant human interleukin-2 polypeptide of embodiment 1, wherein the mutant IL-2 further comprises a second mutation C125A (numbering according to SEQ ID NO: 1). 3. The mutant human interleukin-2 polypeptide of embodiment 1 or 2, wherein the mutant IL-2 comprises one or more additional amino acid mutations that eliminate or reduce the affinity of the mutant IL-2 polypeptide for the high-affinity IL-2 receptor and preserve the affinity of the mutant IL-2 polypeptide for the intermediate-affinity IL-2 receptor, respectively, compared to the wild-type IL-2 polypeptide. 4. The mutant interleukin-2 polypeptide of embodiment 3, wherein said additional amino acid mutation is at a position selected from positions corresponding to residues 35, 38, 42, 43, 45, 62, 68, and 72 (numbering according to SEQ ID NO: 1). 5. The mutant interleukin-2 polypeptide of embodiment 1, 2, 3 or 4, wherein the mutant IL-2 further comprises one or more mutations at one or more positions selected from residues 88, 91, 92, and 126 (numbering according to SEQ ID NO: 1). 6. The additional one or more amino acid mutations are selected from the group consisting of R38A, R38K, R38S, F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, E62A, E62L, E62I, E68A, E68V, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, L72K, N 6. The mutant interleukin-2 polypeptide of embodiment 3, 4, or 5, selected from the group of 88D, N88E, N88F, N88H, N88K, N88L, N88M, N88S, N88T, N88V, N88W, N88Y, N88A, V91K, Q126E, Q126A, Q126D, Q126F, Q126G, Q126H, Q126I, Q126K, Q126L, Q126P, Q126S, Q126T, Q126W and Q126Y (numbering according to SEQ ID NO: 1). 7. The mutant interleukin-2 polypeptide of any one of embodiments 1 to 6, wherein the mutant IL-2 further comprises an amino acid mutation that eliminates the O-glycosylation site of IL-2 at a position corresponding to residue 3 of human IL-2 (numbering according to SEQ ID NO: 1). 8. The mutant interleukin-2 polypeptide of embodiment 1, wherein said mutant IL-2 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 2-33. 9. The mutant interleukin-2 polypeptide of any one of embodiments 1 to 8, wherein said mutant IL-2 polypeptide is linked to a carrier. 10. The mutant interleukin-2 polypeptide of embodiment 9, wherein the carrier comprises an antigen-binding portion. 11. An immunoconjugate comprising a mutant IL-2 polypeptide according to any one of embodiments 1 to 8 and an antigen-binding portion. 12. The immunoconjugate of embodiment 11, wherein said immunoconjugate comprises a first and a second antigen-binding portion. 13. The immunoconjugate of any one of embodiments 11 and 12, wherein said antigen-binding moiety is an antibody or an antibody fragment. 14. The immunoconjugate of embodiment 11 or 12, wherein said antigen-binding portion is selected from a Fab molecule and an scFv molecule. 15. The immunoconjugate of any one of embodiments 11 and 12, wherein said antigen-binding moiety is an immunoglobulin molecule, in particular an IgG molecule. 16. The immunoconjugate of any one of embodiments 11 to 15, wherein the antigen-binding portion is directed against an antigen presented on a tumor cell or in the tumor cell environment. 17. The immunoconjugate of any one of embodiments 11 to 15, wherein the antigen-binding portion is directed against an antigen presented on an immune cell. 18. The immunoconjugate of any one of embodiments 11 to 15, wherein the antigen-binding portion is directed against an antigen presented on T cells, NK cells, or macrophages. 19. The immunoconjugate of embodiment 16, wherein said antigen is selected from the group of fibroblast activation protein (FAP), A1 domain of tenascin-C (TNC A1), A2 domain of tenascin-C (TNC A2), fibronectin extradomain B (EDB), carcinoembryonic antigen (CEA), PSA, 5T4, PDL1, CD47, HER2, CD20, CD38, BCMA, EGFR, CMET, claudin 18.2, claudin 6, CD24, and melanoma-associated chondroitin sulfate proteoglycan (MCSP). 20. The immunoconjugate of embodiment 17 or 18, wherein said antigen is selected from the group of PD-1, PDL1, CD8, Tim-3, LAG-3, TIGIT, SIRP alpha, CTLA-4, CSF1R, NKG2A, NKG2D, CD16A, NKp30, and NKp46. 21. The immunoconjugate of embodiment 12, wherein said antigen is selected from the group consisting of PD-1, CD8, Tim-3, LAG-3, TIGIT, SIRPalpha, CTLA-4, CSF1R, NKG2A, NKG2D, CD16A, NKp30, NKp46, fibroblast activation protein (FAP), the A1 domain of tenascin-C (TNC A1), the A2 domain of tenascin-C (TNC A2), fibronectin extradomain B (EDB), carcinoembryonic antigen (CEA), PSA, 5T4, PDL1, CD47, EGFR, CMET, claudin 18.2, claudin 6, CD24, and melanoma-associated chondroitin sulfate proteoglycan (MCSP). 22. The mutant IL-2 polypeptide or immunoconjugate according to any one of embodiments 1 to 21, wherein said IL-2 polypeptide is masked. 23. The mutant IL-2 polypeptide or immunoconjugate of any one of embodiments 1 to 21, wherein said IL-2 polypeptide is masked by a masking moiety, and said masking moiety comprises the extracellular domain (ECD) of IL-2 receptor beta (IL-2Rβ) or a functional fragment thereof. 24. The mutant IL-2 polypeptide or immunoconjugate of embodiment 23, wherein said IL-2Rβ-ECD comprises the amino acid sequence of SEQ ID NO: 37, or an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO: 37. 25. The mutant IL-2 polypeptide or immunoconjugate of any one of embodiments 1 to 21, wherein the IL-2 polypeptide is masked by a masking moiety, the masking moiety comprises an scFv or Fab, and the scFv or Fab binds to the IL-2 polypeptide and inhibits the biological activity of the IL-2 polypeptide. 26. The mutant IL-2 polypeptide or immunoconjugate of embodiment 23, wherein the masking moiety is an scFv comprising the amino acid sequence of SEQ ID NO: 34, 35, or 36, or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 34, 35, or 36. 27. The mutant IL-2 polypeptide or immunoconjugate of embodiment 23, wherein the masking moiety comprises an scFv or Fab comprising the same heavy chain CDRs and the same light chain CDRs as those of an scFv comprising the amino acid sequence of SEQ ID NO: 34, 35, or 36. 28. The mutant IL-2 polypeptide or immunoconjugate according to any one of embodiments 23 to 27, wherein the masking moiety further comprises a cleavable peptide linker. 29. The immunoconjugate of embodiment 17 or 28, wherein the antigen-binding portion is an antibody or binding fragment thereof against PD-1; and the anti-PD-1 antibody is selected from nivolumab and pembrolizumab. 30. An immunoconjugate comprising two identical light chains and a first heavy chain polypeptide chain and a second heavy chain polypeptide chain; said light chain comprising the amino acid sequence of SEQ ID NO: 38, or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 38; said first heavy chain polypeptide chain comprising an amino acid sequence selected from SEQ ID NOs: 40, 41, 44, and 45, or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 40, 41, 44, or 45; and said second heavy chain polypeptide chain comprising the amino acid sequence of SEQ ID NOs: 42, 43, 46, and 47, or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 42, 43, 46, or 47. 31. An immunoconjugate comprising two identical light chains and two identical heavy polypeptide chains; said light chains comprising the amino acid sequence of SEQ ID NO: 38, or an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 38; and said heavy polypeptide chains comprising an amino acid sequence selected from SEQ ID NOs: 40, 41, 48, 49, 50, and 51, or an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 40, 41, 48, 49, 50, or 51. 32. The immunoconjugate of embodiment 17 or 28, wherein the antigen-binding portion is an antibody to CD8 or a binding fragment thereof; and the anti-CD8 antibody is OKT8 or humanized OKT8, or comprises the same heavy chain CDRs and the same light chain CDRs as those derived from OKT8. 33. The immunoconjugate of embodiment 32, wherein the antigen-binding portion further comprises an additional antigen-binding portion that binds to an antigen on a tumor cell or in the tumor cell environment. 34. The immunoconjugate of embodiment 33, wherein the tumor-associated antigen is selected from the group consisting of fibroblast activation protein (FAP), A1 domain of tenascin-C (TNC A1), A2 domain of tenascin-C (TNC A2), fibronectin extradomain B (EDB), carcinoembryonic antigen (CEA), PSA, 5T4, PDL1, CD47, HER2, CD20, CD38, BCMA, EGFR, CMET, claudin 18.2, claudin 6, CD24, and melanoma-associated chondroitin sulfate proteoglycan (MCSP). 35. An isolated polynucleotide encoding the mutant IL-2 polypeptide or immunoconjugate of any one of embodiments 1 to 34. 36. An expression vector comprising the polynucleotide of embodiment 35. 37. A host cell comprising a polynucleotide according to embodiment 35 or an expression vector according to embodiment 36. 38. A method for producing a mutant IL-2 polypeptide or an immunoconjugate thereof, comprising culturing a host cell according to embodiment 37 under conditions suitable for expression of the mutant IL-2 polypeptide or immunoconjugate. 39. A mutant IL-2 polypeptide or immunoconjugate produced by the method of embodiment 38. 40. A pharmaceutical composition comprising a mutant IL-2 polypeptide or immunoconjugate according to any one of embodiments 1 to 34 or 39 and a pharmaceutically acceptable carrier. 41. A mutant IL-2 polypeptide or immunoconjugate according to any one of embodiments 1 to 34 or 39 for use in treating a disease in an individual in need thereof. 42. The mutant IL-2 polypeptide or immunoconjugate of embodiment 41, wherein the disease is cancer. 43. Use of a mutant IL-2 polypeptide or immunoconjugate according to any one of embodiments 1 to 34 or 39 for the manufacture of a medicament for treating a disease in an individual in need thereof. 44. A method of treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of a composition comprising a mutant IL-2 polypeptide or immunoconjugate of any one of embodiments 1 to 34 or 39, in a pharmaceutically acceptable form. 45. The method of embodiment 44, wherein the disease is cancer. 46. ​​A method of stimulating the immune system of an individual, comprising administering to the individual an effective amount of a composition comprising a mutant IL-2 polypeptide or immunoconjugate of any one of embodiments 1 to 34 or 39, in a pharmaceutically acceptable form. 47. A mutant IL-2 polypeptide or immunoconjugate according to any one of embodiments 1 to 34 or 39, which has a reduced risk of immunogenicity when administered to an animal or patient compared to a corresponding IL-2 polypeptide or immunoconjugate comprising the amino acid L at position 36 (numbering according to SEQ ID NO: 1). 48. The immunoconjugate of embodiment 32, wherein said CD8 antibody comprises a light chain variable domain of SEQ ID NO: 52, or a light chain variable domain that is at least 90% identical to the light chain variable domain of SEQ ID NO: 52, and a heavy chain variable domain of SEQ ID NO: 53 or 54, or a heavy chain variable domain that is at least 90% identical to the sequence set forth in SEQ ID NO: 53 or 54. 49. The immunoconjugate of embodiment 28, 29, 32, 33, 34, or 48, comprising one or more cleavable peptide linkers; wherein said cleavable linkers comprise an amino acid sequence selected from SEQ ID NOs: 55-124.

