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 the same to modulate the immune system in a subject.

Description

[0001] Cross - reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 429,940, filed on December 2, 2022, the content of which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a Sequence Listing that has been electronically submitted in XML format. The 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 part of the specification and is hereby incorporated by reference in its entirety. Background of the Invention

[0005] Interleukin-2 (IL-2) plays a central role in lymphocyte generation, survival, and homeostasis. The IL-2 has 133 amino acids and consists of four anti-parallel, amphipathic α-helices that form a quaternary structure crucial for its function (Smith, Science (1988) 240:1169-76; Bazan, Science (1992) 257:410-13).

[0006] IL-2 exerts its activity by binding to the IL-2 receptor (IL-2R), which consists of up to three separate subunits. Association of the α (CD25 or Tac antigen), β (CD122), and γ (γc, common γ chain or CD132) subunits gives rise to the trimeric high-affinity receptor for IL-2 (KD ∼ 0.01 nM). The dimeric IL-2 receptor composed of the β and γ subunits is referred to as the intermediate-affinity IL-2R (KD ∼ 1 nM). The individual α subunit forms the monomeric low-affinity IL-2 receptor (KD ∼ 10 nM). See, e.g., Kim et al., Cytokine Growth Factor Rev. (2006) 17:349-66. Although the affinity of the dimeric intermediate-affinity IL-2 receptor for binding IL-2 is approximately 1 / 100 of that of the trimeric high-affinity receptor for binding IL-2, 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, while conferring high-affinity binding of the receptor to IL-2, the α subunit does not appear to be essential for IL-2 signal transduction. However, the β and γ subunits are crucial for IL-2 signal transduction (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 constitutively express the highest levels of IL-2Rα (CD25) in vivo (Fontenot et al., Nature Immunol. (2005) 6:1142-51). The trimeric IL-2 receptor is also transiently induced on conventionally activated T cells, whereas in the resting state, these cells express only the dimeric IL-2 receptor.

[0007] Mutated versions of IL-2 have been developed for optimizing the treatment of cancer and autoimmune diseases. However, the immunogenicity of the mutated IL-2 molecules is a potential risk for the clinical development of the molecules. Thus, there is a need to develop IL-2-based therapeutic agents with reduced immunogenicity. SUMMARY OF THE INVENTION

[0008] The present disclosure provides mutant human IL-2 polypeptides that comprise a mutation at position L36 according to SEQ ID NO:1 (e.g., L36I). In one aspect, the present disclosure provides a mutant human IL-2 polypeptide that comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1 and the L36I mutation relative to SEQ ID NO:1. In some embodiments, the mutant IL-2 polypeptide comprises additional mutations further described herein.

[0009] In some embodiments, one or more additional mutations of the IL-2 polypeptide reduce the binding affinity of the polypeptide for CD25. In some embodiments, the IL-2 polypeptide mutation is the C125A mutation relative to SEQ ID NO:1. In some embodiments, the one or more additional mutations are located at positions selected from: T3, where the mutation is the N3A mutation; D20, where the mutation is the D20H, D20K, D20L, D20M, D20N, D20Q, D20R, D20S, D20V or D20Y mutation; R38, where the mutation is the R38A, R38K or R38S; F42, where the mutation is the F42A, F42G, F42I, F42S, F42T, F42Q, F42E, F42N, F42D, F42R or F42K; Y45, where the mutation is the Y45A, Y45G, Y45I, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R or Y45K; E62, where the mutation is the E62L, E62A or E62I; E68, where the mutation is the E68V; L72, where the mutation is the L72G; A73, where the mutation is the A73T; N88, where the mutation is the N88A, N88E, N88F, N88H, N88K, N88T, N88L, N88M, N88S, N88V, N88W or N88Y; N90, where the mutation is the N90T; V91, where the mutation is the V91K, V91A, V91H or V91R; I92; and Q126, where the mutation is the Q126A, Q126D, Q126F, Q126G, Q126H, Q126I, Q126K, Q126L, Q126P, Q126S, Q126T, Q126W or Q126Y (numbered according to SEQ ID NO:1).

[0010] In some embodiments, the mutant IL-2 polypeptide comprises an amino acid sequence selected from SEQ ID NO:3 and 5 - 33, or an amino acid sequence that is at least 95% identical thereto. In other embodiments, the mutant IL-2 polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:2 or 4.

[0011] On the other hand, the present disclosure provides an anti-IL-2 antibody or an antigen-binding fragment thereof, wherein the anti-IL-2 antibody or the antigen-binding fragment thereof comprises heavy chain CDR1-3 shown in SEQ ID NO: 170-172 and light chain CDR1-3 shown in SEQ ID NO: 173-175. In some embodiments, the anti-IL-2 antibody or the antigen-binding fragment thereof herein comprises a light chain variable domain (V L ) and a heavy chain variable domain (V H ), wherein the V L comprises the amino acid sequence of SEQ ID NO: 190 or an amino acid sequence that is at least 95% identical thereto, and the V H comprises the amino acid sequence of SEQ ID NO: 191 or an amino acid sequence that is at least 95% identical thereto; or, the V L comprises the amino acid sequence of SEQ ID NO: 192 or an amino acid sequence that is at least 95% identical thereto, and the V H comprises the amino acid sequence of SEQ ID NO: 193 or an amino acid sequence that is at least 95% identical thereto; or, the V L comprises the amino acid sequence of SEQ ID NO: 194 or an amino acid sequence that is at least 95% identical thereto, and the V H comprises the amino acid sequence of SEQ ID NO: 195 or an amino acid sequence that is at least 95% identical thereto. In some embodiments, the anti-IL-2 antigen-binding fragment herein comprises an amino acid sequence selected from SEQ ID NO: 34, 35, 36, and 250-258 or an amino acid sequence that is at least 90% identical thereto. In some embodiments, the antibody or antigen-binding fragment reduces the binding of the IL-2 polypeptide to IL-2Rβ (CD122) or reduces the binding to the complex of IL-2β and IL-2Rγ (CD132) when binding to the IL-2 polypeptide. In some embodiments, the antibody or antigen-binding fragment enhances the thermal stability of the IL-2 polypeptide when complexed with the human IL-2 polypeptide. In some embodiments, compared with the complex formed by the IL-2 polypeptide and IL-2Rβ, the complex has higher thermal stability or a Tagg temperature; and / or the IL-2 polypeptide in the complex has an increased Tagg temperature. In some embodiments, the Tagg increases by more than 2 °C, more than 5 °C, approximately 10 °C, or more than 10 °C.

[0012] Also provided is an anti-IL-2 antibody or an antigen-binding fragment thereof, wherein the anti-IL-2 antibody or the antigen-binding fragment thereof competes with the specifically exemplified antibodies or antigen-binding fragments herein for binding to human IL-2, or binds to the same epitope as the antibody or antigen-binding fragment.

[0013] On the other hand, the present disclosure provides a prodrug comprising an IL-2 cytokine moiety, a masking moiety, and optionally a carrier moiety, wherein the masking moiety comprises an antibody or antigen-binding fragment herein, and the cytokine moiety comprises SEQ ID NO:1 or an amino acid sequence that is at least 90% identical thereto (e.g., a mutant IL-2 polypeptide herein). In some embodiments, the cytokine moiety comprises a mutant IL-2 polypeptide described herein. In some embodiments, the IL-2 cytokine moiety comprises an amino acid sequence selected from SEQ ID NO:1-33. In some embodiments, the masking moiety is an antibody comprising V H and V L , wherein the V H has SEQ ID NO:191 or an amino acid sequence that is at least 95% identical thereto, and the V L has SEQ ID NO:190 or an amino acid sequence that is at least 95% identical thereto. In some embodiments, the IL-2 cytokine moiety comprises SEQ ID NO:1, wherein in other embodiments, one or more mutations are selected from T3A, L36I, V69A, Q74P, and C125A.

[0014] In a related aspect, the present disclosure also provides a prodrug comprising an IL-2 cytokine moiety, a masking moiety, and optionally a carrier moiety, wherein the masking moiety binds to the cytokine moiety and inhibits the biological activity of the cytokine moiety, and the IL-2 cytokine moiety comprises a mutant IL-2 polypeptide as described herein. In some embodiments, the prodrug comprises an IL-2 cytokine moiety, a masking moiety, and optionally a carrier moiety, wherein the masking moiety binds to the cytokine moiety and inhibits the biological activity of the cytokine moiety, and the IL-2 cytokine moiety comprises the mutant IL-2 polypeptide. In some embodiments, the masking moiety comprises the extracellular domain (ECD) of IL-2Rβ or a functional fragment thereof, or a single-chain antibody (scFv) or Fab. In some embodiments, the masking moiety comprises the IL-2Rβ ECD, and the IL-2Rβ ECD comprises SEQ ID NO:37 or an amino acid sequence that is at least 90% identical thereto. In some embodiments, the mutant IL-2 polypeptide comprises an L36I mutation and may additionally comprise a C125A mutation, and wherein the masking moiety may comprise SEQ ID NO:34 or an amino acid sequence that is 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 that is at least 90% identical thereto. In some embodiments, the masking moiety comprises an anti-IL-2 antibody or an 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.

[0015] In some embodiments, the vector portion comprises an antigen-binding portion that targets an antigen presented on immune cells or cancer cells, wherein the antigen-binding portion can be a bispecific antibody, a single-domain antibody, a Fab, or an scFv. In some embodiments, the antigen-binding portion targets an antigen presented on T cells, NK cells, macrophages, or cells in the tumor microenvironment (TME), wherein the antigen can be selected from 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), extra domain B of fibronectin (EDB), fibronectin (α5β1), vitronectin (αvβ3 and αvβ5 integrins), 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 portion comprises an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the anti-PD-1 antibody can be selected from nivolumab and pembrolizumab. In some embodiments, the antigen-binding portion comprises an anti-CD8 antibody or an antigen-binding fragment thereof, wherein the antigen-binding portion can comprise OKT8 or humanized OKT8, the heavy and light chain CDR1-3 derived from OKT8, and / or the light chain variable domain and the heavy chain variable domain, the light chain variable domain comprising SEQ ID NO:52 or an amino acid sequence that is at least 90% identical thereto, and the heavy chain variable domain comprising SEQ ID NO:53 or 54 or an amino acid sequence that is at least 90% identical thereto. In some embodiments, the vector portion comprises an IgG Fc domain or an IgG antibody, the IgG Fc domain or IgG antibody comprising the L234A and L235A ("LALA") mutations (Eu numbering) and / or knobs-into-holes mutations, wherein the IL-2 cytokine portion and the masking portion are fused to different polypeptide chains of the Fc domain, or to different heavy chains of the IgG antibody, or to the light and heavy chains of the IgG antibody, respectively.

[0016] 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 is fused to the masking moiety via a cleavable peptide linker. In some embodiments, the cleavable peptide linker can be cleaved by one or more proteases located in the tumor microenvironment (TME), and the cleavage activates the prodrug in the TME, wherein the cleavable peptide linker can comprise a substrate sequence of urokinase-type plasminogen activator (uPA), matrix metallopeptidase 2 (MMP2), MMP7, MMP9, MMP14, matrilysin or matriptase, and / or substrate sequences of 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.

[0017] In some embodiments, the present disclosure provides a prodrug comprising a mutant IL-2 polypeptide as 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 that is at least 95% identical thereto, the second heavy chain polypeptide chain comprises SEQ ID NO: 277 or an amino acid sequence that is at least 95% (e.g., at least 96%, 97%, 98%, or 99%) identical thereto, and two identical light chains, the light chain comprising SEQ ID NO: 276 or an amino acid sequence that is at least 95% identical thereto; b. the first heavy chain polypeptide chain comprises SEQ ID NO: 278 or an amino acid sequence that is at least 95% (e.g., at least 96%, 97%, 98%, or 99%) identical thereto, the second heavy chain polypeptide chain comprises SEQ ID NO: 279 or an amino acid sequence that is at least 95% (e.g., at least 96%, 97%, 98%, or 99%) identical thereto, and two identical light chains, the light chain comprising SEQ ID NO: 189 or an amino acid sequence that is at least 95% identical thereto; c. the first heavy chain polypeptide chain and the second heavy chain polypeptide chain comprise SEQ ID NO: 286 or an amino acid sequence that is at least 95% (e.g., at least 96%, 97%, 98%, or 99%) identical thereto, and two identical light chains comprise SEQ ID NO: 276 or an amino acid sequence that is at least 95% identical thereto; d. the first heavy chain polypeptide chain and the second heavy chain polypeptide chain comprise SEQ ID NO: 187 or an amino acid sequence that is at least 95% (e.g., at least 96%, 97%, 98%, or 99%) identical thereto, and two identical light chains comprise SEQ ID NO: 189 or an amino acid sequence that is at least 95% identical thereto; or e. the first heavy chain polypeptide chain comprises SEQ ID NO: 283 or an amino acid sequence that is at least 95% (e.g., at least 96%, 97%, 98%, or 99%) identical thereto, the second heavy chain polypeptide chain comprises SEQ ID NO: 284 or an amino acid sequence that is at least 95% (e.g., at least 96%, 97%, 98%, or 99%) identical thereto, and one light chain, the light chain comprising SEQ ID NO: 189 or an amino acid sequence that is at least 95% identical thereto.

[0018] On the other hand, the present 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 SEQ ID NO: 38 or an amino acid sequence that is at least 90% identical thereto, the first heavy chain comprises SEQ ID NO: 40, 41, 45, 46 or an amino acid sequence that is at least 95% identical thereto, and the second heavy chain comprises SEQ ID NO: 42, 43, 44 or 47 or an amino acid sequence that is at least 95% identical thereto; b. each of the light chains comprises SEQ ID NO: 38 or an amino acid sequence that is at least 90% identical thereto, and the first heavy chain and the second heavy chain each comprise SEQ ID NO: 40, 41, 48, 49, 50 or 51 or an amino acid sequence that is at least 95% identical thereto; or c. each of the light chains comprises SEQ ID NO: 189 or an amino acid sequence that is at least 90% identical thereto, the first heavy chain comprises SEQ ID NO: 185 or an amino acid sequence that is at least 95% identical thereto, and the second heavy chain comprises SEQ ID NO: 186 or an amino acid sequence that is at least 95% identical thereto.

[0019] On the other hand, the present disclosure provides an IL-2 antibody fusion molecule comprising: a. two identical light chains and two identical heavy chains, wherein the light chain and the heavy chain respectively comprise (i) SEQ ID NO: 207 or an amino acid sequence that is at least 95% identical thereto, and SEQ ID NO: 205, 206, 211, 212, 213 or 214 or an amino acid sequence that is at least 95% identical thereto, or (ii) SEQ ID NO: 208 or an amino acid sequence that is at least 95% identical thereto, and SEQ ID NO: 215 or an amino acid sequence that is at least 95% identical thereto; or b. a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain respectively comprise (i) SEQ ID NO: 196 or 197 or an amino acid sequence that is at least 95% identical thereto, and SEQ ID NO: 209 or 210 or an amino acid sequence that is at least 95% identical thereto, or (ii) SEQ ID NO: 198 or an amino acid sequence that is at least 95% identical thereto, and SEQ ID NO: 199, 202, 203 or 204 or an amino acid sequence that is at least 95% identical thereto.

[0020] There is also provided a pharmaceutical composition comprising a mutant human IL-2 polypeptide, a prodrug or an IL-2 antibody fusion molecule herein and a pharmaceutically acceptable excipient.

[0021] One or more polynucleotides are also provided, the polynucleotides encoding the novel proteins herein (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 methods for producing the novel proteins by culturing mammalian host cells permissive for expression of the novel proteins and isolating the expressed novel proteins from the culture.

[0022] On the other hand, the present disclosure provides a method for treating cancer or an infectious disease in a patient in need thereof or modulating (e.g., stimulating) its immune system, the method 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 present invention. Mutant IL-2 polypeptides, prodrugs, IL-2 antibody fusion molecules and pharmaceutical compositions are also provided for use in treating cancer or an infectious disease in a patient in need thereof (e.g., a human patient) or modulating (e.g., stimulating) its immune system; and uses of these mutant IL-2 polypeptides, prodrugs and IL-2 antibody fusion molecules for the preparation of a medicament for treating cancer or an infectious disease in a patient in need thereof or modulating (e.g., stimulating) its immune system. In some embodiments, the patient has a viral infection (e.g., HIV infection); has 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, cholangiocarcinoma, breast cancer and ovarian cancer and medullary thyroid cancer; or has an inflammatory or autoimmune disease, such as one selected from asthma, type I diabetes, rheumatoid arthritis, allergy, systemic lupus erythematosus, organ transplant rejection and graft-versus-host disease. Articles (e.g., kits) are also provided that comprise one or more dosage units of the novel proteins of the present invention.

[0023] Other features, objects, and advantages of the invention will be apparent from the following detailed description. However, it should be understood that the detailed description is given by way of illustration only and not by way of limitation. Various changes and modifications within the scope of the invention will be apparent to those skilled in the art from the detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1A -D shows the results of the blocking assay. The ability of the supernatants of the test candidate scFv clones to block the binding of Fc-IL-2 to Fc-IL-2Rβγ was tested. The blocking was tested using an Octet Red96e kinetic binding assay. Figure 1AIt 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 It is a graph showing the blocking of the binding of IL-2 to IL-2Rβγ. Figure 1C It is a graph showing the partial inhibition of the binding of IL-2 to IL-2Rβγ. Figure 1D It shows the blocking assay of 100 μl of the supernatant of clone K3-23A2, which does not block the binding of IL-2 to IL-2Rβγ.

[0025] Figure 2 It shows the results of the IL-2 activity inhibition assay based on CTLL2 cells using different fusion (scFv-Fc) proteins.

[0026] Figure 3A It is a table showing the heavy chain and expression titer of the scFv clone.

[0027] Figure 3B It is a graph showing the IL-2 activity inhibition assay based on CTLL2 cells using various scFv clones.

[0028] Figure 4A and 4B It shows the binding kinetics of the selected scFv-Fc fusion molecule. The name, binding affinity, and response of each clone are shown.

[0029] Figure 5A -C shows the information of the PD-1 antibody-IL-2 prodrug and the screening data of the prodrug after the CTLL2-based activity assay. Figure 5A It shows the anti-PD-1 antibody (JR11.20.1), the schematic diagrams of the PD-1 antibody-IL-2 fusion molecule (JR11.20.2) and the PD-1 antibody-IL-2 prodrug fusion molecule, wherein the IL-2 part of each prodrug has a mask, and the mask contains an scFv against IL-2 (JR11.20.3 to JR11.20.6) or IL-2Rβ-ECD (JR11.20.7). Figure 5B It is a table showing the name of the sample / molecule, plasmid code, and SEQ ID NO of each molecule. The expression titer and brief description of each molecule are also included. Figure 5C It shows the results of the CTLL2 cell-based activity assay of the prodrug sample before and after activation.

[0030] Figure 6A -D shows the information and data of screening the PD-1 antibody-IL-2 prodrug based on the NK92-based activity assay. Figure 6AA table showing the names, plasmid codes, SEQ ID NOs, and titers of transient expression of additional PD-1-antibody-IL-2 prodrug molecules masked by scFvs screened from a yeast library. Figure 6B A schematic diagram showing anti-PD-1 antibody-IL-2 prodrug fusion molecules, where the IL-2 portion of each prodrug is masked by a mask containing an scFv against IL-2 (JR11.29.1, JR11.31.1-8). Figure 6C SDS-PAGE analysis of prodrug samples JR11.29.1 and JR11.31.1 before activation and all prodrug samples after activation based on protease MMP-2. All samples are capable of being digested by MMP-2. Figure 6D A graph (left panel) showing the results of an NK92 cell-based activity assay that measures the activity of prodrug samples before and after activation by MMP-2. The activity is shown in the right panel.

