WO2025256578A1 - Protéine de fusion d'il12 ciblant des cellules treg et son procédé de préparation, et procédé thérapeutique l'utilisant - Google Patents

Protéine de fusion d'il12 ciblant des cellules treg et son procédé de préparation, et procédé thérapeutique l'utilisant

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Publication number
WO2025256578A1
WO2025256578A1 PCT/CN2025/100595 CN2025100595W WO2025256578A1 WO 2025256578 A1 WO2025256578 A1 WO 2025256578A1 CN 2025100595 W CN2025100595 W CN 2025100595W WO 2025256578 A1 WO2025256578 A1 WO 2025256578A1
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WO
WIPO (PCT)
Prior art keywords
seq
peptide
targeting
targeting module
sequence
Prior art date
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Pending
Application number
PCT/CN2025/100595
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English (en)
Chinese (zh)
Inventor
吴艾伦
吴晓云
徐文
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Immunowake Biologics Hangzhou Co Ltd
Immunowake Biotech Shanghai Co Ltd
Immunowake Inc
Original Assignee
Immunowake Biologics Hangzhou Co Ltd
Immunowake Biotech Shanghai Co Ltd
Immunowake Inc
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Application filed by Immunowake Biologics Hangzhou Co Ltd, Immunowake Biotech Shanghai Co Ltd, Immunowake Inc filed Critical Immunowake Biologics Hangzhou Co Ltd
Publication of WO2025256578A1 publication Critical patent/WO2025256578A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins

Definitions

  • This disclosure relates to IL12 fusion proteins, and more specifically, to Treg cell-targeted IL12 fusion proteins, their preparation, and treatment methods.
  • Tregs are a type of immunosuppressive CD4 + T cells, primarily manifested as CD4 + CD25 + or CD4 + CD25 + FoxP3 + (references 1-2). They play a crucial role in maintaining homeostasis and preventing autoimmune diseases. Treg cells induce immunosuppression through various mechanisms, including secreting cytokines (IL10, IL35, TGFB), disrupting metabolism by regulating the CD39/CD73 ratio, expressing inhibitory molecules (PD1, CTLA4, LAG3, etc.), and depriving the microenvironment of IL2.
  • cytokines IL10, IL35, TGFB
  • Treg cell numbers have been observed in peripheral blood and tumor tissues of patients with various cancers.
  • multiple cancers including NSCLC, melanoma, ovarian cancer, breast cancer, and gastric cancer, a higher proportion of Treg cells is associated with poorer disease-free survival.
  • Treg cells are in a state of suppressive hyperactivation and proliferation.
  • Treg elimination therapy not only eliminates Treg cells in the tumor microenvironment but also systemic Treg cells, which can easily cause systemic immune instability and lead to autoimmune diseases.
  • cell surface molecules highly expressed by tumor-infiltrating Treg cells are also expressed on activated effector T cells, making it difficult to selectively eliminate tumor-infiltrating Treg cells without affecting effector T cells (Reference 3).
  • Treg fragility is defined as maintaining Foxp3 expression but losing immunosuppressive function.
  • Fragile Treg cells produce IFN ⁇ and upregulate the IFN ⁇ receptor and the transcription factor Tbet.
  • Fragile Treg cells exhibit reduced expression of repressive molecules such as CD73 and IL10 and lower immunosuppressive activity in the tumor microenvironment.
  • repressive molecules such as CD73 and IL10 and lower immunosuppressive activity in the tumor microenvironment.
  • T-bet + IFN ⁇ + FOXp3 + Tregs When FOXp3 + Treg cells are exposed to IL-12, they transform into Th1-like Tregs (T-bet + IFN ⁇ + FOXp3 + Tregs; for a detailed definition of Th1-like Tregs, see reference 4).
  • T-bet + Tregs can produce IFN ⁇ independently of IL-12, indicating that T-bet expression itself is sufficient to trigger IFN ⁇ production, and IL-12 can further enhance this effect.
  • IL-12 has unique properties, exhibiting different effects on Tregs and CD4 + Tcon cells (CD4 + FOXp3- T cells) and CD8 + T cells.
  • IL-12 can induce the upregulation of CD25 on activated CD4 + Tcon and CD8 + T cells (reference 5).
  • IL-12 therapy A major limitation of IL-12 therapy is the serious side effects of systemic administration.
  • Clinical studies have shown that the therapeutic window of IL-12 is very narrow, greatly limiting its application in cancer treatment.
  • IL-12 toxicity has been shown to be associated with systemic IFN ⁇ release.
  • severe IL-12 toxicity was accompanied by high levels of systemic IFN ⁇ release, with peak plasma IFN ⁇ concentrations exceeding 24,000 pg/mL.
  • IL-12 toxicity studies based on IFN ⁇ neutralizing antibodies and IFN ⁇ knockout mice have further confirmed that many acute IL-12 toxicities are IFN ⁇ -dependent (References 7-8). Reducing systemic IFN ⁇ release helps improve the safety of IL-12 therapy. However, a decrease in plasma IFN ⁇ concentration is usually accompanied by a decrease in the efficacy of IL-12 therapy (References 9-10). Safe doses of IL-12 therapy have shown very limited clinical efficacy.
  • This disclosure generally relates to a Treg cell-targeting IL12 fusion protein, the corresponding nucleic acid molecule, vector, cell, pharmaceutical composition, manufacturing method, tumor treatment method, and pharmaceutical use.
  • this disclosure provides a Treg cell-targeting IL12 fusion protein.
  • Non-limiting exemplary embodiments of the Treg cell-targeting IL12 fusion protein described in this disclosure may include one or more of the following features.
  • the Treg cell-targeting IL12 fusion protein comprises one or more Treg cell-targeting modules and IL12 directly or indirectly linked, the one or more Treg cell-targeting modules specifically binding to Treg cell surface molecules, and the IL12 comprising one or more mutations with reduced activity compared to wild-type IL12.
  • the one or more activity-reducing mutations are located on the p35 and/or p40 subunits of the IL12.
  • the one or more activity-reducing mutations include one or more mutations occurring at the W15, E59, F60, K84, and K195 positions of the p40 subunit, the positions being located according to SEQ ID NO:5.
  • the one or more activity-reducing mutations include mutations occurring at any of the following sets of positions on the p40 subunit: (1) F60, (2) E59/F60, (3) E59/F60/K84, (4) E59/F60/K84/K195, (5) W15/E59/F60, (6) W15/E59/F60/K84 and (7) W15/E59/F60/K84/K195; preferably E59/F60/K84/K195.
  • the one or more activity-reducing mutations include one or more mutations that change wild-type amino acids to A.
  • the one or more activity-reducing mutations include any of the following groups of mutations of the p40 subunit: (1) F60A, (2) F60E, (3) F60D, (4) E59A/F60A, (5) E59A/F60A/K84A, (6) E59A/F60A/K84A/K195A, (7) W15A/E59A/F60A, (8) W15A/E59A/F60A/K84A, (9) W15A/E59A/F60A/K84A/K195A; preferably including E59A/F60A/K84A/K195A.
  • the p40 subunit comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identity with any one of SEQ ID NO: 9-17; preferably, the p40 subunit comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO: 14.
  • the p35 and p40 subunits of the IL12 are linked by a linker.
  • the IL12 after linker linking comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identity with any one of SEQ ID NO:18-26. More preferably, the IL12 after linker linking comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO:23.
  • the Treg cell surface molecules include one or more selected from the group consisting of CTLA4, CCR8, CCR4, CCR10, CD25, GITR, OX-40, ICOS, and 4-1BB.
  • the Treg cell-targeting IL12 fusion protein further includes a PD1-targeting module that specifically binds to PD1.
  • the Treg cell targeting module and/or the PD1 targeting module includes: a ligand or a ligand fragment, or an antibody or a fragment of an antibody; optionally, the Treg cell targeting module and/or the PD1 targeting module includes a ligand extracellular domain (LECD), Fab, scFab, Fab', (Fab') 2 , Fv, scFv, or VHH.
  • LCD ligand extracellular domain
  • the Treg cell-targeting IL12 fusion protein further includes a first Fc unit and a second Fc unit with a hinge region, wherein the first Fc unit and the second unit dimerize to form a dimer, and optionally, the IL12 is located between the Treg cell-targeting module or the PD1-targeting module and the hinge region of the first Fc unit or the second Fc unit.
  • the Treg cell-targeting IL12 fusion protein comprises a dimer composed of A-[L1] n1 -B-[L2] n2 -C and A'-[L3] n3 -C', wherein:
  • a and A' represent the first targeting module and the second targeting module, respectively, wherein at least one of A and A' specifically binds to Treg cell surface molecules;
  • B represents IL12 composed of p40 subunit-[L4] n4 -p35 subunit or p35 subunit-[L4] n4 -p40 subunit, wherein at least one of the p35 subunit and p40 subunit includes one or more weakened mutations that reduce the affinity of the IL12 for binding to its receptor compared to the wild type;
  • C and C' represent the first Fc unit and the second Fc unit used for dimerization, respectively;
  • L1, L2, L3, and L4 represent connectors, and n1, n2, n3, and n4 are selected from 0 or 1; and,
  • the Treg cell-targeting IL12 fusion protein comprises a dimer composed of A-[L1] n1 -B-[L2] n2 -C and A'-[L3] n3 -B'-[L4] n4 -C', wherein:
  • a and A' represent the first targeting module and the second targeting module, respectively, wherein at least one of A and A' specifically binds to Treg cell surface molecules;
  • B represents the p40 subunit and B' represents the p35 subunit, or B represents the p35 subunit and B' represents the p40 subunit; at least one of the p35 and p40 subunits includes one or more weakened mutations that reduce the affinity of the IL12 for binding to its receptor compared to the wild type;
  • C and C' represent the first Fc unit and the second Fc unit used for dimerization, respectively;
  • L1, L2, L3, and L4 represent connectors, and n1, n2, n3, and n4 are selected from 0 or 1; and,
  • the first targeting module and/or the second targeting module includes a ligand, antibody, or fragment of the ligand or antibody that specifically binds to the target antigen; optionally, the first targeting module and/or the second targeting module includes an extracellular domain (LECD), Fab, scFab, Fab', (Fab') 2 , Fv, scFv, or VHH of a ligand that specifically binds to the target antigen.
  • LCD extracellular domain
  • both the first targeting module and the second targeting module include a Fab.
  • the Treg cell-targeting IL12 fusion protein includes a first peptide, a second peptide, a third peptide, and a fourth peptide.
  • the first peptide includes VH1-(H1-CH1)-[L1] n1 -B-[L2] n2 -C
  • the second peptide includes VH2-(H2-CH1)-[L3] n3 -C'
  • the third peptide includes VL1-(L1-CL)
  • the fourth peptide includes VL2-(L2-CL).
  • the first peptide and the second peptide form a dimer through C and C'.
  • the first peptide and the third peptide form the first targeting module through VH1-(H1-CH1) and VL1-(L1-CL).
  • the second peptide and the fourth peptide form the second targeting module through VH2-(H2-CH1) and VL2-(L2-CL).
  • the first targeting module includes Fab
  • the second targeting module includes VHH or LECD.
  • the Treg cell-targeting IL12 fusion protein comprises a first peptide, a second peptide, and a third peptide.
  • the first peptide comprises VH1-(H1-CH1)-[L1] n1 -B-[L2] n2 -C
  • the second peptide comprises VHH2-[L3] n3 -C' or LECD2-[L3] n3 -C'
  • the third peptide comprises VL1-(L1-CL).
  • the first peptide and the second peptide form a dimer through C and C'.
  • the first peptide and the third peptide form the first targeting module through VH1-(H1-CH1) and VL1-(L1-CL).
  • the second peptide's VHH2 or LECD2 forms the second targeting module.
  • the first targeting module includes VHH or LECD
  • the second targeting module includes Fab
  • the Treg cell-targeting IL12 fusion protein includes a first peptide, a second peptide, and a third peptide.
  • the first peptide includes VHH1-[L1] n1 -B-[L2] n2 -C or LECD1-[L1] n1 -B-[L2] n2 -C
  • the second peptide includes VH2-(H2-CH1)-[L3] n3 -C'
  • the third peptide includes VL2-(L2-CL).
  • the first peptide and the second peptide form a dimer through C and C'.
  • VHH1 or LECD1 of the first peptide forms the first targeting module.
  • the second peptide and the third peptide form the second targeting module through VH1-(H2-CH1) and VL2-(L2-CL).
  • the first targeting module includes VHH or LECD
  • the second targeting module includes VHH or LECD
  • the Treg cell-targeted IL12 fusion protein includes a first peptide and a second peptide, the first peptide including VHH1-[L1] n1 -B-[L2] n2 -C or LECD1-[L1] n1 -B-[L2] n2 -C, and the second peptide including VHH2-[L3] n3 -C' or LECD2-[L3] n3 -C'; the first peptide and the second peptide form a dimer through C and C'; VHH1 or LECD1 of the first peptide forms the first targeting module; and VHH2 or LECD2 of the second peptide forms the second targeting module.
  • the first targeting module specifically binds to Treg cell surface molecules
  • the second targeting module specifically binds to Treg cell surface molecules.
  • the first targeting module specifically binds to CTLA4 and the second targeting module specifically binds to CTLA4, or the first targeting module specifically binds to CCR8 and the second targeting module specifically binds to CCR8, or the first targeting module specifically binds to CTLA4 and the second targeting module specifically binds to CCR8, or the first targeting module specifically binds to CCR8 and the second targeting module specifically binds to CTLA4.
  • the KD value of the first targeting module binding to its target antigen is ⁇ 1E-7M, ⁇ 1E-8M, ⁇ 1E-9M, ⁇ 1E-10M, ⁇ 1E-11M, or ⁇ 1E-12M.
  • the KD value is in the range of 1E-10M to 1E-7M, and more preferably, the KD value is in the range of 1E-9M to 1E-8M.
  • the KD value of the second targeting module binding to its target antigen is ⁇ 1E-7M, ⁇ 1E-8M, ⁇ 1E-9M, ⁇ 1E-10M, ⁇ 1E-11M, or ⁇ 1E-12M.
  • the KD value is in the range of 1E-10M to 1E-7M, and more preferably, the KD value is in the range of 1E-9M to 1E-8M.
  • the first targeting module specifically binds to Treg cell surface molecules, and the second targeting module specifically binds to PD1.
  • the first targeting module specifically binds to CTLA4 or CCR8, and the second targeting module specifically binds to PD1.
  • the KD value of the first targeting module binding to its target antigen is ⁇ 1E-7M, ⁇ 1E-8M, ⁇ 1E-9M, ⁇ 1E-10M, ⁇ 1E-11M, or ⁇ 1E-12M.
  • the KD value is in the range of 1E-10M to 1E-7M, and more preferably, the KD value is in the range of 1E-9M to 1E-8M.
  • the KD value of the second targeting module binding to its target antigen is ⁇ 1E-6M, ⁇ 1E-7M, ⁇ 1E-8M, ⁇ 1E-9M, ⁇ 1E-10M, ⁇ 1E-11M, or ⁇ 1E-12M.
  • the KD value is in the range of 1E-8M to 1E-6M, and more preferably, the KD value is in the range of 1E-7M to 10E-6M, or in the range of 1E-8 to 1E-7.
  • the first targeting module specifically binds to PD1, and the second targeting module specifically binds to Treg cell surface molecules.
  • the first targeting module specifically binds to PD1, and the second targeting module specifically binds to CTLA4 or CCR8.
  • the KD value of the first targeting module binding to its target antigen is ⁇ 1E-6M, ⁇ 1E-7M, ⁇ 1E-8M, ⁇ 1E-9M, ⁇ 1E-10M, ⁇ 1E-11M, or ⁇ 1E-12M.
  • the KD value is in the range of 1E-8M to 1E-6M.
  • the KD value is in the range of 1E-7M to 10E-6M, or in the range of 1E-8 to 1E-7.
  • the KD value of the second targeting module binding to its target antigen is ⁇ 1E-7M, ⁇ 1E-8M, ⁇ 1E-9M, ⁇ 1E-10M, ⁇ 1E-11M, or ⁇ 1E-12M.
  • the KD value is in the range of 1E-10M to 1E-7M. More preferably, the KD value is in the range of 1E-9M to 1E-8M.
  • the KD value of the Treg cell targeting module binding to its target antigen is ⁇ 1E-7M, ⁇ 1E-8M, ⁇ 1E-9M, ⁇ 1E-10M, ⁇ 1E-11M, or ⁇ 1E-12M.
  • the KD value is in the range of 1E-10M to 1E-7M. More preferably, the KD value is in the range of 1E-9M to 1E-8M.
  • the KD value of the PD1 targeting module binding to its target antigen is ⁇ 1E-6M, ⁇ 1E-7M, ⁇ 1E-8M, ⁇ 1E-9M, ⁇ 1E-10M, ⁇ 1E-11M, or ⁇ 1E-12M.
  • the KD value is in the range of 1E-8M to 1E-6M. More preferably, the KD value is in the range of 1E-7M to 10E-6M, or in the range of 1E-8 to 1E-7.
  • the CTLA4 targeting module includes Fab, scFab, Fab', (Fab') 2 , Fv, scFv, or VHH that specifically bind to CTLA4.