[0115] In order that the present invention may be better understood, the following examples are set forth, which are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. [Example]

[0116] [Example]

[0117] Discovery of IL-2 mutant polypeptides with reduced immunogenicity Several human IL-2 mutants were generated, including IL-2V1 / L53I, IL-2V1 / L56I, IL-2V1 / Y45G, IL-2V1 / Y45G / L53I, IL-2V1 / Y45G / L56I, IL-2V1 / L70I, IL-2V1 / L72I, IL-2V1 / Y45G / L80I, and IL-2V1 / Y45G / L118I (all mutations relative to SEQ ID NO: 1). None of these mutations eliminated the immunogenic "hot spot" introduced by the "non-alpha" mutations (data not shown). Surprisingly, introduction of the L36I mutation eliminated most of the "hot spot" introduced by the "non-alpha" mutations (Table 4).

[0118] [Table 4]

[0119] An additional "hot spot" introduced by the C125S mutation was removed using a C125A mutation instead (Table 5). Sequence numbering / amino acid residue positions are according to SEQ ID NO: 1.

[0120] [Table 5]

[0121] Surprisingly, IL-2V1 / L36I / C125A (SEQ ID NO: 184) was shown to pose a lower immunogenic risk than wild-type IL-2. Side-by-side comparisons showed that this IL-2 mutant had fewer "hot spots" than wild-type IL-2 (Table 6; additional "hot spot" binding peptides in wild-type IL-2 are in bold; SEQ ID NO: 184).

[0122] [Table 6] [Example]

[0123] Discovery of an scFv molecule that binds to IL-2 and inhibits the interaction between IL-2 and IL-2Rβγ A. Screening of scFv from the yeast library A human scFv yeast display library was screened for IL-2 binders. IL-2 was labeled with biotin from avi-tag (Acro Biosystems). After two initial rounds of magnetic-activated cell sorting (MACS) using 200 nM biotinylated IL-2 and streptavidin beads and fluorescence-activated cell sorting (FACS) using 200 nM biotinylated IL-2 and APC-streptavidin, the enriched pool was further screened for scFv binders that block the IL-2 / IL-2Rβγ interaction. Single clones were then plated onto six 96-well plates. An iQue® screen was further performed using both 100 nM IL-2 and 100 nM biotinylated IL-2Rα for scFv binders that do not block the IL-2Rα interaction. A second round of iQue® screening was performed using both 100 nM biotinylated IL-2 and 100 nM Fc-IL-2βγ for scFv binders that blocked IL-2Rβγ interactions. Sixty-eight yeast clones possessing both characteristics were collected for further analysis. Culture supernatants of these clones were collected and tested for binding to IL-2 and blocking IL-2Rβγ function using Fortebio. Thirty-six scFv candidates were cloned, fused to an Fc fragment, and expressed in ExpiCHO cells. Purified scFv-Fc homodimers were further characterized using Fortebio and a CTLL2 neutralization assay.