[0031] Figure 7A -C shows information and results of peptide linker optimization experiments. Figure 7A Shows the plasmids and SEQ ID NOs of each prodrug sample. Figure 7B Schematic diagrams showing anti-PD-1 antibody-IL-2 prodrug fusion molecules in which the IL-2 portion (IL-2v) is linked to the heavy chain using different linkers (JR11.101.1-3) or no linker (JR11.101.4). Figure 7C Shows the results of SEC-HPLC analysis.

[0032] Figure 8A -E shows the results of in vitro assays of PD-1 antibody-IL-2 prodrugs using human PBMCs. Figure 8A A table showing the plasmids and SEQ ID NOs of two prodrug molecules, ASKG812K-C7 and ASKG812K-G3, and a control molecule (PD-1 antibody-IL-2v fusion molecule PD1-mab-IL-2vRef without a mask). Prodrug ASKG812K-G3 does not have a cleavable linker. Figure 8B Schematic diagram of a prodrug. IL-2 induces phosphorylation of STAT5 (pSTAT5) in immune cells. Figure 8C Shows induction of pSTAT5 in CD4+ T cells, Figure 8D Shows induction of pSTAT5 in CD8+ T cells, and Figure 8E Shows induction of pSTAT5 in NK cells.

[0033] Figure 9A -D shows sample information and enhanced stability and thermal stability with scFv masks. Figure 9AA table showing the plasmids and SEQ ID NOs of three prodrug molecules, ASKG812K-C7, ASKG812K-F7, and ASKG812K-G3, and a control molecule, which is the ASKG812-β-ECD prodrug masked with the extracellular domain (ECD) of IL-2Rβ. Figure 9B A table showing the thermal stability of the prodrugs. Figure 9C A schematic diagram showing the prodrugs. Figure 9D A graph showing the accelerated stability study of the prodrugs.

[0034] Figure 10A -C shows the sample information and results of the CTLL2 assay for several prodrug molecules. Figure 10A A table showing the plasmids and SEQ ID NOs of the following molecules: PD-1 antibody-IL-2v reference molecule (EB01-08; PD1-mab-IL-2vRef), 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 PD-1 antibody-IL-2v / L36I masked with F7 (masked with JR11.145.4; PD-1mab-IL-2v / L36I_C7 masked). All prodrugs were tested by CTLL2 assay. The assay results are shown in Figure 10B (shown in the left and right figures). Figure 10C A schematic diagram showing the prodrugs.

[0035] Figure 11A -F shows a schematic diagram of the structure of the antibody-cytokine fusion molecule. Figure 11A Shows the structure of ASKG222C7-C, where the cytokine IL-2 and the scFv mask K1-69C7 or "C7" are fused to the C-terminus of the Fc domain. Figure 11B Shows the structure of ASKG222C7-N, where the cytokine IL-2 and the mask C7 are fused to the N-terminus of the Fc domain. Figure 11C Shows the structure of ASKG222A, where the antibody contains the V L and V H identical to V L and V H ; and where the IL-2 cytokine is fused to the N-terminus of each heavy chain. Figure 11D Shows the structure of ASKG222B, where the antibody contains the V L and V H identical to V L and V H; and wherein the IL-2 cytokine is fused to the N-terminus of each light chain. Figure 11E shows the structure of ASKG222G, which is substantially the same as that of ASKG222A( Figure 11C ), except that it has a single Fab. Figure 11F shows the structure of ASKG222H, which is substantially the same as that of ASKG222B( Figure 11D ), except that it has a single Fab.

[0036] Figure 12A shows the plasmid and sequence information of ASKG222C7-C and ASKG222C7-D.

[0037] Figure 12B shows the DRC-HPLC purity of the molecules expressed in CHO cells and purified by protein A affinity chromatography.

[0038] Figure 13A shows the plasmid and sequence information of ASKG222A-C7-Ab and ASKG222B-C7-Ab.

[0039] Figure 13B shows the DRC-HPLC purity of the molecules expressed in CHO cells and purified by protein A affinity chromatography.

[0040] Figure 14A and 14B shows the results of in vitro assays using antibody-cytokine fusion molecules. Figure 14A is a graph showing the activity of the antibody fusion molecule in stimulating the proliferation of Ki67+ regulatory T cells (Tregs). Figure 14B is a graph showing the activity of the antibody fusion molecule in stimulating the proliferation of Ki67+ effector T cells (Teffs).

[0041] Figure 15 is a graph showing the rat PK data of ASKG222A-C7-Ab. The results show that the half-life of ASKG222A-C7-Ab is 66 - 88 hours.

[0042] Figure 16A - 16E shows a schematic diagram of the structure of the antibody-cytokine fusion molecule and the polypeptide chain sequence forming the fusion molecule.

[0043] Figure 17A and 17B shows the in vivo efficacy and safety results of two fusion molecules (812mN-mut4 and 812mW5-mut4) compared with the reference molecule Ref3 and the anti-mouse PD-1 antibody.

[0044] Figure 18Shows the sequence information of the antibody-cytokine fusion molecule.

[0045] Figure 19A and 19B Shows the in vivo efficacy and safety results of two different doses of the fusion molecule (678F3-Fab-B).

[0046] Figure 20 Shows the results of an in vitro cell-based activity assay (HEK Blue reporter gene assay).

[0047] Figure 21 Shows a schematic diagram of the structure of the fusion molecule 678F3-Fab-B. Detailed Description

[0048] As used herein and in the appended claims, the singular forms "a", "or", and "the" include plural referents unless the context clearly indicates otherwise.

[0049] References herein to "about" a value or parameter include (and describe) variations that are specific to that value or parameter itself. For example, a reference to "about X" includes a description of "X". Additionally, the use of "about" before any numerical series includes "about" each of the numbers recited in the series. For example, a reference to "about X, Y, or Z" is intended to describe "about X, about Y, or about Z".

[0050] The term "antigen-binding portion" refers to a polypeptide or a set of interacting polypeptides that specifically binds to an antigen, and includes but is not limited to an antibody (e.g., a monoclonal antibody, a polyclonal antibody, a multispecific antibody, a dual-specific or bispecific antibody, an anti-idiotype antibody, or a bifunctional hybrid antibody) or an antigen-binding fragment thereof (e.g., Fab, Fab', F(ab')2, Fv, disulfide-linked Fv, scFv, a single-domain antibody (dAb), or a diabody), a single-chain antibody, and an Fc-containing polypeptide such as an immunoadhesin or an scFv-Fc. In some embodiments, the antibody can belong to 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 can belong to any light-chain isotype (e.g., κ or λ). The antibody can be human, non-human (e.g., from a mouse, a rat, a rabbit, a goat, or another non-human animal), chimeric (e.g., having non-human variable regions and human constant regions), or humanized (e.g., having non-human CDRs and human framework and constant regions). In some embodiments, the antibody is a derivatized antibody).

[0051] The term "cytokine agonist polypeptide" refers to a wild-type cytokine or an analogue thereof. An analogue of a wild-type cytokine has the same biological specificity as the wild-type cytokine (e.g., binds to the same receptor and activates the same target cells), even if the activity level of the analogue may be different from that of the wild-type cytokine. The analogue can be, for example, a mutant protein (i.e., a mutant polypeptide) of the wild-type cytokine and can contain at least one mutation, at least two mutations, at least three mutations, at least four mutations, at least five mutations, at least six mutations, at least seven mutations, at least eight mutations, at least nine mutations, or at least ten mutations relative to the wild-type cytokine.

[0052] The term "prodrug" as used herein refers to a cytokine fusion protein that contains a cytokine moiety bound by a masking moiety and has not yet been activated to remove the masking moiety from the cytokine moiety. Once the cytokine moiety is unbound, the fusion molecule is "activated".

[0053] The terms "cytokine antagonist", "cytokine masking agent" and "masking moiety" refer to a moiety (e.g., a polypeptide) that binds to a cytokine and thereby inhibits the binding of the cytokine to its receptor on the surface of target cells, and / or a moiety (e.g., a polypeptide) that inhibits the cytokine (when bound to the antagonist or masking agent) from exerting its biological function. Examples of cytokine antagonists or masking agents include, but are not limited to, polypeptides derived from the extracellular domain of the natural receptor of a cytokine that has been in contact with the cytokine, and antibodies or antigen-binding fragments thereof (e.g., scFv) that bind to the cytokine.

[0054] The term "effective amount" or "therapeutically effective amount" refers to the amount of a compound or composition that is sufficient to treat a specific disorder, condition or disease, such as to ameliorate, alleviate, mitigate and / or delay one or more of its symptoms. For a disease such as cancer, an effective amount can be an amount sufficient to delay the development or progression of cancer (e.g., reduce the tumor growth rate and / or delay or prevent tumor angiogenesis, metastasis or infiltration of cancer cells into peripheral organs), reduce the number of epitheloid cells, cause regression of cancer (e.g., shrink or eradicate the tumor) and / or prevent or delay the occurrence or recurrence of cancer. The effective amount can be administered in one or more administrations.

[0055] The term "functional analogue" refers to a molecule that has the same biological specificity (e.g., binds to the same ligand) and / or activity (e.g., activates or inhibits target cells) as a reference molecule.

[0056] The term "fused" or "fusion" with respect to two polypeptide sequences refers to the joining of the two polypeptide sequences by backbone peptide bonds. The two polypeptides can be fused directly or through a peptide linker that is one or more amino acids in length. The fusion polypeptide can be prepared by recombinant techniques from a coding sequence containing the respective coding sequences of the two fusion partners, with or without a coding sequence for a peptide linker therebetween. In some embodiments, fusion encompasses chemical conjugation.

[0057] The term "pharmaceutically acceptable excipient", when used to refer to a component in a composition, means that the excipient is suitable for administration to a subject, including a human subject, without causing unduly harmful side effects to the subject and without affecting the biological activity of the active pharmaceutical ingredient (API).

[0058] The term "subject" refers to a mammal and includes, but is not limited to, humans, pets (e.g., canines or felines), farm animals (e.g., cows or horses), rodents or primates.

[0059] As used herein, "treatment" or "treating" is a method for obtaining a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by a disease, reducing the severity of the disease, improving the disease state, stabilizing the disease (e.g., preventing or delaying the deterioration or progression of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the recurrence of the disease, providing partial or complete remission of the disease, reducing the dosage of one or more other drugs required to treat the disease, improving the quality of life of the patient, and / or prolonging the survival period. The methods of the present disclosure encompass any one or more of these treatment aspects.

[0060] It should be understood that one, some, or all of the properties described in the various embodiments herein can 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 below.

[0061] I. Mutant IL - 2 prodrug

[0062] The present disclosure further provides cytokine prodrugs that are metabolized in vivo at a target site into active cytokine therapeutic agents. The cytokine prodrugs have fewer side effects (including a lower risk of immunogenicity of the cytokine compared to wild-type cytokines), better in vivo PK properties (e.g., longer half-life), and better targeting specificity, and are more effective compared to cytokine therapeutic agents of the prior art. The prodrugs of the present invention comprise a cytokine moiety that comprises a cytokine agonist polypeptide linked to a carrier moiety and masked (bound) by a cytokine antagonist (masking moiety). The cytokine antagonist can be, for example, the extracellular domain of the receptor of the cytokine and is linked to the cytokine moiety or the carrier moiety by a peptide linker (e.g., a cleavable or non-cleavable peptide linker). When the masking agent binds to the cytokine moiety, the masking agent inhibits the biological function of the cytokine moiety. The prodrug can be activated at the target site of the patient (e.g., at the tumor site or the surrounding environment) by cleaving the linker and subsequently releasing the cytokine masking agent from the prodrug, thereby exposing the previously masked cytokine moiety and allowing the cytokine moiety to bind to its 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 by a non-cleavable peptide linker, the prodrugs disclosed herein can bind to the target cell by "cis-binding" of the cytokine receptor and the antigen expressed on the cell surface, such that the activity of the prodrug is increased without cleavage and removal of the masking moiety. The cytokine moiety of the prodrug can increase its activity at the target site (e.g., at the tumor site or the surrounding environment), where the antigen targeted by the carrier and the receptor of the cytokine are expressed on the same cell.

[0063] In some embodiments, the carrier moiety of the prodrug is an antigen-binding moiety, such as an antibody or an antigen-binding fragment thereof that binds an antigen at the target site.

[0064] In some embodiments, the prodrug of the present invention is a pro-inflammatory cytokine prodrug that is metabolized into a pro-inflammatory cytokine at an in vivo target site targeted by the carrier moiety. In additional embodiments, the carrier moiety in the prodrug is an antibody or an antigen-binding fragment thereof that targets a tumor antigen, such that the prodrug is delivered to the tumor site of a patient and is locally metabolized (e.g., inside or near the tumor microenvironment) by cleaving a linker that connects the cytokine masker to the carrier moiety or the cytokine moiety, thereby enabling the pro-inflammatory cytokine moiety to interact with its receptor on target cells and locally stimulate target immune cells.

[0065] The IL-2 prodrug can comprise: a cytokine moiety comprising an IL-2 agonist polypeptide, a carrier moiety, and a masking moiety (IL-2 antagonist), wherein the cytokine moiety is fused directly or via a linker (e.g., a cleavable or non-cleavable peptide linker) to the carrier moiety, 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 can be an IL-2 mutant protein, such as an IL-2 mutant protein derived from human IL-2 as described herein. Compared to wild-type IL-2, the IL-2 mutant protein may have a significantly reduced affinity for CD25 or the trimeric high-affinity IL-2R. In some embodiments, the binding affinity of the IL-2 mutant protein for the high-affinity IL-2R is 1 / 100, 1 / 300, 1 / 500, 1 / 1,000, or 1 / 10,000 of the binding affinity of wild-type IL-2 for the high-affinity IL-2R. Unless otherwise specified, all residue numbering in the IL-2 and IL-2 mutant proteins described herein is consistent with the numbering in SEQ ID NO:1.

[0066] The present disclosure further provides a prodrug comprising a mutant IL-2 polypeptide as described above, and a carrier moiety. In some embodiments, the carrier moiety comprises an antigen-binding moiety, wherein the antigen-binding moiety binds an antigen expressed on an immune cell. In some embodiments, the prodrug comprises an antigen-binding moiety, wherein the antigen-binding moiety binds an antigen expressed on a tumor cell or in a tumor microenvironment. In some embodiments, the carrier moiety comprises two or more antigen-binding moieties, wherein the antigen-binding moieties bind two different antigens, wherein one of the antigens is expressed on an immune cell and the other antigen is expressed on a tumor cell or in a tumor microenvironment.

[0067] In some embodiments, the prodrug further comprises a masking moiety, wherein the masking moiety 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 β (IL-2Rβ) or a functional analogue 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 an antigen-binding fragment thereof, wherein the antibody binds to IL-2. In some embodiments, the masking moiety is a scFv. In some embodiments, the scFv comprises an amino acid sequence selected from SEQ ID NO:34, 35, and 36. In some embodiments, the IL-2 prodrug further comprises a cleavable peptide linker. In some embodiments, the cleavable linker connects the masking moiety to the carrier moiety. In some embodiments, the cleavable linker connects the mutant IL-2 polypeptide to the carrier moiety.

[0068] A. Mutant IL-2 polypeptide

[0069] In the IL-2 prodrugs of the present invention, the IL-2 agonist polypeptide is an IL-2 mutant protein derived from human IL-2. Mutant IL-2s with reduced or eliminated interaction between IL-2 and CD25 have been previously disclosed. For example, WO 2008 / 0034473 relates to mutations R38W and F42K, while WO 2012 / 107417 relates to a mutation at position 72. U.S. Patent Publication 2003 / 0124678 relates to 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 5,229,109) describe the introduction of two mutations, R38A and F42K, to obtain an IL-2 mutant protein with reduced ability to induce natural killer (NK) cells to secrete pro-inflammatory cytokines. EP2639241B1 relates to mutations in stimulating T regIn terms of cells, the effective IL-2 mutant proteins are at most 1 / 1,000 of natural IL-2, and involve IL-2 mutant proteins with mutations selected from the following: 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 Publication 2014 / 0328791 relates to pegylated IL-2 with reduced affinity for CD25. These IL-2 mutants are referred to as "non-α" IL-2 agonist polypeptides and show preferential activation of T effector cells and natural killer (NK) cells compared to regulatory T cells. Such IL-2 mutants may contain one or more mutations selected from the following: 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 (numbered according to SEQ ID NO:1). Unfortunately, such mutations may pose a potential immunogenic risk. For example, the IL-2 mutant containing the mutations T3A / R38S / F42A / Y45A / E62A / C125S (numbered according to SEQ ID NO:1) shows additional hotspots based on MHC class II binding predictions using a T cell epitope prediction tool. Table 1 shows the prediction results of wild-type IL-2. Table 2 shows the results of the IL-2 mutant with T3A / R38S / F42A / Y45A / E62A / C125S (numbered according to SEQ ID NO:1). This mutant is named IL-2V1 and contains the amino acid sequence of SEQ ID NO:176.

[0070] Table 1. MHC class II molecule binding prediction of WT IL-2 using a T cell epitope prediction tool

[0071]

[0072] Table 2. MHC class II molecule binding prediction of the IL-2 mutant with "non-α" mutations, T3A, and C125S (IL-2V1, SEQ ID NO:176) using a T cell epitope prediction tool

[0073]

[0074]

[0075] In some embodiments, the present invention provides novel IL-2 mutant proteins (mutant IL-2 polypeptides) with a lower risk of immunogenicity. In some embodiments, the mutant IL-2 polypeptide comprises a mutation at position L36 (numbered according to SEQ ID NO: 1). In certain embodiments, the mutant IL-2 polypeptide comprises an L36I mutation (numbered 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 an amino acid sequence that is at least 95%, at least 98%, or at least 99% identical to an amino acid sequence selected from SEQ ID NOs: 2-33.

[0076] In some embodiments, the mutant IL-2 polypeptides disclosed herein may further comprise a mutation at A73 (e.g., mutated to T or another amino acid residue) and / or a K35N mutation. Without being bound by theory, the inventors expect that A73 and K35 are potential glycosylation sites on IL-2, and mutations at these glycosylation sites modulate the affinity of the IL-2 mutant protein for the IL-2 receptor. The mutant IL-2 polypeptide will have safer clinical properties and can be used in patients in need of IL-2 activity, such as patients in need of stimulating the immune system (e.g., cancer patients and AIDS patients). The mutant IL-2 polypeptide can be used as a separate entity or in a conjugate (e.g., fused to a carrier, such as in a prodrug of the present invention).

[0077] In some embodiments, the mutant IL-2 polypeptide of the present invention may comprise a mutation at L36 (e.g., L36I) and one or more mutations at positions selected from the following: T3, D20, K35, R38, F42, F44, Y45, E62, E68, L72, A73, N88, N90, C125, and Q126 (numbered according to SEQ ID NO: 1). In certain embodiments, the mutant IL-2 polypeptide comprises mutations at L36, R38, F42, Y45, and A73 (numbered according to SEQ ID NO: 1).

[0078] In some embodiments, the mutant human IL-2 polypeptide of the present invention may comprise the K35N mutation and one or more mutations at positions selected from the following: T3, D20, R38, F42, F44, Y45, E62, E68, L72, A73, N88, N90, C125, and Q126 (numbered according to SEQ ID NO:1). In certain embodiments, the mutant human IL-2 polypeptide comprises the mutation K35N and additional mutations at R38, F42, and Y45, with or without a mutation at A73. In some embodiments, in addition to the mutation at L36, the mutant human IL-2 polypeptide of the present invention may further comprise the K35N mutation and one or more mutations at positions selected from the following: T3, D20, R38, F42, F44, Y45, E62, E68, L72, A73, N88, N90, C125, and Q126 (numbered according to SEQ ID NO:1).