  • CTLA4 the specific binding of CTLA4 to Fab, scFab, Fab', (Fab') 2 , Fv, and scFv includes VH and VL as shown below:
  • the VH includes HCDR1 as shown in SEQ ID NO:79 or 82, HCDR2 as shown in SEQ ID NO:80 or 83, and HCDR3 as shown in SEQ ID NO:81 or 84;
  • the VL includes LCDR1 as shown in SEQ ID NO:85 or 88, LCDR2 as shown in SEQ ID NO:86, and LCDR3 as shown in SEQ ID NO:87;
  • the VH includes HCDR1 as shown in SEQ ID NO:89, HCDR2 as shown in SEQ ID NO:90 and HCDR3 as shown in SEQ ID NO:91;
  • the VL includes LCDR1 as shown in SEQ ID NO:92, LCDR2 as shown in SEQ ID NO:93 and LCDR3 as shown in SEQ ID NO:94;
  • the VH includes HCDR1 as shown in SEQ ID NO:95, HCDR2 as shown in SEQ ID NO:96 and HCDR3 as shown in SEQ ID NO:97; the VL includes LCDR1 as shown in SEQ ID NO:98, LCDR2 as shown in SEQ ID NO:99 and LCDR3 as shown in SEQ ID NO:100;
  • the VH includes HCDR1 as shown in SEQ ID NO:134, HCDR2 as shown in SEQ ID NO:135, and HCDR3 as shown in SEQ ID NO:136;
  • the VL includes LCDR1 as shown in SEQ ID NO:137, LCDR2 as shown in SEQ ID NO:138, and LCDR3 as shown in SEQ ID NO:139;
  • the VH includes HCDR1 as shown in SEQ ID NO:149, HCDR2 as shown in SEQ ID NO:150, and HCDR3 as shown in SEQ ID NO:151;
  • the VL includes LCDR1 as shown in SEQ ID NO:152, LCDR2 as shown in SEQ ID NO:153, and LCDR3 as shown in SEQ ID NO:154;
  • the VH includes HCDR1 as shown in SEQ ID NO:155, HCDR2 as shown in SEQ ID NO:156, and HCDR3 as shown in SEQ ID NO:157;
  • the VL includes LCDR1 as shown in SEQ ID NO:158, LCDR2 as shown in SEQ ID NO:159, and LCDR3 as shown in SEQ ID NO:160;
  • the VH includes HCDR1 as shown in SEQ ID NO:161, HCDR2 as shown in SEQ ID NO:162 and HCDR3 as shown in SEQ ID NO:163;
  • the VL includes LCDR1 as shown in SEQ ID NO:164, LCDR2 as shown in SEQ ID NO:165 and LCDR3 as shown in SEQ ID NO:166;
  • the VH includes HCDR1 as shown in SEQ ID NO:167, HCDR2 as shown in SEQ ID NO:168, and HCDR3 as shown in SEQ ID NO:169;
  • the VL includes LCDR1 as shown in SEQ ID NO:170, LCDR2 as shown in SEQ ID NO:171, and LCDR3 as shown in SEQ ID NO:172;
  • the VH includes HCDR1 as shown in SEQ ID NO:173, HCDR2 as shown in SEQ ID NO:174 and HCDR3 as shown in SEQ ID NO:175;
  • the VL includes LCDR1 as shown in SEQ ID NO:176, LCDR2 as shown in SEQ ID NO:177 and LCDR3 as shown in SEQ ID NO:178;
  • the VH includes HCDR1 as shown in SEQ ID NO:179, HCDR2 as shown in SEQ ID NO:180 and HCDR3 as shown in SEQ ID NO:181; the VL includes LCDR1 as shown in SEQ ID NO:182, LCDR2 as shown in SEQ ID NO:183 and LCDR3 as shown in SEQ ID NO:184;
  • the VH includes HCDR1 as shown in SEQ ID NO:188, HCDR2 as shown in SEQ ID NO:189, and HCDR3 as shown in SEQ ID NO:190;
  • the VL includes LCDR1 as shown in SEQ ID NO:191, LCDR2 as shown in SEQ ID NO:192, and LCDR3 as shown in SEQ ID NO:193;
  • the VH includes HCDR1 as shown in SEQ ID NO:194, HCDR2 as shown in SEQ ID NO:195 and HCDR3 as shown in SEQ ID NO:196; the VL includes LCDR1 as shown in SEQ ID NO:197, LCDR2 as shown in SEQ ID NO:198 and LCDR3 as shown in SEQ ID NO:199;
  • the VH includes HCDR1 as shown in SEQ ID NO:200, HCDR2 as shown in SEQ ID NO:201 and HCDR3 as shown in SEQ ID NO:202; the VL includes LCDR1 as shown in SEQ ID NO:203, LCDR2 as shown in SEQ ID NO:204 and LCDR3 as shown in SEQ ID NO:205;
  • the VH includes HCDR1 as shown in SEQ ID NO:206, HCDR2 as shown in SEQ ID NO:207, and HCDR3 as shown in SEQ ID NO:208; the VL includes LCDR1 as shown in SEQ ID NO:209, LCDR2 as shown in SEQ ID NO:210, and LCDR3 as shown in SEQ ID NO:211; or,
  • the VH includes HCDR1 as shown in SEQ ID NO:212, HCDR2 as shown in SEQ ID NO:213 and HCDR3 as shown in SEQ ID NO:214;
  • the VL includes LCDR1 as shown in SEQ ID NO:215, LCDR2 as shown in SEQ ID NO:216 and LCDR3 as shown in SEQ ID NO:217;
  • the VHH that specifically binds to CTLA4 includes:
  • HCDR1 as shown in SEQ ID NO:101
  • HCDR2 as shown in SEQ ID NO:102
  • HCDR3 as shown in SEQ ID NO:103;
  • HCDR1 as shown in SEQ ID NO:104
  • HCDR2 as shown in SEQ ID NO:105
  • HCDR3 as shown in SEQ ID NO:106;
  • HCDR1 as shown in SEQ ID NO:107
  • HCDR2 as shown in SEQ ID NO:108
  • HCDR3 as shown in SEQ ID NO:109;
  • HCDR1 as shown in SEQ ID NO:110, HCDR2 as shown in SEQ ID NO:111 and HCDR3 as shown in SEQ ID NO:112;
  • HCDR1 as shown in SEQ ID NO:113
  • HCDR2 as shown in SEQ ID NO:114
  • HCDR3 as shown in SEQ ID NO:115;
  • HCDR1 as shown in SEQ ID NO:116
  • HCDR2 as shown in SEQ ID NO:117
  • HCDR3 as shown in SEQ ID NO:118;
  • HCDR1 as shown in SEQ ID NO:119
  • HCDR2 as shown in SEQ ID NO:120
  • HCDR3 as shown in SEQ ID NO:121;
  • HCDR1 as shown in SEQ ID NO:122, HCDR2 as shown in SEQ ID NO:123 and HCDR3 as shown in SEQ ID NO:124;
  • HCDR1 as shown in SEQ ID NO:125
  • HCDR2 as shown in SEQ ID NO:126
  • HCDR3 as shown in SEQ ID NO:127;
  • HCDR1 as shown in SEQ ID NO:128, HCDR2 as shown in SEQ ID NO:129 and HCDR3 as shown in SEQ ID NO:130;
  • HCDR1 as shown in SEQ ID NO:131
  • HCDR2 as shown in SEQ ID NO:132
  • HCDR3 as shown in SEQ ID NO:133;
  • HCDR1 as shown in SEQ ID NO:140
  • HCDR2 as shown in SEQ ID NO:141
  • HCDR3 as shown in SEQ ID NO:142;
  • HCDR1 as shown in SEQ ID NO:143
  • HCDR2 as shown in SEQ ID NO:144
  • HCDR3 as shown in SEQ ID NO:145;
  • HCDR1 as shown in SEQ ID NO:146
  • HCDR2 as shown in SEQ ID NO:147
  • HCDR3 as shown in SEQ ID NO:148
  • HCDR1 as shown in SEQ ID NO:185
  • HCDR2 as shown in SEQ ID NO:186
  • HCDR3 as shown in SEQ ID NO:187.
  • the specific binding of CTLA4 to Fab, scFab, Fab', (Fab') 2 , Fv, and scFv includes VH and VL as shown below:
  • VH includes any of the sequences shown in SEQ ID NO:31-33, and the VL includes the sequences shown in SEQ ID NO:34 or 35;
  • VH includes the sequence shown in SEQ ID NO:36
  • VL includes the sequence shown in SEQ ID NO:37
  • VH includes the sequence shown in SEQ ID NO:38, and the VL includes the sequence shown in SEQ ID NO:39;
  • VH comprises the sequence shown in SEQ ID NO:51
  • VL comprises the sequence shown in SEQ ID NO:52;
  • VH comprises the sequence shown in SEQ ID NO:56
  • VL comprises the sequence shown in SEQ ID NO:57
  • VH comprises the sequence shown in SEQ ID NO:58
  • VL comprises the sequence shown in SEQ ID NO:59
  • VH comprises the sequence shown in SEQ ID NO:60
  • VL comprises the sequence shown in SEQ ID NO:61
  • VH comprises the sequence shown in SEQ ID NO:62
  • VL comprises the sequence shown in SEQ ID NO:63
  • VH comprises the sequence shown in SEQ ID NO:64
  • VL comprises the sequence shown in SEQ ID NO:65
  • VH comprises the sequence shown in SEQ ID NO:66
  • VL comprises the sequence shown in SEQ ID NO:67;
  • VH comprises the sequence shown in SEQ ID NO:69
  • VL comprises the sequence shown in SEQ ID NO:70
  • VH comprises the sequence shown in SEQ ID NO:71
  • VL comprises the sequence shown in SEQ ID NO:72;
  • VH comprises the sequence shown in SEQ ID NO:73
  • VL comprises the sequence shown in SEQ ID NO:74
  • VH comprises the sequence shown in SEQ ID NO:75
  • VL comprises the sequence shown in SEQ ID NO:76
  • VH comprises the sequence shown in SEQ ID NO:77
  • VL comprises the sequence shown in SEQ ID NO:78; or
  • (16) has an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99% or 100% identical to the VH and/or VL shown in any of groups (1)-(15).
  • the VHH that specifically binds to CTLA4 includes:
  • the CCR8 targeting module includes Fab, scFab, Fab', (Fab') 2 , Fv, scFv, or VHH that specifically bind to CCR8;
  • the specific binding of CCR8 to Fab, scFab, Fab', (Fab') 2 , Fv, and scFv includes VH and VL as shown below:
  • HCDR1 as shown in SEQ ID NO:265, HCDR2 as shown in SEQ ID NO:266, and HCDR3 as shown in SEQ ID NO:267;
  • the VL includes LCDR1 as shown in SEQ ID NO:268, LCDR2 as shown in SEQ ID NO:269, and LCDR3 as shown in SEQ ID NO:270; or,
  • the VH includes HCDR1 as shown in SEQ ID NO:271, HCDR2 as shown in SEQ ID NO:272 and HCDR3 as shown in SEQ ID NO:273; the VL includes LCDR1 as shown in SEQ ID NO:274, LCDR2 as shown in SEQ ID NO:275 and LCDR3 as shown in SEQ ID NO:276;
  • the VHH that specifically binds to CCR8 includes:
  • HCDR1 as shown in SEQ ID NO:259
  • HCDR2 as shown in SEQ ID NO:260
  • HCDR3 as shown in SEQ ID NO:261
  • HCDR1 as shown in SEQ ID NO:277
  • HCDR2 as shown in SEQ ID NO:278
  • HCDR3 as shown in SEQ ID NO:279.
  • the specific binding of CCR8 to Fab, scFab, Fab', (Fab') 2 , Fv, and scFv includes VH and VL, as shown below:
  • VH includes the sequence shown in SEQ ID NO:254, and the VL includes the sequence shown in SEQ ID NO:255;
  • VH comprises the sequence shown in SEQ ID NO:256
  • VL comprises the sequence shown in SEQ ID NO:257; or
  • the VHH that specifically binds to CCR8 includes:
  • the PD1 targeting module includes Fab, scFab, Fab', (Fab') 2 , Fv, scFv, or VHH that specifically bind to PD1;
  • the specific binding of PD1 to Fab, scFab, Fab', (Fab') 2 , Fv, and scFv includes VH and VL as shown below:
  • the VH includes HCDR1 as shown in SEQ ID NO:234, HCDR2 as shown in SEQ ID NO:235 and HCDR3 as shown in SEQ ID NO:236, and the VL includes LCDR1 as shown in SEQ ID NO:237, LCDR2 as shown in SEQ ID NO:238 and LCDR3 as shown in SEQ ID NO:239;
  • the VH includes HCDR1 as shown in SEQ ID NO:240, HCDR2 as shown in SEQ ID NO:241 and HCDR3 as shown in SEQ ID NO:242, and the VL includes LCDR1 as shown in SEQ ID NO:243, LCDR2 as shown in SEQ ID NO:244 and LCDR3 as shown in SEQ ID NO:245;
  • the VH includes HCDR1 as shown in SEQ ID NO:246, HCDR2 as shown in SEQ ID NO:247, and HCDR3 as shown in SEQ ID NO:248;
  • the VL includes LCDR1 as shown in SEQ ID NO:249, LCDR2 as shown in SEQ ID NO:250, and LCDR3 as shown in SEQ ID NO:251; or,
  • the VH includes HCDR1 as shown in SEQ ID NO:293, HCDR2 as shown in SEQ ID NO:294 and HCDR3 as shown in SEQ ID NO:295, and the VL includes LCDR1 as shown in SEQ ID NO:296, LCDR2 as shown in SEQ ID NO:297 and LCDR3 as shown in SEQ ID NO:298;
  • the VHH that specifically binds to PD1 includes:
  • HCDR1 as shown in SEQ ID NO:228, HCDR2 as shown in SEQ ID NO:229, and HCDR3 as shown in SEQ ID NO:230; or,
  • HCDR1 as shown in SEQ ID NO:231, HCDR2 as shown in SEQ ID NO:232 and HCDR3 as shown in SEQ ID NO:233.
  • the specific binding of PD1 to Fab, scFab, Fab', (Fab') 2 , Fv, and scFv includes VH and VL as shown below:
  • VH includes the sequence shown in SEQ ID NO:218, and the VL includes the sequence shown in SEQ ID NO:219;
  • VH includes the sequence shown in SEQ ID NO:222, and the VL includes the sequence shown in SEQ ID NO:223;
  • VH includes the sequence shown in SEQ ID NO:224, and the VL includes the sequence shown in SEQ ID NO:225;
  • VH comprises the sequence shown in SEQ ID NO:226, and the VL comprises the sequence shown in SEQ ID NO:227; or,
  • the specific binding of PD1 to VHH includes:
  • the PD1 targeting module includes a PDL1 extracellular domain and/or a PDL2 extracellular domain;
  • the PD1 targeting module includes:
  • the first Fc unit and the second Fc unit include Knob mutations and Hole mutations to form a Knob-in-Hole structure.
  • the Knob mutation is selected from S354C, T366W, preferably S354C/T366W, and the Hole mutation is selected from Y349C, T366S, L368A, and Y349C, preferably Y349C/T366S/L368A/Y349C.
  • the first Fc unit and the second Fc unit include mutations that reduce or eliminate effector functionality
  • the mutations that reduce or eliminate effector function include L234A/L235A mutations.
  • the first Fc unit includes:
  • the second Fc unit includes:
  • the connector is selected from the sequence shown in any one of SEQ ID NO:280-292.
  • the Treg-targeting IL12 fusion protein includes:
  • a first peptide, a second peptide, a third peptide, and a fourth peptide wherein the first peptide comprises a sequence having at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO:307 or 310, the second peptide comprises a sequence having at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO:308, the third peptide comprises a sequence having at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO:309, and the fourth peptide comprises a sequence having at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO:309;
  • first peptide comprises a sequence having at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO:307 or 310
  • second peptide comprises a sequence having at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO:311
  • third peptide comprises a sequence having at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO:309;
  • a first peptide, a second peptide, and a third peptide wherein the first peptide comprises a sequence having at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO:312 or 313, the second peptide comprises a sequence having at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO:308, and the third peptide comprises a sequence having at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO:309.
  • this disclosure provides an isolated nucleic acid molecule comprising one or more peptide chains encoding the Treg cell-targeting IL12 fusion protein described in the first aspect of this disclosure.
  • the nucleic acid is DNA or RNA.
  • this disclosure provides a vector comprising the isolated nucleic acid molecules described in the second aspect of this disclosure.
  • this disclosure provides a cell comprising the isolated nucleic acid molecule described in the second aspect of this disclosure or the carrier described in the third aspect.
  • the cell is a prokaryotic cell or a eukaryotic cell, preferably a eukaryotic cell, more preferably a CHO cell, such as a CHO-K1 cell.
  • this disclosure provides a production method comprising culturing the cells described in the fourth aspect of this disclosure to express the Treg cell-targeting IL12 fusion protein described in the first aspect, and isolating and purifying the Treg cell-targeting IL12 fusion protein.
  • this disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising: (1) the Treg cell-targeting IL12 fusion protein of the first aspect, the nucleic acid of the second aspect, the carrier of the third aspect, the cell of the fourth aspect, or the product obtained by the method of the fifth aspect; and (2) a pharmaceutically acceptable carrier.
  • this disclosure provides a tumor treatment method comprising administering to a subject an effective amount of the Treg cell-targeting IL12 fusion protein of the first aspect, the isolated nucleic acid molecule of the second aspect, the carrier of the third aspect, the cell of the fourth aspect, the product obtained by the method of the fifth aspect, or the pharmaceutical composition of the sixth aspect, which induces intratumoral Treg cells to secrete IFN ⁇ , thereby weakening or relieving the immunosuppressive effect of the intratumoral Treg cells.
  • the Treg cells induce intratumoral Treg cells to transform into Th1-like Treg cells or fragile Treg cells.
  • the Treg cells targeting the IL12 fusion protein also induce CD8 + T cells or CD4 + Tcon cells in the tumor microenvironment to: (1) secrete IFN ⁇ ; (2) upregulate the expression level of T-bet; and/or upregulate the expression of CD25.
  • the Treg-targeting IL12 fusion protein is administered systemically, such as via intravenous or subcutaneous injection.
  • it is a method of administration as a single therapy or in combination with other therapies.
  • the other therapies are selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, toxin therapy, and surgery.
  • the Treg cells targeting the IL12 fusion protein, the isolated nucleic acid molecule, the vector, the cells, the product obtained by the method, or the pharmaceutical composition are administered before, after, or simultaneously with the other therapies.
  • the subject is a patient resistant to an immune checkpoint inhibitor, and preferably, the immune checkpoint inhibitor is a PD1 antibody.
  • the tumor is a solid tumor; preferably, the solid tumor is selected from the group consisting of melanoma, colorectal cancer, prostate cancer, lung cancer, liver cancer, pancreatic cancer, esophageal cancer, gastric cancer, kidney cancer, breast cancer, ovarian cancer, uterine cancer, bladder cancer, head and neck cancer, and glioma.
  • this disclosure provides the use of the Treg cell-targeting IL12 fusion protein of the first aspect, the isolated nucleic acid molecule of the second aspect, the carrier of the third aspect, the cell of the fourth aspect, the product obtained by the method of the fifth aspect, or the pharmaceutical composition of the sixth aspect in the preparation of a tumor therapeutic drug, wherein the Treg cell-targeting IL12 fusion protein induces intratumoral Treg cells to secrete IFN ⁇ , thereby weakening or relieving the immunosuppressive effect of the Treg cells.
  • the Treg cells induce intratumoral Treg cells to transform into Th1-like Treg cells or fragile Treg cells.
  • the Treg cells targeting the IL12 fusion protein also induce CD8 + T cells or CD4 + Tcon cells in the tumor microenvironment to: (1) secrete IFN ⁇ ; (2) upregulate the expression level of T-bet; and/or upregulate the expression of CD25.
  • the Treg-targeting IL12 fusion protein is administered systemically, such as via intravenous or subcutaneous injection.
  • the Treg-targeting IL12 fusion protein is administered as a monotherapy or in combination with other therapies.
  • the other therapies are selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, toxin therapy, and surgery.
  • the Treg cells targeting the IL12 fusion protein, the isolated nucleic acid molecule, the vector, the cells, the product obtained by the method, or the pharmaceutical composition are administered before, after, or simultaneously with other therapies.
  • the subject is a patient resistant to an immune checkpoint inhibitor, and preferably, the immune checkpoint inhibitor is a PD1 antibody.
  • the tumor is a solid tumor; preferably, the solid tumor is selected from the group consisting of melanoma, colorectal cancer, prostate cancer, lung cancer, liver cancer, pancreatic cancer, esophageal cancer, gastric cancer, kidney cancer, breast cancer, ovarian cancer, uterine cancer, bladder cancer, head and neck cancer, and glioma.
  • Figure 1A Comparison of amino acid sequences of human p35 subunit (Query_10001), monkey p35 subunit (Query_10002), mouse p35 subunit (Query_10003), and dog p35 subunit (Query_10004).
  • Figure 1B Comparison of amino acid sequences of human p40 subunit (Query_10001), monkey p40 subunit (Query_10002), mouse p40 subunit (Query_10003), and dog p40 subunit (Query_10004).
  • Figures 2A-2B Percentage of IL12R ⁇ 1 + cells and PD1 + cells in spleen and tumor-derived lymphocyte subsets; Lymphocyte subset classification: CD4 + Tcon cells (CD45 + CD3 + CD4 + FOXp3- ), CD4 + Treg cells (CD45 + CD3 + CD4 + FOXp3 + ), CD8 + T cells (CD45 + CD3 + CD8 + ), NK cells (CD45 + NK1.1 + CD3- ), NKT cells (CD45 + NK1.1 + CD3 + ).