[0124] B. Blocking assay for IL-2 binders selected by screening from a human ScFv yeast library Supernatants from candidate clones were tested for their ability to block IL-2 binding to IL2Rβγ. Blocking was tested using the Octet® Red96e Kinetics binding assay. Biotinylated IL-2Rβγ was loaded onto a Streptavidin (SA) sensor at 1 μg / ml. The binding affinity of IL-2 was first confirmed by binding three-fold serial dilutions of Fc-IL-2 (JR8_48.3, containing SEQ ID NOs: 261 and 262, starting at 1 μg / ml) to the SA sensor loaded with IL-2Rβγ (FIG. 1A). Fc-IL-2 (JR8.48.3, containing SEQ ID NOs: 261 and 262) was added to the SA sensor loaded with IL-2Rβγ at 1 μg / ml, 0.3 μg / ml, and 0.1 μg / ml.

[0125] A one-to-one fitting curve was applied to the kinetic results to obtain the indicated KDs for the corresponding binding patterns. Addition of 100 μl of a control Fc-scFv (JR8.113.3, SEQ ID NO: 249) to each of the IL-2 dilutions resulted in complete blocking of IL-2 binding to IL-2Rβγ (Figure 1B). Addition of 100 μl of yeast culture supernatant from clone K1-69A8 to each dilution of Fc-IL-2 partially blocked IL-2 binding to IL-2Rβγ (Figure 1C). In contrast, the supernatant from clone K3-23A2 did not block IL-2 binding to IL-2Rβγ (Figure 1D). The results of the blocking assays of scFv clones from the yeast library are summarized in Table 7.

[0126] [Table 7]

[0127] C. CTLL2 cell-based activity assay Nine scFvs (Table 7, numbers 1 to 9) were cloned and fused to a human Fc domain. The fusion proteins were expressed as homodimers in Expi293 cells and purified by ProA chromatography. The purified proteins were tested in a cell-based inhibition assay using the CTLL2 cell line (Figure 2).

[0128] CTLL2 cells were grown in RPMI 1640 medium supplemented with L-glutamine, 10% fetal bovine serum, 10% non-essential amino acids, 10% sodium pyruvate, and 55 μM β-mercaptoethanol. CTLL2 cells were nonadherent and grown at 5 × 10 cells / well in medium containing 100 ng / ml IL-2. 4 ~1×10 6 Cells were maintained at 5000 cells / ml. Cells were typically split twice weekly. For bioassays, cells were optimally used at least 48 hours post-passage. Samples were diluted 2x in 50 μl / well in 96-well plates. IL-2 standards were titrated from 20 ng / ml (2x concentration) across 12 wells of 3-fold serial dilutions. Samples were titrated accordingly. CTLL2 cells were washed five times to remove IL-2, dispensed at 5000 cells / well in 50 μl, and cultured with samples overnight or for at least 18 hours. Then, 100 μl / well of Cell Titer Glo® Reagent (Promega) was added, and luminescence was measured.

[0129] During the CTLL2 assay, 50 nM of IL-2 was added to CTLL2 cells. Purified scFv-Fc fusion proteins were added to the cells along with a positive control (JR8.113.3). The dose-dependent inhibition of IL-2 activity by the purified fusion proteins is shown in the figure. The scFv-Fc homodimers K1-69-C7, K1-69-H5, and K3-30-H7 exhibited strong inhibitory effects on IL-2, with IC values ​​of 7.0 nM, 11.3 nM, and 10.2 nM, respectively. 50 (Figure 2).

[0130] Nine additional scFv-Fc (Table 7, numbers 10 to 18) plasmids were constructed, expressed in ExpiCHO™ cells, and purified using ProA chromatography. Four of the scFv-Fc failed to express. The purified scFv-Fc were tested in the CTLL2 assay for neutralization of 50 nM PD1-IL-2v (Figures 3A and 3B). Their inhibitory activity was compared to that of clone K1-69-C7. Several additional clones potently inhibited IL-2 activity. L4-39-B9, K1-69-A8, K3-23-A2, K3-30-G9, and K3-30-H9 showed potent neutralization, with IC values ​​of 100 and 150, respectively. 50 = 6.2 nM, 11.8 nM, 28.5 nM, 15.4 nM and 4.4 nM.

[0131] The sequences of scFv clones selected from the CTLL2 cell-based activity assay are shown in SEQ ID NOs: 34-36 and 250-270. [Example]

[0132] Binding affinity of anti-IL-2 ScFv-Fc The binding affinity of selected scFv-Fc to IL-2 was tested using Fortebio (Figures 4A and 4B). The results are summarized in Table 8.

[0133] [Table 8] [Example]

[0134] Expression and activity testing of PD-1 antibody-IL-2 prodrug molecules CTLL2 Assay. The scFvs C7, H5, and B12 were fused to the anti-PD1 antibody HC to mask mutant human IL-2 (IL-2v), as shown in the diagram in Figure 5C. The molecules were transiently expressed in the ExpiCHO™ system as shown in Figure 5A and purified by ProA affinity chromatography. The purified proteins were activated by hMMP2 digestion, and their IL-2 activity was tested in a CTLL2 assay (Figure 5B). Similar to β-ECD, the C7, H5, and B12 scFvs were all able to mask IL-2v activity, and the IL-2v activity of each prodrug was restored after activation with hMMP2 (Figure 5C). Specifically, the prodrug molecules JR11.20.3, JR11.20.6, and JR11.20.7, which have masked C7 (or K1-69C7), H5, and β-ECD, showed little activity before activation and significantly improved activity after protease-based activation (Figure 5C).

[0135] NK92 Assay. In a separate experiment, a total of eight prodrug molecules (Figure 6A) were expressed and purified by protein A affinity chromatography. The prodrugs before and after protease digestion were tested by NK92 cell-based activity assay. Briefly, NK92 cells were grown in RPMI 1640 medium supplemented with L-glutamine, 10% fetal bovine serum, 10% non-essential amino acids, 10% sodium pyruvate, and 55 pM β-mercaptoethanol. NK92 cells were nonadherent and cultured at 1 x 10 cells / mL in medium containing 100 ng / ml IL-2. 5 ~1×10 6The cells were maintained at 100 cells / ml. Typically, cells were split twice weekly. For bioassays, cells aged 48 hours or more were optimally used. Purified proteins were activated by digestion with hMMP2 and analyzed by SDS-PAGE (Figure 6B). IL-2 activity was tested in the NK92 assay (Figure 6C). K1-69C7, K3-30F7, K1-69A8, K3-30H9, and L4-39B9 strongly masked IL-2v. K3-30G9 and K3-30-B12 partially masked IL-2 activity. K3-30F6 did not. [Example]

[0136] Linker optimization Linkers between the anti-PD-1 antibody HC and the scFv portion of an anti-PD-1 antibody-carried prodrug were tested in Example 4. Both the linkers GGGGSGGGGSGPLGVRGGGGSGGGGS (SEQ ID NO: 268) (JR11.20.3) and GGGGSGGGGSGPLGVRGGGGS (SEQ ID NO: 269) (JR11.20.5) performed well in molecular assembly and function.