[0079] 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 (numbered 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 (numbered according to SEQ ID NO:1). Additional mutations at T3 and / or C125 may also be included.

[0080] B. Masking moiety of the prodrug

[0081] The cytokine antagonist (i.e., the masking moiety) in the immunoconjugates of the present invention may comprise a peptide, an antibody, or an 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, wherein the masking moiety binds to the mutant IL-2 polypeptide disclosed herein and inhibits the biological activity of the mutant IL-2 polypeptide.

[0082] For example, IL-2 antagonists can include peptides and antibodies that bind to IL-2 and interfere with the binding of the IL-2 moiety to its receptor, resulting in a decrease in the biological activity of the IL-2 moiety when it is masked. In some embodiments, the IL-2 antagonist comprises the extracellular domain of IL-2Rβ or IL-2Rγ or a functional analogue thereof, such as a domain 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 that is 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 an antigen-binding fragment thereof. In certain embodiments, the IL-2 antagonist comprises an antibody or a fragment thereof that blocks the binding of IL-2 or an IL-2 mutant protein to the IL-2 receptor. In certain embodiments, the IL-2 antagonist comprises a scFv, Fab, or single-chain Fab having the same CDR sequences as a scFv comprising an amino acid sequence selected from SEQ ID NOs: 34, 35, and 36.

[0083] In some other embodiments, the prodrug further comprises a peptide linker (e.g., cleavable or non-cleavable), wherein the peptide linker connects the masking moiety to the carrier moiety. In some other embodiments, the peptide linker connects the mutant IL-2 polypeptide to the carrier moiety.

[0084] C. Carrier moiety of the prodrug

[0085] The carrier moiety of the prodrug of the present invention can be an antigen-binding moiety or a moiety that does not bind to an antigen. The carrier moiety can improve the PK properties of the cytokine agonist polypeptide, such as the serum half-life, and can also target the cytokine agonist polypeptide to a target site in the body, such as a tumor site.

[0086] 1. Antigen-binding carrier moiety

[0087] The carrier moiety can be an antibody or an 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, an F(ab')2 fragment, an Fv fragment, a disulfide-linked Fv fragment, a single-domain antibody, a nanobody, or a single-chain antibody (scFv). In some embodiments, the antigen-binding moiety is a bispecific antigen-binding moiety and can bind to two different antigens or two different epitopes on the same antigen. The antigen-binding moiety can provide additional and potentially synergistic therapeutic efficacy for the cytokine agonist polypeptide.

[0088] Cytokine agonist polypeptides and their masks can be fused to the N-terminus and / or C-terminus of the light chain and / or heavy chain of the antigen-binding portion. For example, cytokine agonist polypeptides and their masks can be fused to the heavy chain of an antibody or an antigen-binding fragment thereof. In some embodiments, cytokine agonist polypeptides and their masks can be fused to the light chain of an antibody or an antigen-binding fragment thereof. 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 portion) 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 of the heavy chains 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, wherein the two heavy chains contain mutations that allow specific pairing of the two heavy chains.

[0089] Strategies for forming heterodimers are well known (see, e.g., Spies et al., Mol. Imm. (2015) 67(2)(A):95-106). For example, two heavy chain polypeptides in a prodrug can form a stable heterodimer through "knobs-into-holes" mutations. The "knobs-into-holes" mutations are made to facilitate the formation of heterodimers of antibody heavy chains, and such mutations are commonly used to prepare bispecific antibodies (see, e.g., U.S. Patent 8,642,745). For example, the Fc domain of an antibody can comprise a T366W mutation in the CH3 domain of the "knob chain" and T366S, L368A, and / or Y407V mutations in the CH3 domain of the "hole chain". Additional interchain disulfide bonds between CH3 domains can also be used, such as by introducing a Y349C mutation into the CH3 domain of the "knob chain" and an E356C or S354C mutation into 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 comprise 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 can 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, where the additional Y349C mutation is in one CH3 domain and the additional E356C or S354C mutation is in the other CH3 domain, thereby forming an interchain disulfide bond (always numbered 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-holes techniques can alternatively or additionally be used, such as the knobs-into-holes techniques described in EP1870459A1. Thus, another example of a knobs-into-holes mutation of an antibody portion is having an R409D / K370E mutation in the CH3 domain of the "knob chain" and a D399K / E357K mutation (Eu numbering) in the CH3 domain of the "hole chain".

[0090] In some embodiments, the antibody portion of the prodrug contains L234A and L235A (“LALA”) mutations in its Fc domain. The LALA mutations abrogate complement binding and fixation as well as Fcγ-dependent ADCC (see, e.g., Hezareh et al., Journal of Virology (J. Virol.) (2001) 75(24):12161-8). In additional embodiments, in the antibody portion, in addition to the hinge and CH2 mutations, there are also LALA mutations.

[0091] In some embodiments, the antibody portion contains M252Y / S254T / T256E (“YTE”) mutations in its Fc domain. The YTE mutations allow simultaneous modulation of serum half-life, tissue distribution, and the activity of IgG1 (see Dall'Acqua et al., Journal of Biological Chemistry (J Biol Chem.) (2006) 281:23514-24; and Robbie et al., Antimicrobial Agents and Chemotherapy (Antimicrob Agents Chemother.) (2013) 57(12):6147-53). In additional embodiments, in the antibody portion, in addition to the hinge and CH2 mutations, there are also YTE mutations. In certain embodiments, the antibody portion has YTE, LALA, and hinge and CH2 mutations or any combination thereof.

[0092] The antigen-binding portion can bind to an antigen on the surface of cells such as immune cells (e.g., T cells, NK cells, and macrophages). In other cases, the antigen-binding portion can bind to a cytokine. For example, the antigen-binding portion can be an antibody or an antigen-binding fragment thereof that binds to: PD-1, LAG-3, TIM-3, TIGIT, SIRPα, 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), the extra domain B of fibronectin (EDB), PSA, 5T4, CD47, CMET, Claudin 6, CD24, or melanoma-associated chondroitin sulfate proteoglycan (MCSP) or TGF-β. The antibody may have the ability to activate immune cells and enhance their anti-cancer activity.

[0093] The antigen-binding portion can be an antibody or an antigen-binding fragment thereof that binds to an antigen on the surface of a tumor cell. For example, the antigen-binding portion can bind to FAPα, 5T4, Trop-2, PD-L1, HER-2, EGFR, Claudin 18.2, or carcinoembryonic antigen (CEA). The antibody may or may not have antibody-dependent cell cytotoxicity (ADCC) activity. The antibody can also be conjugated to a cytotoxic drug.

[0094] In some embodiments, the antigen-binding portion is an anti-PD1 antibody. In some embodiments, the anti-PD-1 antibody is pembrolizumab or nivolumab. In some embodiments, the antigen-binding portion is an anti-CD8 antibody or a CD8-binding fragment thereof. In some embodiments, the antigen-binding portion is an anti-NKG2A, NKG2D, CD16A, NKp30, NKP44 or NKP46 antibody or a binding fragment thereof.

[0095] In some embodiments, the PD-1 binding portion comprises an antibody or a 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 its antigen-binding portion specifically binds to PD-1. For example, antibodies that target PD-1 and can be found for use in the present invention include, but are not limited to, nivolumab (BMS-936558, Bristol-Myers Squibb), pembrolizumab (lambrolizumab, MK03475 or MK-3475, Merck), humanized anti-PD-1 antibody JS001 (Shanghai Junshi Biosciences Co., Ltd.), monoclonal anti-PD-1 antibody TSR-042 (Tesaro), 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 Medicine Co., Ltd.), human monoclonal antibody REGN2810 (Regeneron Pharmaceuticals), human monoclonal antibody MDX-1106 (Bristol-Myers Squibb), and / or humanized anti-PD-1 IgG4 antibody PDR001 (Novartis). In some embodiments, the PD-1 antibody is 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 portion comprises a single-domain antibody or a nanobody. In some embodiments, the single-domain antibody comprises those antibodies disclosed in WO 2019 / 137541.

[0096] In some embodiments, the antigen-binding portion binds to guanylate 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), phosphatidylinositol glycan-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 portion binds to the epidermal growth factor (EGF)-like domain of DLL3. In some embodiments, the antigen-binding portion binds to the delta / serrate / Lag2 (DSL)-like domain of DLL3. In some embodiments, the antigen-binding portion binds to an epitope located after the 374th amino acid of GPC3. In some embodiments, the antigen-binding portion binds to the heparan sulfate of GPC3. In some embodiments, the antigen-binding portion binds to Claudin 18.2 and not to Claudin 18.1. In some embodiments, the antigen-binding portion binds to Claudin 18.1 with a binding affinity that is at least 10-fold weaker than that for Claudin 18.2.

[0097] Exemplary antigen-binding portions include trastuzumab, rituximab, brentuximab, cetuximab, panitumumab, GC33 (or its humanized version), anti-EGFR antibody mAb806 (or its humanized version), anti-dPNAG antibody F598, and antigen-binding fragments thereof. In some embodiments, the antigen-binding portion has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to trastuzumab, rituximab, brentuximab, cetuximab, or panitumumab, GC33 (or its humanized version), anti-EGFR antibody mAb806 (or its humanized version), anti-dPNAG antibody F598, or a fragment thereof. In some embodiments, the antigen-binding portion has an antibody heavy chain with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the antibody heavy chain of: trastuzumab, rituximab, brentuximab, cetuximab, panitumumab, GC33 (or its humanized version), anti-EGFR antibody mAb806 (or its humanized version), anti-dPNAG antibody F598, or a fragment thereof. In some embodiments, the antigen-binding portion has an antibody light chain with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the antibody light chain of: trastuzumab, rituximab, brentuximab, cetuximab, panitumumab, GC33 (or its humanized version), anti-EGFR antibody mAb806 (or its humanized version), 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.

[0098] Many CDR depictions are known in the art and are encompassed herein. One of ordinary skill in the art can readily determine the CDRs of a given depiction based on the sequences of the heavy or light chain variable regions. "Kabat" CDRs are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)). "Chothia" CDRs refer to the positions of structural loops (Chothia and Lesk, Canonical structures for the hypervariable regions of immunoglobulins, J. Mol. Biol. 196:901-917 (1987)). "AbM" CDRs represent a compromise between Kabat CDRs and Chothia structural loops and are used by the AbM antibody modeling software of Oxford Molecular. "Contact" CDRs are based on the analysis of available complex crystal structures. Residues from each of these CDRs are noted in Table 3 below. Unless otherwise specified herein, the amino acid numbering of an antibody refers to the Kabat numbering scheme described in Kabat et al., supra, including in cases where CDR depictions are made 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 that correspond to framework regions (FRs) of the variable domain or shortening or insertions into the CDRs. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and insertion residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat residue numbering of a given antibody can be determined by aligning the homologous region of the antibody sequence with the "standard" Kabat numbered sequence.

[0099] Table 3. CDR Depictions According to Various Schemes

[0100] CDR Kabat AbM Chothia Contact VL - CDR1 L24—L34 L24—L34 L26—L32 L30—L36 VL - CDR2 L50—L56 L50—L56 L50—L52 L46—L55 VL - CDR3 L89—L97 L89—L97 L91—L96 L89—L96 VH - CDR1 (Kabat numbering) H31—H35B H26—H35B H26—H32 H30—H35B VH - CDR1 (Chothia numbering) H31—H35 H26—H35 H26—H32 H30—H35 VH - CDR2 H50—H65 H50—H58 H53—H55 H47—H58 VH - CDR3 H95—H102 H95—H102 H95—H101 H93—H101

[0101] In some embodiments, the CDR is an "extended CDR" and encompasses regions that start or end according to different schemes. For example, the extended CDR can be as follows: L24—L36, L26—L34, or L26—L36 (VL-CDR1); L46—L52, L46—L56, or L50—L55 (VL-CDR2); L91—L97 (VL-CDR3); H47—H55, H47—H65, H50—H55, H53—H58, or H53—H65 (VH-CDR2); and / or H93—H102 (VH-CDR3).

[0102] In some embodiments, the antigen-binding portion binds to HER2 and comprises a light chain or a fragment thereof having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 126, and a heavy chain or a fragment thereof having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 127. 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.

[0103] In some embodiments, the antigen-binding portion binds to CD20 and comprises a light chain or a fragment thereof having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 128, and a heavy chain or a fragment thereof having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 129. 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.

[0104] In some embodiments, the antigen-binding portion binds to CD30 and comprises a light chain or a fragment thereof having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 130, and a heavy chain or a fragment thereof having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 131. 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.

[0105] In some embodiments, the antigen-binding portion binds to EGFR and comprises a light chain or a fragment thereof having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 132, and a heavy chain or a fragment thereof having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 133. 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.

[0106] In some embodiments, the antigen-binding portion binds to EGFR and comprises a light chain or a fragment thereof having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 134, and a heavy chain or a fragment thereof having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 135. 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.

[0107] In some embodiments, the antigen-binding portion binds to c-MET and comprises a light chain or a fragment thereof having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 136, and a heavy chain or a fragment thereof having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 137. 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.

[0108] In some embodiments, the antigen-binding portion binds to GPC3 and comprises a light chain or a fragment thereof having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 138, and a heavy chain or a fragment thereof having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 139. 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.

[0109] In some embodiments, the antigen-binding portion binds to Claudin 18.2 and comprises a light chain or a fragment thereof having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 140, and a heavy chain or a fragment thereof having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 141. 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.

[0110] In some embodiments, the antigen-binding portion binds to FAPα and comprises a light chain or a fragment thereof having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 158 or 159, and a heavy chain or a fragment thereof having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 160. 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 FAPα and comprises a light chain variable domain having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 161, and a heavy chain variable domain having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity 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 shown in SEQ ID NO: 158 or 159 and the heavy chain amino acid sequence shown in SEQ ID NO: 160.

[0111] In some embodiments, the antigen-binding portion binds to carcinoembryonic antigen (CEA) and can be derived from the antibody PR1A3 (U.S. Patent 8,642,742). The anti-CEA antibody comprises a light chain or a fragment thereof having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 156, and a heavy chain or a fragment thereof having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 157. 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 that comprises the light chain variable domain amino acid sequence shown in SEQ ID NO: 156 and the heavy chain variable domain amino acid sequence shown in SEQ ID NO: 157.

[0112] In some embodiments, the antigen-binding portion binds to PDL1 and comprises a light chain or a fragment thereof having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 167, and a heavy chain or a fragment thereof having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 168. 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.

[0113] In some embodiments, the antigen-binding portion binds to 5T4 and comprises a light chain variable domain having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 165 or 166, and a heavy chain variable domain or a fragment thereof having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity 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.

[0114] In some embodiments, the antigen-binding portion binds to Trop-2 and comprises a light chain variable region and a heavy chain variable region. The light chain variable region comprises CDR1, CDR2, and CDR3. CDR1 comprises the amino acid sequence of KASQDVSIAVA (SEQ ID NO:142), CDR2 comprises the amino acid sequence of SASYRYT (SEQ ID NO:143), and CDR3 comprises the amino acid sequence of QQHYITPLT (SEQ ID NO:144). The heavy chain variable region comprises CDR1, CDR2, and CDR3. CDR1 comprises the amino acid sequence of NYGMN (SEQ ID NO:145), CDR2 comprises the amino acid sequence of WINTYTGEPTYTDDFKG (SEQ ID NO:146), and CDR3 comprises the amino acid sequence of GGFGSSYWYFDV (SEQ ID NO:147).

[0115] In some embodiments, the antigen-binding portion binds to mesothelin and comprises a light chain variable region and a heavy chain variable region. The light chain variable region comprises CDR1, CDR2, and CDR3. CDR1 comprises the amino acid sequence of SASSSVSYMH (SEQ ID NO:148), CDR2 comprises the amino acid sequence of DTSKLAS (SEQ ID NO:149), and CDR3 comprises the amino acid sequence of QQWSGYPLT (SEQ ID NO:150). The heavy chain variable region comprises CDR1, CDR2, and CDR3. CDR1 comprises the amino acid sequence of GYTMN (SEQ ID NO:151), CDR2 comprises the amino acid sequence of LITPYNGASSYNQKFRG (SEQ ID NO:152), and CDR3 comprises the amino acid sequence of GGYDGRGFDY (SEQ ID NO:153).

[0116] In some embodiments, the antigen-binding portion comprises one, two, or three antigen-binding domains. For example, the antigen-binding portion is bispecific and binds to two different antigens selected from the group consisting of HER2, HER3, EGFR, 5T4, FAPα, Trop-2, GPC3, VEGFR2, Claudin 18.2, and PD-L1. In some embodiments, the bispecific antigen-binding portion binds to two different epitopes of HER2.

[0117] 2. Other vector portions

[0118] Other non-antigen-binding carrier moieties can be used for the prodrugs of the present invention. For example, antibody Fc domains (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc), polymers (e.g., PEG), albumin (e.g., human albumin), or fragments or nanoparticles thereof can be used.

[0119] For example, cytokine agonist polypeptides and their antagonists 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 through a peptide linker) to the C-terminus or N-terminus of one of the Fc domain polypeptide chains, and the cytokine masker is fused to the C-terminus or N-terminus of the other Fc domain polypeptide chain through a cleavable or non-cleavable peptide linker, wherein the two Fc domain polypeptide chains contain mutations that allow specific pairing of the two different Fc chains. In some embodiments, the Fc domain contains the above-mentioned knobs-into-holes mutations. In additional embodiments, the Fc domain can further contain the above-mentioned YTE and / or LALA mutations.

[0120] The carrier moiety of the prodrug can comprise albumin (e.g., human serum albumin) or a fragment thereof. An exemplary sequence of albumin is shown 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.

[0121] In some embodiments, the carrier moiety comprises an albumin fragment (e.g., a human serum albumin fragment) having a length of: about 10 or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino acids, 50 or more amino acids, 60 or more amino acids, 70 or more amino acids, 80 or more amino acids, 90 or more amino acids, 100 or more amino acids, 120 or more amino acids, 140 or more amino acids, 160 or more amino acids, 180 or more amino acids, 200 or more amino acids, 250 or more amino acids, 300 or more amino acids, 350 or more amino acids, 400 or more amino acids, 450 or more amino acids, 500 or more amino acids, or 550 or more amino acids. In some embodiments, the length of the albumin fragment ranges from about 10 amino acids to about 584 amino acids (such as ranging from about 10 amino acids to about 20 amino acids, from about 20 amino acids to about 40 amino acids, from about 40 amino acids to about 80 amino acids, from about 80 amino acids to about 160 amino acids, from about 160 amino acids to about 250 amino acids, from about 250 amino acids to about 350 amino acids, from about 350 amino acids to about 450 amino acids, or from about 450 amino acids to about 550 amino acids). In some embodiments, the albumin fragment comprises a Sudlow I domain or a fragment thereof, or a Sudlow II domain or a fragment thereof.

[0122] D. Linker Component of Prodrug

[0123] The IL-2 agonist polypeptide can be fused to the carrier moiety with or without a peptide linker. The peptide linker can 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), GGGGSGGGGSGGGGS (SEQ ID NO:181), GGGGSAAGGGGAGGGGA (SEQ ID NO:182), or GGGGSGGGGSAAGGGGSGGGGS (SEQ ID NO:183).

[0124] The IL-2 masking agent can be fused to the cytokine moiety or the vector moiety via a cleavable linker. The cleavable linker can contain one or more (e.g., two or three) cleavable moieties (CMs). Each CM can be a substrate of an enzyme or protease selected from the group consisting of: 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 amino acid sequences comprising an amino acid sequence selected from SEQ ID NOs: 55-124.