  • Figures 3A-3B Percentage of CTLA4 + or CCR8 + cells in spleen and tumor-derived T lymphocyte subsets; Lymphocyte subset classification: CD3 + T cells (CD45 + CD3 + ), Tcon cells (CD45 + CD3 + CD4 + CD25- ), Treg cells (CD45 + CD3 + CD4 + CD25 + ), CD8 + T cells (CD45 + CD3 + CD8 + ).
  • Figure 4 Percentage of PD1 + cells, CTLA4 + cells, and CCR8 + cells in tumor-infiltrating lymphocyte subsets; Lymphocyte subset classification: CD3 + T cells (CD45 + CD3 + ); Tcon cells (CD45 + CD3 + CD4 + CD25- ); Treg cells (CD45 + CD3 + CD4 + CD25 + ); CD8 + T cells (CD45 + CD3 + CD8 + ).
  • Figure 5A Schematic diagram of Treg cells targeting the IL12 fusion protein, where ABD represents the antigen-binding domain, the p35 and p40 subunits of IL12 are not shown separately, and the linker is not shown.
  • Figure 5B Schematic diagram of Treg cells targeting the IL12 fusion protein, where ABD represents the antigen-binding domain, the p35 and p40 subunits of IL12 are shown, and the linker is not shown.
  • Figure 6 A specific example of the schematic diagram shown in Figure 5A (targeting CTLA4).
  • Figure 7 A specific example of the schematic diagram shown in Figure 5A (targeting CCR8).
  • Figures 8A-8C IL12 activity detection results of hIL12(WT)-Fc//Fc, hIL12(4A)-Fc//Fc and Ipi-hIL12(4A)//hPDL2.
  • Figure 9 Detection results of CTLA4 targeting IL12 fusion protein (Ipi).
  • FIGS 10A-10C Activity assay results of CTLA4 targeting IL12 fusion proteins (VH146, HL32, Ipi).
  • Figures 11A-11E Activity assay results of CCR8 targeting IL12 fusion protein.
  • Figures 12A-12B Table 7 shows the growth-inhibiting effect of Treg cells targeting IL12 fusion protein on B16 melanoma (modeling and administration: hCTLA4/hPD1 humanized mice (purchased from Jicui Yaokang), cancer cell inoculation amount: 1E5 cells/mouse, group: 4 mice/group, intraperitoneal injection, dose: 5 mg/kg, administration time: days 4, 7, 11 and 15 after inoculation).
  • Figures 13A-13B Table 8 shows the growth inhibition effect of Treg cells targeting IL12 fusion protein on B16 melanoma (modeling and administration: hCTLA4 humanized mice (purchased from Biocytogen), cancer cell inoculation amount: 2.5E5/mouse, group: 7 mice/group, administration via tail vein injection, dose of 5mpk, administration time: days 5, 8, 11 and 14 post-inoculation).
  • Figures 14A-14B Table 9 shows the growth inhibitory effect of Treg cells targeting IL12 fusion protein on RM1 prostate tumors (modeling and administration: hCTLA4/hPD1 humanized mice (purchased from Jicui Yaokang), cancer cell inoculation amount: 2E5 cells/mouse, grouping: 5 mice/group, intraperitoneal injection, dose of 5mpk, administration time: 6, 9, 12 and 15 days after inoculation).
  • Figures 15A-15D Table 10 shows the growth inhibitory effect of Treg cells targeting IL12 fusion protein on RM1 prostate tumors (modeling and administration: hCTLA4 humanized mice (purchased from Biocytogen), cancer cell inoculation amount: 2E5 cells/mouse, grouping: 5 mice/group, intraperitoneal injection, dose of 5 mpk, administration time: days 6, 9, 12, and 15 post-inoculation).
  • Figure 16A IL12 activity detection results of 9D9-mIL12(4A)//mPDL2.
  • Figure 16B IL12 activity detection results of Ipi-hIL12(4A)//hPDL2.
  • Figure 17 Growth inhibitory effect of the tested drugs on MC38 colorectal cancer as shown in Table 12 (modeling and administration: wild-type C57BL/6 mice, cancer cell inoculation amount: 5E6 cells/mouse, grouping: 20 mice/group, intraperitoneal injection, dose of 5mpk, administration time: days 9, 13, 16 and 20 after inoculation).
  • Figure 18 Growth inhibitory effect of the tested drugs on EMT6 breast tumors as shown in Table 12 (modeling and administration: wild-type Balb/c mice, cancer cell inoculation amount: 2E6 cells/mouse, grouping: 20 mice/group, intraperitoneal injection, dose of 5 mpk, administration time: days 6, 9, 12 and 15 after inoculation).
  • Figure 19 Growth inhibitory effect of the tested drugs on B16 melanoma as shown in Table 12 (modeling and administration: wild-type Balb/c mice, cancer cell inoculation amount: 2E5 cells/mouse, group: 7 mice/group, intraperitoneal injection, except for mIpi which was 5 mpk, all others were 10 mpk, administration time: days 4, 8, 11 and 14 after inoculation).
  • Figure 20 The growth inhibitory effect of the tested drugs on MC38 colorectal tumors as shown in Table 13 (modeling and administration: wild-type C57BL/6 mice, cancer cell inoculation amount: 5E5/mouse, grouping: 6 mice/group, intraperitoneal injection, dose of 1.5mpk, administration time: days 7, 12, 17 and 21 after inoculation).
  • Figure 21 The growth inhibitory effect of the tested drugs on B16 melanoma as shown in Table 14 (modeling and administration: wild-type C57BL/6 mice, cancer cell inoculation amount: 2E5 cells/mouse, grouping: 7 mice/group, administration via tail vein injection, dose of 5 mpk, administration time on days 5, 8, 12 and 15 after inoculation).
  • Figures 22A-22C Tables 15 and 16 show the growth inhibitory effect of the tested drugs on CT26 colorectal tumors (modeling and administration: wild-type Balb/c mice, cancer cell inoculation amount: 2.5E5/mouse, grouping: 6 mice/group, intraperitoneal injection, dose of 5mpk, administration time is 4, 7, 11 and 14 days after inoculation).
  • FIGS 23A-23B Results of serum IFN ⁇ levels in mice after drug administration.
  • FIGS 24A-24B Changes in mouse body weight after drug administration.
  • Figure 25 Results of serum IFN ⁇ levels in cynomolgus monkeys after drug administration.
  • FIG. 26 TDLN cell count in mice after drug administration.
  • Figure 27 Percentage of IFN ⁇ + cells in mouse spleen, TDLN, and tumor-derived Treg cells after drug administration.
  • Figure 28 Percentage of IFN ⁇ + cells in mouse spleen, TDLN and tumor-derived Tcon cells after drug administration.
  • Figure 29 Percentage of IFN ⁇ + cells in mouse spleen, TDLN, and tumor-derived CD8 T cells after drug administration.
  • Figure 30 Percentage of T-bet+ cells in mouse spleen, TDLN and tumor-derived Treg cells after drug administration.
  • Figure 31 Percentage of T-bet+ cells in mouse spleen, TDLN and tumor-derived Tcon cells after drug administration.
  • Figure 32 Percentage of T-bet+ cells in mouse spleen, TDLN and tumor-derived CD8 T cells after drug administration.
  • Figure 33 Percentage of CD25+ cells in mouse spleen, TDLN and tumor-derived CD8 T cells after drug administration.
  • Figure 34 Percentage of CD25+ cells in mouse spleen, TDLN and tumor-derived Tcon cells after drug administration.
  • Figure 35 Summary of information on the 9D9-mIL12(4A)//mPDL2 dosing groups in Figures 27-34.
  • Figure 36 IFN ⁇ content detection results in the culture medium supernatant after 72 h of drug treatment.
  • Figure 37A Percentage of IFN ⁇ + cells in PBMC-derived Treg cells after 72 h of drug treatment.
  • Figure 37B Percentage of IFN ⁇ + cells in PBMC-derived Tcon cells after 72 h of drug treatment.
  • Figure 37C Percentage of IFN ⁇ + cells in PBMC-derived CD8 T cells after 72 h of drug treatment.
  • Treg elimination therapies while eliminating Treg cells in the tumor microenvironment, also eliminate systemic Treg cells, triggering immune-related adverse events (irAEs) and autoimmune-related toxicities, and eliminate effector T cells activated in the tumor microenvironment, inhibiting specific anti-tumor responses.
  • Current IL12 therapies also have significant limitations; their excessively high systemic toxicity and narrow therapeutic window restrict their clinical application.
  • the Treg cell-targeting IL12 fusion protein comprises one or more Treg cell-targeting modules and IL12 directly or indirectly linked, wherein the one or more Treg cell-targeting modules specifically bind to Treg cell surface molecules, and the IL12 contains one or more mutations with reduced activity compared to wild-type IL12.
  • This disclosure also provides a novel tumor treatment method comprising administering an effective amount of the Treg cell-targeting IL12 fusion protein to a subject, which induces intratumoral Treg cells to secrete IFN ⁇ , thereby attenuating or relieving the immunosuppressive effect of the intratumoral Treg cells.
  • Treg cell-targeted IL12 fusion protein and tumor treatment methods described in this disclosure have at least one of the following advantages:
  • This disclosure adopts a targeted IL12 delivery strategy, which is different from the existing Treg depletion strategy and will not cause the clearance of systemic Treg cells and the clearance of tumor microenvironment effector T cells.
  • Treg cells especially intratumoral Treg cells
  • IFN ⁇ immunosuppressive effect in the tumor microenvironment
  • the Treg cell-targeting IL12 fusion protein has target antigen-dependent IL12 activity, which selectively acts in the tumor microenvironment and acts less (to a minimum) on the peripheral immune system, thereby reducing the toxic side effects of IL12 and expanding the therapeutic window.
  • the Treg cell-targeting IL12 fusion protein described in this disclosure not only binds to Treg cells (e.g. via CTLA4 and CCR8), but also binds to tumor microenvironment effector T cells (e.g. via PD1). While inducing tumor microenvironment Treg cells to secrete IFN ⁇ , it can also activate intratumoral effector T cells (e.g., CD8 + T cells and Tcon) to a certain extent.
  • intratumoral effector T cells e.g., CD8 + T cells and Tcon
  • certain preferred mutations or certain preferred structural features can further enhance the target antigen dependence of the Treg cell on the IL12 fusion protein, which helps to achieve a balance between therapeutic efficacy and safety.
  • a cell includes one or more cells, including mixtures thereof.
  • fusion protein refers to a protein comprising at least one heterozygous peptide, which includes protein domains derived from at least two different proteins. These protein domains from at least two different proteins may be directly linked by peptide bonds or indirectly linked by linkers or other domains. Fusion proteins can be generated by any method known in the art.
  • the fusion proteins provided in this disclosure can be generated by recombinant protein expression and purification, which is particularly suitable for fusion proteins containing linkers. Methods for recombinant protein expression and purification are well known, including those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.
  • amino acid mutation in a protein, polypeptide, or fragment thereof, it means the substitution, deletion, or insertion of a wild-type amino acid residue that normally resides at that position.
  • a “wild-type amino acid residue” generally refers to the amino acid residue that the typical native form of the protein or polypeptide should have at that position. Typically, this typical native form is generally agreed upon in the art.
  • amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. It is also conceivable that methods other than genetic engineering (such as chemical modification) could be used to alter amino acid side chain groups.
  • substitution mutation includes substitution with a selection from twenty standard amino acids, and also includes substitution with non-naturally occurring amino acids or naturally occurring amino acid derivatives of the aforementioned twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine).
  • the substitution mutation may be a substitution of a conserved amino acid residue or a non-conserved amino acid residue.
  • Constant amino acid residue substitution refers to the replacement of an amino acid residue with another amino acid residue of the same class. This class is defined by common physicochemical properties of the amino acid side chains and high substitution frequencies found in naturally occurring homologous proteins, for example, through a standard Dayhoff frequency exchange matrix or a BLOSUM matrix.
  • amino acids can be classified into six classes based on their side chain groups: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gln, Glu); Class IV (His, Arg, Lys); Class V (Ile, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For instance, replacing Asp with another Class III residue such as Asn, Gln, or Glu is a conserved substitution.
  • non-conservative amino acid residue substitution refers to replacing an amino acid residue of one class with an amino acid residue of another class; for example, replacing Ala (a class II residue) with a class III residue such as Asp, Asn, Glu, or Gln.
  • the slash “/” is used to indicate “and” when linking mutations.
  • “E59/F60” indicates that the polypeptide and/or protein have mutations at positions 59 and 60 of the corresponding reference sequence.
  • “E59A/F60A” indicates that the polypeptide and/or protein have a mutation from E to A at position 59 of the corresponding reference sequence, and a mutation from F to A at position 60 of the corresponding reference sequence.
  • mutant is a set of relative concepts in this disclosure.
  • a mutant should be understood as a protein or polypeptide containing an amino acid mutation.
  • the wild type is identical to its corresponding mutant in all other respects, except that the wild type form has wild-type amino acids at every amino acid position of the mutant. For example, if the mutant is the full-length form with a signal peptide, then its wild type is the full-length form. If the mutant is the processed mature form without a signal peptide, then its corresponding wild type is also the processed mature form. If its mutant is fused with other polypeptides or conjugated with other substances, then its wild type is also the form fused with other polypeptides or conjugated with other substances.
  • sequence identity refers to the degree (percentage) to which two sequences share the same amino acids/nucleic acids at equivalent positions when optimally aligned. During alignment, gaps may be introduced where necessary to achieve the maximum percentage of sequence identity, but any conserved substitutions are not considered part of the sequence identity. To determine the percentage of sequence identity, alignment can be performed using techniques known in the art, such as publicly available computer software like BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.
  • KD Koff/Kon, where Koff represents the dissociation rate constant and Kon represents the binding rate constant.
  • KD values can be determined using methods known in the art. For example, biosensing systems such as systems measuring surface plasmon resonance (e.g., Biacore) or solution equilibrium titration (SET) can be used to measure affinity in solution.
  • ligands especially ligand extracellular domains
  • antibodies, or fragments thereof bind to the target antigen.
  • antibody includes, but is not limited to, typical four-chain antibodies, heavy chain antibodies (HCAbs), and immunoglobulin neoantigen receptors (IgNARs).
  • HCAbs heavy chain antibodies
  • IgNARs immunoglobulin neoantigen receptors
  • a typical "quadruple-chain antibody” refers to an immunoglobulin composed of two heavy chains (HC) and two light chains (LC).
  • the heavy chain is a polypeptide chain that consists of a heavy chain variable region, a heavy chain constant region CH1 domain, a hinge region, a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the direction from the N-terminus to the C-terminus.
  • the full-length antibody is an IgE isotype, it also includes a heavy chain constant region CH4 domain.
  • the light chain is a polypeptide chain composed of a light chain variable region and a light chain constant region in the direction from the N-terminus to the C-terminus.
  • the heavy chains are linked to each other and to each other with light chains by disulfide bonds, forming a "Y"-shaped structure.
  • Heavy chain antibodies are antibodies produced by species of the Camelidae family (including camels, llama, and alpacas) that consist solely of heavy chains and lack light chains. Typically, their heavy chains, from the N-terminus to the C-terminus, consist of a heavy chain variable region, a hinge region, a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain.
  • the heavy chain variable region of a heavy chain antibody is referred to as the VHH (variable domain of heavy chain of heavy-chain antibody).
  • Immunoglobulin neoantigen receptor refers to a class of antibodies derived from the shark immune spectrum, which consists of a homodimer of a variable neoantigen receptor (VNAR) domain and five constant neoantigen receptor (CNAR) domains.
  • VNAR variable neoantigen receptor
  • CNAR constant neoantigen receptor
  • antibody fragment refers to an antibody-derived fragment with antigen-binding function.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , scFab (single chain Fab), Fv, scFv (single chain Fv), and VHH (Variable Domain of Heavy Chain of HCAb).
  • Fab fragments can be obtained by digesting full-length antibodies with papain.
  • the heavy chain fragment (VH-CH1) and light chain fragment (VL-CL) of the Fab fragment are linked by linkers to produce a single protein chain, forming a single chain Fab (scFab).
  • F(ab') 2 a dimer of Fab', which is a divalent antibody fragment.
  • F(ab') 2 can be reduced under neutral conditions by breaking the disulfide bonds in the hinge region, thereby converting the F(ab') 2 dimer into Fab' monomers.
  • Fab' monomers are essentially Fab fragments with hinge regions.
  • the Fv fragment consists of the VL and VH domains of the antibody single arm.
  • the two domains, VL and VH, of the Fv fragment can be encoded by independent genes, or they can be generated as a single protein chain by using a recombinant method and linking the two domains with a linker. In the single protein chain, the VL and VH regions pair up to form a single-chain Fv (scFv).
  • a “variable region” is the amino-terminal domain of an antibody's heavy or light chain that recognizes and binds to antigens. The composition and arrangement of amino acids in this region determine the antibody's specificity in recognizing antigens.
  • the heavy chain variable domain can be called "VH,” and the light chain variable domain can be called “VL.”
  • Each of the heavy and light chain variable regions consists of three complementarity-determining regions (CDRs) (also known as hypervariable regions) connected by four framework regions (FRs).
  • the complementarity-determining region (CDR) or CDR is a region within the antibody variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and/or contains antigen contact residues ("antigen contact sites").
  • the CDR is primarily responsible for binding to antigen epitopes.
  • the CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus.
  • CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while those located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3.
  • each CDR can be determined using any one or a combination of many known antibody CDR assignment schemes, including, for example: Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)), based on antibody sequence variability; and Kabat et al. (1997) based on antibody sequence variability.
  • the definitions of sequences of Proteins of Immunological Interest 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.
  • CDR CDR sequence
  • the CDR region of an antibody can be defined as described in Table A.
  • the CDR region of an antibody may include the following “extended CDRs”: 24-36 or 24-34 (LCDR1), 46-56 or 50-56 (LCDR2), and 89-97 or 89-96 (LCDR3) in VL, and 26-35 (HCDR1), 50-65 or 49-65 (HCDR2), and 93-102, 94-102, or 95-102 (HCDR3) in VH.
  • LCDR1 extended CDRs
  • variable domain residue numbering in Kabat or “amino acid position numbering in Kabat” and their variations refer to the numbering system used for heavy chain or light chain variable domains in the antibody compilation of Kabat et al. (mentioned above). Using this numbering system, the actual linear amino acid sequence may contain fewer or more amino acids, corresponding to a shortening or insertion of the FR or HVR of the variable domain.
  • a heavy chain variable domain may include a single amino acid inserted after residue 52 of H2 (according to residue 52a in Kabat) and residues inserted after FR residue 82 of the heavy chain (e.g., according to residues 82a, 82b, and 82c in Kabat).
  • the Kabat residue number of a given antibody can be determined by comparing the homologous regions of the antibody sequence with the "standard" Kabat numbered sequence.
  • residue numbers in the antibody heavy chain are EU index numbers, as in Kabat et al. above. "EU index in Kabat” refers to the residue numbers of the human IgG1 EU antibody.
  • online tools can be used to define the CDR region of an antibody. These online tools include, but are not limited to:
  • Hinge region refers to the portion of an immunoglobulin heavy chain polypeptide that connects the CH1 and CH2 domains. Hinge domains are structurally diverse, varying in sequence and length across immunoglobulin classes and subclasses. Heavy chains are interconnected via disulfide bonds within the hinge region. Based on crystallographic studies, the immunoglobulin hinge region can be further subdivided structurally and functionally into three regions: the upper hinge, the core, and the lower hinge. See Shin et al., Immunological Reviews 130:87 (1992).