[0137] To optimize the production and purity of the prodrug molecules, we tested prodrug molecules with various linker lengths between the anti-PD1 antibody HC and IL-2v moieties (Figure 7A). The linker between the heavy chain (HC) and cytokine moiety was GGGGSGGGGSGGGGS (SEQ ID NO: 181), i.e., a 3x G4S linker; GGGGSGGGGS (SEQ ID NO: 180), i.e., a 2x G4S linker; or GGGGS (SEQ ID NO: 178), i.e., a 1x G4S linker; or no peptide linker was present. The molecules were transiently expressed in the ExpiCHO™ system and purified by ProA affinity chromatography. The purified proteins were analyzed by SDS-PAGE gel and SEC-HPLC (Figure 7B). The data showed that molecules with short (2x G4S or 1x G4S) or no peptide linker had higher main peak purity (improved purity) and reduced aggregation. The 2x G4S linker was selected for subsequent prodrug design. [Example]

[0138] Ex vivo assay using human PBMCs Human PBMCs were first cultured in 6-well plates containing anti-CD3 antibody (1 μg / ml) (pre-coated) and anti-CD28 antibody (1 μg / ml) for 72 hours to induce PD-1 expression on T cells. PBMCs were collected, washed, resuspended in RPMI 1640 complete medium, and incubated on ice for 4 hours. The rested cells were divided into two portions. PBMCs used in experiments / curves 1a-6a were further pretreated with 40 μg / ml PD-1 antibody to internalize PD-1 expressed on T cells. A portion of the cells was incubated with anti-PD1 antibody (56 μg / ml) on ice for 45 minutes to mask PD-1 expression. After incubation with anti-PD1 antibody, the cells were washed to remove free anti-PD1 antibody. Activated PBMCs, with or without anti-PD1 antibody pretreatment, were incubated with serially diluted test articles in a 96-well plate at 37°C for 15 minutes. The cells were then immediately fixed with BD Cytofix™ fixation buffer and permeabilized with BD Phosflow™ Perm Buffer IV (0.5x), followed by incubation with PE-conjugated anti-p-Stat5 antibody.

[0139] IL-2 induces STAT5 phosphorylation (pSTAT5) in immune cells. Stat5 phosphorylation was measured in CD4+ T cells, CD8+ T cells, and CD3-CD56+ NK cells by flow cytometry. Data were graphed using GraphPad Prism 9 software. PBMCs were then treated with anti-CD3 and anti-CD8 antibodies to induce PD-1 expression in T cells. These results are shown in Figure 8B for CD4+ T cells, Figure 8C for CD8+ T cells, and Figure 8D for NK cells. A C7-masked PD-1 antibody-IL-2 prodrug (ASKG812K-C7) showed very low activity with the cells, regardless of pretreatment with the PD-1 antibody. However, the PD-1 antibody-IL-2 prodrug masked by C7 showed strong activity after activation (ASKG812K-C7 Act; Tests 2 and 2a in Figures 8B, 8C, and 8D) in all cell types, but the activity was significantly higher in T cells than in NK cells. Furthermore, T cells not pretreated with a PD-1 antibody showed significantly higher activity than cells pretreated with a PD-1 antibody. Treatment with anti-CD3 and anti-CD8 induced PD-1 expression in T cells. Pretreatment with a PD-1 antibody led to the internalization of PD-1 expressed on T cells. Our results indicate that the PD-1 antibody-IL-2 prodrug selectively activated PD-1-expressing T cells after activation. This T cell targeting was likely achieved by a "cis-activation" mechanism, i.e., the activated prodrug bound to T cells through both the PD-1-binding domain and the cytokine domain. Similar observations were made using a reference PD-1 antibody-IL-2 fusion reference molecule (Tests 3 and 3a in Figures 8B and 8C). This was not observed with NK cells, as NK cells typically do not express high levels of PD-1 (Tests 3 and 5a in Figures 8B and 8C). "Cis-activation" was not observed with PD-1 antibodies (Tests 5 and 5a in Figures 8B and 8C) or Fc-IL-2v molecules (Tests 4 and 4a in Figures 8B and 8C).We observed that the prodrugs masked by scFv G3 (Tests 6 and 6a in Figures 8B and 8C) exhibited "cis-activation" toward both CD4+ and CD8+ T cells, even though they did not contain a non-cleavable linker. [Example]

[0140] Improving the heat resistance of prodrug molecules Thermal stability testing of prodrug molecules was performed using an Unchained Labs UNcle instrument. Briefly, 8.8 μL of protein sample was loaded in triplicate into a microcuvette ("Uni"). A temperature scan was performed from 25 °C to 95 °C at a rate of 1 °C / min, and intrinsic fluorescence and static light scattering (SLS) were measured. The results (analyzed using UNcle Analysis 4.01) for triplicate runs of Tm (melting point), Tonset (onset of unfolding), Tagg-266 nm (onset of aggregation detected at 266 nm), and Tagg-473 nm (onset of aggregation detected at 473 nm) were manually inspected and averaged after removing outliers. Surprisingly, the results showed that prodrugs masked with mask C7 (ASKG812K-C7) exhibited significantly higher thermal stability than prodrugs masked with other masks (Figure 9B). The Tonset and Tm1 were 10°C and 12°C higher than those masked with IL-2Rβ-ECD, and the Tagg was 22°C higher than those masked with IL-2Rβ-ECD. The prodrugs masked with F7 and G3 also showed higher Tagg than those masked with IL-2Rβ-ECD.