[0125] Specific non-limiting examples of cytokine agonist polypeptides, cytokine masking agents, vectors, peptide linkers, and prodrugs are shown in the sequence section below. Further, the prodrugs and novel IL-2 mutant proteins of the present disclosure can be prepared by well-known recombinant techniques. For example, one or more expression vectors comprising the coding sequence of a polypeptide chain comprising a prodrug can be transfected into a mammalian host cell (e.g., CHO cells), and the cells can be cultured under conditions that permit expression of the coding sequence and assembly of the expressed polypeptide into a prodrug complex. To keep the prodrug inactive, host cells that do not express or express little 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.

[0126] II. Examples of prodrugs

[0127] In some embodiments, the prodrugs presented herein comprise an antibody fused to one or two of the above mutant IL-2 polypeptides.

[0128] In some embodiments, the prodrug comprises one mutant IL-2 polypeptide. By way of example, the prodrug comprises two identical light chains, a first heavy chain polypeptide chain, and a second heavy chain polypeptide chain, the two identical light chains having the amino acid sequence of SEQ ID NO: 38 or an amino acid sequence that is at least 90% identical thereto, the first heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 39 or an amino acid sequence that is at least 90% identical thereto, and the second heavy chain polypeptide chain having the amino acid sequence of SEQ ID NO: 40 or 41 or an amino acid sequence that is at least 90% identical thereto.

[0129] In some embodiments, the prodrug comprises a mutant IL-2 polypeptide. For example, the prodrug comprises two identical light chains, a first heavy chain polypeptide chain, and a second heavy chain polypeptide chain, wherein the two identical light chains have the amino acid sequence of SEQ ID NO: 38 or an amino acid sequence that is at least 90% identical thereto, the first heavy chain polypeptide chain comprises the amino acid sequence of SEQ ID NO: 47 or an amino acid sequence that is at least 90% identical thereto, and the second heavy chain polypeptide chain has the amino acid sequence of SEQ ID NO: 45 or 46 or an amino acid sequence that is at least 90% identical thereto.

[0130] In some embodiments, the prodrug comprises two mutant IL-2 polypeptides. For example, the prodrug comprises two identical light chains and two identical heavy chain polypeptide chains, wherein the two identical light chains have the amino acid sequence of SEQ ID NO: 38 or an amino acid sequence that is at least 90% identical thereto, and the two identical heavy chain polypeptide chains have the amino acid sequence of SEQ ID NO: 40 or 41 or an amino acid sequence that is at least 90% identical thereto.

[0131] For example, the prodrug comprises two identical light chains, a first heavy chain polypeptide chain, and a second heavy chain polypeptide chain, wherein the two identical light chains have the amino acid sequence of SEQ ID NO: 38 or an amino acid sequence that is at least 90% identical thereto, the first heavy chain polypeptide chain comprises the amino acid sequence of SEQ ID NO: 42, 43, or 44 or an amino acid sequence that is at least 90% identical thereto, and the second heavy chain polypeptide chain has the amino acid sequence of SEQ ID NO: 40, 41, or 125 or an amino acid sequence that is at least 90% identical thereto.

[0132] For example, the prodrug comprises two identical light chains and two identical heavy chain polypeptide chains, wherein the two identical light chains have the amino acid sequence of SEQ ID NO: 38 or an amino acid sequence that is at least 90% identical thereto, and the two identical heavy chain polypeptide chains have the amino acid sequence of SEQ ID NO: 48, 49, 50, or 51 or an amino acid sequence that is at least 90% identical thereto.

[0133] III. Pharmaceutical composition

[0134] A pharmaceutical composition comprising a prodrug and a mutant protein (i.e., an active pharmaceutical ingredient or API) of the present disclosure can be prepared by mixing an API of desired purity with one or more optional pharmaceutically acceptable excipients in the form of a lyophilized formulation or an aqueous solution (see, e.g., Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. ed. (1980)). Pharmaceutically acceptable excipients (or carriers) are generally non-toxic to the recipient 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 (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol, or benzyl alcohol; alkyl parabens, such as methyl paraben 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 dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG).

[0135] Buffers are used to control the pH within a range that optimizes the therapeutic effect, especially where stability is pH-dependent. The buffer is preferably present at a concentration in the range of about 50 mM to about 250 mM. Suitable buffers for use with the present invention include organic and inorganic acids and their salts, such as citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, and acetate. Additionally, the buffer can include histidine and trimethylamine salts, such as Tris.

[0136] Preservatives are added to slow microbial growth and are generally present in the range of 0.2% - 1.0% (w / v). Suitable preservatives for use with the present invention include octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butanol, or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

[0137] There are tonicity agents, sometimes referred to as "stabilizers", to adjust or maintain the tonicity of the liquid in the composition. When used with large charged biomolecules such as proteins and antibodies, tonicity agents are often referred to as "stabilizers" because they can interact with the charged groups of amino acid side chains, thereby reducing the likelihood of intermolecular and intramolecular interactions. Depending on the relative amounts of the other components, the tonicity agent can be present in any amount between 0.1% by weight and 25% by weight, or more preferably between 1% by weight and 5% by weight. Preferred tonicity agents include polyhydric alcohols, preferably trihydric or higher polyhydric alcohols such as glycerol, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0138] There is a non-ionic surfactant or detergent (also referred to as a "wetting agent") to assist in dissolving the therapeutic agent and to protect the therapeutic protein from agitation-induced aggregation, which also allows the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. The non-ionic surfactant is present in the 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.

[0139] Suitable non-ionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (184, 188, etc.), polyhydric alcohols, polyoxyethylene sorbitan monoethers ( etc.), lauromacrogol 400, polyethylene glycol 40 stearate, polyoxyethylene hydrogenated castor oil 10, polyoxyethylene hydrogenated castor oil 50, polyoxyethylene hydrogenated castor oil 60, glyceryl monostearate, sucrose fatty acid ester, methylcellulose, and carboxymethylcellulose. Anionic detergents that can be used include sodium lauryl sulfate, sodium dioctyl sulfosuccinate, and sodium dioctyl sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0140] The choice of pharmaceutical carrier, excipient, or diluent can be selected according to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition can additionally contain any suitable binder, lubricant, suspending agent, coating agent, or solubilizing agent.

[0141] Depending on the different delivery systems, there may be different composition / formulation requirements. For example, the pharmaceutical compositions useful in the present invention can be formulated for administration using a micropump or via a mucosal route, such as a nasal spray or aerosol for inhalation or an ingestible solution, or the composition can be formulated in an injectable form for parenteral delivery, via, for example, an intravenous route, an intramuscular route, or a subcutaneous route.

[0142] In some embodiments, the pharmaceutical compositions of the present disclosure are lyophilized protein formulations. In other embodiments, the pharmaceutical composition can be an aqueous liquid formulation.

[0143] IV. Treatment method

[0144] The prodrugs and novel IL-2 mutant proteins (mutant IL-2 polypeptides) of the present invention can be used to treat diseases. In some embodiments, the prodrug or the mutant IL-2 polypeptide is used to treat cancer. In some embodiments, the prodrug or the mutant IL-2 polypeptide is used to treat an infection, such as when the drug molecule is an antibacterial or antiviral agent.

[0145] In some embodiments, a method of treating a disease (such as cancer, viral infection or bacterial infection) in a subject comprises administering to the subject an effective amount of a prodrug or a mutant IL-2 polypeptide disclosed herein.

[0146] In some embodiments, the cancer is a solid tumor. 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.

[0147] In some embodiments, the prodrug or the mutant IL-2 polypeptide is used to treat a bacterial infection, such as sepsis. In some embodiments, the bacterium causing the bacterial infection is a drug-resistant bacterium. In some embodiments, the antigen-binding portion (vector portion) disclosed herein binds to a bacterial antigen.

[0148] In some embodiments, the prodrug or the mutant IL-2 polypeptide is used to treat a viral infection. In some embodiments, the virus causing the viral infection is hepatitis C (HCV), hepatitis B (HBV), human immunodeficiency virus (HIV), human papillomavirus (HPV). In some embodiments, the antigen-binding portion disclosed herein binds to a viral antigen.

[0149] Typically, the dosage and route of administration of the pharmaceutical composition of the present invention are determined according to standard pharmaceutical practice based on the body weight and condition of the subject. In some embodiments, the pharmaceutical composition is administered to the subject by any route, including oral administration, transdermal administration, administration by inhalation, intravenous administration, intra-arterial administration, intramuscular administration, direct application to the wound site, application to the surgical site, intraperitoneal administration, administration by suppository, subcutaneous administration, intradermal administration, transdermal administration, administration by nebulization, intrapleural administration, intraventricular administration, intra-articular administration, intraocular administration, intracranial administration or intraspinal administration. In some embodiments, the composition is administered intravenously to the subject.

[0150] In some embodiments, the dosage of the pharmaceutical composition is a single dose or multiple doses. In some embodiments, the dosage is administered to the subject once a day, twice a day, three times a day, or four times a day or more. In some embodiments, about 1 or more (e.g., about 2, 3, 4, 5, 6, 7 or more) doses are administered within a week. In some embodiments, the pharmaceutical composition is administered once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once a week for 2 weeks out of 3 weeks or once a week for 3 weeks out of 4 weeks. In some embodiments, multiple doses are administered over the course of several days, weeks, months or years. In some embodiments, a treatment course is about 1 dose or more (e.g., about 2 doses, 3 doses, 4 doses, 5 doses, 7 doses, 10 doses, 15 doses or 20 or more doses).

[0151] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by one of ordinary skill in the art. Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, shall control. Further, unless the context otherwise requires, singular terms shall include plural meanings and plural terms shall include singular meanings. Throughout the specification and examples, the words "have", "comprise", or variations such as "has", "having", "comprises", or "comprising" shall be understood to imply the inclusion of the 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 many documents are cited herein, such citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. As used herein, when applied to one or more values of interest, the term "about" or "approximately" refers to a value similar to the reference value. In certain embodiments, unless otherwise specified or otherwise apparent from the context, the term refers to a range of values that are 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater than or less than) of the specified reference value.

[0152] According to the present disclosure, reverse reference in dependent claims means a shorthand for the direct and explicit disclosure of each and every combination of the claims indicated by the reverse reference. Further, the headings herein are created for convenience of organization and are not intended to limit in any way the scope of the claimed invention.

[0153] V. Exemplary embodiments

[0154] Additional specific embodiments of the present disclosure are described below. These embodiments are intended to illustrate the compositions and methods described in the present disclosure and are not intended to limit the scope of the present disclosure.

[0155] 1. A mutant human interleukin-2 (IL-2) polypeptide comprising the mutation L36I (numbered 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 shown in SEQ ID NO:1.

[0156] 2. The mutant human interleukin-2 polypeptide according to embodiment 1, wherein the mutant IL-2 further comprises a second mutation C125A (numbered according to SEQ ID NO:1).

[0157] 3. The mutant human interleukin-2 polypeptide according to embodiment 1 or 2, wherein the mutant IL-2 comprises one or more additional amino acid mutations, each of which, compared to the wild-type IL-2 polypeptide, eliminates or reduces the affinity of the mutant IL-2 polypeptide for the high-affinity IL-2 receptor and maintains the affinity of the mutant IL-2 polypeptide for the medium-affinity IL-2 receptor.

[0158] 4. The mutant interleukin-2 polypeptide according to embodiment 3, wherein the additional amino acid mutations are located at positions selected from the positions corresponding to residues 35, 38, 42, 43, 45, 62, 68, and 72 (numbered according to SEQ ID NO:1).

[0159] 5. The mutant interleukin-2 polypeptide according to embodiment 1, 2, 3, or 4, wherein the mutant IL-2 further comprises mutations at one or more positions selected from residues 88, 91, 92, and 126 (numbered according to SEQ ID NO:1).

[0160] 6. The mutant interleukin-2 polypeptide according to embodiment 3, 4, or 5, wherein the one or more additional 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, N88D, 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 (numbered according to SEQ ID NO:1).

[0161] 7. The mutant interleukin-2 polypeptide according to 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 the position corresponding to residue 3 of human IL-2 (numbered according to SEQ ID NO:1).

[0162] 8. The mutant interleukin-2 polypeptide according to embodiment 1, wherein the mutant IL-2 polypeptide comprises an amino acid sequence selected from SEQ ID NO: 2-33.

[0163] 9. The mutant interleukin-2 polypeptide according to any one of embodiments 1 to 8, wherein the mutant IL-2 polypeptide is linked to a carrier.

[0164] 10. The mutant interleukin-2 polypeptide according to embodiment 9, wherein the carrier comprises an antigen-binding portion.

[0165] 11. An immunoconjugate comprising the mutant IL-2 polypeptide according to any one of embodiments 1 to 8, and an antigen-binding portion.

[0166] 12. The immunoconjugate according to embodiment 11, wherein the immunoconjugate comprises a first antigen-binding portion and a second antigen-binding portion.

[0167] 13. The immunoconjugate according to any one of embodiments 11 and 12, wherein the antigen-binding portion is an antibody or an antibody fragment.

[0168] 14. The immunoconjugate according to embodiment 11 or 12, wherein the antigen-binding portion is selected from Fab molecules and scFv molecules.

[0169] 15. The immunoconjugate according to any one of embodiments 11 and 12, wherein the antigen-binding portion is an immunoglobulin molecule, specifically an IgG molecule.

[0170] 16. The immunoconjugate according to any one of embodiments 11 to 15, wherein the antigen-binding portion is directed against an antigen presented on or in the tumor cell environment of tumor cells.

[0171] 17. The immunoconjugate according to any one of embodiments 11 to 15, wherein the antigen-binding portion is directed against an antigen presented on immune cells.

[0172] 18. The immunoconjugate according to 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.

[0173] 19. The immunoconjugate according to embodiment 16, wherein the antigen is selected from the group consisting of fibroblast activation protein (FAP), the A1 domain of tenascin C (TNC A1), the A2 domain of tenascin C (TNC A2), extra domain B of fibronectin (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).

[0174] 20. The immunoconjugate according to embodiment 17 or 18, wherein the antigen is selected from the group consisting of PD-1, PDL1, CD8, Tim-3, LAG-3, TIGIT, SIRPα, CTLA-4, CSF1R, NKG2A, NKG2D, CD16A, NKp30, and NKp46.

[0175] 21. The immunoconjugate according to embodiment 12, wherein the antigen is selected from the group consisting of PD-1, CD8, Tim-3, LAG-3, TIGIT, SIRPα, 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), extra domain B of fibronectin (EDB), carcinoembryonic antigen (CEA), PSA, 5T4, PDL1, CD47, EGFR, CMET, Claudin 18.2, Claudin 6, CD24, and melanoma-associated chondroitin sulfate proteoglycan (MCSP).

[0176] 22. The mutant IL-2 polypeptide or immunoconjugate according to any one of embodiments 1 to 21, wherein the IL-2 polypeptide is masked.

[0177] 23. The mutant IL-2 polypeptide or immunoconjugate according to any one of embodiments 1 to 21, wherein the IL-2 polypeptide is partially masked by a masking moiety, and the masking moiety comprises the extracellular domain (ECD) of interleukin-2 receptor beta (IL-2Rβ) or a functional fragment thereof.

[0178] 24. The mutant IL-2 polypeptide or immunoconjugate according to embodiment 23, wherein the IL-2Rβ-ECD comprises the amino acid sequence of SEQ ID NO:37 or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:37.

[0179] 25. The mutant IL-2 polypeptide or immunoconjugate according to any one of embodiments 1 to 21, wherein the IL-2 polypeptide is masked by a masking moiety, wherein the masking moiety comprises an scFv or a Fab, and wherein the scFv or Fab binds to the IL-2 polypeptide and inhibits the biological activity of the IL-2 polypeptide.

[0180] 26. The mutant IL-2 polypeptide or immunoconjugate according to 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 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 34, 35 or 36.

[0181] 27. The mutant IL-2 polypeptide or immunoconjugate according to embodiment 23, wherein the masking moiety comprises an scFv or a Fab that comprises the same heavy chain CDRs and the same light chain CDRs as the scFv having the amino acid sequence of SEQ ID NO: 34, 35 or 36.

[0182] 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.

[0183] 29. The immunoconjugate according to embodiment 17 or 28, wherein the antigen-binding moiety is an antibody or a binding fragment thereof against PD-1; and wherein the anti-PD-1 antibody is selected from nivolumab and pembrolizumab.

[0184] 30. An immunoconjugate comprising two identical light chains and a first heavy chain polypeptide chain and a second heavy chain polypeptide chain; wherein the light chain comprises the amino acid sequence of SEQ ID NO: 38 or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 38; the first heavy chain polypeptide chain comprises an amino acid sequence selected from SEQ ID NO: 40, 41, 44 and 45, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 40, 41, 44 or 45; and the second heavy chain polypeptide chain comprises the amino acid sequence of SEQ ID NO: 42, 43, 46 and 47 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 42, 43, 46 or 47.

[0185] 31. An immunoconjugate comprising two identical light chains and two identical heavy chain polypeptide chains; wherein the light chain comprises the amino acid sequence of SEQ ID NO: 38 or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 38; and the heavy chain polypeptide chain comprises an amino acid sequence selected from SEQ ID NO: 40, 41, 48, 49, 50, and 51, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 40, 41, 48, 49, 50, or 51.

[0186] 32. The immunoconjugate according to embodiment 17 or 28, wherein the antigen-binding portion is an antibody against CD8 or a binding fragment thereof; and wherein the anti-CD8 antibody is OKT8 or a humanized OKT8, or comprises a heavy chain CDR derived from OKT8 and an identical light chain CDR.

[0187] 33. The immunoconjugate according to embodiment 32, wherein the antigen-binding portion further comprises an additional antigen-binding portion that binds to an antigen on or in the tumor cell environment.

[0188] 34. The immunoconjugate according to embodiment 33, wherein the tumor-associated antigen is selected from the group consisting of fibroblast activation protein (FAP), tenascin C domain A1 (TNC A1), tenascin C domain A2 (TNC A2), fibronectin extra domain 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).

[0189] 35. An isolated polynucleotide encoding a mutant IL-2 polypeptide or an immunoconjugate according to any one of embodiments 1 to 34.

[0190] 36. An expression vector comprising the polynucleotide according to embodiment 35.

[0191] 37. A host cell comprising the polynucleotide according to embodiment 35 or the expression vector according to embodiment 36.

[0192] 38. A method for producing a mutant IL-2 polypeptide or an immunoconjugate thereof, the method comprising culturing the host cell according to embodiment 37 under conditions suitable for expressing the mutant IL-2 polypeptide or the immunoconjugate.

[0193] 39. A mutant IL-2 polypeptide or immunoconjugate produced by the method according to Example 38.

[0194] 40. A pharmaceutical composition comprising a mutant IL-2 polypeptide or immunoconjugate according to any one of Examples 1 to 34 or 39, and a pharmaceutically acceptable carrier.

[0195] 41. A mutant IL-2 polypeptide or immunoconjugate according to any one of Examples 1 to 34 or 39 for treating a disease in an individual in need thereof.

[0196] 42. The mutant IL-2 polypeptide or immunoconjugate according to Example 41, wherein the disease is cancer.

[0197] 43. Use of a mutant IL-2 polypeptide or immunoconjugate according to any one of Examples 1 to 34 or 39 for the preparation of a medicament for treating a disease in an individual in need thereof.

[0198] 44. A method for treating a disease in an individual, the method comprising administering to the individual a therapeutically effective amount of a composition in a pharmaceutically acceptable form comprising a mutant IL-2 polypeptide or immunoconjugate according to any one of Examples 1 to 34 or 39.