  • the upper hinge comprises the amino acids from the carboxyl terminus of CH1 to the first motility-restricting residue in the hinge, typically the first cysteine residue that forms an interchain disulfide bond between the two heavy chains.
  • the length of the upper hinge region is related to the segmental flexibility of the antibody.
  • the core hinge region contains the interchain disulfide bonds.
  • the lower hinge region connects to the amino terminus of the CH2 domain and includes residues therein.
  • the protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan).
  • a compensating cavity of the same or similar size as the protrusion is created at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).
  • Protrusions and cavities can be prepared by altering the nucleic acids encoding the polypeptide, for example, through site-specific mutagenesis or peptide synthesis.
  • the clasp-modified subunit containing one of the two subunits of the Fc domain has an amino acid substitution of T366W, while the mortar-modified subunit contains amino acids substitutions of T366S, L368A, and Y407V.
  • the clasp-modified subunit of the Fc domain further contains an amino acid substitution of S354C, while the mortar-modified subunit of the Fc domain further contains an amino acid substitution of Y349C.
  • EC50 refers to the concentration of the active substance at which 50% of the maximum response is induced (i.e., half of the maximum response between the baseline and the baseline).
  • Teff refers to non-regulatory T cells and includes helper T cells and cytotoxic T cells.
  • Teff includes Tcon and CD8 + T cells.
  • Tcon and CD4 + Tcon are used interchangeably, and in some embodiments, Tcon is defined as CD45 + CD3 + CD4 + FOXp3- or CD45 + CD3 + CD4 + CD25- .
  • CD8 + T cells are defined as CD45 + CD3 + CD8 + .
  • Tregs regulatory T cells
  • Treg cells may be defined as CD4 + CD25 + T cells, CD4 + FoxP3 + T cells, or CD4 + CD25 + FoxP3 + T cells.
  • Th1-like Tregs refers to T-bet + IFN ⁇ + FoxP3 + Treg cells. Further description of Th1-like Tregs can be found in Reference 4, which is incorporated herein by reference in its entirety.
  • fragment Treg cells is defined as Treg cells that maintain Foxp3 expression but have lost their immunosuppressive function.
  • fragile Treg cells see reference 11, which is incorporated herein by reference in its entirety.
  • effector Treg cells refers to Treg cells that differentiate from nTregs (naive Treg cells) upon stimulation.
  • effector Treg cells exhibit CD45RA - FOXP3 hi CD25 hi CD4 + . Further description of eTreg cells can be found in Reference 7, which is incorporated herein by reference in its entirety.
  • this disclosure provides a Treg cell-targeting IL12 fusion protein.
  • the Treg cell-targeting IL12 fusion protein comprises one or more Treg cell-targeting modules and IL12 directly or indirectly linked, the one or more Treg cell-targeting modules specifically binding to Treg cell surface molecules, and the IL12 comprising one or more mutations with reduced activity compared to wild-type IL12.
  • a module should be understood as a unit or element with a certain function, and should not be limited to a specific structure.
  • a targeting module should be understood as a module that specifically binds to a target antigen.
  • the targeting module includes, but is not limited to, a ligand or fragment thereof that specifically binds to a target antigen, an antibody or fragment thereof.
  • the targeting module includes an antibody fragment that specifically binds to a target antigen, said antibody fragment including, but not limited to, Fab, Fab', F(ab')2, scFab, Fv, scFv, and VHH.
  • the targeting module includes a ligand fragment that specifically binds to a target antigen, said ligand fragment including a ligand extracellular domain.
  • Treg cell targeting modules are modules that specifically bind to molecules on the surface of Treg cells.
  • the Treg cell surface molecules include, but are not limited to, target antigens selected from the group consisting of: CTLA4, CCR8, CCR4, CCR10, CD25, GITR, OX-40, ICOS, and 4-1BB.
  • the KD value of the Treg cell targeting module binding to its target antigen is ⁇ 1E-7M, ⁇ 1E-8M, ⁇ 1E-9M, ⁇ 1E-10M, ⁇ 1E-11M, or ⁇ 1E-12M.
  • the KD value is in the range of 1E-10M to 1E-7M. More preferably, the KD value is in the range of 1E-9M to 1E-8M.
  • the CTLA4 targeting module includes, but is not limited to, its ligands CD80, CD86, or ligand fragments (e.g., the CD80 extracellular domain or the CD86 extracellular domain), CTLA4 antibody, or antibody fragment.
  • the CTLA4 binding module includes an anti-CTLA4 antibody or a fragment thereof.
  • the CTLA4 antibody or a fragment thereof is selected from Ipilimumab (Ipi), Tremelimumab, Tuvonralimab, Quavonlimab, Botensilimab, Lorigerlimab, Porustobart, Cadonilimab, Vudalimab, YH-001, JS-007, firastotug, Muzastotug, Erfonrilimab, or 9D9, or antibody fragments of the aforementioned antibodies, or antibodies or antibody fragments derived therefrom.
  • the CTLA4 antibody is selected from CTLA4 antibodies and fragments thereof disclosed in patents CN116535506A, CN116478289A, CN111153999B, CN106188297B, and WO2019152413A1 (the entire patent is incorporated herein by reference), or antibodies and fragments thereof derived therefrom.
  • the antibody fragment that specifically binds to CTLA-4 includes Fab, scFab, Fab', (Fab') 2 , Fv, scFv, or VHH.
  • the specific binding of CTLA4 to Fab, scFab, Fab', (Fab') 2 , Fv, and scFv includes VH and VL as shown below:
  • the VH includes HCDR1 as shown in SEQ ID NO:79, HCDR2 as shown in SEQ ID NO:80, and HCDR3 as shown in SEQ ID NO:81; the VL includes LCDR1 as shown in SEQ ID NO:85, LCDR2 as shown in SEQ ID NO:86, and LCDR3 as shown in SEQ ID NO:87; or,
  • the VH includes HCDR1 as shown in SEQ ID NO:82, HCDR2 as shown in SEQ ID NO:83, and HCDR3 as shown in SEQ ID NO:81;
  • the VL includes LCDR1 as shown in SEQ ID NO:88, LCDR2 as shown in SEQ ID NO:86, and LCDR3 as shown in SEQ ID NO:87; or,
  • the VH includes HCDR1 as shown in SEQ ID NO:82, HCDR2 as shown in SEQ ID NO:83, and HCDR3 as shown in SEQ ID NO:84;
  • the VL includes LCDR1 as shown in SEQ ID NO:88, LCDR2 as shown in SEQ ID NO:86, and LCDR3 as shown in SEQ ID NO:87;
  • the VH includes HCDR1 as shown in SEQ ID NO:89, HCDR2 as shown in SEQ ID NO:90 and HCDR3 as shown in SEQ ID NO:91;
  • the VL includes LCDR1 as shown in SEQ ID NO:92, LCDR2 as shown in SEQ ID NO:93 and LCDR3 as shown in SEQ ID NO:94;
  • the VH includes HCDR1 as shown in SEQ ID NO:95, HCDR2 as shown in SEQ ID NO:96 and HCDR3 as shown in SEQ ID NO:97; the VL includes LCDR1 as shown in SEQ ID NO:98, LCDR2 as shown in SEQ ID NO:99 and LCDR3 as shown in SEQ ID NO:100;
  • the VH includes HCDR1 as shown in SEQ ID NO:134, HCDR2 as shown in SEQ ID NO:135, and HCDR3 as shown in SEQ ID NO:136;
  • the VL includes LCDR1 as shown in SEQ ID NO:137, LCDR2 as shown in SEQ ID NO:138, and LCDR3 as shown in SEQ ID NO:139;
  • the VH includes HCDR1 as shown in SEQ ID NO:149, HCDR2 as shown in SEQ ID NO:150, and HCDR3 as shown in SEQ ID NO:151;
  • the VL includes LCDR1 as shown in SEQ ID NO:152, LCDR2 as shown in SEQ ID NO:153, and LCDR3 as shown in SEQ ID NO:154;
  • the VH includes HCDR1 as shown in SEQ ID NO:155, HCDR2 as shown in SEQ ID NO:156, and HCDR3 as shown in SEQ ID NO:157;
  • the VL includes LCDR1 as shown in SEQ ID NO:158, LCDR2 as shown in SEQ ID NO:159, and LCDR3 as shown in SEQ ID NO:160;
  • the VH includes HCDR1 as shown in SEQ ID NO:161, HCDR2 as shown in SEQ ID NO:162 and HCDR3 as shown in SEQ ID NO:163;
  • the VL includes LCDR1 as shown in SEQ ID NO:164, LCDR2 as shown in SEQ ID NO:165 and LCDR3 as shown in SEQ ID NO:166;
  • the VH includes HCDR1 as shown in SEQ ID NO:167, HCDR2 as shown in SEQ ID NO:168, and HCDR3 as shown in SEQ ID NO:169;
  • the VL includes LCDR1 as shown in SEQ ID NO:170, LCDR2 as shown in SEQ ID NO:171, and LCDR3 as shown in SEQ ID NO:172;
  • the VH includes HCDR1 as shown in SEQ ID NO:173, HCDR2 as shown in SEQ ID NO:174 and HCDR3 as shown in SEQ ID NO:175;
  • the VL includes LCDR1 as shown in SEQ ID NO:176, LCDR2 as shown in SEQ ID NO:177 and LCDR3 as shown in SEQ ID NO:178;
  • the VH includes HCDR1 as shown in SEQ ID NO:179, HCDR2 as shown in SEQ ID NO:180 and HCDR3 as shown in SEQ ID NO:181; the VL includes LCDR1 as shown in SEQ ID NO:182, LCDR2 as shown in SEQ ID NO:183 and LCDR3 as shown in SEQ ID NO:184;
  • the VH includes HCDR1 as shown in SEQ ID NO:188, HCDR2 as shown in SEQ ID NO:189, and HCDR3 as shown in SEQ ID NO:190;
  • the VL includes LCDR1 as shown in SEQ ID NO:191, LCDR2 as shown in SEQ ID NO:192, and LCDR3 as shown in SEQ ID NO:193;
  • the VH includes HCDR1 as shown in SEQ ID NO:194, HCDR2 as shown in SEQ ID NO:195 and HCDR3 as shown in SEQ ID NO:196; the VL includes LCDR1 as shown in SEQ ID NO:197, LCDR2 as shown in SEQ ID NO:198 and LCDR3 as shown in SEQ ID NO:199;
  • the VH includes HCDR1 as shown in SEQ ID NO:200, HCDR2 as shown in SEQ ID NO:201 and HCDR3 as shown in SEQ ID NO:202; the VL includes LCDR1 as shown in SEQ ID NO:203, LCDR2 as shown in SEQ ID NO:204 and LCDR3 as shown in SEQ ID NO:205;
  • the VH includes HCDR1 as shown in SEQ ID NO:206, HCDR2 as shown in SEQ ID NO:207, and HCDR3 as shown in SEQ ID NO:208; the VL includes LCDR1 as shown in SEQ ID NO:209, LCDR2 as shown in SEQ ID NO:210, and LCDR3 as shown in SEQ ID NO:211; or,
  • the VH includes HCDR1 as shown in SEQ ID NO:212, HCDR2 as shown in SEQ ID NO:213 and HCDR3 as shown in SEQ ID NO:214; the VL includes LCDR1 as shown in SEQ ID NO:215, LCDR2 as shown in SEQ ID NO:216 and LCDR3 as shown in SEQ ID NO:217.
  • the VHH that specifically binds to CTLA4 includes:
  • HCDR1 as shown in SEQ ID NO:101
  • HCDR2 as shown in SEQ ID NO:102
  • HCDR3 as shown in SEQ ID NO:103;
  • HCDR1 as shown in SEQ ID NO:104
  • HCDR2 as shown in SEQ ID NO:105
  • HCDR3 as shown in SEQ ID NO:106;
  • HCDR1 as shown in SEQ ID NO:107
  • HCDR2 as shown in SEQ ID NO:108
  • HCDR3 as shown in SEQ ID NO:109;
  • HCDR1 as shown in SEQ ID NO:110, HCDR2 as shown in SEQ ID NO:111 and HCDR3 as shown in SEQ ID NO:112;
  • HCDR1 as shown in SEQ ID NO:113
  • HCDR2 as shown in SEQ ID NO:114
  • HCDR3 as shown in SEQ ID NO:115;
  • HCDR1 as shown in SEQ ID NO:116
  • HCDR2 as shown in SEQ ID NO:117
  • HCDR3 as shown in SEQ ID NO:118;
  • HCDR1 as shown in SEQ ID NO:119
  • HCDR2 as shown in SEQ ID NO:120
  • HCDR3 as shown in SEQ ID NO:121;
  • HCDR1 as shown in SEQ ID NO:122, HCDR2 as shown in SEQ ID NO:123 and HCDR3 as shown in SEQ ID NO:124;
  • HCDR1 as shown in SEQ ID NO:125
  • HCDR2 as shown in SEQ ID NO:126
  • HCDR3 as shown in SEQ ID NO:127;
  • HCDR1 as shown in SEQ ID NO:128, HCDR2 as shown in SEQ ID NO:129 and HCDR3 as shown in SEQ ID NO:130;
  • HCDR1 as shown in SEQ ID NO:131
  • HCDR2 as shown in SEQ ID NO:132
  • HCDR3 as shown in SEQ ID NO:133;
  • HCDR1 as shown in SEQ ID NO:140
  • HCDR2 as shown in SEQ ID NO:141
  • HCDR3 as shown in SEQ ID NO:142;
  • HCDR1 as shown in SEQ ID NO:143
  • HCDR2 as shown in SEQ ID NO:144
  • HCDR3 as shown in SEQ ID NO:145;
  • HCDR1 as shown in SEQ ID NO:146
  • HCDR2 as shown in SEQ ID NO:147
  • HCDR3 as shown in SEQ ID NO:148
  • HCDR1 as shown in SEQ ID NO:185
  • HCDR2 as shown in SEQ ID NO:186
  • HCDR3 as shown in SEQ ID NO:187.
  • the specific binding of CTLA4 to Fab, scFab, Fab', (Fab') 2 , Fv, and scFv includes VH and VL as shown below:
  • VH comprises the sequence shown in SEQ ID NO:31
  • VL comprises the sequence shown in SEQ ID NO:34; or
  • the VH comprises the sequence shown in SEQ ID NO:32, and the VL comprises the sequence shown in SEQ ID NO:35; or,
  • the VH includes the sequence shown in SEQ ID NO:33, and the VL includes the sequence shown in SEQ ID NO:35;
  • VH includes the sequence shown in SEQ ID NO:36
  • VL includes the sequence shown in SEQ ID NO:37
  • VH includes the sequence shown in SEQ ID NO:38, and the VL includes the sequence shown in SEQ ID NO:39;
  • VH comprises the sequence shown in SEQ ID NO:51
  • VL comprises the sequence shown in SEQ ID NO:52;
  • VH comprises the sequence shown in SEQ ID NO:56
  • VL comprises the sequence shown in SEQ ID NO:57
  • VH comprises the sequence shown in SEQ ID NO:58
  • VL comprises the sequence shown in SEQ ID NO:59
  • VH comprises the sequence shown in SEQ ID NO:60
  • VL comprises the sequence shown in SEQ ID NO:61
  • VH comprises the sequence shown in SEQ ID NO:62
  • VL comprises the sequence shown in SEQ ID NO:63
  • VH comprises the sequence shown in SEQ ID NO:64
  • VL comprises the sequence shown in SEQ ID NO:65
  • VH comprises the sequence shown in SEQ ID NO:66
  • VL comprises the sequence shown in SEQ ID NO:67;
  • VH comprises the sequence shown in SEQ ID NO:69
  • VL comprises the sequence shown in SEQ ID NO:70
  • VH comprises the sequence shown in SEQ ID NO:71
  • VL comprises the sequence shown in SEQ ID NO:72;
  • VH comprises the sequence shown in SEQ ID NO:73
  • VL comprises the sequence shown in SEQ ID NO:74
  • VH comprises the sequence shown in SEQ ID NO:75
  • VL comprises the sequence shown in SEQ ID NO:76
  • VH comprises the sequence shown in SEQ ID NO:77
  • VL comprises the sequence shown in SEQ ID NO:78; or
  • (16) has an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99% or 100% identical to the VH and/or VL shown in any of groups (1)-(15).
  • the VHH that specifically binds to CTLA4 includes:
  • the CCR8 targeting module includes, but is not limited to, CCR8 ligands CCL1, CCL8, CCL16, CCL18 or ligand fragments (e.g., extracellular domains of CCL1, CCL8, CCL16, and CCL18), anti-CCR8 antibodies or fragments thereof.
  • the CCR8 targeting module includes an anti-CCR8 antibody or a fragment thereof.
  • the CCR8 antibody or fragment thereof is selected from BMS-986340, LM-108, S-531011, ABBV-514, AMG-355, BAY3375968, BGB-A3055, CM369, HBM-1022, PSB-114, SRF-114, 2MW4691, CHS-3318, FG-3175, G B2101, HFB-101110, IMD-2408, IPG0521, JTX-1811, PM-1024, PM-1092, REMD-355, BCG-005, GNUV-202, CTM-033, FG-3163, IPGA05, PM-1008, FPA 157, or antibody fragments of the aforementioned antibodies, or antibodies or antibody fragments derived therefrom.
  • the CCR8 antibody or its fragments are selected from the CCR8 antibodies and their fragments disclosed in the following patents, or their derived antibodies and their fragment
  • the antibody fragment that specifically binds to CCR8 includes Fab, scFab, Fab', (Fab') 2 , Fv, scFv, or VHH.
  • the specific binding of CCR8 to Fab, scFab, Fab', (Fab') 2 , Fv, and scFv includes VH and VL as shown below:
  • HCDR1 as shown in SEQ ID NO:265, HCDR2 as shown in SEQ ID NO:266, and HCDR3 as shown in SEQ ID NO:267;
  • the VL includes LCDR1 as shown in SEQ ID NO:268, LCDR2 as shown in SEQ ID NO:269, and LCDR3 as shown in SEQ ID NO:270; or,
  • the VH includes HCDR1 as shown in SEQ ID NO:271, HCDR2 as shown in SEQ ID NO:272 and HCDR3 as shown in SEQ ID NO:273; the VL includes LCDR1 as shown in SEQ ID NO:274, LCDR2 as shown in SEQ ID NO:275 and LCDR3 as shown in SEQ ID NO:276.
  • the VHH that specifically binds to CCR8 includes:
  • HCDR1 as shown in SEQ ID NO:259
  • HCDR2 as shown in SEQ ID NO:260
  • HCDR3 as shown in SEQ ID NO:261
  • HCDR1 as shown in SEQ ID NO:277
  • HCDR2 as shown in SEQ ID NO:278
  • HCDR3 as shown in SEQ ID NO:279.
  • the specific binding of CCR8 to Fab, scFab, Fab', (Fab') 2 , Fv, and scFv includes VH and VL as shown below:
  • VH includes the sequence shown in SEQ ID NO:254, and the VL includes the sequence shown in SEQ ID NO:255;
  • VH comprises the sequence shown in SEQ ID NO:256
  • VL comprises the sequence shown in SEQ ID NO:257; or
  • the VHH that specifically binds to CCR8 includes:
  • the CCR4 targeting module includes, but is not limited to, CCR4 ligands CCL2, CCL4, CCL5, CCL17, CCL22 or ligand fragments (e.g., extracellular domains of CCL2, CCL4, CCL5, CCL17, and CCL22), anti-CCR4 antibodies, or antibody fragments.