[0141] Accelerated stability studies of the prodrugs were also performed. The purity of samples stored at 25°C and 40°C was tested by SEC-HPLC. Briefly, 3 μg of sample was injected into a size-exclusion chromatography system equipped with a Phenomenex BioZen® dSEC-2 column (3 μm particles, 4.6 × 300 mm, part number 00H-4788-E0) on an Agilent 1260 UHPLC system controlled by Chromeleon 7.2 software. The column temperature was set at 30°C. The mobile phase was 200 mM potassium phosphate, pH 6.2, containing 250 mM potassium chloride, and was run at 0.35 mL / min. Protein signals were detected by UV at 220 nm. Surprisingly, the C7-masked prodrug showed no decrease in its main peak percentage, while all other prodrugs showed a significant decrease in their main peak percentage (Figure 9C). Both the F7 and G3 masked prodrugs were more stable than those masked with IL-2Rβ-ECD. IL-2 was less thermostable and prone to aggregation during storage under accelerated conditions. Mask C7 could potentially increase the Tm of IL-2 by more than 10°C, significantly enhancing the stability of IL-2. [Example]

[0142] MaskF7 can mask IL-2v with the L36I mutation The PD-1 antibody-IL-2v reference molecule (EB01-08), PD-1 antibody-IL-2v (LL24-68), PD-1 antibody-IL-2v / L36I (JR11.145.2), and a prodrug containing PD-1 antibody-IL-2v / L36I masked with F7 (JR11.145.4 masked) were expressed and purified. The samples were tested using the CTLL2 assay. Sample information is shown in Figure 10A. The results are shown in Figure 10B. The data showed that PD-1 antibody-IL-2v / L36I had similar activity to the reference molecule. IL-2v / L36I and the prodrug with mask F7 showed little activity in the CTLL2 assay, indicating that mask F7 can efficiently mask IL-2v / L36I. Introducing the L36I mutation reduces the potential risk of immunogenicity, but the mutation does not prevent masking by F7. [Example]

[0143] Construction, expression, and ex vivo activity analysis of antibody-cytokine fusion molecules Several antibody-IL-2 cytokine fusion molecules (ASKG222) were designed with the structures shown in Figures 11A-11F. The IL-2 in the fusion molecules was designed to have no or little binding activity to IL-2Rβ, but most retained affinity for IL-2Rα and IL-2Rγ. This class of molecules was designed to selectively stimulate Tregs over Teff cells and NK cells.

[0144] A comparison of the C-terminus and N-terminus of the K1-69C7 Fc fragment fusion is shown in Figures 12A-12B. The Fc fusion molecules (shown in Figures 10A and 10B and Figure 12A) were expressed in ExpiCHO cells and purified on a Protein A affinity column. The SEC-HPLC purity of the two molecules in Figure 12A is shown in Figure 12B.

[0145] An antibody containing the same VH and VL domains as scFv K1-69C7 was constructed and fused to an IL-2 polypeptide (Figure 13A). The antibody-cytokine fusion molecule was expressed in ExpiCHO cells and purified on a Protein A affinity column. The SEC-HPLC purity of the two molecules in Figure 13A is shown in Figure 13B.

[0146] Three samples, ASKG222C7-C, ASKG222C7-N, and ASKG222A-C7-ab, were further purified and tested in ex vivo assays. Reference samples (R1, an analog of AMG592, and R2, an analog of PT101), wild-type IL-2, and a negative control (human IgG) were also included. To examine the ability of the antibody-cytokine fusion molecules to selectively stimulate the expansion of Treg cells, an ex vivo assay was performed using human PBMCs. Induction of Ki67 was used as the readout for this activity assay.

[0147] Briefly, ASKG222 molecular assay dilutions were prepared, and 100 μl of each sample was added to plate wells. 100 μl of PBMCs (400–500 k cells / well) were added to wells containing the diluted test article and cultured for 3 days. For cell surface staining, cells were stained with different fluorochrome-conjugated anti-CD3, anti-CD4, anti-CD8, anti-CD25, and anti-CD161a antibodies. CD3-CD161a+ cells were defined as NK cells. Cells were further treated with Thermo Fisher Invitrogen Foxp3 fixation / permeabilization buffer and stained with anti-Foxp3 and anti-Ki67. Samples were analyzed by flow cytometry, and data were graphed using GraphPad Prism 9 software. IL-2 was added as a positive control, and human IgG (hIgG) was added as a negative control. Two reference molecules, R1 (an analog of Amgen's AMG592) and R2 (an analog of Padion Therapeutics' PT101), were also included. The results are shown in Figure 14A (Treg cells) and Figure 14B (Teff cells). The results indicated that ASKG222C7-C and ASKG222C7-N were more selective than R1 for stimulating Treg cells over Teff cells, while ASKG222A-C7-ab was more selective for Treg cells than R2. Both R1 and R2 are currently in clinical trials for autoimmune indications. [Example]

[0148] PK / PD in rats Three male Sprague-Dawley rats, aged 53-58 days, were injected with purified ASKG222A-C7-ab (Lot No. LL28-149) at 2 mg / kg IV via the jugular vein cannula (Envigo). Blood samples were collected at time point (TO) and 1, 2, 4, 6, 10, 24, 48, 72, 96, 120, and 144 hours post-dose for PK determination. Flow cytometric PD analysis was performed on the TO, 72, and 120 hour samples, and plasma cytokine levels were determined for the TO, 24, 48, 72, 96, 120, and 144 hour samples.

[0149] Test article concentrations in plasma were determined by ELISA. ELISA plates were coated with 100 μL / well of 2 μg / mL F(ab')2 goat anti-human IgG Fcγ (Jackson Immuno-Research, no. 109-006-170) in PBS and test article was captured overnight at 4°C. Plates were blocked with 100 μL of PBS / 10% goat serum for 1 hour and washed with DI water. Plasma samples were serially diluted 1:10 in 3 x 8 wells, and test article standards were diluted 2 μg / mL in PBS / 10% goat serum in 100 μL / well in 3 x 12 wells. Plates were incubated for 1 hour and washed with DI water. For test article detection, 100 μL / well of anti-IL2 biotin (eBioscience, #13-7028-85) was added at 0.5 μg / mL in PBS / 10% goat serum, incubated for 1 hour, and washed with DI water. Streptavidin-HRP (Jackson Immuno-Research) 1:1000, 100 μL / well, was added in PBS / 10% goat serum and incubated for 1 hour. After washing with DI water, TMB substrate (Thermo Scientific) was added at 100 μL / well. Color development was stopped with 100 μL of H2SO4, and OD 450 was measured using a microplate spectrophotometer (Molecular Devices). Figure 15 shows the PK results of ASKG222A-C7-ab. Three rats were administered a single IV dose of 2 mg / kg. Drug concentrations in serum samples were shown at each time point and measured by ELISA. The data indicate that the half-life of the molecule is approximately 68 to 88 hours, which is typical for an antibody. [Example]

[0150] In vivo efficacy studies using the Colon 26 syngenic tumor model In vivo efficacy studies were performed using the colon 26 syngenic tumor model. Briefly, 7-8 week old female Balb / C mice were inoculated with 5 × 10 5 Colon 26 tumor cells were implanted and delivered subcutaneously across the abdomen. The average tumor volume was approximately 82 mm 3Once tumor volume reached 100 μL, mice were randomly assigned to treatment groups so that the mean tumor volume in each group was approximately the same. Mice were treated twice with the test article or vehicle by intraperitoneal injection in 100 μL of PBS, 3 days apart. Tumor volume was calculated by measuring the longest diameter (a) and the shortest diameter (b) and calculating the tumor volume using the formula: ab 2 The volume was calculated using π / 6.

[0151] The fusion molecule 812mN-mut4 contains two identical light chains of an anti-mouse PD-1 antibody and two identical heavy polypeptide chains, which contain, from N- to C-terminus, the heavy chain of the anti-mouse PD-1 antibody, an IL-2-binding mask, and an IL-2 mutein. The structure of this molecule is shown in Figure 16A, and its sequence information is shown in Figure 16D.