[0199] 45. The method according to Example 44, wherein the disease is cancer.

[0200] 46. A method for stimulating the immune system of an individual, the method comprising administering to the individual an effective amount of a composition in a pharmaceutically acceptable form comprising a mutant IL-2 polypeptide or immunoconjugate according to any one of Examples 1 to 34 or 39.

[0201] 47. A mutant IL-2 polypeptide or immunoconjugate according to any one of Examples 1 to 34 or 39, which has a reduced immunogenicity risk upon administration to an animal or a patient as compared to the corresponding IL-2 polypeptide or immunoconjugate comprising amino acid L at position 36 (numbered according to SEQ ID NO:1).

[0202] 48. The immunoconjugate according to Example 32, wherein the CD8 antibody comprises the 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 the 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 sequences shown in SEQ ID NO:53 or 54.

[0203] 49. The immunoconjugate according to embodiment 28, 29, 32, 33, 34 or 48, wherein the immunoconjugate comprises one or more cleavable peptide linkers; wherein the cleavable linker comprises an amino acid sequence selected from SEQ ID NOs: 55-124.

[0204] To better understand the present invention, the following examples are set forth. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

[0205] Examples

[0206] Example 1: Discovery of IL-2 mutant polypeptides with reduced immunogenicity

[0207] A number of human IL-2 variants were prepared, 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 are relative to SEQ ID NO: 1). None of these mutations removed the immunogenic "hot spots" introduced by the "non-α" mutations (data not shown). Surprisingly, the introduction of the mutation L36I removed most of the "hot spots" introduced by the "non-α" mutations (Table 4).

[0208] Table 4. MHC class II molecule binding prediction of IL-2V1 (SEQ ID NO: 176) with the mutation L36I (IL-2V1 / L36I) using a T cell epitope prediction tool

[0209]

[0210] Additional "hot spots" introduced by the C125S mutation were removed by using the C125A mutation (Table 5). The sequence number / amino acid residue position is based on SEQ ID NO: 1.

[0211] Table 5. MHC class II molecule binding prediction of the IL-2 mutant with L36I, C125A, and "non-α" mutations (IL-2V1 / L36I / C125A) using a T cell epitope prediction tool

[0212]

[0213]

[0214] Surprisingly, IL-2V1 / L36I / C125A (SEQ ID NO:184) showed a lower immunogenicity risk compared to wild-type IL-2. Side-by-side comparison showed that this IL-2 mutant had fewer "hot spots" compared to wild-type IL-2 (Table 6; additional "hot spot" binding peptides in wild-type IL-2 are shown in bold; SEQ: SEQ ID NO).

[0215] Table 6. Comparison of "hot spots" in IL-2 mutant IL-2V1 / L36I / C125A and WT IL-2

[0216]

[0217] Example 2: Discovery of scFv molecules that bind to IL-2 and inhibit the interaction between IL-2 and IL-2Rβγ

[0218] A. Screening of scFv from a yeast library

[0219] IL-2 binders were screened from a human scFv yeast display library. IL-2 was biotinylated on an avi tag (AcroBiosystems). After two rounds of initial magnetic-activated cell sorting (MACS) using 200 nM biotinylated IL-2 and streptavidin beads and two rounds of fluorescence-activated cell sorting (FACS) using 200 nM biotinylated IL-2 and APC-streptavidin, scFv binders that blocked the IL-2 / IL-2Rβγ interaction in the further sorted enriched pool were sorted. Then individual clones were plated into six 96-well plates. scFv binders that did not block the IL-2Rα interaction were further screened using 100 nM IL-2 and 100 nM biotinylated IL-2Rα. scFv binders that blocked the IL-2Rβγ interaction were secondarily screened using 100 nM biotinylated IL-2 and 100 nM Fc-IL-2βγ. Sixty-eight yeast clones with both of these properties were collected for further analysis. Supernatants of these clone cultures were collected and their binding to IL-2 and IL-2Rβγ blocking function were tested using Fortebio. Thirty-six scFv candidates fused to an Fc fragment were cloned and expressed in ExpiCHO cells. Purified scFv-Fc homodimers were further characterized using Fortebio and CTLL2 neutralization assays.

[0220]

[0221] B. Blocking assay of selected IL-2 binders screened from a human ScFv yeast library

[0221] The ability of the supernatant of the test candidate clone to block the binding of IL-2 to IL2Rβγ was tested. Using the Red96e kinetic binding assay to test the blockade. Biotinylated IL-2Rβγ was loaded onto the streptavidin (SA) sensor at 1 μg / ml. First, the binding affinity of IL-2 was confirmed by associating a three-fold serial dilution of Fc-IL-2 (JR8_48.3, containing SEQ ID NO: 261 and 262, with an initial concentration of 1 μg / ml) to the SA sensor loaded with IL-2Rβγ ( Figure 1A ). Fc-IL-2 (JR8.48.3, containing SEQ ID NO: 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.

[0222] A one-to-one fitting curve was fitted to the kinetic results, generating the indicated KD with the corresponding binding mode. 100 μl of control Fc-scFv (JR8.113.3, SEQ ID NO: 249) was added to each IL-2 dilution, which completely blocked the binding of IL-2 to IL-2Rβγ ( Figure 1B ). 100 μl of the yeast culture supernatant of clone K1-69A8 was added to each dilution of Fc-IL-2, which partially blocked the binding of IL-2 to IL-2Rβγ ( Figure 1C ). In contrast, the supernatant of clone K3-23A2 did not block the binding of IL-2 to IL-2Rβγ ( Figure 1D ). The results of the blockade assay of scFv clones from the yeast library are summarized in Table 7.

[0223] Table 7. Results of the ForteBio blockade assay

[0224]

[0225]

[0226] *NA: Not applicable.

[0227] C. CTLL2 cell-based activity assay

[0228] Nine scFvs (#1-#9 in Table 7) were cloned and their genes were fused with the 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 ).

[0229] Grow CTLL2 cells 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 are non-adherent cells and are maintained at 5×10 4 -1×10 6 cells / ml in medium with 100 ng / ml of IL-2. Typically, the cells divide twice a week. For bioassays, it is preferable to use the cells no less than 48 hours after subculture. Dilute the samples in 50 μl / well at 2× concentration in a 96-well plate. Titrate the IL-2 standard from 20 ng / ml (2× concentration) to 3× serial dilutions in 12 wells. Perform a titer test on the samples when appropriate. Wash the CTLL2 cells 5 times to remove IL-2, dispense 5000 cells / well in 50 μl and culture overnight or at least 18 hours with the samples. Subsequently, add 100 μl / well of CellTiter reagent (Promega) and measure luminescence.

[0230] During the CTLL2 assay, add 50 nM IL-2 to the CTLL2 cells. Add the purified scFv-Fc fusion protein together with a positive control (JR8.113.3) to the cells. The dose-dependent inhibition of IL-2 activity by the purified fusion protein is shown in the figure. The scFv-Fc homodimers K1-69-C7, K1-69-H5, and K3-30-H7 showed strong inhibitory effects on IL-2, and their IC 50 values were 7.0 nM, 11.3 nM, and 10.2 nM, respectively ( Figure 2 ).

[0231] Nine additional scFv-Fc (#10 - #18 in Table 7) plasmids were constructed, expressed in ExpiCHO TM cells, and purified using ProA chromatography. Four scFv-Fc were not expressed. Test the purified scFv-Fc in the CTLL2 assay to neutralize 50 nM PD1-IL-2v ( Figure 3A and 3B ). Compare their inhibitory activities with clone K1-69-C7. Several additional clones showed strong inhibition of IL-2 activity. L4-39-B9, K1-69-A8, K3-23-A2, K3-30-G9, and K3-30-H9 showed strong neutralization, and their IC 50 values were 6.2 nM, 11.8 nM, 28.5 nM, 15.4 nM, and 4.4 nM, respectively.

[0232] The sequences of the scFv clones selected from the CTLL2 cell-based activity assay are shown in SEQ ID NO: 34 - 36 and 250 - 270.

[0233] Example 3: Binding Affinity of Anti-IL-2 ScFv-Fc

[0234] The binding affinity of the selected scFv-Fc to IL-2 was tested using Fortebio ( Figure 4A and 4B ). The results are summarized in Table 8.

[0235] Table 8. Results of ForteBio Blocking Assay

[0236] ScFv clone Purified sample <![CDATA[K D KD]]> Response K1 - 69G3 JR8.156.5K1 - 69G3 <1 pM 0.6 K3 - 30B12 JR8.156.6K3 - 30B12 605pM 3.1 K3 - 30F7 JR8.144.1K3 - 30F7 <1 pM 0.3 K3 - 30H7 JR8.144.2K3 - 30H7 <1 pM 1.2 K1 - 69H5 JR8.144.3K1 - 69H5 <1 pM 3.0 K3 - 15F7 JR8.144.4K3 - 15F7 <1 pM 3.0 K1 - 69C7 JR8.153.1K1 - 69C7 117pM 3.5 L4 - 39D7 JR8.153.2L4 - 39D7 771pM 5.6 K4 - 39B3 JR8.156.1K4 - 39B3 1.82nM 2.5 K4 - 39G4 JR8.156.2K4 - 39G4 1.22nM 7.9 L4 - 39A5 JR8.156.3L4 - 39A5 <1pM 4.4 K3 - 30F6 JR8.156.4K3 - 30F6 <1 pM 3.9

[0237] Example 4: Expression and Activity Testing of PD-1 Antibody-IL-2 Prodrug Molecules

[0238] CTLL2 assay. The scFvs (C7, H5, and B12) were fused with the anti-PD1 antibody HC to mask mutant human IL-2 (IL-2v), as shown in the figure in Figure 5C . As shown in Figure 5A , the molecules were transiently expressed in the ExpiCHO TM system and purified by ProA affinity chromatography. The purified proteins were activated by hMMP2 digestion and their IL-2 activity was tested in the CTLL2 assay ( Figure 5B ). All of the C7, H5, and B12 scFvs were able to mask the IL-2v activity like β-ECD, and the IL-2v activity of each prodrug was restored after activation by hMMP2 ( Figure 5C ). Specifically, the prodrug molecules JR11.20.3, JR11.20.6, and JR11.20.7 with the masks C7 (or K1-69C7), H5, and β-ECD showed little activity before activation, while their activity increased significantly after protease-based activation ( Figure 5C ).

[0239] NK92 assay. In another experiment, a total of eight prodrug molecules ( Figure 6A ) were expressed and purified by protein A affinity chromatography. The prodrugs were tested before and after protease digestion by an 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 are non-adherent cells and were maintained at 1×10 5 -1×106 cells / ml. Typically, the cells divide twice a week. For the bioassay, it is preferable to use the cells not less than 48 hours after subculture. The purified protein is activated by digestion with hMMP2 and analyzed by SDS-PAGE ( Figure 6B ), and the IL-2 activity is tested in the NK92 assay ( Figure 6C ). K1-69C7, K3-30F7, K1-69A8, K3-30H9, and L4-39B9 strongly mask IL-2v. K3-30G9 and K3-30-B12 can partially mask IL-2 activity. K3-30F6 does not mask well.

[0240] Example 5: Linker Optimization

[0241] In Example 4, the linker between the anti-PD1 antibody HC and the scFv portion of the prodrug was tested, where the prodrug uses the anti-PD-1 antibody as a carrier. Two linkers, GGGGSGGGGSGPLGVRGGGGSGGGGS (SEQ ID NO:268) (JR11.20.3) and GGGGSGGGGSGPLGVRGGGGS (SEQ ID NO:269) (JR11.20.5), work well for molecular assembly and function.

[0242] To optimize the yield and purity of the prodrug molecule, prodrug molecules with different length linkers between the anti-PD1 antibody HC and the IL-2v portion were tested ( Figure 7A ). The linker between the heavy chain (HC) and the cytokine portion is GGGGSGGGGSGGGGS (SEQ ID NO:181) or 3x G4S linker, GGGGSGGGGS (SEQ ID NO:180) or 2x G4S linker, or GGGGS (SEQ ID NO:178) or 1x G4S linker; or no peptide linker. The molecules were transiently expressed in the ExpiCHO TM system and purified by ProA affinity chromatography. The purified proteins were analyzed by SDS-PAGE gel and SEC-HPLC ( Figure 7B ). The data show that molecules with shorter peptide linkers (2xG4S or 1xG4S) or no peptide linker have higher main peak purity (improved purity) and reduced aggregation. The 2xG4S linker was selected for subsequent prodrug design.

[0243] Example 6: In Vitro Assay Using Human PBMC

[0244] Human PBMCs were first cultured in a 6-well plate with anti-CD3 antibody (1 μg / ml) (pre-coated) and anti-CD28 antibody (1 μg / ml) for 72 hours to induce the expression of PD-1 on T cells. The PBMCs were collected, washed, and re-suspended in RPMI 1640 complete medium that had been left standing on ice for 4 hours. The standing cells were divided into two parts. The PBMCs used in experiments / curves 1a-6a were further pretreated with 40 μg / ml of PD-1 antibody, which caused the internalization of PD-1 expressed on T cells. One part 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 the anti-PD1 antibody, the cells were then washed to remove the free anti-PD1 antibody. The activated PBMCs pretreated or not pretreated with anti-PD1 antibody were incubated with serial dilutions of the test article in a 96-well plate in a 37 °C incubator for 15 minutes. The cells were then immediately fixed with BD Cytofix TM fixation buffer and permeabilized with BD Phosflow TM Perm buffer IV (0.5x). The cells were then incubated with PE-conjugated anti-p-Stat5 antibody.

[0245] IL-2 induces phosphorylation of STAT5 (pSTAT5) in immune cells. Phosphorylation of Stat5 in CD4+ T cells, CD8+ T cells, and CD3-CD56+ NK cells was measured by flow cytometry. The data were plotted using GraphPad Prism 9 software. The PBMCs were then treated with anti-CD3 antibody and anti-CD8 antibody to induce the expression of PD-1 on T cells. The results for CD4+ T cells are shown in Figure 8B and the results for CD8+ T cells are shown in Figure 8C and the results for NK cells are shown in Figure 8D . The C7-masked PD-1 antibody-IL-2 prodrug (ASKG812K-C7) showed very low activity in cells pretreated or not pretreated with PD-1 antibody. However, the C7-masked PD-1 antibody-IL-2 prodrug showed strong activity against all cell types after activation (ASKG812K-C7 activity; Figure 8B 、 8CIn Tests 2 and 2a) of 8D, although the activity of T cells was significantly higher than that of NK cells. Additionally, T cells not pre-treated with the PD-1 antibody showed significantly higher activity than cells pre-treated with the PD-1 antibody. Treatment with anti-CD3 and anti-CD8 induced the expression of PD-1 on T cells. Pre-treatment with the PD-1 antibody caused the internalization of PD-1 expressed on T cells. The results showed that the PD-1 antibody-IL-2 prodrug selectively activated PD-1-expressing T cells. This targeted stimulation of T cells may be achieved through a "cis-activation" mechanism, i.e., the activated prodrug binds to T cells through the PD-1 binding domain as well as the cytokine domain. Similar observations were made with a reference PD-1 antibody-IL-2 fusion reference molecule ( Figure 8B and 8C in Tests 3 and 3a). This was not observed in NK cells because they generally do not express high levels of PD-1( Figure 8B and 8C in Tests 3 and 5a). No "cis-activation" was observed with the PD-1 antibody( Figure 8B and 8C in Tests 5 and 5a) or the Fc-IL-2v molecule( Figure 8B and 8C in Tests 4 and 4a). "Cis-activation" was observed for the prodrug masked with scFv G3( Figure 8B and 8C in Trials 6 and 6a) for CD4+ T cells and CD8+ T cells, even though the prodrug did not contain a cleavable linker.

[0246] Example 7: Enhanced thermal stability of the prodrug molecule

[0247] Thermal stability studies of the prodrug molecule were performed on an Unchained Labs UNcle instrument. Briefly, 8.8 μL of the protein sample was loaded in triplicate into a microcuvette ("Uni"). A temperature scan from 25 °C to 95 °C was performed at a rate of 1 °C / minute, and intrinsic fluorescence as well as static light scattering (SLS) were measured. The results of Tm (melting temperature), Tonset (onset of unfolding), Tagg-266nm (onset of aggregation detected at 266 nm), and Tagg-473nm (onset of aggregation detected at 473 nm) for the three runs were manually inspected (analyzed by UNcle Analysis 4.01), and the average was taken after removing outliers. Surprisingly, the results showed that the prodrug masked with the mask C7 (ASKG812K-C7) showed significantly higher thermal stability than prodrugs masked with other masks( Figure 9B)。The onset temperature (Tonset) and Tm1 are 10 °C and 12 °C higher than those masked with IL-2Rβ-ECD, while Tagg is 22 °C higher than that masked with IL-2Rβ-ECD. The two prodrugs masked with F7 and G3 also showed a higher Tagg than the prodrug masked with IL-2Rβ-ECD.

[0248] Accelerated stability studies were also conducted on the prodrugs. The purity of the samples stored at 25 °C and 40 °C was tested by the SEC-HPLC method. Briefly, 3 μg of the sample was injected onto a size exclusion chromatography system containing a dSEC-2 column (3 μm particles, 4.6 × 300 mm, part number 00H-4788-E0), which was installed on an Agilent 1260UHPLC 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 the flow rate was 0.35 mL / min. The protein signal was detected at UV 220 nm. Surprisingly, it was observed that the prodrug masked with C7 showed no decrease in its main peak percentage, while all other prodrugs showed a significant decrease in their main peak percentage ( Figure 9C ). The two prodrugs masked with F7 and G3 also exhibited higher stability than the prodrug masked with IL-2Rβ-ECD. IL-2 showed poor thermal stability and was prone to aggregation during storage under accelerated conditions. The mask C7 may potentially enhance the Tm of IL-2 by more than 10 °C and significantly enhance the stability of IL-2.

[0249] Example 8: The mask F7 is capable of masking IL-2v with the L36I mutation

[0250] 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 the 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. The 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 an activity similar to that of the reference molecule. The prodrug with IL-2v / L36I and the mask F7 showed little activity in the CTLL2 assay, indicating that the mask F7 was able to effectively mask IL-2v / L36I. Although the introduction of the L36I mutation reduced the potential risk of immunogenicity, the mutation did not interfere with the masking by F7.

[0251] Example 9: Construction, Expression, and In Vitro Activity Analysis of Antibody-Cytokine Fusion Molecules

[0252] Many antibody-IL-2 cytokine fusion molecules (ASKG222) were designed to have structures as shown in Figure 11A - 11F . The IL-2 of the fusion molecule was designed to have no or little activity in binding to IL-2Rβ, while mostly retaining its affinity for IL-2Rα and IL-2Rγ. Such molecules were designed to be able to selectively stimulate Tregs on Teff and NK cells.

[0253] The comparison of the C-terminus and N-terminus of the Fc fragment fusion of K1-69C7 is shown in Figure 12A - 12B . Fc fusion molecules (such as those shown in Figure 10A and 10B and Figure 12A ) were expressed in ExpiCHO cells and purified by protein A affinity column. Figure 12A The SEC-HPLC purity of the two molecules in Figure 12B is shown in

[0254] Antibodies were constructed containing V H and V L domains identical to those of scFv K1-69C7 and fused with IL-2 polypeptide ( Figure 13A ). The antibody-cytokine fusion molecules were expressed in ExpiCHO cells and purified by protein A affinity column. Figure 13A The SEC-HPLC purity of the two molecules in Figure 13B is shown in

[0255] Three samples of ASKG222C7-C, ASKG222C7-N, and ASKG222A-C7-ab were further purified to high purity and tested in in vitro assays. Reference samples (analogs R1 of AMG592 and R2 of PT101), wild-type IL-2, and a negative control (human IgG) were also included. In vitro assays were performed using human PBMCs to detect the ability of the antibody-cytokine fusion molecules to selectively stimulate the expansion of Treg cells. The induction of Ki67 was used as a readout for this activity assay.