  • CCR4 ligands CCL2, CCL4, CCL5, CCL17, CCL22 or ligand fragments e.g., extracellular domains of CCL2, CCL4, CCL5, CCL17, and CCL22
  • anti-CCR4 antibodies e.g., anti-CCR4 antibodies, or antibody fragments.
  • the CCR4 targeting module includes an anti-CCR4 antibody or a fragment thereof.
  • the anti-CCR4 antibody or a fragment thereof is selected from Mogamulizumab, KM 2760, TQB-2619 or GNR-015, or antibody fragments of the aforementioned antibodies, or antibodies or antibody fragments derived therefrom.
  • the CCR10 targeting module includes, but is not limited to, CCR10 ligands CCL27, CCL28 or ligand fragments (e.g., CCL27 extracellular domain, CCL28 extracellular domain), anti-CCR10 antibodies or antibody fragments.
  • the CD25 targeting module includes an anti-CD25 antibody or a fragment thereof.
  • the anti-CD25 antibody or a fragment thereof is selected from dacliximab, basiliximab, daclizumab, inolimomab, camidanlumab, daclizumab, BA 1106, RO-7296682, vopikitug, 9MW3911, ALD2510, DO2, H11E11-2V1, H3F14V2, IBIO-101, INV-1013, INV 321, INV 322, TST 010, 33B3.1, RM-1995, or antibody fragments of the aforementioned antibodies, or antibodies or antibody fragments derived therefrom.
  • the GITR targeting module includes, but is not limited to, the GITR ligand GITRL or ligand fragments (e.g., the GITRL extracellular domain), anti-GITR antibodies or antibody fragments.
  • the GITR targeting module includes an anti-GITR antibody or a fragment thereof.
  • the anti-GITR antibody or a fragment thereof is selected from TRX-518, BMS-986156, ASP1951, MK-4166, REGN-6569, IBI-102, BCD-166, IBI37G5, SHR-1705, MFA 021, MMB-102, AMG228, Ragifilimab, LVGN4680, LY 3844583, MK1248, ATOR 1144, CK-302, DTA-1, GWN323, or antibody fragments of the aforementioned antibodies, or antibodies or antibody fragments derived therefrom.
  • the OX40 targeting module includes, but is not limited to, the OX40 ligand OX40L or ligand fragments (e.g., the OX40L extracellular domain), anti-OX40 antibodies or antibody fragments.
  • the OX40 targeting module includes an anti-OX40 antibody or a fragment thereof.
  • the anti-OX40 antibody or a fragment thereof is selected from Rocatinlimab, Revdofilimab, Ivuxolimab, Tavolixizumab, Vonderolizumab, ES-102, INCAGN-1949, MEDI-6469, YH-002, BAT-6026, GEN1055, IMG-007, BMS-986178, EMB09, FS-120, HFB-3010, HLX-51, I BI-101, SAR446422, CS-01, DF-004, IBI-327, ILB-2107, STAR 0310, MEDI-1109, MIL-96, GSK3174998, KN052, LVGN4506, SCTB03, Telazorlimab, APVO-603, ATOR-1015, ZL-1101, YH006, SHR-1806, KY-B-602 or antibody fragments
  • the ICOS targeting module includes, but is not limited to, the ICOS ligand ICOS L or ligand fragments (e.g., the ICOSL extracellular domain), anti- ICOS antibodies or antibody fragments.
  • the ICOS targeting module includes an anti- ICOS antibody or a fragment thereof.
  • the anti- ICOS antibody or a fragment thereof is selected from Acazicolcept, Izuralimab, Vopratelimab, Alomfilimab, BMS-986226, Feladilimab, KY1055, MEDI-570, or antibody fragments of the aforementioned antibodies, or antibodies or antibody fragments derived therefrom.
  • the 4-1BB targeting module includes, but is not limited to, the 4-1BB ligand 4-1BB L or ligand fragments (e.g., the 4-1BB extracellular domain), anti- 4-1BB antibodies or antibody fragments.
  • the 4-1BB targeting module includes an anti- 4-1BB antibody or a fragment thereof.
  • the anti -4-1BB antibody or a fragment thereof is selected from Acasunlimab, Cinrebafusp alfa, Enristomig, Urelumab, Utomilumab, Sytalizumab, evunzekibart, Exlinkibart, HLX-35, QL-301, QLF-31907, YH-004, ABL-105, ADG-106, EU-101, PE-0116, ADG206, AGEN-2373, BC3425, CTX-471, DF003, FTL-001, WBP-3425, ZG-033, or antibody fragments of the aforementioned antibodies, or antibodies or antibody fragments derived therefrom.
  • the Treg cell-targeting IL12 fusion protein described in this disclosure may further include a PD1-targeting module that specifically binds to PD1.
  • the PD1-targeting module described in this disclosure includes, but is not limited to, PD1 ligands PDL1, PDL2, or fragments thereof (e.g., the extracellular domain of PDL1, the extracellular domain of PDL2), and anti-PD1 antibodies or fragments thereof.
  • the PD1 targeting module includes its ligands PDL1 or PDL2 and fragments thereof.
  • the PD1 targeting module includes a PDL1 extracellular domain. More specifically, the PDL1 extracellular domain includes a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:27. In some specific embodiments, the PDL1 extracellular domain includes the sequence shown in SEQ ID NO:27.
  • the PD1 targeting module is the PDL2 extracellular domain. More specifically, the PDL2 extracellular domain includes a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:28. In some specific embodiments, the PDL2 extracellular domain includes the sequence shown in SEQ ID NO:28.
  • the PD1 targeting module is the PDL2 extracellular domain. More specifically, the PDL2 extracellular domain includes a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:29. In some specific embodiments, the PDL2 extracellular domain includes the sequence shown in SEQ ID NO:29.
  • the PD1 antibody or its fragment is selected from: Retifanlimab, Pucotenlimab, Cadonilimab, Serplulimab, Zimberelimab, Penpulimab, Dostarlimab-gxly, Prolgolimab, Tislelizumab, Camrelizumab, Sintilimab, Toripalimab, Cemiplimab-RWLC, Pembrolizumab, and Nivolumab.
  • Monoclonal antibodies (Nivolumab), Balstilimab, Finotonlimab, Iparomlimab, ivonescimab, SG-001, Cetrelimab, Ezabenlimab, Genolimzumab, Nofazinlimab, Rilvegostomig, Sasanlimab, Spartalizumab, Tebotelimab, Volrustomig, Budigalimab, ABBV-1882, BAT-1306, Danvilostomig, HX-009, IBI363, Izuralimab, Lomvastomig, Lor igerlimab, LZM-009, Peramprizumab, Peresolimab, Pidilizumab, Rosnilimab, SSGJ-707, Tombestomig, TQB-2868, Vudalimab, Acrixolimab, EMB-02, Fidasimtamab, IAP-0971, JS-201,
  • the PD1 antibody or fragment is selected from the PD1 antibody and fragment disclosed in patents CN111699200B and WO2023205754A (the patents are incorporated herein by reference in their entirety), or their derivative antibodies and fragments.
  • the antibody fragment that specifically binds to PD1 includes Fab, scFab, Fab', (Fab') 2 , Fv, scFv, or VHH.
  • the specific binding of PD1 to Fab, scFab, Fab', (Fab') 2 , Fv, and scFv includes VH and VL as shown below:
  • the VH includes HCDR1 as shown in SEQ ID NO:234, HCDR2 as shown in SEQ ID NO:235 and HCDR3 as shown in SEQ ID NO:236, and the VL includes LCDR1 as shown in SEQ ID NO:237, LCDR2 as shown in SEQ ID NO:238 and LCDR3 as shown in SEQ ID NO:239;
  • the VH includes HCDR1 as shown in SEQ ID NO:240, HCDR2 as shown in SEQ ID NO:241 and HCDR3 as shown in SEQ ID NO:242, and the VL includes LCDR1 as shown in SEQ ID NO:243, LCDR2 as shown in SEQ ID NO:244 and LCDR3 as shown in SEQ ID NO:245;
  • the VH includes HCDR1 as shown in SEQ ID NO:246, HCDR2 as shown in SEQ ID NO:247, and HCDR3 as shown in SEQ ID NO:248;
  • the VL includes LCDR1 as shown in SEQ ID NO:249, LCDR2 as shown in SEQ ID NO:250, and LCDR3 as shown in SEQ ID NO:251; or,
  • the VH includes HCDR1 as shown in SEQ ID NO:293, HCDR2 as shown in SEQ ID NO:294 and HCDR3 as shown in SEQ ID NO:295, and the VL includes LCDR1 as shown in SEQ ID NO:296, LCDR2 as shown in SEQ ID NO:297 and LCDR3 as shown in SEQ ID NO:298.
  • the VHH that specifically binds to PD1 includes:
  • HCDR1 as shown in SEQ ID NO:228, HCDR2 as shown in SEQ ID NO:229, and HCDR3 as shown in SEQ ID NO:230; or,
  • HCDR1 as shown in SEQ ID NO:231, HCDR2 as shown in SEQ ID NO:232 and HCDR3 as shown in SEQ ID NO:233.
  • the specific binding of PD1 to Fab, scFab, Fab', (Fab') 2 , Fv, and scFv includes VH and VL as shown below:
  • VH includes the sequence shown in SEQ ID NO:218, and the VL includes the sequence shown in SEQ ID NO:219;
  • VH includes the sequence shown in SEQ ID NO:222, and the VL includes the sequence shown in SEQ ID NO:223;
  • VH includes the sequence shown in SEQ ID NO:224, and the VL includes the sequence shown in SEQ ID NO:225;
  • VH comprises the sequence shown in SEQ ID NO:226, and the VL comprises the sequence shown in SEQ ID NO:227; or,
  • the VHH that specifically binds to PD1 includes:
  • IL12 comprises p35 and p40 subunits.
  • the p35 and p40 subunits can be connected in ways known in the art, including but not limited to: as in natural IL12, the p35 and p40 subunits are connected by forming interchain disulfide bonds; or, the p35 and p40 subunits are connected by linkers; or, the p35 and p40 subunits are connected to other functional modules respectively, for example, by dimerization of Fc to achieve spatial proximity and dimerization of p35 and p40.
  • the connection methods of the p35 subunit and p40 subunit disclosed in 2021236676A1, WO2022156773A1, CN108250303B, WO2022094046A1, WO2023114775A2, WO2023115033A2 and WO2022129313A1 are incorporated herein by reference in their entirety.
  • connection from the N end to the C end is as follows: p40 subunit - connector - p35 subunit.
  • connection from the N end to the C end is as follows: p35 subunit - connector - p40 subunit.
  • the p35 subunit is the ⁇ subunit shared by IL-12 and IL-35.
  • the p35 subunit dimerizes with the p40 subunit to form IL-12, while it dimerizes with Ebi3 to form IL-35.
  • the p35 subunits described in this disclosure are diverse in length, including but not limited to: full-length p35 subunits of unresected signal peptides, mature p35 subunits after signal peptide resection, isoforms, and fragments thereof.
  • the p35 subunits described in this disclosure include p35 from different species.
  • the p35 subunits are of vertebrate origin, including but not limited to primates (e.g., humans, non-human primates (such as monkeys)), rodents (e.g., mice, rats, rabbits), and domesticated pets or agricultural mammals (e.g., cats, dogs, horses, cattle, sheep).
  • primates e.g., humans, non-human primates (such as monkeys)
  • rodents e.g., mice, rats, rabbits
  • domesticated pets or agricultural mammals e.g., cats, dogs, horses, cattle, sheep.
  • Vertebrate-derived p35 subunits include, but are not limited to: UniProt: P29459, Homo sapiens (Human); UniProt: A0A2K5V4U3, Macaca fascicularis (Crab-eating macaque); UniProt: P43431, Mus musculus (Mouse); UniProt: Q9R103, Rattus norvegicus (Rat); UniProt: Q28267, Canis lupus familiaris (Dog); UniProt: O02743, Felis catus (Cat); UniProt: O02814, Capra hircus (Goat); UniProt: P54349, Bos taurus (Bovine); UniProt: Q9XSQ6, Equus cabalus (Horse).
  • the p35 subunit described in this disclosure can be wild-type.
  • sequences of typical wild-type human, monkey, mouse and dog p35 subunits are shown in SEQ ID NO:1-4, and sequence alignment results show that they are conserved (see Figure 1A).
  • the p35 subunit described in this disclosure can also be a mutant, which includes one or more mutations.
  • the p35 subunit mutants include human p35 subunit mutants and non-human animal (e.g., monkey, dog, mouse, etc.) p35 subunit mutants. Mutations can be obtained by methods known in the art.
  • Patents WO2020072821A2, WO2021067863A2, WO2022094046A1, WO2023004282A2, WO2023043978A2, WO2023279085A1, WO2022155263A2, US20230051304A1, and WO2023133540A1 disclose mutations occurring on the p35 subunit and methods for obtaining these mutations, and are incorporated herein by reference in their entirety.
  • the mutation positions of the human p35 subunit mutants and non-human p35 subunit mutants described in this disclosure are determined based on the mature human wild-type IL12 p35 subunit (SEQ ID NO:1). For example, when this disclosure mentions a mutation at position 60, it means that the human p35 subunit mutants and non-human p35 subunit mutants of this invention have a mutation at the position corresponding to position 60 of the mature human wild-type IL12 p35 subunit (SEQ ID NO:1), without requiring that the mutation occur at the 60th amino acid residue position of the mutant (thus excluding positional confusion that may be caused by species differences, signal peptides, truncation, etc.). The correspondence of positions can be determined by sequence alignment, for example, as shown in Figure 1A.
  • one or more of these mutations reduce the affinity of the p35 subunit or the corresponding IL12 for binding to its receptor.
  • the location of the one or more p35 subunit mutations is selected from the group consisting of the following (the mutation locations are located according to SEQ ID NO:1): Q20, N21, Q35, T36, L37, E38, F39, Y40, P41, T43, S44, E45, E46, I47, D48, H49, E50, K54, D55, T59, V60, E61, C63, L64, P65, E67, L68, N71, S73, C74, L75, N76, E79, T80, F82, N85, L89, F96, M97.
  • the one or more p35 subunit mutations are transformed from wild-type to A.
  • the one or more p35 subunit mutations are selected from the group consisting of (the mutation sites are located according to SEQ ID NO:1): N21D, Q35D, T36A, L37E, L37G, L37S, E38A, E38D, E38F, E38G, E38H, E38I, E38K, E38L, E38M, E38N, E38P, E38Q, E38R.
  • the p35 subunits described in this disclosure are human or non-human animal (e.g., monkey, dog, or mouse) p35 subunits; the p35 subunits may be wild-type or human p35 subunit mutants or non-human p35 subunit mutants containing the aforementioned mutations; optionally, the p35 subunits described in this invention have at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NO: 1-4.
  • IL12 p40 is a ⁇ subunit shared by IL-12 and IL-23.
  • the p40 subunit dimerizes with the p35 subunit to form the cytokine IL-12, while the p40 subunit dimerizes with the p19 subunit to form the cytokine IL-23.
  • the p40 subunits described in this disclosure are diverse in length, including but not limited to: full-length p40 subunits of unresected signal peptides, mature p40 subunits after signal peptide resection, isoforms, and fragments thereof.
  • the p40 subunits described in this disclosure include p40 from different species.
  • the p40 subunits described in this disclosure are of vertebrate origin, including but not limited to primates (e.g., humans, non-human primates (such as monkeys)), rodents (e.g., mice, rats, rabbits), domesticated pets, or agricultural mammals (e.g., cats, dogs, horses, cattle, sheep).
  • Vertebrate-derived p40 subunits include, but are not limited to: UniProt: P29460, Homo sapiens (Human); UniProt: A0A8J8Y9U3, Macaca fascicularis (Crab-eating macaque); UniProt: P43432, Mus musculus (Mouse); UniProt: E9PU71, Rattus norvegicus (Rat); UniProt: Q28268, Canis lupus familiaris (Dog); UniProt: O02744, Felis catus (Cat); UniProt: P68221, Capra hircus (Goat); UniProt: P46282, Bos taurus (Bovine); UniProt: Q9XSQ5 (Horse).
  • the p40 subunit described in this disclosure can be wild-type.
  • sequences of typical wild-type human, monkey, mouse and dog p40 subunits are shown in SEQ ID NO:5-8, and sequence alignment results show that they are conserved (see Figure 1B).
  • the p40 subunit described in this disclosure can also be a mutant, comprising one or more mutations.
  • the p40 subunit mutants include human p40 subunit mutants and non-human animal (e.g., monkey, dog, mouse, etc.) p40 subunit mutants. Mutations can be obtained according to methods known in the art.
  • the mutation positions of the human p40 subunit mutants and non-human p40 subunit mutants described in this disclosure are determined based on the mature human wild-type IL12 p40 subunit (SEQ ID NO:5).
  • a mutation at position 60 it means that the human p40 subunit mutants and non-human p40 subunit mutants of this invention have a mutation at the position corresponding to position 60 of the mature human wild-type IL12 p40 subunit (SEQ ID NO:5), without requiring the mutation to occur at the 60th amino acid residue position of the mutant (thus excluding positional confusion that may occur due to species differences, signal peptides, truncation, etc.).
  • the correspondence of positions can be determined by sequence alignment, for example, as shown in Figure 1B.
  • one or more of these mutations reduce the affinity of the p40 subunit or the corresponding IL12 for binding to its receptor.
  • the location of the one or more mutated p40 subunits is selected from the group consisting of the following (the location of the mutation is located according to SEQ ID NO:5): E3, K6, D7, E12, D14, W15, P17, D18, A19, P20, G21, E22, M23, D29, E32, E33, D34, L40, D41, Q42, S43, E45, L4 7.
  • the location of one or more p40 subunit mutations shown is selected from (the location of the mutation is located according to SEQ ID NO:5): (1) W15; (2) P17; (3) D18; (4) E59; (5) F60; (6) K84; (7) D87; (8) K195; (9) K197; (10) E59/F60; (11) W15/E59/F60; (12) E59/F60/K84; (13) E59/F60/K84/K197; (14) E59/F60/K84/K195; (15) E59/F60/K84/E86/D93; (17) P17/D18/E59/F60.
  • the one or more p40 subunit mutations are mutated from wild-type to A.
  • the one or more p40 subunit mutations are selected from the group consisting of (the location of the mutations is located according to SEQ ID NO:5): K6A, W15A, W15H, W15K, W15R, P17A, D18A, D18G, D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45K, E45Q, Q56E, K58H, K58W, E59A, E 59F, E59H, E59K, E59L, E59Q, E59S, E59D, E59G, E59R, F60A, F60D, F60E, F60K, F60R, F60V, D62N, D62 H, D62I, D62N, E73Q, K84A, K84N, K84Q, K84T, K84R, K84E, K84I, K84L, K84V, K84W, K
  • the one or more p40 subunit mutations shown are selected from: (1) W15A; (2) P17A; (3) D18A; (4) E59A; (5) F60A; (6) F60E; (7) F60D; (8) K84A; (9) D87A; (10) K195A; (11) K197A; (12) E59A/F60A; (13) W15A/E59A/F60A; (14) E59A/F60A/K84A; (15) E59A/F60A/K84A/K197A; (16) E59A/F60A/K8 4A/K195A; (17)E59A/F60A/K84A/E86A/D93A; (18)E59F/F60A; (19)E59K/F60A; (20)E59L/F60A; (21)E59H/F60A; (22)E59S/F 60A; (23)E59A/F60A/K84N; (24)E59A/F60A/
  • the p40 subunits described in this disclosure are human or non-human animal (e.g., monkey, dog, or mouse) p40 subunits; the p40 subunits may be wild-type or human p40 subunit mutants or non-human p40 subunit mutants containing the aforementioned mutations; optionally, the p40 subunits described in this invention have at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NO: 5-8.