[0152] The fusion molecule 812mW5-mut4 has the structure shown in Figure 16B. 812mN-mut4 is similar to 812mN-mut4, except that 812mN-mut4 contains only one mask and one IL-2 mutein at the C-terminus of the heavy chain of the anti-mouse PD-1 antibody.

[0153] Ref3 is an anti-PD-1 antibody-IL-2 mutein fusion molecule with the structure shown in Figure 16C. It is the murine analog of eciskafusp alfa, which is currently in clinical development.

[0154] All three molecules share the same anti-PD-1 antibody as the mPD-1 molecule. 812mN-mut4 contains two copies of the masked IL-2 mutein. The human version of the fusion molecule 812mN-mut4 (i.e., 812KN-mut4, with an anti-human PD-1 antibody) exhibited stronger in vitro cell-based bioactivity than the human version of 812mW5-mut4 (812KW5-mut4), which contains only one copy of the same masked IL-2 mutein (Figure 20). The IL-2 mutein in Ref3 is unmasked. It has the strongest in vitro cell-based IL-2 activity of the three molecules (Figure 20). Therefore, 812KW5-mut4 is less active than 812KN-mut4 and the reference molecule Ref3. The IL-2 site is the same in the human and mouse versions.

[0155] The in vivo efficacy and safety results of two chimeric molecules, Ref3 and an anti-mouse PD-1 antibody, are shown in Figures 17A and 17B. Surprisingly, 812mW5-mut4 has similar antitumor efficacy to Ref3 (Figure 17A), but is safer than Ref3, as significant weight loss was observed with Ref3, whereas no weight loss was observed with 812mW5-mut4 (Figure 17B). Furthermore, the observation that 812mW5-mut4 has stronger in vivo efficacy than 812mN-mut4 was surprising, as the latter was predicted to have stronger in vitro cell-based activity. [Example]

[0156] In vivo efficacy studies using the CT26 syngenic tumor model In vivo efficacy studies were performed using the CT26 syngenic tumor model. Briefly, female 7-8 week old Balb / C mice were inoculated with 1 × 10 6 CT26 tumor cells were implanted and delivered subcutaneously across the abdomen. The average tumor volume was approximately 94 mm 3Once tumor volume reached 100 mL, mice were randomly assigned to treatment groups so that the mean tumor volume in each group was approximately the same. Mice were treated twice with the test article or vehicle by intraperitoneal injection in 100 mL of PBS, 3 days apart. Tumor volume was determined by measuring the longest diameter (a) and the shortest diameter (b), where volume = ab 2 Calculated using the π / 6 formula.

[0157] 678F3-Fab-B and Ref3 were tested in the CT26 model. 678F3-Fab-B at doses of 9.93 mg / kg and 29.8 mg / kg demonstrated superior efficacy and safety compared to Ref3 (Figures 19A and 19B). Sequence information for 678F3-Fab-B and Ref3 is shown in Figure 18. An example structure of 678F3-Fab-B is shown in Figure 21.

[0158] array In the sequences below, boxed residues indicate mutations. Underlined sequences in the cleavable linker indicate protease substrate sequences. Underlined and bolded sequences refer to CDRs. [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5] [Table 9-6] [Table 9-7] [Table 9-8] [Table 9-9] Table 9-10 Table 9-11 Table 9-12 Table 9-13 Table 9-14 Table 9-15 Table 9-16 Table 9-17 Table 9-18 Table 9-19 Table 9-20 Table 9-21 Table 9-22 Table 9-23 Table 9-24 Table 9-25 Table 9-26 Table 9-27 Table 9-28 Table 9-29 Table 9-30 Table 9-31 Table 9-32 Table 9-33 Table 9-34 Table 9-35 Table 9-36 Table 9-37

Claims

1. A mutant human IL-2 polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO:1 and an L36I mutation relative to SEQ ID NO:

1.

2. 10. The mutant human IL-2 polypeptide of claim 1, further comprising one or more additional mutations that reduce the binding affinity of the polypeptide to CD25.

3. 2. The mutant human IL-2 polypeptide of claim 1, further comprising a C125A mutation relative to SEQ ID NO:

1.

4. T3, where the mutation may be N3A; D20, the mutation of which may be D20H, D20K, D20L, D20M, D20N, D20Q, D20R, D20S, D20V, or D20Y; R38, where the mutation may be R38A, R38K, or R38S; F42, the mutation of which may be F42A, F42G, F42I, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, or F42K; Y45, which mutation may be Y45A, Y45G, Y45I, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, or Y45K; E62, the mutation of which may be E62L, E62A, or E62I; E68, where the mutation may be E68V; L72, where the mutation may be L72G; A73, where the mutation may be A73T; N88, where the mutation may be N88A, N88E, N88F, N88H, N88K, N88T, N88L, N88M, N88S, N88V, N88W, or N88Y; N90, where the mutation may be N90T; V91, the mutation of which may be V91K, V91A, V91H, or V91R; I92; and Q126 (numbering according to SEQ ID NO: 1), wherein the mutation may be Q126A, Q126D, Q126F, Q126G, Q126H, Q126I, Q126K, Q126L, Q126P, Q126S, Q126T, Q126W, or Q126Y.

4. The mutant human IL-2 polypeptide of any one of claims 1 to 3, further comprising one or more additional mutations at positions selected from:

5. 5. The mutant human IL-2 polypeptide of any one of claims 1 to 4, comprising an amino acid sequence selected from SEQ ID NOs: 3 and 5 to 33, or an amino acid sequence at least 95% identical thereto.

6. A mutant human IL-2 polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:2 or SEQ ID NO:

4.

7. An anti-IL-2 antibody or antigen-binding fragment thereof, comprising heavy chain CDR1-3 of SEQ ID NOs: 170-172, respectively, and light chain CDR1-3 of SEQ ID NOs: 173-175, respectively.

8. A light chain variable domain (V) comprising an amino acid sequence of SEQ ID NO: 190 or at least 95% identical thereto. Ｌ ), and a heavy chain variable domain (V) comprising an amino acid sequence of SEQ ID NO: 191 or at least 95% identical thereto Ｈ ); V comprising an amino acid sequence of SEQ ID NO: 192 or at least 95% identical thereto Ｌ and V comprising an amino acid sequence of SEQ ID NO: 193 or at least 95% identical thereto. Ｈ ;or V comprising an amino acid sequence of SEQ ID NO: 194 or at least 95% identical thereto Ｌ and V comprising an amino acid sequence of SEQ ID NO: 195 or at least 95% identical thereto. Ｈ An anti-IL-2 antibody or antigen-binding fragment thereof comprising:

9. An anti-IL-2 antigen-binding fragment comprising an amino acid sequence selected from SEQ ID NOs: 34, 35, 36, and 250-258, or an amino acid sequence at least 90% identical thereto.