[0256] Briefly, prepare the assay diluent for the ASKG222 molecule, and add 100 μl of the 2× sample to the wells of the plate. Add 100 μl of PBMC (400 - 500 k cells / well) to the wells with the diluted test article, and culture for 3 days. Stain the cells with anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD25 antibody, and anti-CD161a antibody labeled with different fluorescent dyes for cell surface staining. CD3-CD161a+ cells are defined as NK cells. Further process the cells with the Foxp3 Fixation / Permeabilization Buffer from Thermo Fisher Invitrogen while performing anti-Foxp3 and anti-Ki67 staining. Analyze the samples by flow cytometry and plot the data using GraphPad Prism 9 software. Add IL-2 as a positive control and add human antibody (hIgG) as a negative control. Also include two reference molecules, R1 (an analogue of AMG592 from Amgen) and R2 (an analogue of PT101 from Padion Therapeutics). The results are shown in Figure 14A (Treg cells) and Figure 14B (Teff cells). The results show that, compared to R1, ASKG222C7-C and ASKG222C7-N have better stimulation selectivity for Treg cells than for Teff cells, while compared to R2, ASKG222A-C7-ab has better selectivity for Treg cells. Both R1 and R2 are currently in clinical trials for autoimmune indications.

[0257] Example 10: Rat PK / PD

[0258] Intravenously (I.V.) inject 3 male Sprague-Dawley rats, 53 - 58 days old, with jugular vein cannulas (Envigo), with 2 mg / Kg of purified ASKG222A-C7-ab (lot number LL28-149). Collect blood samples at T0 (before dosing), 1 hour, 2 hours, 4 hours, 6 hours, 10 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, and 144 hours after dosing for PK determination. Perform flow cytometry PD analysis on the samples at T0, 72 hours, and 120 hours, and determine plasma cytokine levels for the samples at T0, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, and 144 hours.

[0259] The concentration of the test article in plasma was determined by ELISA. The ELISA plates were coated with 100 μL / well of F(ab')2 goat anti-human IgG Fcγ (Jackson Immuno-Research, #109-006-170) at 2 μg / mL in PBS overnight at 4 °C for test article capture. The plates were blocked with 100 μL PBS / 10% goat serum for 1 hour and washed with deionized water (D.I.). Plasma samples were serially diluted from 1:103 in 8 wells, and test article standards were diluted from 2 μg / mL in 12 wells with 100 μL / well PBS / 10% goat serum. The plates were incubated for 1 hour and washed with deionized water. For test article detection, 100 μL / well of anti-IL2 biotin (eBioscience, #13-7028-85) at 0.5 μg / mL in PBS / 10% goat serum was added, incubated for 1 hour, and washed with deionized water. Streptavidin-HRP (Jackson Immuno-Research) at 1:1000, 100 μL / well was added in PBS / 10% goat serum and incubated for 1 hour. After washing with deionized water, TMB substrate (Thermo Scientific) was added at 100 μL / well. The color development was stopped with 100 μL H2SO4, and the OD was measured with a microplate spectrophotometer (Molecular Devices). 450 。 Figure 15 PK results of ASKG222A-C7-ab are shown. Three rats were administered 2 mg / kg intravenously once. The drug concentration in serum samples was shown at different time points and measured by ELISA. The data showed that the half-life of the molecule was about 68 to 88 hours, which is typical for an antibody.

[0260] Example 11: In Vivo Efficacy Study Using Colon 26 Syngeneic Tumor Model

[0261] An in vivo efficacy study was conducted using a colon 26 syngeneic tumor model. Briefly, 5×10 5 colon 26 tumor cells were implanted subcutaneously above the abdomen in 7-8-week-old female Balb / C mice. When the average tumor volume reached approximately 82 mm 3 , the mice were randomly assigned to treatment groups such that each group had approximately the same average tumor volume. The mice were treated by intraperitoneal injection of the test article or vehicle in 100 μL PBS, administered at 3-day intervals for a total of two treatments. The tumor volume was determined by measuring the major axis (a) and minor axis (b) and using the formula volume = ab 2 π / 6.

[0262] The fusion molecule 812mN-mut4 comprises two identical light chains of an anti-mouse PD-1 antibody and two identical heavy chain polypeptide chains; wherein the heavy chain polypeptide chain comprises, from the N-terminus to the C-terminus, the heavy chain of an anti-mouse PD-1 antibody, a mask that binds to IL-2, and an IL-2 mutant protein. The structure of this molecule is shown in Figure 16A and its sequence information is shown in Figure 16D .

[0263] The fusion molecule 812mW5-mut4 has the structure shown in Figure 16B . The fusion molecule is similar to 812mN-mut4, except that it contains only one mask and one IL-2 mutant protein at the C-terminus of the heavy chain of the anti-mouse PD-1 antibody.

[0264] Ref3 is an anti-PD-1 antibody-IL-2 mutant protein fusion molecule, which has the structure shown in Figure 16C . It is a murine analogue of eciskafusp alfa, which is currently in clinical development.

[0265] All three molecules have the same anti-PD-1 antibody as the mPD-1 molecule. 812mN-mut4 contains two copies of the masked IL-2 mutant protein. The human version of the fusion molecule 812mN-mut4 (i.e., having an anti-human PD-1 antibody, 812KN-mut4) shows stronger in vitro cell-based bioactivity than the human version of 812mW5-mut4 (812KW5-mut4), and the human version of 812mW5-mut4 contains only one copy of the same masked IL-2 mutant protein ( Figure 20 ). The IL-2 mutant protein in Ref3 is not masked. It has the strongest in vitro cell-based IL-2 activity among the three molecules ( Figure 20 ). Therefore, 812KW5-mut4 has weaker activity than 812KN-mut4 and the reference molecule Ref3. The IL-2 portion is the same between the human and murine versions.

[0266] The in vivo efficacy and safety results of two chimeric molecules, Ref3 and the anti-mouse PD-1 antibody, are shown in Figure 17A and Figure 17B . Surprisingly, 812mW5-mut4 has an anti-tumor efficacy similar to that of Ref3 ( Figure 17A ), yet 812mW5-mut4 is safer than Ref3 because 812mW5-mut4 does not have weight loss, but significant weight loss was observed with Ref3 ( Figure 17B)。Surprisingly, 812mW5-mut4 was also observed to have stronger in vivo efficacy than 812mN-mut4, as the latter was expected to have stronger in vitro cell-based activity.

[0267] Example 12: In Vivo Efficacy Study Using CT26 Syngeneic Tumor Model

[0268] An in vivo efficacy study was conducted using the CT26 syngeneic tumor model. Briefly, 1×10 6 CT26 tumor cells were implanted into 7-8-week-old female Balb / C mice, delivered subcutaneously above the abdomen. When the average tumor volume reached approximately 94 mm 3 , the mice were randomly assigned to treatment groups such that each group had approximately the same average tumor volume. The mice were treated with the test article or vehicle in 100 mL PBS by intraperitoneal injection, administered at 3-day intervals for a total of 2 treatments. Tumor volume was determined by measuring the long diameter (a) and short diameter (b) and using the formula volume = ab 2 π / 6.

[0269] 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 showed better efficacy and safety than Ref3 ( Figure 19A and 19B ). The sequence information of 678F3-Fab-B and Ref3 is shown in Figure 18 . A diagram of the structure of 678F3-Fab-B is shown in Figure 21 .

[0270] Sequence

[0271] In the following sequences, the boxed residues indicate mutations. The underlines in the cleavable linker indicate protease substrate sequences. The underlined and bold sequences refer to the CDRs.

[0272] SEQ ID NO:1 – Human IL-2

[0273] APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE

[0274] EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR

[0275] WITFCQSIIS TLT

[0276] SEQ ID NO:2 - IL-2 agonist polypeptide

[0277] APX aa3 SSSTKKT QLQLEHLLL X aa20 LQMILNGINN YKNPKLTX aa38 ML TX aa42 KFX aa45 MPKKATELKHLQCLE EX aa62 LKPLEX aa68 VL NLX aa73 QSKNFHL RPRDLISX aa88 IX aa90 VIVLELKGSETTFMCEYADE TATIVEFLNR WITFX aa125 X aa126 SIIS TLT

[0278] where X aa3 is N or A; where X aa125 is C or S; where X aa20 is selected from D, H, K, L, M, N, Q, R, S, V, and Y; where X aa38 is selected from A, K, and S; where X aa42 is selected from A, G, I, S, T, Q, E, N, D, R, and K; where X aa45 is selected from A, G, S, T, Q, E, N, D, R, and K; where X aa62 is selected from E, L, A, and I; where X aa68 is E or V; where X aa73 is A or T; where X aa88 is selected from N, A, E, F, H, K, L, M, S, T, V, W, and Y; where X aa90 is N or T; and where X aa126 is selected from A, D, F, G, H, I, K, L, P, S, T, W, and Y; and where X aa20 , X aa88 , X aa90 and X aa126 and at least one of X

[0279] SEQ ID NO:3 - IL-2 agonist polypeptide

[0280] APX aa3 SSSTKKT QLQLEHLLLD LQMILNGINN YKNPX aa35 ITRMLTX aa42 KFX aa45MPKKATELKHLQCLE EELKPLEEVL NX aa72 X aa73 QSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADETATIVEFLNR WITFX aa125 X aa126 SIIS TLT

[0281] wherein X aa3 is N or A; wherein X aa125 is C or A; wherein X aa35 is selected from K and N; wherein X aa42 is selected from A, G, S, T, Q, E, N, D, R, and K; wherein X aa45 is selected from A, G, S, T, Q, E, N, D, R, and K; wherein X aa72 is selected from A, G, S, T, Q, E, N, D, R, and K; wherein X aa73 is selected from A and T; and wherein X aa126 is selected from A, D, F, G, H, I, K, L, P, S, T, W, and Y.

[0282] SEQ ID NO:4 - IL-2 agonist polypeptide with L72G

[0283]

[0284] SEQ ID NO:5 - IL-2 agonist polypeptide mutant protein with T3A / C125A-L36I / R38S / F42A / Y45A / E62A

[0285]

[0286] SEQ ID NO:6 - IL-2 agonist polypeptide mutant protein with C125A-L36I R38S / F42A / Y45A / E62A

[0287]

[0288] SEQ ID NO:7 - IL-2 agonist polypeptide mutant protein with T3A / C125A-L36I R38S / F42A / Y45A / E62L

[0289]

[0290] SEQ ID NO:8 - IL-2 agonist polypeptide mutant protein with T3A / C125A-L36I R38S / F42A / Y45A / E62L / E68V

[0291]

[0292] SEQ ID NO: 9 - IL-2 agonist polypeptide mutant protein with T3A / C125A-L36I R38S / F42 I / Y45A / E62A

[0293]

[0294] SEQ ID NO: 10 - IL-2 agonist polypeptide mutant protein with T3A / C125A-L36I R38S / F42K / Y45A / E62A

[0295]

[0296] SEQ ID NO: 11 - IL-2 agonist polypeptide mutant protein with T3A / C125A-L36I R38S / F42K / Y45N / E62A

[0297]

[0298] SEQ ID NO: 12 - IL-2 agonist polypeptide mutant protein with T3A / C125A-L36I R38S / F42A / Y45R / E62A

[0299]

[0300] SEQ ID NO: 13 - IL-2 agonist polypeptide mutant protein with T3A / C125A-L36I R38S / F42K / Y45A / E62A / E68V

[0301]

[0302] SEQ ID NO: 14 - IL-2 agonist polypeptide mutant protein with T3A / C125A-L36I R38S / F42A / Y45N / E62A / E68V

[0303]

[0304] SEQ ID NO: 15 - IL-2 agonist polypeptide mutant protein with T3A / C125A-L36I R38S / F42A / Y45R / E62A / E68V

[0305]

[0306] SEQ ID NO:16 - IL-2 agonist polypeptide mutant protein with T3A / L36I R38S / F42A / Y45A / A73T / C125A

[0307]

[0308] SEQ ID NO:17 - IL-2 agonist polypeptide mutant protein with T3A / K35N / L36I R38S / F42A / Y45A / A73T / C125A

[0309]

[0310] SEQ ID NO:18 - IL-2 agonist polypeptide mutant protein with T3A / L36I R38S / F42I / Y45A / A73T / C125A

[0311]

[0312] SEQ ID NO:19 - IL-2 agonist polypeptide mutant protein with T3A / K35N / L36I R38S / F42I / Y45A / A73T / C125A

[0313]

[0314] SEQ ID NO:20 - IL-2 agonist polypeptide mutant protein with T3A / L36I R38S / F42K / Y45A / A73T / C125A

[0315]

[0316] SEQ ID NO:21 - IL-2 agonist polypeptide mutant protein with T3A / K35N / L36I R38S / F42K / Y45A / A73T / C125A

[0317]

[0318] SEQ ID NO:22 - IL-2 agonist polypeptide mutant protein with T3A / L36I R38S / F42A / Y45N / A73T / C125A

[0319]

[0320]

[0321] SEQ ID NO:23 - IL-2 agonist polypeptide mutant protein with T3A / L36I R38S / F42A / Y45R / A73T / C125A

[0322]

[0323] SEQ ID NO:24 - IL-2 agonist polypeptide mutant protein with T3A / L36I R38S / F42A / Y45A / E62A / C125A / Q126W

[0324]

[0325] SEQ ID NO:25 - IL-2 agonist polypeptide mutant protein with T3A / L36I R38S / F42K / Y45A / E62A / A73T / C125A

[0326]

[0327] SEQ ID NO:26 - IL-2 agonist polypeptide mutant protein with T3A / L36I R38S / F42A / Y45N / E62A / A73T / C125A

[0328]

[0329] SEQ ID NO:27 - IL-2 agonist polypeptide mutant protein with T3A / L36I R38S / F42A / Y45R / E62A / A73T / C125A

[0330]

[0331] SEQ ID NO:28 - IL-2 agonist polypeptide mutant protein with T3A / K35N / L36I R38S / F42A / Y45N / A73T / C125A

[0332]

[0333] SEQ ID NO:29 - IL-2 agonist polypeptide mutant protein with T3A / K35N / L36I R38S / F42A / Y45R / A73T / C125A

[0334]

[0335] SEQ ID NO:30 - IL-2 agonist polypeptide mutant protein with T3A / C125A-L36I R38S / F42A / Y45A / E62A / Q126H

[0336]

[0337]

[0338] SEQ ID NO:31 - IL-2 agonist polypeptide mutant protein with T3A / C125A - L36I R38S / F42A / Y45A / E62A / Q126A

[0339]

[0340] SEQ ID NO:32 - IL-2 agonist polypeptide mutant protein with T3A / C125A - L36I R38S / F42A / Y45A / E62A / Q126H

[0341]

[0342] SEQ ID NO:33 - IL-2 agonist polypeptide mutant protein with T3A / C125A - L36I R38S / F42A / Y45A / E62A / Q126H

[0343]

[0344] SEQ ID NO:34 - Anti-IL-2 scFv C7

[0345]

[0346] SEQ ID NO:35 - Anti-IL-2 scFv F7

[0347]

[0348] SEQ ID NO:36 - Anti-IL-2 scFv G3

[0349]

[0350] SEQ ID NO:37 - Extracellular domain of human IL-2 receptor beta subunit (https: / / www.uniprot.org / uniprot / P14784)

[0351]

[0352] SEQ ID NO:38 – Pembrolizumab light chain

[0353] CX11.56.4LC

[0354]

[0355]

[0356] SEQ ID NO:39 - Pembrolizumab HC, IgG1, L234A, L235A, G237A CX11.56.5

[0357]

[0358] SEQ ID NO:40 - ASKG812K - PD1 L234A, L235A, G237A - IL2vvQ126T / L36I 2xG4 S

[0359]

[0360] SEQ ID NO:41 - ASKG812K - PD1 L234A, L235A, G237A - IL2vvQ126A / L36I 2xG4 S

[0361]

[0362] SEQ ID NO:42 - ASKG812K - HC L234A, L235A, G237A - IL2 scFv - C7 cleavable

[0363] SEQ ID NO:43 - ASKG812K - HC L234A, L235A, G237A - IL2 scFv - C7 double cleavable

[0364] SEQ ID NO:44 - ASKG812K - HC L234A, L235A, G237A - IL2 scFv - C7 double cleavable

[0365] SEQ ID NO:45 - ASKG812K - PD1 L234A, L235A, G237A - IL2vv Q126T / L36I pestle - 2xG4S

[0366]

[0367] SEQ ID NO:46 - ASKG812K - PD1 L234A, L235A, G237A - IL2vvQ126A / L36I pestle - 2xG4S

[0368]

[0369]

[0370] SEQ ID NO:47 – ASKG812K-HC L234A, L235A, G237A, mortar

[0371]

[0372] SEQ ID NO:48 – ASKG812K-HC-L234A, L235A, G237A-IL2vvQ126T / L36I-Clv-C7

[0373]

[0374] SEQ ID NO:49 – ASKG812K-HC-L234A, L235A, G237A-IL2vvQ126A / L36I-Clv-C7

[0375]

[0376] SEQ ID NO:50 – ASKG812K-HC-L234A, L235A, G237A-G3-IL2vvQ126T / L36I

[0377]

[0378]

[0379] SEQ ID NO:51 – ASKG812K-HC-L234A, L235A, G237A-F7-IL2vvQ126T / L36I

[0380]

[0381] SEQ ID NO:52 – OKT8 L1

[0382]

[0383] SEQ ID NO:53 – OKT8 H1

[0384]

[0385] SEQ ID NO:54 – OKT8 H2

[0386]

[0387] SEQ ID NO:55-124 - Cleavable peptide linker

[0388] SGRSA (SEQ ID NO:55)

[0389] GGGGS ISSGLLSS GGSGGS LGGSGRSANAILE GGGGSGGG GS (SEQ ID NO:56)

[0390] GGGGS ISSGLLSS GGS LGGSGRSANAILE G GGGS (SEQ ID NO:57)

[0391] GGGGS LGGSGRSANAILE GG SGGS ISSGLLSS GGGGS (SEQ ID NO:58)

[0392] GGGGS LGGSGRSANAILE GG S ISSGLLSS G GGGS (SEQ ID NO:59)

[0393] GGGGS LGGSGRSANAILE GG SGGS ISSGLLSS GGGGSGGG GS (SEQ ID NO:60)

[0394] GGGGS LGGSGRSANAILE GG GGSGGGGSGG GGS (SEQ ID NO:61)

[0395] GGGGSGGGGS GGGGS ISSGLLSS GGGGS (SEQ ID NO:62)

[0396] GGGGS LSGRSDNH GGGGS (SEQ ID NO:63)

[0397] (GGGGS) n GGWHTGRN (GGGGS) m (SEQ ID NO:64)

[0398] (GGGGS) n TGRGPSWV (GGGGS) m (SEQ ID NO:65)

[0399] (GGGGS) n SARGPSRW (GGGGS) m (SEQ ID NO:66)

[0400] (GGGGS) n TARGPSFK(GGGGS) m (SEQ ID NO:67)

[0401] (GGGGS) n TARGPSW(GGGGS) m (SEQ ID NO:68)

[0402] (GGGGS) n LSGRSDNH(GGGGS) m (SEQ ID NO:69)

[0403] (GGGGS) n LGGSGRSANAILE GPLGVR(GGGGS) m (SEQ ID NO:70)