  • the targeted IL12 fusion protein described in this disclosure further includes a first Fc unit and a second Fc unit, wherein the first Fc unit and the second Fc unit are dimerized.
  • Fc unit or "Fc region” is used to define a carboxyl-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. This term includes native sequence Fc regions and variant Fc regions.
  • the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain.
  • antibodies produced by host cells can undergo post-translational cleavage of one or more (particularly one or two) amino acids from the carboxyl terminus of the heavy chain.
  • antibodies produced by host cells by expressing a specific nucleic acid molecule encoding the full-length heavy chain can comprise the full-length heavy chain, or the antibody can comprise a cleaved variant of the full-length heavy chain.
  • amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described by Kabat et al. (Sequences of Proteins of Immunological Interest, 5th edition, U.S. Department of Health and Human Services, National Institutes of Health, Bethesda, MD, 1991).
  • the Fc unit or Fc region described in this disclosure includes a hinge region.
  • the hinge area is a complete hinge area or a segment thereof.
  • the hinge region of the human IgG1 antibody corresponds to amino acid positions 216-230, or 226-230, according to the EU numbering specified in Kabat.
  • the core hinge region of human IgG1 contains the sequence Cys-Pro-Pro-Cys, which, upon dimerization via disulfide bonds, produces a cyclic octapeptide believed to act as a pivot, thus conferring flexibility. Conformational changes permitted by the structure and flexibility of the immunoglobulin hinge region polypeptide sequence may affect the effector function of the antibody's Fc moiety. Hinge regions of other IgG subclasses can be determined by aligning them with the cysteine residues of the hinge region of the IgG1 subclass sequence.
  • the Fc unit or Fc region contains a mutation design that is conducive to the formation of heterodimers.
  • Representative examples include the "Knob-into-Hole” form proposed by Cater et al.; the formation of Fc-containing heterodimers by Amgen engineers using electrostatic steering (US20100286374 A1); the formation of heterodimers (SEEDbodies) through IgG/Ig chain exchange proposed by Jonathan H.
  • the Fc unit or Fc region contains mutations that reduce or eliminate effector functions (e.g., ADCC or ADCP functions).
  • Reduced or eliminated effector function can be achieved by mutations in the Fc region of the antibody, and has been described in the art, including but not limited to LALA and N297A (Strohl, W., 2009, Curr. Opin. Biotechnol. vol. 20(6): 685-691); and D265A (Baudino et al., 2008, J. Immunol, 181: 6664-69; Strohl, W., ibid.); and DAPA (D265A and P329A) (Shields RL., J Biol Chem. 2001, 276(9): 6591-604; US2015/0320880).
  • LALA mutants containing L234A and L235A mutations in the IgG1 Fc amino acid sequence include LALA mutants containing L234A and L235A mutations in the IgG1 Fc amino acid sequence, DAPA (D265A, P329A) (see, for example, US 6,737,056), N297A, DANAPA (D265A, N297A, and P329A) and/or LALADANAPS (L234A, L235A, D265A, N297A, and P331S).
  • DAPA D265A, P329A
  • N297A DANAPA
  • LALADANAPS L234A, L235A, D265A, N297A, and P331S
  • non-limiting exemplary embodiments that reduce or eliminate effector function include LALGA (L234A, L235A, and G237A), LALASKPA (L234A, L235A, S267K, and P329A), DAPASK (D265A, P329A, and S267K), GADAPA (G237A, D265A, and P329A), GADAPASK (G237A, D265A, P329A, and S267K), LALAPG (L234A, L235A, and P329G), and LALAPA (L234A, L235A, and P329A), wherein the amino acid residues are numbered according to the EU numbering system.
  • the first Fc unit includes a Knob mutation
  • the second Fc unit includes a Hole mutation
  • the first Fc unit includes a Hole mutation
  • the second Fc unit includes a Knob mutation
  • the first Fc unit includes the L234A/L235A mutation
  • the second Fc unit includes the L234A/L235A mutation
  • the first Fc unit includes the sequence shown in SEQ ID NO:299
  • the second Fc unit includes the sequence shown in SEQ ID NO:301.
  • the first Fc unit includes the sequence shown in SEQ ID NO:299
  • the second Fc unit includes the sequence shown in SEQ ID NO:302.
  • the first Fc unit includes the sequence shown in SEQ ID NO:300
  • the second Fc unit includes the sequence shown in SEQ ID NO:301.
  • the first Fc unit includes the sequence shown in SEQ ID NO:300
  • the second Fc unit includes the sequence shown in SEQ ID NO:302.
  • the first Fc unit includes the sequence shown in SEQ ID NO:300
  • the second Fc unit includes the sequence shown in SEQ ID NO:303.
  • linker is synonymous with “linking peptide” or “peptide adapter,” referring to a connecting unit used to connect two structural domains.
  • Linkers typically possess a degree of flexibility, and their use helps the structural domain maintain its original spatial conformation and function.
  • the linker is enriched with glycine and/or serine.
  • the linker may be selected from (G4S)x, where x is an integer of 1 or greater than 1.
  • the linker comprises one or more amino acids, typically about 1-30, 2-24, or 3-15 amino acids.
  • Exemplary connectors include, but are not limited to: GGGGSGGGSGGG, GGPGGGGSGGGSGGGGGSG, SGGGGS, GGSG, SGGSG, PGGGSG, SGGGGSGGGGS, GGGGS, GGSGGS, GGGSG, SGGGSG, TGGSG.
  • the IL2 fusion protein targeting Treg cells described in this disclosure can be in any reasonable conformation.
  • the conformations shown in the embodiments of this disclosure should not be construed as limiting this disclosure.
  • the Treg cell targeting module of the Treg cell targeting IL12 fusion protein described in this disclosure can be linked to IL12 in any way possible in the art, including but not limited to direct linking via peptide bonds, linking via linkers, or non-covalent linking between peptide chains.
  • Figures 6 and 7 further illustrate the structure shown in Figure 5A, describing the possible configurations of the Treg cell-targeted IL12 fusion protein for the CTLA and CCR8 targets, respectively.
  • Figures 6 and 7 further illustrate the structure shown in Figure 5A, describing the possible configurations of the Treg cell-targeted IL12 fusion protein for the CTLA and CCR8 targets, respectively.
  • these are not exhaustive and should not be construed as limiting the present disclosure.
  • the Treg cell-targeting IL12 fusion protein further includes a first Fc unit and a second Fc unit with a hinge region, wherein the first Fc unit and the second unit dimerize to form a dimer, and optionally, the IL12 is located between the Treg cell-targeting module or the PD1-targeting module and the hinge region of the first Fc unit or the second Fc unit.
  • the Treg cell-targeting IL12 fusion protein comprises a dimer composed of A-[L1] n1 -B-[L2] n2 -C and A'-[L3] n3 -C', wherein:
  • a and A' represent the first targeting module and the second targeting module, respectively, wherein at least one of A and A' specifically binds to Treg cell surface molecules;
  • B represents IL12 composed of p40 subunit-[L4] n4 -p35 subunit or p35 subunit-[L4] n4 -p40 subunit, wherein at least one of the p35 subunit and p40 subunit includes one or more weakened mutations that reduce the affinity of the IL12 for binding to its receptor compared to the wild type;
  • L1, L2, L3, and L4 represent connectors, and n1, n2, n3, and n4 are selected from 0 or 1; and,
  • the Treg cell-targeting IL12 fusion protein comprises a dimer composed of A-[L1] n1 -B-[L2] n2 -C and A'-[L3] n3 -B'-[L4] n4 -C', wherein:
  • a and A' represent the first targeting module and the second targeting module, respectively, wherein at least one of A and A' specifically binds to Treg cell surface molecules;
  • B represents the p40 subunit and B' represents the p35 subunit, or B represents the p35 subunit and B' represents the p40 subunit; at least one of the p35 and p40 subunits includes one or more weakened mutations that reduce the affinity of the IL12 for binding to its receptor compared to the wild type;
  • L1, L2, L3, and L4 represent connectors, and n1, n2, n3, and n4 are selected from 0 or 1; and,
  • the first targeting module and/or the second targeting module includes a ligand, antibody, or fragment of the ligand or antibody that specifically binds to the target antigen; optionally, the first targeting module and/or the second targeting module includes an extracellular domain (LECD), Fab, scFab, Fab', (Fab') 2 , Fv, scFv, or VHH of a ligand that specifically binds to the target antigen.
  • LCD extracellular domain
  • both the first targeting module and the second targeting module include a Fab.
  • the Treg cell-targeting IL12 fusion protein includes a first peptide, a second peptide, a third peptide, and a fourth peptide.
  • the first peptide includes VH1-(H1-CH1)-[L1] n1 -B-[L2] n2 -C
  • the second peptide includes VH2-(H2-CH1)-[L3] n3 -C'
  • the third peptide includes VL1-(L1-CL)
  • the fourth peptide includes VL2-(L2-CL).
  • the first peptide and the second peptide form a dimer through C and C'.
  • the first targeting module includes Fab
  • the second targeting module includes VHH or LECD.
  • the Treg cell-targeting IL12 fusion protein comprises a first peptide, a second peptide, and a third peptide.
  • the first peptide comprises VH1-(H1-CH1)-[L1] n1 -B-[L2] n2 -C
  • the second peptide comprises VHH2-[L3] n3 -C' or LECD2-[L3] n3 -C'
  • the third peptide comprises VL1-(L1-CL).
  • the first peptide and the second peptide form a dimer through C and C'.
  • the first peptide and the third peptide form the first targeting module through VH1-(H1-CH1) and VL1-(L1-CL).
  • the second peptide's VHH2 or LECD2 forms the second targeting module.
  • the first targeting module includes VHH or LECD
  • the second targeting module includes Fab
  • the Treg cell-targeting IL12 fusion protein includes a first peptide, a second peptide, and a third peptide.
  • the first peptide includes VHH1-[L1] n1 -B-[L2] n2 -C or LECD1-[L1] n1 -B-[L2] n2 -C
  • the second peptide includes VH2-(H2-CH1)-[L3] n3 -C'
  • the third peptide includes VL2-(L2-CL).
  • the first peptide and the second peptide form a dimer through C and C'.
  • VHH1 or LECD1 of the first peptide forms the first targeting module.
  • the second peptide and the third peptide form the second targeting module through VH1-(H2-CH1) and VL2-(L2-CL).
  • the first targeting module includes VHH or LECD
  • the second targeting module includes VHH or LECD
  • the Treg cell-targeted IL12 fusion protein includes a first peptide and a second peptide, the first peptide including VHH1-[L1] n1 -B-[L2] n2 -C or LECD1-[L1] n1 -B-[L2] n2 -C, and the second peptide including VHH2-[L3] n3 -C' or LECD2-[L3] n3 -C'; the first peptide and the second peptide form a dimer through C and C'; VHH1 or LECD1 of the first peptide forms the first targeting module; and VHH2 or LECD2 of the second peptide forms the second targeting module.
  • VH2 and H2-CH1 represent VH (heavy chain variable region) and CH1 (heavy chain first constant region structural domain) corresponding to the second targeting module, respectively
  • VL2 and L2-CL represent VL (light chain variable region) and CL (light chain constant region structural domain) corresponding to the second targeting module, respectively.
  • VHH1 represents the VHH (heavy chain variable region of heavy chain antibody) corresponding to the first targeting module.
  • LECD1 represents the LECD (ligand extracellular domain) corresponding to the first targeting module.
  • LECD2 represents the LECD (ligand extracellular domain) corresponding to the second targeting module.
  • the first targeting module specifically binds to Treg cell surface molecules, and the second targeting module specifically binds to PD1.
  • the first targeting module specifically binds to CTLA4 or CCR8, and the second targeting module specifically binds to PD1.
  • the first targeting module specifically binds to PD1, and the second targeting module specifically binds to Treg cell surface molecules.
  • the first targeting module specifically binds to PD1
  • the second targeting module specifically binds to CTLA4 or CCR8.
  • a first peptide, a second peptide, a third peptide and a fourth peptide wherein the first peptide comprises the sequence shown in SEQ ID NO:307 or 310, the second peptide comprises the sequence shown in SEQ ID NO:308, the third peptide comprises the sequence shown in SEQ ID NO:309, and the fourth peptide comprises the sequence shown in SEQ ID NO:309.
  • a first peptide, a second peptide and a third peptide wherein the first peptide comprises the sequence shown in SEQ ID NO:307 or 310, the second peptide comprises the sequence shown in SEQ ID NO:311, and the third peptide comprises the sequence shown in SEQ ID NO:309;
  • a first peptide, a second peptide and a third peptide wherein the first peptide comprises the sequence shown in SEQ ID NO:312 or 313, the second peptide comprises the sequence shown in SEQ ID NO:308, and the third peptide comprises the sequence shown in SEQ ID NO:309.
  • this disclosure provides an isolated nucleic acid molecule comprising a nucleotide fragment encoding one or more polypeptides encoding a Treg cell-targeting IL12 fusion protein.
  • nucleic acid molecule and “polynucleotide” are used interchangeably in this disclosure and refer to both RNA and DNA molecules, including nucleic acid molecules comprising: cDNA, genomic DNA, synthetic DNA, and DNA or RNA molecules containing nucleic acid analogs. Nucleic acid molecules can be double-stranded or single-stranded (e.g., sense or antisense strands). Nucleic acid molecules can contain unconventional or modified nucleotides. As used herein, the terms “polynucleotide sequence” and “nucleic acid sequence” refer interchangeably to the sequence of a polynucleotide molecule.
  • the nucleotide sequence is incorporated into an expression cassette or expression vector.
  • an expression cassette typically contains a construct of genetic material containing a coding sequence and sufficient regulatory information to guide the coding sequence to be properly transcribed and/or translated in recipient cells in vivo and/or in vitro.
  • the expression cassette can be inserted into a vector and/or individual for targeting a desired host cell.
  • the expression cassette of this disclosure contains a coding sequence for a Treg cell-targeting IL12 fusion protein as previously described, the coding sequence being operatively linked to expression control elements such as promoters, and any one or a combination of other nucleic acid sequences optionally influencing the transcription or translation of the coding sequence.
  • the nucleotide sequence is incorporated into an expression vector.
  • vector generally refers to a recombinant polynucleotide construct designed for transfer between host cells and for transformation purposes, such as introducing heterologous DNA into a host cell. Therefore, in some embodiments, the vector may be a replicon, such as a plasmid, bacteriophage, or granule, into which another DNA segment can be inserted to induce replication of the inserted segment.
  • the expression vector may be an integration vector.
  • the expression vector may be a viral vector.
  • viral vector is widely used to refer to nucleic acid molecules (e.g., transfer plasmids) that include viral-derived nucleic acid elements that typically facilitate the transfer or integration of nucleic acid molecules into the genome of a cell, or to viral particles that mediate nucleic acid transfer. Viral particles will typically contain a variety of viral components and sometimes also contain host cell components in addition to one or more nucleic acids.
  • the term viral vector may refer to a virus or viral particle capable of transferring nucleic acid into a cell, or to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and/or functional genetic elements primarily derived from viruses.
  • the viral vector is a bacculorival vector, a retroviral vector, or a lentiviral vector.
  • retroviral vector refers to a viral vector or plasmid containing structural and functional genetic elements or portions thereof primarily derived from retroviruses.
  • lentiviral vector refers to a viral vector or plasmid containing structural and functional genetic elements or portions thereof (including LTRs) primarily derived from lentiviruses (a genus of retroviruses).
  • this disclosure also provides vectors, plasmids, or viruses containing one or more nucleic acid molecules encoding the Treg cell-targeting IL12 fusion protein of this disclosure.
  • the nucleic acid molecules may be contained within a vector capable of guiding the expression of the nucleic acid molecules in cells, for example, cells that have been transformed/transduced with the vector.
  • Suitable vectors for eukaryotic and prokaryotic cells are known in the art and are commercially available or readily prepared by a skilled craftsman.
  • DNA vectors can be introduced into eukaryotic cells via conventional transformation or transfection techniques.
  • Appropriate methods for transforming or transfecting cells can be found in Sambrook et al. (2012, ibid.) and other standard molecular biology laboratory manuals, such as calcium phosphate transfection, DEAE-glucan-mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scratch loading, ballistic introduction, nuclear perforation, hydrodynamic shock, and infection.
  • Viral vectors that may be used in this disclosure include, for example, baculovirus vectors, retrovirus vectors, adenovirus vectors and adeno-associated virus vectors, lentivirus vectors, herpesviruses, simian virus 40 (SV40) and bovine papillomavirus vectors (see, for example, Gluzman (ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.).
  • nucleic acid molecules provided in this disclosure may contain naturally occurring sequences, or sequences that differ from naturally occurring sequences but encode the same polypeptide (e.g., antibody) due to the degeneracy of the genetic code.
  • These nucleic acid molecules may consist of combinations or modifications of nucleotides within RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA such as DNA produced through phosphoramide-based synthesis) or these types of nucleic acids.
  • the nucleic acid molecules may be double-stranded or single-stranded (e.g., sense strand or antisense strand).
  • the expression cassette or expression vector may be one or more.
  • the expression cassette or expression vector is a single expression cassette or expression vector that encodes all peptide chains of the targeted IL12 fusion protein, with the peptide chains linked by self-cleaving peptides (e.g., P2A, T2A) or open reading frames (ORFs) encoding the peptide chains linked by IRES.
  • the expression cassette or expression vector is multiple, with each expression cassette or expression vector independently expressing each peptide chain of the targeted IL12 fusion protein.
  • the expression cassette or expression vector is multiple, including an expression cassette or expression vector expressing a single peptide chain of the targeted IL12 fusion protein, and an expression cassette or expression vector including an expression cassette or expression vector expressing multiple peptide chains of the targeted IL12 fusion protein, with the multiple peptide chains linked by self-cleaving peptides (e.g., P2A, T2A) or open reading frames (ORFs) encoding the multiple peptide chains linked by IRES.
  • self-cleaving peptides e.g., P2A, T2A
  • ORFs open reading frames
  • This disclosure also provides a cell comprising the aforementioned isolated nucleic acid molecule, the nucleic acid molecule encoding a nucleotide fragment sequence of one or more polypeptides of the aforementioned Treg cell-targeting IL12 fusion protein.
  • the terms "cell,” “cell culture,” and “cell line” refer not only to the specific subject cell, cell culture, or cell line, but also to the offspring or potential offspring of such cells, cell cultures, or cell lines, regardless of the number of transfers or passages in culture. It should be understood that not all offspring are identical to the parent cells. This is because certain modifications may occur in offspring due to mutations (e.g., intentional or unintentional mutations) or environmental influences (e.g., methylation or other epigenetic modifications), making the offspring potentially different from the parent cells, but still included within the scope of the terminology used herein, provided that the offspring retain the same function as the original cell, cell culture, or cell line.