10. 10. The anti-IL-2 antibody or antigen-binding fragment of claim 7, 8, or 9, which, when bound to an IL-2 polypeptide, reduces binding of the IL-2 polypeptide to IL-2Rβ (CD122) or to the complex of IL-2β and IL-2Rγ (CD132).

11. enhances the thermal stability of the IL-2 polypeptide when in a complex with the human IL-2 polypeptide, and optionally the complex has a higher thermal stability or Tag temperature than the complex formed by said IL-2 polypeptide and IL-2Rβ; and / or The anti-IL-2 antibody or antigen-binding fragment of claim 9 or 10, wherein the IL-2 polypeptide in the complex has an increased Tag temperature.

12. 12. The anti-IL-2 antibody or antigen-binding fragment of claim 11, wherein the Tag increases by more than 2°C, more than 5°C, more than about 10°C, or more than 10°C.

13. An anti-IL-2 antibody or antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment of any one of claims 7 to 12 for binding to human IL-2 or binds to the same epitope as the antibody or antigen-binding fragment thereof.

14. A prodrug comprising an IL-2 cytokine moiety, a masking moiety, and optionally a carrier moiety, The masking moiety comprises an antibody or antigen-binding fragment according to any one of claims 7 to 13, A prodrug wherein the cytokine portion comprises SEQ ID NO: 1, or an amino acid sequence at least 90% identical thereto.

15. The prodrug of claim 14, wherein the cytokine moiety comprises a mutant IL-2 polypeptide of any one of claims 1 to 6.

16. 15. The prodrug of claim 14, wherein the IL-2 cytokine moiety comprises an amino acid sequence selected from SEQ ID NOs: 1-33.

17. The masking portion is V of SEQ ID NO: 191 Ｈ or an amino acid sequence at least 95% identical thereto, and V of SEQ ID NO: 190 Ｌ Or, the prodrug according to any one of claims 14 to 16, which is an antibody comprising an amino acid sequence at least 95% identical thereto.

18. 18. The prodrug of claim 17, wherein the IL-2 cytokine moiety comprises SEQ ID NO: 1, optionally with one or more mutations selected from T3A, L36I, V69A, Q74P, and C125A.

19. A prodrug comprising an IL-2 cytokine moiety, a masking moiety, and optionally a carrier moiety, the masking moiety binds to the cytokine moiety and inhibits the biological activity of the cytokine moiety; A prodrug wherein the IL-2 cytokine moiety comprises a mutant IL-2 polypeptide according to any one of claims 1 to 6.

20. The masking part is the extracellular domain (ECD) of IL-2Rβ or a functional fragment thereof, or Single chain variable fragment (scFv) or Fab 20. The prodrug of claim 19, comprising:

21. 21. The prodrug of claim 20, wherein the masking moiety comprises an IL-2Rβ ECD comprising SEQ ID NO: 37 or an amino acid sequence at least 90% identical thereto.

22. The mutant IL-2 polypeptide is L36I mutation, Optional C125A mutation Including, The prodrug of any one of claims 14, 15, and 17-20, wherein the masking moiety may comprise SEQ ID NO: 34 or an amino acid sequence at least 90% identical thereto.

23. The prodrug of any one of claims 19 to 22, wherein the masking moiety comprises an anti-IL-2 antibody or antigen-binding fragment of any one of claims 7 to 13.

24. 24. The prodrug of any one of claims 19 to 23, comprising a carrier moiety selected from an antigen-binding moiety, an Fc domain, albumin or a fragment thereof, and PEG.

25. 25. The prodrug of claim 24, wherein the carrier moiety comprises an antigen-binding moiety that targets an antigen presented on an immune cell or a cancer cell, and the antigen-binding moiety can be a bispecific antibody, a single domain antibody, a Fab, or an scFv.

26. The antigen-binding portion targets an antigen presented on a T cell, an NK cell, a macrophage, or a cell within the tumor microenvironment (TME), and the antigen is selected from the group consisting of PD-1, CD3, CD4, CD8, Tim-3, LAG-3, TIGIT, HER2, signal regulatory protein alpha (SIRPα), CTLA-4, CSF1R, NKG2A, NKG2D, CD16A, NKp30, NKp46, ILT2, ILT4, CD40, CD163, LRRC15, fibroblast activation protein (FAP), the A1 domain of tenascin C (TNC A1), the A2 domain of tenascin C (TNC A2), and the A3 domain of tenascin C (TNC B1). A2), fibronectin extra domain B (EDB), fibronectin (α5β1), vitronectin (αvβ3 integrin and αvβ5 integrin), carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), 5T4, BCMA, PD-L1, CD47, epidermal growth factor receptor (EGFR), c-MET, claudin 18.2, claudin 6, CD20, CD24, CD38, CD47, GPC3, mesothelin, ROR1, and melanoma-associated chondroitin sulfate proteoglycan (MCSP).

27. 26. The prodrug of claim 25, wherein the antigen-binding portion comprises an anti-PD-1 antibody or antigen-binding fragment thereof, optionally selected from nivolumab and pembrolizumab.

28. the antigen-binding portion comprises an anti-CD8 antibody or antigen-binding fragment thereof, OKT8 or humanized OKT8, heavy and light chain CDRs 1-3 from OKT8, and / or a light chain variable domain comprising an amino acid sequence of SEQ ID NO: 52 or at least 90% identical thereto, and a heavy chain variable domain comprising an amino acid sequence of SEQ ID NO: 53 or 54 or at least 90% identical thereto.

26. The prodrug of claim 25, which may comprise:

29. The carrier portion is L234A and L235A (“LALA”) mutations (Eu numbering), and / or Knob-into-hole mutation and wherein the IL-2 cytokine moiety and the masking moiety are fused to different polypeptide chains of the Fc domain, different heavy chains of the IgG antibody, or the light and heavy chains, respectively, of the IgG antibody.

30. The prodrug of any one of claims 14 to 29, comprising one or more cleavable and / or non-cleavable peptide linkers.

31. 31. The prodrug of any one of claims 14 to 30, wherein the masking moiety is fused to the carrier moiety via a cleavable or non-cleavable peptide linker, optionally via a cleavable peptide linker.

32. 32. The prodrug of any one of claims 14 to 31, wherein the cytokine moiety is fused to the carrier moiety via a cleavable or non-cleavable peptide linker or to the masking moiety via a cleavable peptide linker.

33. the cleavable peptide linker is cleavable by one or more proteases located in the tumor microenvironment (TME), and cleavage results in activation of the prodrug in the TME, and the cleavable peptide linker a substrate sequence for urokinase-type plasminogen activator (uPA), matrix metallopeptidase 2 (MMP2), MMP7, MMP9, MMP14, legumain, or matriptase, and / or Substrate sequences for two, three, four, or more proteases preferentially expressed in the TME The prodrug of any one of claims 30 to 32, which may comprise:

34. The prodrug of any one of claims 30 to 33, wherein the cleavable peptide linker comprises an amino acid sequence selected from SEQ ID NOs: 55-124, 268, and 269.