[0404] (GGGGS) n LGGSGRSANAILE GGSGPLGVR(GGGGS) m (SEQ ID NO:71)

[0405] (GGGGS) n LGGSGRSANAILE GGSGGSGPLGVR(GGGGS) m (SEQ ID NO:72)

[0406] (GGGGS) n ISSGLLSSLSGRSDNH (GGGGS) m (SEQ ID NO:73)

[0407] (GGGGS) n ISSGLLSS GGS LSGRSDNH (GGGGS) m (SEQ ID NO:74)

[0408] (GGGGS) n ISSGLLSS GGSGGGS LSGRSDNH (GGGGS) m (SEQ ID NO:75)

[0409] where n = 0, 1, 2, 3 or 4; and where m = 0, 1, 2, 3 or 4

[0410] VNGGGGSGPLGVRAAQPA(SEQ ID NO:76)

[0411] GGGGSGPLGVRGGGGS (SEQ ID NO:77)

[0412] GGGGSGPLGVRGGS (SEQ ID NO:78)

[0413] (GGGGS)n1(QGQSGQ)n2 PLGL(GGGGS)n3 (SEQ ID NO:79)

[0414] (GGGGS)n1(QGQSGQ)n2 HTGRSGAL(GGGGS)n3 (SEQ ID NO:80)

[0415] (GGGGS)n1(QGQSGQ)n2 PLTGRSGG(GGGGS)n3 (SEQ ID NO:81)

[0416] (GGGGS)n1(QGQSGQ)n2 AARGPAIH(GGGGS)n3 (SEQ ID NO:82)

[0417] (GGGGS)n1(QGQSGQ)n2 RGPAFNPM(GGGGS)n3 (SEQ ID NO:83)

[0418] (GGGGS)n1(QGQSGQ)n2 SSRGPAYL(GGGGS)n3 (SEQ ID NO:84)

[0419] (GGGGS)n1(QGQSGQ)n2 RGPATPIM(GGGGS)n3 (SEQ ID NO:85)

[0420] (GGGGS)n1(QGQSGQ)n2 RGPA(GGGGS)n3 (SEQ ID NO:86)

[0421] (GGGGS)n1(QGQSGQ)n2 GGQPSGMWGW(GGGGS)n3 (SEQ ID NO:87)

[0422] (GGGGS)n1(QGQSGQ)n2 FPRPLGITGL(GGGGS)n3 (SEQ ID NO:88)

[0423] (GGGGS)n1(QGQSGQ)n2 VILMPLGFLGP(GGGGS)n3 (SEQ ID NO:89)

[0424] (GGGGS)n1(QGQSGQ)n2 SPLTGRSG(GGGGS)n3(SEQ ID NO:90)

[0425] (GGGGS)n1(QGQSGQ)n2 SAGFSLPA(GGGGS)n3(SEQ ID NO:91)

[0426] (GGGGS)n1(QGQSGQ)n2 LAPLGLQRR(GGGGS)n3(SEQ ID NO:92)

[0427] (GGGGS)n1(QGQSGQ)n2 SGGPLGVR(GGGGS)n3(SEQ ID NO:93)

[0428] (GGGGS)n1(QGQSGQ)n2 GPLGVR(GGGGS)n3(SEQ ID NO:94)

[0429] (GGGGS)n1(QGQSGQ)n2 ISSGLLSS(GGGGS)n3(SEQ ID NO:95)

[0430] (GGGGS)n1(QGQSGQ)n2 QNQALRMA(GGGGS)n3(SEQ ID NO:96)

[0431] (GGGGS)n1(QGQSGQ)n2 AQNLLGMV(GGGGS)n3(SEQ ID NO:97)

[0432] (GGGGS)n1(QGQSGQ)n2 STFPFGMF(GGGGS)n3(SEQ ID NO:98)

[0433] (GGGGS)n1(QGQSGQ)n2 PVGYTSSL(GGGGS)n3(SEQ ID NO:99)

[0434] (GGGGS)n1(QGQSGQ)n2 DWLYWPGI(GGGGS)n3(SEQ ID NO:100)

[0435] (GGGGS)n1(QGQSGQ)n2 MIAPVAYR(GGGGS)n3(SEQ ID NO:101)

[0436] (GGGGS)n1(QGQSGQ)n2 RPSPMWAY(GGGGS)n3(SEQ ID NO:102)

[0437] (GGGGS)n1(QGQSGQ)n2 WATPRPMR(GGGGS)n3(SEQ ID NO:103)

[0438] (GGGGS)n1(QGQSGQ)n2 FRLLDWQW(GGGGS)n3(SEQ ID NO:104)

[0439] (GGGGS)n1(QGQSGQ)n2 LKAAPRWA(GGGGS)n3(SEQ ID NO:105)

[0440] (GGGGS)n1(QGQSGQ)n2 GPSHLVLT(GGGGS)n3(SEQ ID NO:106)

[0441] (GGGGS)n1(QGQSGQ)n2 LPGGLSPW(GGGGS)n3(SEQ ID NO:107)

[0442] (GGGGS)n1(QGQSGQ)n2 MGLFSEAG(GGGGS)n3(SEQ ID NO:108)

[0443] (GGGGS)n1(QGQSGQ)n2 SPLPLRVP(GGGGS)n3(SEQ ID NO:109)

[0444] (GGGGS)n1(QGQSGQ)n2 RMHLRSLG(GGGGS)n3(SEQ ID NO:110)

[0445] (GGGGS)n1(QGQSGQ)n2 LAAPLGLL(GGGGS)n3(SEQ ID NO:111)

[0446] (GGGGS)n1(QGQSGQ)n2 AVGLLAPP(GGGGS)n3(SEQ ID NO:112)

[0447] (GGGGS)n1(QGQSGQ)n2 LLAPSHRA(GGGGS)n3(SEQ ID NO:113)

[0448] (GGGGS)n1(QGQSGQ)n2 PAGLWLDP(GGGGS)n3(SEQ ID NO:114)

[0449] (GGGGS)n1(QGQSGQ)n2 ISSGLSS(GGGGS)n3(SEQ ID NO:115)

[0450] (GGGGS)n1 ISSGLLSSGGSGGSLSGRSDNH(GGGGS)n3 (SEQ ID NO:116)

[0451] (GGGGS)n1 LSGRSDNHGGSGGSISSGLLSS(GGGGS)n3 (SEQ ID NO:117)

[0452] (GGGGS)n1(QGQSGQ)n2 LSGRSDNH(GGGGS)n3 (SEQ ID NO:118)

[0453] (GGGGS)n1(QGQSGQ)n2 TARGPSFK(GGGGS)n3 (SEQ ID NO:119)

[0454] where n1 = 0, 1, 2, 3 or 4; n2 = 0 or 1; and n3 = 0, 1, 2, 3 or 4

[0455] VPLSLYSGRSA (SEQ ID NO:120)

[0456] PLGLAG (SEQ ID NO:121)

[0457] LSGRSDNH (SEQ ID NO:122)

[0458] ISSGLLSS (SEQ ID NO:123)

[0459] GPLGVR (SEQ ID NO:124)

[0460] SEQ ID NO:125 – ASKG812K-PD1 L234A, L235A, G237A-IL2v / L36I 2xG4S

[0461]

[0462] SEQ ID NO:126 – Trastuzumab light chain

[0463]

[0464] SEQ ID NO:127 – Trastuzumab heavy chain

[0465]

[0466] SEQ ID NO:128 – Rituximab light chain

[0467]

[0468]

[0469] SEQ ID NO:129 - Rituximab heavy chain

[0470]

[0471] SEQ ID NO:130 - Brentuximab vedotin light chain

[0472]

[0473] SEQ ID NO:131 - Brentuximab vedotin heavy chain

[0474]

[0475] SEQ ID NO:132 - Cetuximab light chain

[0476]

[0477] SEQ ID NO:133 - Cetuximab heavy chain

[0478]

[0479] SEQ ID NO:134 - Panitumumab light chain

[0480]

[0481] SEQ ID NO:135 - Panitumumab heavy chain

[0482]

[0483] SEQ ID NO:136 - Anti-c-MET antibody light chain

[0484]

[0485] SEQ ID NO:137 - Anti-c-MET antibody heavy chain

[0486]

[0487] SEQ ID NO:138 - Anti-GPC3 antibody light chain

[0488]

[0489] SEQ ID NO:139 - Anti-GPC3 antibody heavy chain

[0490]

[0491] SEQ ID NO:140 - Anti-Claudin 18.2 antibody light chain

[0492]

[0493] SEQ ID NO:141 - Anti-Claudin 18.2 antibody heavy chain

[0494]

[0495]

[0496] SEQ ID NO:142 - Anti-Trop-2 antibody light chain CDR1

[0497] KASQDVSIAVA

[0498] SEQ ID NO:143 - Anti-Trop-2 antibody light chain CDR2

[0499] SASYRYT

[0500] SEQ ID NO:144 - Anti-Trop-2 antibody light chain CDR3

[0501] QQHYITPLT

[0502] SEQ ID NO:145 - Anti-Trop-2 antibody heavy chain CDR1

[0503] NYGMN

[0504] SEQ ID NO:146 - Anti-Trop-2 antibody heavy chain CDR2

[0505] WINTYTGEPTYTDDFKG

[0506] SEQ ID NO:147 - Anti-Trop-2 antibody heavy chain CDR3

[0507] GGFGSSYWYFDV

[0508] SEQ ID NO:148 - Anti-mesothelin antibody light chain CDR1

[0509] SASSSVSYMH

[0510] SEQ ID NO:149 - Anti-mesothelin antibody light chain CDR2

[0511] DTSKLAS

[0512] SEQ ID NO:150 - Anti-mesothelin antibody light chain CDR3

[0513] QQWSGYPLT

[0514] SEQ ID NO:151 - Anti-mesothelin antibody heavy chain CDR1

[0515] GYTMN

[0516] SEQ ID NO:152 - Anti-mesothelin antibody heavy chain CDR2

[0517] LITPYNGASSYNQKFRG

[0518] SEQ ID NO:153 - Anti-mesothelin antibody heavy chain CDR3

[0519] GGYDGRGFDY

[0520] SEQ ID NO:154 - Light chain variable domain of PR1A3

[0521]

[0522] SEQ ID NO:155 - Heavy chain variable domain of PR1A3

[0523]

[0524] SEQ ID NO:156 - Humanized light chain variable domain of PR1A3

[0525]

[0526] SEQ ID NO:157 - Humanized heavy chain variable domain of PR1A3

[0527]

[0528] SEQ ID NO:158 - Anti-FAP LC version 1 (protein sequence)

[0529]

[0530] SEQ ID NO:159 - Anti-FAP LC version 2 (protein sequence)

[0531]

[0532] SEQ ID NO:160 - Anti-FAP VH (protein sequence)

[0533]

[0534] SEQ ID NO:161 - Humanized light chain variable domain of FAPα antibody BIBH1

[0535]

[0536] SEQ ID NO:162 - Humanized heavy chain variable domain of FAPα antibody BIBH1

[0537]

[0538] SEQ ID NO:163 - Humanized H8 anti - 5T4 VH version 1 (protein sequence)

[0539]

[0540] SEQ ID NO:164 - Humanized H8 anti - 5T4 VH version 2 (protein sequence)

[0541]

[0542] SEQ ID NO:165 - Humanized H8 anti - 5T4 VL version 1 (protein sequence)

[0543]

[0544] SEQ ID NO:166 - Humanized H8 anti - 5T4 VL version 2 (protein sequence)

[0545]

[0546] SEQ ID NO:167 - Anti - PDL1 atezolizumab LC

[0547]

[0548] SEQ ID NO:168 - Anti - PDL1 nivolumab HC (protein sequence)

[0549]

[0550] SEQ ID NO:169 – Human albumin

[0551]

[0552] SEQ ID NO:170 – Anti - IL - 2 antibody heavy chain CDR1

[0553] TSYAI

[0554] SEQ ID NO:171 – Anti - IL - 2 antibody heavy chain CDR2

[0555] RIIPIIGRVD YAQKFQG

[0556] SEQ ID NO:172 - Anti-IL-2 antibody heavy chain CDR3

[0557] FVGGFDV

[0558] SEQ ID NO:173 - Anti-IL-2 antibody light chain CDR1

[0559] RASQDISNYLN

[0560] SEQ ID NO:174 - Anti-IL-2 antibody light chain CDR2

[0561] GASSLQS

[0562] SEQ ID NO:175 - Anti-IL-2 antibody light chain CDR3

[0563] QTYTIPL

[0564] SEQ ID NO:176 - IL-2 agonist polypeptide mutant protein with T3A / R38S / F42A / Y45A / E62A / C125S

[0565]

[0566] SEQ ID NO:177 - 183 - Non-cleavable linker

[0567] GGS(SEQ ID NO:177)

[0568] GGGGS(SEQ ID NO:178)

[0569] GGSGGS(SEQ ID NO:179)

[0570] GGGGSGGGGS(SEQ ID NO:180)

[0571] GGGGSGGGGS GGGGS(SEQ ID NO:181)

[0572] GGGGSAAGGG GAGGGGA(SEQ ID NO:182)

[0573] GGGGSGGGGS AAGGGGSGGG GS(SEQ ID NO:183)

[0574] SEQ ID NO:184 - IL-2 agonist polypeptide mutant protein with T3A / L36I / R38S / F42A / Y45A / E62A / C125A

[0575]

[0576] SEQ ID NO:185–ASKG812N-PD1 L234A, L235A, G237A-IL2vL36 I 2xG4 S

[0577]

[0578] SEQ ID NO:186–ASKG812N-HC L234A, L235A, G237A-IL2 scFv-C7 cleavable (CDRs shown in bold and underlined)

[0579]

[0580] SEQ ID NO:187-812N-mut4

[0581]

[0582]

[0583] SEQ ID NO:188-Cleavable peptide linker

[0584]

[0585] SEQ ID NO:189–PD1-nivolumab-LCCX3.73.3

[0586]

[0587] SEQ ID NO:190-Anti-IL-2VL

[0588]

[0589] SEQ ID NO:191-Anti-IL-2VH

[0590]

[0591] SEQ ID NO:192-Anti-IL-2VL

[0592]

[0593] SEQ ID NO:193-Anti-IL-2VH

[0594]

[0595] SEQ ID NO:194 - Anti - IL - 2VL

[0596]

[0597] SEQ ID NO:195 - Anti - IL - 2VH

[0598]

[0599] SEQ ID NO:196 - Fc - Igg4 - ji - scFv C7(K1 - 69), ASKG222.CX12.48.1

[0600]

[0601] SEQ ID NO:197 - Fc - Igg4 - ji - scFv H5(K1 - 69); ASKG222.CX12.48.2

[0602]

[0603] SEQ ID NO:198 - scFv - C7 - IGG4 YTEji; ASKG222.CX12.50.1

[0604]

[0605] SEQ ID NO:199 - IL - 2 - IGG4 Fc, YTE, chu; ASKG222.CX12.7.5

[0606]

[0607] SEQ ID NO:200 - ASKD982 - D1, ji chain (without γECD; only βECD), ASKG222D1; ASKD982.CX7.56.6,

[0608]

[0609] SEQ ID NO:201 - ASKD982 - C, chu chain (only C125A mutation); ASKD982.CX7.56.5

[0610]

[0611] SEQ ID NO:202 - IL-2w C125A-IGG4 YTE with H435R, Y436F 2xG4S; ASKG222.CX12.25.1

[0612]

[0613] SEQ ID NO:203 - IL-2w C125A-IGG4 YTE with H435R, Y436F 1xG4S;

[0614] ASKG222.CX12.25.2

[0615]

[0616] SEQ ID NO:204 - IL-2w C125A-IGG4 YTE with H435R, Y436F;

[0617] ASKG222.CX12.25.3

[0618]

[0619] SEQ ID NO:205 - IGG1-LALA-YTE IL-2-T3A / C125A-C7HC 4xG4S; ASKG222.CX12.65.1

[0620]

[0621] SEQ ID NO:206 - IGG1-LALA-YTE IL-2-T3A / C125A-C7HC 2xG4S; ASKG222.CX12.65.2

[0622]

[0623]

[0624] SEQ ID NO:207 - C7-LC ASKG222.CX12.65.3

[0625]

[0626] SEQ ID NO:208 - IL-2-T3A / C125A-C7 LC 4xG4S; ASKG222.CX12.65.4

[0627]

[0628] SEQ ID NO:209-IGG4 Fc-YTE-杵-IL2 T3A、C125A H435R、Y436F 2xG4S;ASKG222.CX12.66.1

[0629]

[0630] SEQ ID NO:210-IGG4-Fc YTE-杵-IL2 T3A、C125A H435R、Y436F 0xG4S;ASKG222.CX12.66.2

[0631]

[0632] SEQ ID NO:211-IL-2-C125A-C7HC 4xG4S;ASKG222.CX12.76.1

[0633]

[0634]

[0635] SEQ ID NO:212-IGG1-LALA-YTE IL-2-N88D / C125A-C7HC 4xG4S;ASKG222.CX12.76.2

[0636]

[0637] SEQ ID NO:213-IGG1-LALA-YTE IL-2-V69A / Q74 P / C125A-C7HC 4xG4S;ASKG222.CX12.76.3

[0638]

[0639] SEQ ID NO:214-IGG1-LALA-YTE IL-2-V69A / Q74 P / N88D / C125A-C7HC 4xG4S;ASKG222.CX12.76.4

[0640]

[0641] SEQ ID NO:215-C7HC;ASKG222.CX12.76.5

[0642]

[0643]

[0644] SEQ ID NO:216 - ASKG812K - PD1 - IL2v H435R, Y436FYTE.3xG4S; ASKG812K.CX13.7.5

[0645]

[0646] SEQ ID NO:217 - ASKG812K - PD1 - IL2v H435R, Y436F YTE.2xG4S; ASKG812K.CX13.7.6

[0647]

[0648] SEQ ID NO:218 - ASKG812K - PD1 - IL2v H435R, Y436FYTE.1xG4S; ASKG812K.CX13.7.7

[0649]

[0650] SEQ ID NO:219 - ASKG812K - PD1 - IL2v H435R, Y436FYTE.0xG4S; ASKG812K.CX13.7.8

[0651]

[0652]

[0653] SEQ ID NO:220 - ASKG812K - PD1 - Pestle - IL2v YTE.3xG4S; ASKG812K.CX13.13.1

[0654]

[0655] SEQ ID NO:221 - ASKG812K - HC - IL2 scFv - C7 YTE Cleavable 4xG4S; ASKG812K.CX13.55.1 (CDRs are in bold and underlined)

[0656]

[0657] SEQ ID NO:222 - ASKG812K - HC - IL2 scFv - B12 YTE Non - Cleavable 4xG4S; ASKG812K.CX13.55.2 (CDRs are in bold and underlined)

[0658]

[0659] SEQ ID NO: 223 - ASKG812K - HC - scFv - C7 YTE Cleavable 3xG4S;

[0660] ASKG812K.CX13.60.1

[0661]

[0662]

[0663] SEQ ID NO: 224 - ASKG812K - HC - scFv - H5 YTE Cleavable 3xG4S; ASKG812K.CX13.60.2 (CDRs are in bold and underlined)

[0664]

[0665] SEQ ID NO: 225 - ASKG812K - HC - scFv#3K1 - 69 - G3 YTE Cleavable 3xG4S; ASKG812K.CX13.61.1 (CDRs are in bold and underlined)

[0666]

[0667] SEQ ID NO: 226 - ASKG812K - HC - scFv#6K3 - 30 - F6 YTE Cleavable 3xG4S;

[0668] ASKG812K.CX13.61.2 (CDRs are in bold and underlined)