  • nucleic acid molecules of this disclosure into cells can be performed by methods known to those skilled in the art, such as viral infection, transfection, conjugation, protoplast fusion, liposome transfection, electroporation, nuclear transfection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, and nanoparticle-mediated nucleic acid delivery.
  • PKI polyethyleneimine
  • the nucleic acid molecules can be delivered using viral or non-viral delivery vectors known in the art.
  • the nucleic acid molecules can be stably integrated into the genome of the recombinant cell, or can be replicated as episomes, or exist in the recombinant cell as microcircular expression vectors for transient expression.
  • nucleic acid molecules are maintained and replicated as episome units in the recombinant host cell.
  • nucleic acid molecules are stably integrated into the genome of the recombinant cell. Stable integration can be achieved using classical random genomic recombination techniques or using more precise techniques such as RNA-guided CRISPR/Cas9 genome editing, DNA-guided endonuclease genome editing using NgAgo (Argonaute), or TALEN genome editing (transcription activator-like effector nucleases).
  • nucleic acid molecules are present in the recombinant cell as microcircular expression vectors for transient expression.
  • nucleic acid molecules can be encapsulated in viral capsids or lipid nanoparticles, or delivered via viral or non-viral delivery means and methods known in the art, such as electroporation.
  • nucleic acid can be introduced into cells via viral transduction.
  • baculoviruses or adeno-associated viruses can be engineered to deliver nucleic acids to target cells via viral transduction.
  • AAV serotypes have been described, and all known serotypes can infect cells from a wide variety of tissue types. AAVs are capable of transducing a broad range of species and tissues in vivo without signs of toxicity, and they elicit relatively mild innate and adaptive immune responses.
  • host cells can be genetically engineered (e.g., transduced, transformed, or transfected) using vector constructs such as those described in this application.
  • vector constructs can be, for example, viral vectors or vectors for homologous recombination (comprising nucleic acid sequences homologous to a portion of the host cell genome), or expression vectors for expressing a target polypeptide.
  • the host cell can be an untransformed cell or a cell already transfected with at least one nucleic acid molecule.
  • the cells are prokaryotic or eukaryotic cells, such as bacteria (Escherichia coli), fungi (yeast), insect cells, or mammalian cells (e.g., CHO cell line, HEK293 cell line).
  • bacteria Esscherichia coli
  • fungi fungi
  • insect cells or mammalian cells (e.g., CHO cell line, HEK293 cell line).
  • mammalian cells e.g., CHO cell line, HEK293 cell line.
  • this disclosure provides cell cultures comprising at least one recombinant cell as described herein and a culture medium.
  • the culture medium can be any suitable medium used for culturing the cells described herein.
  • Techniques for transforming the wide variety of cells and species mentioned above are known in the art and described in the technical and scientific literature. Therefore, cell cultures comprising at least one recombinant cell as disclosed herein are also within the scope of this disclosure. Methods and systems suitable for producing and maintaining cell cultures are known in the art.
  • compositions can be incorporated into compositions (including pharmaceutical compositions).
  • Such compositions typically comprise one or more of the Treg cell-targeting IL12 fusion proteins, nucleic acids, recombinant cells, and/or cell cultures provided and described herein, as well as pharmaceutically acceptable excipients (e.g., carriers).
  • the pharmaceutical compositions of this disclosure are formulated for treating, preventing, improving diseases (such as cancer), or for reducing or delaying the onset of diseases.
  • compositions comprising: (a) the Treg cell-targeting IL12 fusion protein, recombinant nucleic acid, recombinant cells, or cell cultures disclosed herein; and (b) a pharmaceutically acceptable carrier.
  • “Pharmaceutically acceptable carrier” includes any and all physiologically compatible solvents, dispersion media, coatings, antimicrobial agents, and antifungal agents, etc.
  • this disclosure relates to various methods for producing the Treg cell-targeting IL12 fusion protein of this disclosure, said methods comprising: (a) providing one or more recombinant cells disclosed herein; and culturing said one or more recombinant cells in a culture medium such that said cells produce the Treg cell-targeting IL12 fusion protein encoded by a recombinant nucleic acid molecule. Therefore, the Treg cell-targeting IL12 fusion protein produced by the methods disclosed herein is also within the scope of this disclosure.
  • the method further includes isolating and/or purifying the resulting Treg cell-targeting IL12 fusion protein. In some embodiments, the method further includes structurally modifying (e.g., PEGylation) the resulting Treg cell-targeting IL12 fusion protein to increase its half-life.
  • structurally modifying e.g., PEGylation
  • this disclosure relates to a treatment method for tumors, the method comprising administering to a subject an effective amount of the Treg cell-targeting IL12 fusion protein provided in this disclosure, the IL12 fusion protein inducing intratumoral Treg cells to secrete IFN ⁇ thereby attenuating or relieving their immunosuppressive effects.
  • treatment refers to a clinical intervention that attempts to alter the natural course of the disease in the treated individual, and may be performed for prevention or during the course of clinicopathological progression.
  • desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and mitigating or improving prognosis.
  • the antibodies disclosed herein are used to delay the development of disease or slow its progression.
  • application means the delivery of a bioactive composition or preparation via routes of administration including, but not limited to, intravenous, intra-arterial, intramuscular, intraperitoneal, subcutaneous, intramuscular, and local application, or combinations thereof. This includes, but is not limited to, application by a medical professional and self-application.
  • subject includes animals, such as humans and non-human animals.
  • subject is a patient under the care of a physician.
  • the subject can be a person, patient, or individual who has, is at risk of having, or is suspected of having one or more symptoms of the intended disease (e.g., cancer) and/or disease.
  • the subject can also be an individual who is diagnosed at or after diagnosis as being at risk of having the intended condition.
  • non-human animal includes all vertebrates, such as mammals, for example, rodents such as mice, non-human primates, and other mammals such as sheep, dogs, cattle, chickens, and non-mammals such as amphibians, reptiles, etc.
  • effective amount means an amount of composition sufficient to accomplish the stated purpose (e.g., to achieve the effect of administration, to treat a disease, to reduce a signaling pathway, or to alleviate one or more symptoms of a disease or health condition) relative to the absence of the composition.
  • effective amount are amounts sufficient to promote the treatment, prevention, or alleviation of one or more symptoms of a disease, which may also be referred to as “therapeutic effective amount.”
  • “Amelioration” of symptoms means a reduction in the severity or frequency of one or more symptoms, or the elimination of one or more symptoms.
  • compositions including the “therapeutic effective amount” will depend on the purpose of treatment and can be determined by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (Vols. 1–3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, edited by Gennaro, Lippincott, Williams & Wilkins).
  • the Treg cells induce intratumoral Treg cells to transform into Th1-like Treg cells or fragile Treg cells.
  • the Treg cells targeting the IL12 fusion protein also induce CD8 + T cells or CD4 + Tcon cells in the tumor microenvironment to: (1) secrete IFN ⁇ ; (2) upregulate the expression level of T-bet; and/or upregulate the expression of CD25.
  • the Treg cells are administered to the IL12 fusion protein via systemic administration, such as intravenous or subcutaneous injection.
  • the other therapies may be selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, toxin therapy, and surgery.
  • the Treg cells targeting the IL12 fusion protein are administered before, after, or simultaneously with the other therapies.
  • the subject is a patient resistant to immune checkpoint inhibitors, such as a patient resistant to PD1 antibodies.
  • the tumor is a solid tumor; preferably, the solid tumor is selected from the group consisting of: melanoma, colorectal cancer, prostate cancer, lung cancer, liver cancer, pancreatic cancer, esophageal cancer, gastric cancer, kidney cancer, breast cancer, ovarian cancer, uterine cancer, bladder cancer, head and neck cancer, and glioma.
  • this disclosure relates to the use of the aforementioned Treg cell-targeting IL12 fusion protein, the aforementioned isolated nucleic acid molecule, the aforementioned vector, the aforementioned recombinant cell, the product obtained by the aforementioned production method, or the aforementioned pharmaceutical composition in the preparation of a tumor therapeutic drug, wherein the Treg cell-targeting IL12 fusion protein induces intratumoral Treg cells to secrete IFN ⁇ , thereby weakening or relieving the immunosuppressive effect of the Treg cells.
  • the Treg cells induce intratumoral Treg cells to transform into Th1-like Treg cells or fragile Treg cells.
  • the Treg cells targeting the IL12 fusion protein also induce CD8 + T cells or CD4 + Tcon cells in the tumor microenvironment to: (1) secrete IFN ⁇ ; (2) upregulate the expression level of T-bet; and/or upregulate the expression of CD25.
  • the Treg-targeting IL12 fusion protein is administered systemically, such as via intravenous or subcutaneous injection.
  • the Treg-targeting IL12 fusion protein is administered as a monotherapy or in combination with other therapies.
  • the other therapies are selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, toxin therapy, and surgery.
  • the Treg cell-targeting IL12 fusion protein is administered before, after, or simultaneously with the other therapies.
  • the tumor is a solid tumor; preferably, the solid tumor is selected from the group consisting of: melanoma, colorectal cancer, prostate cancer, lung cancer, liver cancer, pancreatic cancer, esophageal cancer, gastric cancer, kidney cancer, breast cancer, ovarian cancer, uterine cancer, bladder cancer, head and neck cancer, and glioma.
  • Example 1 Differential expression of IL12R ⁇ 1, PD1, CTLA4, and CCR8 in tumor-infiltrating lymphocyte subsets or splenic lymphocyte subsets
  • Spleen and tumor tissue were harvested from MC38 colorectal cancer tumor model mice (C57BL/6) with tumors measuring 200-400 mm3 or 300-500 mm3.
  • Splenic lymphocytes and tumor-infiltrating lymphocytes (TILs) were isolated. Immunophenotyping of the isolated lymphocytes was performed using flow cytometry-activated cell sorting (FACS), and the expression levels of IL12R ⁇ 1, PD1, CTLA-4, and/or CCR8 on different lymphocyte subsets were detected. Positive and negative gating was determined using fluorescence minus one (FMO) staining. Results are detailed in Figures 2-4.
  • the proportion of IL12R ⁇ 1 + cells in the splenic lymphocyte subset is extremely low; in contrast, the proportion of IL12R ⁇ 1 + cells in the tumor-infiltrating lymphocyte subset is generally significantly higher than that in the corresponding splenic immune cell subset.
  • the proportion of PD1 + cells in the tumor-infiltrating T cell subset was significantly higher than that in the corresponding splenic T cell subset.
  • the proportion of CTLA4 + cells and CCR8 + cells in tumor-infiltrating Treg cells is much higher than that of spleen-derived Treg cells or other tumor-infiltrating lymphocytes besides Treg cells, demonstrating the specificity of the target antigens CTLA4 and CCR8 in tumor-infiltrating Treg cells.
  • PD1 + , CTLA4 + , and CCR8 + cells were compared among tumor-infiltrating T lymphocyte subsets. As shown in Figure 4, PD1 was highly expressed in all intratumoral T cell subsets, while CTLA4 and CCR8 were only highly expressed in intratumoral Treg cell subsets, indicating that PD1 is specific to tumor-infiltrating T lymphocytes, while CTLA4 and CCR8 are specific to tumor-infiltrating Treg cells.
  • Treg cell-targeting IL12 fusion protein which may have the structure shown in Figure 5A or Figure 5B.
  • Figure 5A [ABD1]-[connector]-[p40 subunit]-[connector]-[p35 subunit]-[connector]-[Fc1 with hinge region]; and [ABD2]-[connector]-[Fc2 with hinge region], and form a heterodimer through Fc1 and Fc2.
  • Figure 5B [ABD1]-[connector]-[p35 subunit]-[connector]-[Fc1 with hinge region]; and [ABD2]-[connector]-[p40 subunit]-[Fc2 with hinge region], forming a heterodimer through Fc1 and Fc2.
  • ABS Antigen-binding domain
  • ABD stands for Antigen Binding Domain, which can be selected from a ligand or a fragment thereof that specifically binds to the target antigen (e.g., the extracellular domain of the ligand), an antibody or a fragment thereof (e.g., Fab, scFab, Fab', (Fab') 2 , Fv, scFv, or VHH).
  • target antigen e.g., the extracellular domain of the ligand
  • an antibody or a fragment thereof e.g., Fab, scFab, Fab', (Fab') 2 , Fv, scFv, or VHH.
  • the IL12 fusion protein can be single-target specific, with ABD1 and ABD2 binding to the same target antigen.
  • This target antigen can be the Treg cell-specific target antigen shown in Example 1: CTLA4 or CCR8. It can also be other similar target antigens known in the art, such as CCR4, CCR10, CD25, GITR, OX-40, ICOS, and 4-1BB.
  • the IL12 fusion protein can also be dual-target specific, with ABD1 and ABD2 binding to different target antigens.
  • Both targets can be selected from Treg cell-specific target antigens, such as CTLA4, CCR8, CCR4, CCR10, CD25, GITR, OX-40, ICOS, and 4-1BB; or, one target can be selected from the aforementioned Treg cell-specific target antigens, and the other target can be selected from tumor-infiltrating T lymphocyte-specific target antigens, such as PD1.
  • the dual targets can be selected from the following groups: (1) CTLA4 and CCR8, (2) CTLA4 and PD1, (3) CCR8 and PD1.
  • CTLA4/PD1 dual-target or CCR8/PD1 dual-target Treg cell-targeted IL12 fusion proteins they are mainly divided into two categories: (1) contralateral structures , that is, IL12 is located on the opposite side of the PD1 binding domain, including Figures 6B, 6E, 6H, 6J, 7B, 7E, 7H, 7J, and 7M; (2) ipsilateral structures , that is, IL12 is located on the same side of the PD1 binding domain, including Figures 6C, 6F, 6I, 6K, 7C, 7F, 7I, 7K, and 7L.
  • contralateral structures that is, IL12 is located on the opposite side of the PD1 binding domain, including Figures 6B, 6E, 6H, 6J, 7B, 7E, 7H, 7J, and 7M
  • ipsilateral structures that is, IL12 is located on the same side of the PD1 binding domain, including Figures 6C, 6F, 6I, 6K,
  • the IL12 in this IL12 fusion protein may undergo a weakening mutation.
  • This weakening mutation results in a decreased ability of IL12 to bind to and activate its receptor compared to wild-type amino acid residues.
  • This weakening mutation may occur on the p35 subunit and/or the p40 subunit. As an example, the weakening mutation occurs on the p40 subunit.
  • the weakening mutation may be selected from the following group: (1) F60A, (2) F60E, (3) F60D, (4) E59A/F60A, (5) E59A/F60A/K84A, (6) E59A/F60A/K84A/K195A, (7) W15A/E59A/F60A, (8) W15A/E59A/F60A/K84A, (9) W15A/E59A/F60A/K84A/K195A.
  • Example 3 A general method for preparing the target protein (vector construction, protein expression, and purification)
  • the target protein e.g., the Treg cell-targeting IL12 fusion protein shown in Example 2
  • a corresponding expression vector was constructed, and the target protein was expressed, isolated, and purified:
  • the DNA fragment corresponding to the target protein was chemically synthesized, and this DNA fragment was loaded into the expression vector PKS001 (purchased from Kang Sheng Biotechnology, catalog number: A13201) using molecular cloning techniques well-known to those skilled in the art, such as enzyme digestion, ligation, and transformation.
  • the plasmid was extracted and sequenced. After sequencing verification, the plasmid expressing the target protein was electroporated into CHO K1Q host cells (purchased from Kang Sheng Biotechnology, catalog number: A13101) in suspension culture.
  • Example 4 A general method for detecting and testing molecular IL12 activity using target cells or non-target cells.
  • IL12 reporter cell line HEK293/IL12 i.e., non-target cells.
  • the STAT4-induced luciferase reporter gene was introduced into HEK293 cells using a lentiviral vector, and a selection agent was added to obtain a stable monoclonal cell line expressing the target gene. Subsequently, the IL12R ⁇ 2-IL12R ⁇ 1 and STAT4 genes were sequentially introduced into this monoclonal cell line, and a stable cell line was obtained, which is the IL12 reporter cell line (non-target cells), named HEK293/IL12R.
  • This IL12 reporter cell line possesses the complete downstream IL12 signaling pathway. IL12 activates the IL12 receptor on the surface of this reporter cell and its downstream JAK2 and STAT4, inducing luciferase expression. The activity of IL12 is reflected by the detectable luciferase activity.
  • the hCTLA4, hPD1, or hCTLA4/hPD1 genes were introduced into the cells via lentiviral vectors, followed by pressure selection to obtain monoclonal cell lines (i.e., target cells) stably expressing hCTLA4, hPD1, or hCTLA4/hPD1.
  • the introduced CTLA4 gene was modified to omit the segment encoding the intracellular signal transduction region.
  • monoclonal cell lines i.e., target cells
  • construct monoclonal cell lines i.e., target cells
  • mCLTA4, mPD1, or mCTLA4/mPD1 mCLTA4, mPD1, or mCTLA4/mPD1.
  • monoclonal cell lines i.e., target cells
  • hCCR8, hPD1, or hCCR8/hPD1 were constructed.
  • test proteins were designed, and corresponding expression vectors were constructed.
  • the test proteins were transiently or stably expressed in CHO K1Q cells and secreted into the culture medium.
  • the culture medium supernatant was collected for later use, or the supernatant was purified for later use.
  • test samples or PBS controls were incubated with the aforementioned target cells or non-target cells (150,000 cells) at 37°C and 5% CO2 for 24 hours. After incubation, the supernatant was mixed with an equal volume of luciferase substrate and incubated at room temperature for 3-5 minutes. The chemiluminescence signal was then detected using a microplate reader. The detection value of the test supernatant was used as the signal value, and the detection value of the PBS control was used as the background value.
  • the signal-to-noise ratio (signal value/background value) reflects the activity of luciferase and IL12.
  • Example 5 The activity of Treg cells targeting the IL12 fusion protein is highly dependent on the expression of the target antigen.
  • the IL12 fusion protein was designed as shown in Table 1.
  • the activation effect of the test protein on IL12 receptors on target and non-target cells was detected according to Example 4.
  • the results are detailed in Figures 8A-8C and Tables 2-3.
  • hIL12(4A)-Fc//Fc exhibits a 4A mutation in the IL 12p40 subunit.
  • the EC50 of hIL12(WT)-Fc//Fc ranges from 0.09 to 0.031 nM, while that of hIL12(4A)-Fc//Fc ranges from 1.16 to 5.28 nM. The difference is significant, indicating that the mutation greatly reduces IL12 activity.
  • the restoration of IL12 activity depends on the target antigen.
  • CTLA4-targeting IL12 fusion proteins generally exhibit target antigen-dependent activation, and this target antigen-dependent activation is even more pronounced in the "contralateral structure" and 4A mutation.
  • CTLA4-targeting IL12 fusion proteins were constructed (see Tables 4-5 for details). The activation ability of these IL12-targeting fusion proteins on the surface of target cells or non-target cells was tested according to Example 4. Specific test results are shown in Figures 9-10.
  • CTLA4-targeting IL12 fusion proteins generally exhibit good targeting and activation effects.
  • Table 3-4 shows the diversity and differentiation of targeted IL12 fusion proteins in terms of target combinations, formats, IL12 mutations, and CTLA4 antibodies.
  • the response curves of all targeted IL12 fusion proteins on CTLA4 + or CTLA4 + PD1 + target cells showed a significant left shift relative to the non-target cell HEK293/IL12R, indicating a recovery of IL12 activity and demonstrating good target antigen dependence.