35. 10. A prodrug comprising the mutant IL-2 polypeptide of claim 1, comprising a first heavy polypeptide chain, a second heavy polypeptide chain, and one or two light chains; a. a first heavy polypeptide chain comprises SEQ ID NO:275 or an amino acid sequence at least 95% identical thereto, a second heavy polypeptide chain comprises SEQ ID NO:277 or an amino acid sequence at least 95% identical thereto, and two identical light chains comprise SEQ ID NO:276 or an amino acid sequence at least 95% identical thereto; b. a first heavy polypeptide chain comprises an amino acid sequence set forth in SEQ ID NO:278 or at least 95% identical thereto, a second heavy polypeptide chain comprises an amino acid sequence set forth in SEQ ID NO:279 or at least 95% identical thereto, and two identical light chains comprise an amino acid sequence set forth in SEQ ID NO:189 or at least 95% identical thereto; c. the first and second heavy polypeptide chains comprise an amino acid sequence set forth in SEQ ID NO:286, or at least 95% identical thereto, and the two identical light chains comprise an amino acid sequence set forth in SEQ ID NO:276, or at least 95% identical thereto; d. the first and second heavy polypeptide chains comprise an amino acid sequence set forth in SEQ ID NO: 187, or at least 95% identical thereto, and the two identical light chains comprise an amino acid sequence set forth in SEQ ID NO: 189, or at least 95% identical thereto; or e. A prodrug wherein a first heavy polypeptide chain comprises an amino acid sequence set forth in SEQ ID NO:283 or at least 95% identical thereto, a second heavy polypeptide chain comprises an amino acid sequence set forth in SEQ ID NO:284 or at least 95% identical thereto, and one light chain comprises an amino acid sequence set forth in SEQ ID NO:189 or at least 95% identical thereto.

36. 1. An IL-2 antibody fusion molecule comprising two identical antibody light chains, a first antibody heavy chain, and a second antibody heavy chain, a. each of the light chains comprises an amino acid sequence set forth in SEQ ID NO: 38 or at least 90% identical thereto, the first heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 40, 41, 45, 46 or at least 95% identical thereto, and the second heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 42, 43, 44, or 47 or at least 95% identical thereto; or b. each of the light chains comprises an amino acid sequence set forth in SEQ ID NO: 38, or at least 90% identical thereto, and each of the first and second heavy chains comprises an amino acid sequence set forth in SEQ ID NO: 40, 41, 48, 49, 50, or 51, or at least 95% identical thereto; c. An IL-2 antibody fusion molecule wherein each light chain comprises an amino acid sequence set forth in SEQ ID NO: 189 or at least 90% identical thereto, the first heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 185 or at least 95% identical thereto, and the second heavy chain comprises an amino acid sequence set forth in SEQ ID NO: 186 or at least 95% identical thereto.

37. a. two identical light chains and two identical heavy chains, each of the light and heavy chains being: (i) SEQ ID NO: 207 or an amino acid sequence at least 95% identical thereto, and SEQ ID NO: 205, 206, 211, 212, 213, or 214 or an amino acid sequence at least 95% identical thereto; or (ii) SEQ ID NO: 208 or an amino acid sequence at least 95% identical thereto, and SEQ ID NO: 215 or an amino acid sequence at least 95% identical thereto. two identical light chains and two identical heavy chains, comprising: b. a first polypeptide chain and a second polypeptide chain, wherein each of the first and second polypeptide chains comprises: (i) SEQ ID NO: 196 or 197, or an amino acid sequence at least 95% identical thereto, and SEQ ID NO: 209 or 210, or an amino acid sequence at least 95% identical thereto; or (ii) SEQ ID NO: 198 or an amino acid sequence at least 95% identical thereto, and SEQ ID NO: 199, 202, 203, or 204 or an amino acid sequence at least 95% identical thereto. a first polypeptide chain and a second polypeptide chain comprising 1. An IL-2 antibody fusion molecule comprising:

38. 38. A pharmaceutical composition comprising a mutant human IL-2 polypeptide according to any one of claims 1 to 6, a prodrug according to any one of claims 14 to 35, or an IL-2 antibody fusion molecule according to claim 36 or 37, and a pharmaceutically acceptable excipient.

39. One or more polynucleotides encoding the mutant human IL-2 polypeptide of any one of claims 1 to 6, the anti-IL-2 antibody or antigen-binding fragment of any one of claims 7 to 13, the prodrug of any one of claims 14 to 35, or the IL-2 antibody fusion molecule of claim 36 or 37.

40. 40. One or more expression vectors comprising the polynucleotide of claim 39.

41. 41. A host cell comprising the expression vector of claim 40, which may be a mammalian cell, wherein genes encoding matriptase, uPA, MMP-2, MMP-9, and / or MMP14 are knocked out in the host cell.

42. 1. A method of making a protein, comprising: Culturing the host cell of claim 41, which is a mammalian cell, under conditions that allow expression of the mutant IL-2 polypeptide, anti-IL-2 antibody or antigen-binding fragment thereof, prodrug, or IL-2 antibody fusion molecule; Isolating the expressed protein from the culture. A method comprising:

43. 39. A method of treating cancer or an infectious disease or modulating the immune system in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a mutant IL-2 polypeptide of any one of claims 1-6, a prodrug of any one of claims 14-35, an IL-2 antibody fusion molecule of claim 36 or 37, or a pharmaceutical composition of claim 38.

44. 39. A mutant IL-2 polypeptide according to any one of claims 1 to 6, a prodrug according to any one of claims 14 to 35, an IL-2 antibody fusion molecule according to claim 36 or 37, or a pharmaceutical composition according to claim 38, for use in treating cancer or an infectious disease or modulating the immune system in a patient in need thereof.

45. 38. Use of a mutant IL-2 polypeptide according to any one of claims 1 to 6, a prodrug according to any one of claims 14 to 35, or an IL-2 antibody fusion molecule according to claim 36 or 37 for the manufacture of a medicament for treating cancer or an infectious disease or modulating the immune system in a patient in need thereof.

46. The patient, Have HIV infection? have a cancer selected from the group consisting of leukemia, lymphoma, kidney cancer, bladder cancer, urinary tract cancer, cervical cancer, brain cancer, head and neck cancer, skin cancer, uterine cancer, testicular cancer, esophageal cancer, liver cancer, colorectal cancer, stomach cancer, squamous cell carcinoma, prostate cancer, pancreatic cancer, lung cancer including non-small cell lung cancer, bile duct cancer, breast cancer, and ovarian cancer, and medullary thyroid cancer; or 46. ​​The method of claim 42, the mutant IL-2 polypeptide, prodrug, IL-2 antibody fusion molecule, or pharmaceutical composition for use according to claim 44, or the use according to claim 45, in a patient with an inflammatory or autoimmune disease, as appropriate selected from asthma, type 1 diabetes, rheumatoid arthritis, allergy, systemic lupus erythematosus, organ transplant rejection, and graft versus host disease.

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