[0669]

[0670]

[0671] SEQ ID NO: 227 - ASKG812K - HC - scFv#7K3 - 30 - F7 YTE Cleavable 3xG4S; ASKG812K.CX13.61.3 (CDRs are in bold and underlined)

[0672]

[0673] SEQ ID NO: 228 - ASKG812K - HC - scFv#9K3 - 30 - B12 YTE Cleavable 3xG4S; ASKG812K.CX13.61.4 (CDRs are in bold and underlined)

[0674]

[0675] SEQ ID NO:229 - ASKG812K - HC - scFv#10K1 - 69 - A8 YTE Cleavable 3xG4S; ASKG812K.CX13.61.5 (CDRs in bold and underlined)

[0676]

[0677] SEQ ID NO:230 - ASKG812K - HC - scFv#13K3 - 30 - G9 YTE Cleavable 3xG4S; ASKG812K.CX13.61.6 (CDRs in bold and underlined)

[0678]

[0679] SEQ ID NO:231 - ASKG812K - HC - scFv#14K3 - 30 - H9 YTE Cleavable 3xG4S; ASKG812K.CX13.61.7 (CDRs in bold and underlined)

[0680]

[0681] SEQ ID NO:232 - ASKG812K - HC - scFv#18L4 - 39 - B9 YTE Cleavable 3xG4S; ASKG812K.CX13.61.8 (CDRs in bold and underlined)

[0682]

[0683] SEQ ID NO:233 - ASKG812K - HC - IL2 scFv - K3 - 30 - F7 YTE Non - Cleavable 4xG4S; ASKG812K.CX13.96.1 (CDRs in bold and underlined)

[0684]

[0685]

[0686] SEQ ID NO:234 - ASKG812K - HC - IL2 scFv - K1 - 69 - G3 YTE Non - Cleavable 4xG4S; ASKG812K.CX13.96.2 (CDRs in bold and underlined)

[0687]

[0688] SEQ ID NO:235 - ASKG812K - HC - IL2 scFv - L - 69 - B9 YTE non - cleavable 4xG4S; ASKG812K.CX13.96.3 (CDRs are in bold and underlined)

[0689]

[0690] SEQ ID NO:236 - ASKG812K.CX13.98.1

[0691]

[0692] SEQ ID NO:237 - ASKG812K - PD1 - IL2v L36 I H435R, Y436F YTE.3xG4S;

[0693] ASKG812K.CX13.98.2

[0694]

[0695] SEQ ID NO:238 - ASKG812K - PD1 - IL2v L36 I YTE.3xG4S; ASKG812K.CX13.98.3

[0696]

[0697] SEQ ID NO:239 - ASKG812K - PD1 - IL2v YTE.2xG4S; ASKG812K.CX13.119.1

[0698]

[0699] SEQ ID NO:240 - ASKG812K - PD1 - IL2v L36 I YTE.2xG4S; ASKG812K.CX13.119.2

[0700]

[0701] SEQ ID NO:241 - ASKG812K - PD1 - IL2v YTE.0xG4S; ASKG812K.CX13.124.1

[0702]

[0703]

[0704] SEQ ID NO:242 - ASKG812K - PD1 - IL2v L36I YTE.0xG4S; ASKG812K.CX13.124.2

[0705]

[0706] SEQ ID NO:243 - ASKG812K - HC - IL2 scFv - C7 YTE Cleavable 4xG4S; ASKG812K.CX13.129.1 (CDRs in bold and underlined)

[0707]

[0708] SEQ ID NO:244 - ASKG812K - HC - IL2scFv - C7 YTE Non - Cleavable 4xG4S; ASKG812K.CX13.129.2

[0709]

[0710] SEQ ID NO:245 - ASKG812K - PD1 - IL2wt T3A, C126A YTE.2xG4 S;

[0711] ASKG812 K.CX13.129.3

[0712]

[0713] SEQ ID NO:246 - Reference Molecule > PD1 - IL2v_pestle (S354C / T366W) (IL2, T3A, Y45A, L72G, C125A) Fc mutations to remove effector function: L234A, L235A, P329G;

[0714] ASKG812.CX13.1.1

[0715]

[0716] SEQ ID NO:247 - Reference Molecule > PD1 - HC_mortar2 (Y349C, T366S, L368A and Y410V) plus H435R, Y436F; ASKG812.CX13.1.3

[0717]

[0718] SEQ ID NO:248 - Reference Molecule > PD1 - LC; ASKG812.CX13.1.4

[0719]

[0720] SEQ ID NO:249 - Fc - IL - 2ScFv2, VL - VH: Positive control molecule;

[0721] ASKD215.CX7.108.2

[0722]

[0723]

[0724] SEQ ID NO:250 - AG111 - #20 - K1 - 69 - H5

[0725]

[0726] SEQ ID NO:251 - AG111 - T3 - K1 - 69 - A8

[0727]

[0728] SEQ ID NO:252 - AG111 - T12 - K3 - 30 - H9

[0729]

[0730] SEQ ID NO:253 - AG111 - #4 - K3 - 30 - G9

[0731]

[0732] SEQ ID NO:254 - AG111 - #21 - L4 - 39 - B9

[0733]

[0734] SEQ ID NO:255 - AG111 - T 9 - K3 - 30 - B12

[0735]

[0736] SEQ ID NO:256 - AG111 - #3 - K3 - 30 - F6

[0737]

[0738] SEQ ID NO:257 - AG111 - #22 - K3 - 15 - F7

[0739]

[0740]

[0741] SEQ ID NO:258-AG111-#93-K1-69-D2

[0742]

[0743] SEQ ID NO:259-AG111-#44-K3-30-E12

[0744]

[0745] SEQ ID NO:260-ASKG812K-PD1-mortine-YTE RF mutation; ASKG812K.CX13.13.2

[0746]

[0747] SEQ ID NO:261 - IL-2wC125A-IGG4 Fc knob [R8.48.3: CX7.106.1 and CX7.70.1]; ASKD215.CX7.106.1

[0748]

[0749] SEQ ID NO:262-ASKD215.CX7.70.1

[0750]

[0751] SEQ ID NO:263 - LC of PD-1 antibody with novel signal peptide (CX11.114.1)

[0752]

[0753] SEQ ID NO:264-PD1-HC-IL2V KNOB; ASKB1296.CX3.58.1

[0754]

[0755]

[0756] SEQ ID NO:265-PD1HC-β-acetyl long linker; ASKB1296.CX3.58.3

[0757]

[0758] SEQ ID NO:266-PD1 HC mortar; ASKB1296.CX3.58.4

[0759]

[0760] SEQ ID NO:267 - ASKG812K - β - ECD CX13.7.4

[0761]

[0762] SEQ ID NO:268

[0763]

[0764] SEQ ID NO:269

[0765]

[0766] SEQ ID NO:270 - mPD1 - mIgG2 - L234A / L2356A / P329G CX11_158_3

[0767]

[0768]

[0769] SEQ ID NO:271 - mDX400 - LC CX11_154_1

[0770]

[0771] SEQ ID NO:272 - mPD1 - mIgG2 - L234A / L2356A / P329G、K409E / K439D IL - 2vRASKG812_CX15_154_2

[0772]

[0773] SEQ ID NO:273 - mPD1 - mIgG2 - L234A / L2356A / P329G、E356K / D399KCX11_158_2

[0774]

[0775] SEQ ID NO:274 - mPD1 - mIgG2 - L234A / L2356A / P329G、K409E / K439D - F7 - IL - 2v - mut4

[0776] ASKG812_CX21_132_2

[0777]

[0778]

[0779] SEQ ID NO:275-ASKG812K_CX21_134_4

[0780]

[0781] SEQ ID NO:276-ASKG812K_CX13_157_4

[0782]

[0783] SEQ ID NO:277-812K-HC L234A, L235A, G237A, ASKG812K_CX13_152_5

[0784]

[0785] SEQ ID NO:278-ASKG812NCX22_31_1

[0786]

[0787]

[0788] SEQ ID NO:279-812N-HC L234A, L235A, G237A

[0789] CX22_31_2

[0790]

[0791] SEQ ID NO:280-IL-2vL-mPD1-Fab-NX2-Fc-mIgG2 L234A / L2356A / P329GK409E / K439D

[0792] ASKG678_CX18_30_1

[0793]

[0794] SEQ ID NO:281-C7-Donkey-mIgG2-Fc-L234A / L2356A / P329G、E356K / D399KASKG812_CX21_32_1

[0795]

[0796] SEQ ID NO:282—Cleavable linker sequence

[0797]

[0798] SEQ ID NO:283-812NP-A IL-2vL-Nivolumab-Fab-NX2-Fc-Igg1-knob-YTE

[0799]

[0800]

[0801] SEQ ID NO:284-C7-Uncleavable-hIgG1 LALA G237A, YTE, ASKG812_CX21_27_2

[0802]

[0803] SEQ ID NO:285-CX21_87_4

[0804]

[0805] SEQ ID NO:286-812KN-mut4

[0806]

[0807] SEQ ID NO:287

[0808] RWITFSQSI

[0809] SEQ ID NO:288

[0810] NPKLTSMLT

[0811] SEQ ID NO:289

[0812] LISNINVIV

[0813] SEQ ID NO:290

[0814] HLRPRDLIS

[0815] SEQ ID NO:291

[0816] LNLAQSKNF

[0817] SEQ ID NO:292

[0818] LAQSKNFHL

[0819] SEQ ID NO:293

[0820] LLLDLQMIL

[0821] SEQ ID NO:294

[0822] QLQLEHLLL

[0823] SEQ ID NO:295

[0824] LEEVLNLAQ

[0825] SEQ ID NO:296

[0826] FCQSIISTL

[0827] SEQ ID NO:297

[0828] WITFCQSII

[0829] SEQ ID NO:298

[0830] LTSMLTAKF

[0831] SEQ ID NO:299

[0832] FSQSIISTL。

Claims

1. A mutant human IL-2 polypeptide 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.

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

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

1.

4. The mutant human IL-2 polypeptide according to any one of claims 1 to 3, further comprising one or more additional mutations at positions selected from the following: T3, optionally wherein the mutation is N3A; D20, optionally wherein the mutation is D20H, D20K, D20L, D20M, D20N, D20Q, D20R, D20S, D20V or D20Y; R38, optionally wherein the mutation is R38A, R38K or R38S; F42, optionally wherein the mutation is F42A, F42G, F42I, F42S, F42T, F42Q, F42E, F42N, F42D, F42R or F42K; Y45, optionally wherein the mutation is Y45A, Y45G, Y45I, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R or Y45K; E62, optionally wherein the mutation is E62L, E62A or E62I; E68, optionally wherein the mutation is E68V; L72, optionally wherein the mutation is L72G; A73, optionally wherein the mutation is A73T; N88, optionally wherein the mutation is N88A, N88E, N88F, N88H, N88K, N88T, N88L, N88M, N88S, N88V, N88W or N88Y; N90, optionally wherein the mutation is N90T; V91, optionally wherein the mutation is V91K, V91A, V91H or V91R; I92； and Q126, optionally wherein the mutation is Q126A, Q126D, Q126F, Q126G, Q126H, Q126I, Q126K, Q126L, Q126P, Q126S, Q126T, Q126W or Q126Y (numbered according to SEQ ID NO:1).

5. The mutant human IL-2 polypeptide according to any one of claims 1 to 4, wherein the mutant IL-2 polypeptide comprises an amino acid sequence selected from SEQ ID NO:3 and 5 - 33 or an amino acid sequence that is 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 4.

7. An anti-IL-2 antibody or an antigen-binding fragment thereof, wherein the anti-IL-2 antibody or the antigen-binding fragment thereof comprises heavy chain CDR1-3 shown in SEQ ID NO: 170-172 and light chain CDR1-3 shown in SEQ ID NO: 173-175.

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

9. An anti-IL-2 antigen-binding fragment, which comprises an amino acid sequence selected from SEQ ID NO: 34, 35, 36 and 250-258 or an amino acid sequence that is at least 90% identical thereto.

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

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

12. The anti-IL-2 antibody or antigen-binding fragment according to claim 11, wherein the increase in Tagg is more than 2 °C, more than 5 °C, about 10 °C or more than 10 °C.

13. An anti-IL-2 antibody or an antigen-binding fragment thereof, which competes with the antibody or antigen-binding fragment according to 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.

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

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

16. The prodrug according to claim 14, wherein the IL-2 cytokine moiety comprises an amino acid sequence selected from SEQ ID NO: 1-33.

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

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

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

20. The prodrug according to claim 19, wherein the masking moiety comprises the extracellular domain (ECD) of IL-2Rβ or a functional fragment thereof, or a single-chain antibody (scFv) or Fab.

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

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

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

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

25. The prodrug according to claim 24, wherein the carrier moiety comprises an antigen-binding moiety that targets an antigen presented on an immune cell or a cancer cell, optionally wherein the antigen-binding moiety is a bispecific antibody, a single-domain antibody, a Fab or an scFv.

26. The prodrug according to claim 25, wherein the antigen-binding moiety targets an antigen presented on a T cell, an NK cell, a macrophage or a cell in the tumor microenvironment (TME), optionally wherein the antigen is selected from PD-1, CD3, CD4, CD8, Tim-3, LAG-3, TIGIT, HER2, signal regulatory protein α (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), the extra domain B of fibronectin (EDB), fibronectin (α5β1), vitronectin (αvβ3 and αvβ5 integrins), carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), 5T4, BCMA, PD-L1, CD47, epidermal growth factor receptor (EGFR), c-MET, Claudin 18.2, Claudin6, CD20, CD24, CD38, CD47, GPC3, mesothelin, ROR1 and melanoma-associated chondroitin sulfate proteoglycan (MCSP).

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

28. The prodrug according to claim 25, wherein the antigen-binding portion comprises an anti-CD8 antibody or an antigen-binding fragment thereof, optionally wherein the antigen-binding portion comprises OKT8 or humanized OKT8, the heavy chain and light chain CDR1-3 derived from OKT8, and / or the light chain variable domain and the heavy chain variable domain, the light chain variable domain comprising the amino acid sequence of SEQ ID NO:52 or an amino acid sequence at least 90% identical thereto, and the heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:53 or 54 or an amino acid sequence at least 90% identical thereto.

29. The prodrug according to any one of claims 24 to 28, wherein the carrier portion comprises an IgG Fc domain or an IgG antibody, the IgG Fc domain or IgG antibody comprising: L234A and L235A ("LALA") mutations (Eu numbering), and / or a hinge mutation, wherein the IL-2 cytokine portion and the masking portion are respectively fused to different polypeptide chains of the Fc domain, or to different heavy chains of the IgG antibody, or to the light chain and heavy chain of the IgG antibody.

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

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

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

33. The prodrug according to any one of claims 30 to 32, wherein the cleavable peptide linker is capable of being cleaved by one or more proteases present in the tumor microenvironment (TME), and the cleavage activates the prodrug in the TME, optionally wherein the cleavable peptide linker comprises a substrate sequence of urokinase-type plasminogen activator (uPA), matrix metallopeptidase 2 (MMP2), MMP7, MMP9, MMP14, podoplanin or matriptase, and / or substrate sequences of two, three, four or more proteases preferentially expressed in the TME.

34. The prodrug according to 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. A prodrug comprising the mutant IL-2 polypeptide according to claim 1, 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 that is at least 95% identical thereto, the second heavy chain polypeptide chain comprises SEQ ID NO: 277 or an amino acid sequence that is at least 95% identical thereto, and two identical light chains, the light chains comprising SEQ ID NO: 276 or an amino acid sequence that is at least 95% identical thereto; b. the first heavy chain polypeptide chain comprises SEQ ID NO: 278 or an amino acid sequence that is at least 95% identical thereto, the second heavy chain polypeptide chain comprises SEQ ID NO: 279 or an amino acid sequence that is at least 95% identical thereto, and two identical light chains, the light chains comprising SEQ ID NO: 189 or an amino acid sequence that is at least 95% identical thereto; c. the first heavy chain polypeptide chain and the second heavy chain polypeptide chain comprise SEQ ID NO: 286 or an amino acid sequence that is at least 95% identical thereto, and two identical light chains comprise SEQ ID NO: 276 or an amino acid sequence that is at least 95% identical thereto; d. the first heavy chain polypeptide chain and the second heavy chain polypeptide chain comprise SEQ ID NO: 187 or an amino acid sequence that is at least 95% identical thereto, and two identical light chains comprise SEQ ID NO: 189 or an amino acid sequence that is at least 95% identical thereto; or e. the first heavy chain polypeptide chain comprises SEQ ID NO: 283 or an amino acid sequence that is at least 95% identical thereto, the second heavy chain polypeptide chain comprises SEQ ID NO: 284 or an amino acid sequence that is at least 95% identical thereto, and one light chain, the light chain comprising SEQ ID NO: 189 or an amino acid sequence that is at least 95% identical thereto.

36. 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. the light chains each comprise SEQ ID NO: 38 or an amino acid sequence that is at least 90% identical thereto, the first heavy chain comprises SEQ ID NO: 40, 41, 45, 46 or an amino acid sequence that is at least 95% identical thereto, and the second heavy chain comprises SEQ ID NO: 42, 43, 44 or 47 or an amino acid sequence that is at least 95% identical thereto; or b. the light chains each comprise SEQ ID NO: 38 or an amino acid sequence that is at least 90% identical thereto, and the first heavy chain and the second heavy chain each comprise SEQ ID NO: 40, 41, 48, 49, 50 or 51 or an amino acid sequence that is at least 95% identical thereto; c. Each of the light chains comprises the amino acid sequence of SEQ ID NO: 189 or an amino acid sequence that is at least 90% identical thereto, the first heavy chain comprises the amino acid sequence of SEQ ID NO: 185 or an amino acid sequence that is at least 95% identical thereto, and the second heavy chain comprises the amino acid sequence of SEQ ID NO: 186 or an amino acid sequence that is at least 95% identical thereto.

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

38. A pharmaceutical composition, comprising the mutant human IL-2 polypeptide according to any one of claims 1 to 6, the prodrug according to any one of claims 14 to 35 or the 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 according to any one of claims 1 to 6, the anti-IL-2 antibody or antigen-binding fragment according to any one of claims 7 to 13, the prodrug according to any one of claims 14 to 35 or the IL-2 antibody fusion molecule according to claim 36 or 37.

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

41. A host cell comprising the expression vector according to claim 40, optionally wherein the host cell is a mammalian cell and the genes encoding proteinase, uPA, MMP-2, MMP-9 and / or MMP14 are knocked out in the host cell.

42. A method for preparing a protein, the method comprising: culturing the host cell according to claim 41 under conditions allowing the expression of a mutant IL-2 polypeptide, an anti-IL-2 antibody or its antigen-binding fragment, a prodrug or an IL-2 antibody fusion molecule, wherein the host cell is a mammalian cell, and isolating the expressed protein from the culture.

43. A method for treating cancer or an infectious disease or modulating the immune system of a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount 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, an IL-2 antibody fusion molecule according to claim 36 or 37, or a pharmaceutical composition according to claim 38.

44. 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 of a patient in need thereof.

45. 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 of a patient in need thereof.

46. The method according to claim 42, the use of the mutant IL-2 polypeptide, prodrug, IL-2 antibody fusion molecule or pharmaceutical composition according to claim 44, or the use according to claim 45, wherein the patient has an HIV infection; has 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, cholangiocarcinoma, breast cancer and ovarian cancer, and medullary thyroid cancer, or has an inflammatory or autoimmune disease, which is optionally selected from asthma, type I diabetes, rheumatoid arthritis, allergy, systemic lupus erythematosus, organ transplant rejection and graft-versus-host disease.

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