  • CTLA4 targeting IL12 fusion proteins generally exhibits good targeting and activation effects, rather than being limited to specific target combinations, formats, mutation types, or the CDR region of CTLA4 antibodies.
  • the "contralateral structure” exhibits superior target antigen-dependent activity compared to the "ipsilateral structure” ( Figure 6B v. Figure 6C).
  • Isolateral structures Fig. 6C: hPDL2-hIL12(2A)//Ipi, hPDL2-hIL12(4A)//Ipi.
  • the contralateral structure is very similar to the ipsilateral structure.
  • the leftward shift of the IL12 activity curve from non-target cells HEK293/IL12R to CTLA4 + or CTLA4 + /PD1 + target cells
  • the leftward shift of the contralateral structure is significantly greater than that of the ipsilateral structure. This phenomenon is particularly pronounced in the 2A mutation. Therefore, it is evident that the contralateral structure possesses stronger target antigen-dependent activity compared to the ipsilateral structure.
  • the 4A mutation has stronger target antigen-dependent activity than the 2A mutation.
  • the targeting IL12 fusion protein with 4A mutations exhibits significantly lower activation of the IL12 receptor on non-target cells compared to its counterparts with 2A mutations (E59A/F60A).
  • its IL12 activity can be restored to levels comparable to those of its counterparts with 2A mutations.
  • This demonstrates a surprisingly high target antigen dependence in the activity of targeting the IL12 fusion protein (4A mutation) with Treg cells, suggesting the potential for a good balance between antitumor efficacy and safety.
  • Example 7 CCR8-targeting IL12 fusion proteins generally possess target antigen-dependent activity, and the "contralateral structure" exhibits stronger target antigen-dependent activity than the "ipsilateral structure.”
  • a CCR8-targeting IL12 fusion protein was constructed (see Table 6 for details), and its activation effect on IL12R on the surface of target cells and non-target cells was tested according to Example 4. The results are shown in Figures 11A-11E.
  • CCR8-targeting IL12 fusion proteins generally possess target antigen-dependent activity.
  • Table 6 shows that Treg cell-targeting IL12 fusion proteins exhibit diversity and differentiation in target combinations, formats, and PD1 binding domains. However, as shown in Figures 11A-11E, all Treg cell-targeting IL12 fusion proteins in Table 6 show IL12 activity dependent on either CTLA4 + or CTLA4 + PD1 + target cells. This indicates that CCR8-based targeting Treg cell-targeting IL12 fusion proteins generally possess target antigen-dependent activity.
  • the "contralateral structure” has stronger target antigen-dependent activity than the "ipsilateral structure”.
  • Isolateral structure (Fig. 7I): mPDL2-mhIL12(4A)//17-64.
  • the contralateral structure compared with the ipsilateral structure, the contralateral structure exhibited lower non-specific activity on non-target cells, but stronger activity on target cells CTLA4 + and CTLA4 + PD1 + cells (see Emax). This indicates that IL12 on the contralateral structure has stronger target antigen-dependent activity than that on the ipsilateral structure.
  • Example 8 A General Method for Constructing Mouse Tumor Models and Efficacy Evaluation
  • mouse tumor model and efficacy evaluation involved in the embodiments of this disclosure are carried out in accordance with the following methods:
  • Tumor cells were subcutaneously inoculated on the right back of mice. Once the tumor reached a certain size, mice of suitable weight were selected, randomly assigned to groups, and administered the test drug or PBS control according to the prescribed dosing regimen. Mouse weight and tumor volume were measured and recorded three times a week. If necessary, the release of IFN ⁇ in peripheral blood of mice (IFN ⁇ Kit, purchased from Xinbosheng, catalog number: EMC101g.96.10) was measured 48 hours after drug administration. At the end of the experiment, mice were euthanized, tumors were removed, and weighed to calculate the relative tumor inhibition value (TGI). TGI calculation method:
  • RTV Relative Tumor Volume
  • Vt the tumor volume at the end of one experimental cycle (usually around 30 days).
  • V0 the tumor volume at the start of the experiment.
  • the experimental group and the control group each have one RTV.
  • TGI [1 - RTV(experimental group) / RTV(control group)] * 100%.
  • Example 9 Evaluation of the antitumor effect of CTLA4-targeting IL12 fusion protein in a humanized mouse tumor model.
  • Example 8 the growth-inhibiting effect of the targeted IL12 fusion protein shown in Tables 7-10 on melanoma or prostate tumors was evaluated in CTLA4 humanized mouse models or tumor models established by CTLA4/PD1 humanized mice. The specific results are shown in Figures 12-15.
  • Targeted IL12 fusion proteins based on Ipi or its variants have growth-inhibiting effects on both melanoma and prostate tumors.
  • the CTLA4 antibody is an Ipi variant (Ipi.105 or Ipi.106), or the target combination is a single CTLA4 target
  • targeted Treg cells also exhibit significant antitumor activity targeting the IL12 fusion protein (see Figures 12B, 13B, 14B, and 15A).
  • Targeted Treg cells with the 2A mutation showed stronger antitumor activity targeting the IL12 fusion protein than those with the 3A or 4A mutations ( Figure 14A), possibly because the 2A mutation has the lowest attenuation level, retaining the strongest IL12 activity.
  • Ipi variants see reference 12.
  • CTLA4-targeting IL12 fusion protein The antitumor activity of CTLA4-targeting IL12 fusion protein is independent of specific CTLA4 antibodies or CDR regions.
  • Treg cells when targeted Treg cells target the IL12 fusion protein using CTLA4 antibodies other than Ipi or its variants, they also exhibit significant antitumor activity. This demonstrates that CTLA4-based targeted Treg cells targeting the IL12 fusion protein generally possess antitumor activity, independent of specific CTLA4 antibodies or CDR regions.
  • the targeted Treg cell-targeting IL12 fusion protein shown in Table 7-10 did not cause a significant decrease in mouse body weight after administration, and no obvious toxic side effects were observed (specific data not shown).
  • the levels of IFN ⁇ in the peripheral blood of mice were also measured after administration of some test molecules, and the results showed that they were generally at low levels ( ⁇ 500 pg/mL, specific data not shown).
  • Example 10 Ipi-hIL12(4A)//hPDL2 and its alternative molecules have similar target antigen-dependent activities.
  • Ipilimumab (Ipi) binds only to human CTLA4 and not to mouse CTLA4.
  • 9D9 antibody is commonly used in the art as an alternative molecule for Ipi to evaluate its performance in wild-type mouse models (Reference 13).
  • 9D9 can also serve as an alternative molecule for Ipi in the Treg cell-targeting IL12 fusion protein disclosed in this disclosure, the Treg cell-targeting IL12 fusion protein shown in Table 11 was constructed, and its activation effects on target and non-target cells were examined. The results are detailed in Figures 16A-16B.
  • 9D9-mIL12(4A)//mPDL2 possessed similar target antigen-dependent activity to Ipi-hIL12(4A)//hPDL2. Therefore, 9D9-mIL12(4A)//mPDL2 could serve as an alternative molecule to Ipi-hIL12(4A)//hPDL2 for efficacy evaluation or mechanism of action exploration in wild-type mouse tumor models.
  • Example 11 Evaluation of the antitumor effect of CTLA4-targeting Treg cells targeting IL12 fusion protein in a wild-type mouse tumor model.
  • Example 9 in a tumor model established in wild-type mice with a healthy immune system, the growth-inhibiting effect of Treg cells targeting IL12 fusion protein as shown in Tables 12-15 on colorectal cancer, breast cancer, and melanoma was evaluated. The specific test results are shown in Figures 17-22A.
  • CTLA4-targeted IL12 fusion protein (9D9) exhibits growth-inhibiting effects against colorectal cancer, breast cancer, and melanoma, and also interacts with PD-1 inhibitors. Compared with monoclonal or mIpi antibodies, it showed good performance in tumor models sensitive to PD1 antibodies (MC38) and tumor models resistant to PD1 antibodies (B16, EMT6). Anti-tumor effect
  • the 9D9-based Treg cell-targeting IL12 fusion protein exhibits significant tumor growth inhibition against various tumors, including colorectal cancer, breast cancer, and melanoma.
  • CTLA4-targeting IL12 fusion protein The antitumor activity of CTLA4-targeting IL12 fusion protein is independent of specific CTLA4 antibodies or CDR regions.
  • Treg cells targeting the IL12 fusion protein used various CTLA4 antibodies other than 9D9, all of which exhibited varying degrees of antitumor activity. This demonstrates that the antitumor effect of CTLA4-targeted IL12 fusion proteins is universal and not limited to specific CTLA4 antibodies or CDR regions.
  • the "contralateral structure” showed superior antitumor effects compared to the "ipsilateral structure”.
  • Tables 14-15 show that Treg cells with "contralateral structures" ( Figures 6B and 6E) targeting IL12 fusion proteins exhibit significantly better tumor growth inhibition than their corresponding "ipsilateral structures” ( Figures 6C and 6F), demonstrating a stronger anti-tumor effect.
  • the Treg cell-targeting IL12 fusion protein shown in Tables 12-15 did not cause a significant decrease in mouse body weight after administration, and no obvious toxic side effects were observed (specific data not shown).
  • the serum IFN ⁇ levels in mice were also measured after administration of some test molecules, and the results showed that they were generally at low levels ( ⁇ 500 pg/mL, specific data not shown).
  • Example 12 Evaluation of the antitumor effect of CCR8 targeting IL12 fusion protein in wild-type mice
  • Example 9 the anti-tumor effect of Treg cells targeting the IL12 fusion protein, as shown in Table 16, was evaluated in a tumor model established in wild-type mice with intact immune systems. Specific test results are shown in Figures 22B-22C.
  • mice in the test group shown in Table 16 did not experience significant weight loss or other toxic side effects after administration (related data not shown).
  • mice of suitable weight were randomly divided into groups of three. On days 5, 8, 11, and 14 after grouping, mice were injected with either PBS control or different doses of the test molecules listed in Table 17. Serum IFN ⁇ levels were measured at 24, 48, and 72 hours after the first administration. Mouse weight was measured and recorded twice weekly. Animals were euthanized at the end of the experiment. Results are shown in Figures 23A-23B and 24A-24B.
  • mIL12(WT)-Fc exhibited a significant dose-dependent IFN ⁇ release compared to 9D9-mIL12(4A)//mPDL2.
  • Figures 24A-24B show the weight monitoring results of mice during the four administrations. Compared to 9D9-mIL12(4A)//mPDL2, mIL12(WT)-Fc showed a significant dose-dependent weight loss. More importantly, mortality was observed in mIL12(WT)-Fc starting at 0.1 mpk, while no mortality was observed in 9D9-mIL12(4A)//mPDL2 at a dosage of 15 mpk.
  • 9D9-mIL12(4A)//mPDL2 exhibits high safety and significantly reduced toxicity compared to the mIL12(WT)-Fc fusion protein. Mice can tolerate doses up to 15 mpk without significant toxicity.
  • Example 14 Monkey toxicology assay of Treg cells targeting IL12 fusion protein
  • the toxicology of the test molecules shown in Table 18 was evaluated in cynomolgus monkeys: Four cynomolgus monkeys were administered the test molecules shown in Table 18 once weekly for four consecutive weeks.
  • the dosage of Ipi-hIL12(2A)//hPDL2 and hPDL2-hIL12(4A)//Ipi was 5 mpk, while the dosage of Ipi-hIL12(4A)//hPDL2 was 5 mpk or 15 mpk.
  • the release of the peripheral blood cytokine IFN ⁇ and changes in blood immunology were detected in the cynomolgus monkeys.
  • the results showed that Ipi-hIL12(2A)//hPDL2 was highly safe in cynomolgus monkeys, and the monkeys tolerated a dose of 15 mpk without significant toxicity. Specific results are as follows:
  • Figure 25 shows the results of serum IFN ⁇ detection in cynomolgus monkeys after the first administration.
  • the peripheral blood IFN ⁇ in the Ipi-hIL12(4A)//hPDL2 group was lower than that in the Ipi-hIL12(2A)//hPDL2 group and the hPDL2-hIL12(4A)//Ipi group.
  • Hematological parameters including ALT, AST, CK, TBIL, WBC, NEU, MONO, LYM, and EOS, were measured on the day of administration and on days 2, 6, 9, 13, 16, 20, 23, and 29. Results showed that after administration of the assay molecules shown in Table 18, the hematological parameters of the cynomolgus monkeys were all within the normal range, or exceeded the normal range at a few time points but quickly returned to the normal range (this may be due to multiple blood samplings) (related data not shown).
  • the no-observed adverse reaction dose level (NOAEL) for Ipi-hIL12(2A)//hPDL2 and hPDL2-hIL12(4A)//Ipi was 5 mg/kg
  • the NOAEL for Ipi-hIL12(4A)//hPDL2 was 15 mg/kg.
  • Examples 15-16 Analysis of the mechanism of action of Treg cells targeting IL12 fusion protein
  • Example 15 Treg cells selectively activate TDLNs and tumor-derived T lymphocytes by targeting the IL12 fusion protein, exhibiting a different mechanism of action than PD1 antibodies and CTLA4 antibodies.
  • mice Five B16 melanoma cells (2.5E each) were subcutaneously injected into the right back of wild-type C57BL/6 mice. On day 5 post-inoculation, mouse weight was measured, and mice of suitable weight were randomly assigned to groups of 20. On days 5 and 8 post-inoculation, mice were intraperitoneally injected with either PBS (control) or the test molecules shown in Table 19 (dose: 5 mpk). Tumor volume and body weight were measured and recorded three times weekly. On day 10 post-inoculation, when the tumor volume reached approximately 300-500 mm3 , the mice were euthanized, and the spleen, draining lymph nodes (TDLN), and tumor tissue were harvested.
  • TDLN draining lymph nodes
  • T cells were classified using FACS: CD4 Tcon cells (CD45 + CD3 + CD4 + FOXp3 - ); CD4 Treg cells (CD45 + CD3 + CD4 + FOXp3 + ); and CD8 T cells (CD45 + CD3 + CD8 + ). IFN ⁇ secretion was detected (after 4 hours of stimulation with PMA/ionomycin/BFA before staining). The expression of transcription factor T-bet and the cytokine IL2 receptor CD25 was also detected (staining of T-bet and CD25 did not require further stimulation). Specific results are shown in Figures 26-34, with a summary of key information for the 9D9-mIL12(4A)//mPDL2 group shown in Figure 35.
  • Treg cells targeting the IL12 fusion protein 9D9-mIL12(4A)//mPDL2 exhibited different activation levels in mouse spleen, TDLN, and tumor-derived T cell subsets: its activation of splenic T cells was weak or non-existent, while it had a strong activation effect on tumor-derived T cells (TILs), and its activation of TDLN-derived T cells, which have "crosstalk" with tumor immune cells, was intermediate between that of splenic T cells and TILs.
  • TILs tumor-derived T cells
  • Treg cells targeting the IL12 fusion protein stimulate intratumoral Tregs and TDLN Tregs to express T-bet and secrete IFN ⁇ , converting them into fragile Tregs and weakening or relieving the immunosuppressive effect of Tregs. This may be the core mechanism of action of Treg cells targeting the IL12 fusion protein.
  • Treg cells targeting the IL12 fusion protein also induce intratumoral CD8 T cells and Tcon cells to express T-bet and secrete IFN ⁇ , enhancing the killing effect of T cells.
  • Treg cells targeting the IL12 fusion protein also upregulate the expression of CD25 in CD8 T cells and Tcon cells, enhancing their utilization of the cytokine IL2 and promoting the proliferation of CD8 T cells and Tcon cells.
  • 9D9-mIL12(4A)//mPDL2 also stimulated a significant increase in TDLN size, a phenomenon not observed in the mPD1 antibody group or the mIpi group.
  • Treg cells selectively induce activated Treg cells to secrete IFN ⁇ by targeting the IL12 fusion protein.
  • CD3+ T cells and Treg cells were isolated from human PBMCs using the EasySep TM Human CD4+CD127lowCD25+Regulatory T Cell Isolation Kit.
  • the isolated Treg cells were labeled with CSFE.
  • CD3+ T cells and CSFE-labeled Treg cells were mixed at a 95%:5% ratio.
  • the mixed cells were seeded into 96-well plates at a rate of 2.5E6 cells/well and co-incubated with Ipi-hIL12(4A)//hPDL2 as shown in Table 20 under either stimulated (1 ⁇ g/ml coated anti-CD3 antibody, 1 ⁇ g/ml soluble anti-CD28 antibody) or non-stimulated conditions.
  • the non-stimulated condition simulated the peripheral immune system, while the stimulated condition simulated the tumor microenvironment (inflammatory). After 72 hours of incubation, the concentration of IFN ⁇ in the supernatant was detected by ELISA. T cell subsets were classified using FACS (Tregs: (CSFE + CD3 + CD4 + FOXp3 + ); CD8: (CSFE - CD3 + CD8 + ) ; Tcon: (CSFE - CD3 + CD4 + FOXp3 - )) and intracellular IFN ⁇ expression in T cell subsets was detected. The specific detection results are shown in Figures 36-37.
  • Ipi-hIL12(4A)//hPDL2 does not induce the release of IFN ⁇ from unactivated PBMCs, demonstrating high safety.
  • the ELISA results showed that unstimulated PBMC cells did not release IFN ⁇ even at a concentration of 100 nM, indicating that Ipi-hIL12(4A)//hPDL2 had a very low activation level of immune cells in systemic peripheral tissues and exhibited high safety; while stimulated PBMC cells released a large amount of IFN ⁇ , indicating that the environment of activated PBMCs was highly inflammatory (TME).
  • Ipi-hIL12(4A)//hPDL2 induced Treg cells to release IFN ⁇ .
  • Ipi-hIL12(4A)//hPDL2 significantly induced the release of IFN ⁇ from activated Treg cells, and showed a slight but measurable dose-dependent increase in the release of IFN ⁇ from activated CD8 and Tcon cells.
  • Human p40 subunit (E59A/F60A)-linker-human p35 subunit (SEQ ID NO:21)
  • Human p40 subunit (E59A/F60A/K84A)-linker-human p35 subunit (SEQ ID NO:22)
  • Human p40 subunit (E59A/F60A/K84A/K195A)-linker-human p35 subunit (SEQ ID NO:23)
  • Wild-type human PDL2 extracellular domains lack the C-terminal "HPT” (SEQ ID NO:29)
  • Ipi.105 or Ipi.106VL anti-CTLA4 (SEQ ID NO:35)

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Abstract

La présente invention concerne une protéine de fusion d'IL12 ciblant les cellules Treg, une molécule d'acide nucléique, un vecteur, une cellule et une composition pharmaceutique correspondants, un procédé de production correspondant, un procédé correspondant pour traiter des tumeurs, et l'utilisation pharmaceutique. Par rapport à un système immunitaire systémique, la protéine de fusion d'IL12 ciblant les cellules Treg de la présente invention active sélectivement le microenvironnement tumoral, en particulier induisant la sécrétion d'IFNg par des cellules Treg intratumorales, ce qui permet de réduire ou d'éliminer son effet immunosuppresseur.
PCT/CN2025/100595 2024-06-12 2025-06-12 Protéine de fusion d'il12 ciblant des cellules treg et son procédé de préparation, et procédé thérapeutique l'utilisant Pending WO2025256578A1 (fr)

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CN112638938A (zh) * 2018-04-25 2021-04-09 免疫靶向有限公司 白介素12融合蛋白及其组合物和治疗方法
US20210355185A1 (en) * 2019-10-03 2021-11-18 Xencor, Inc. Targeted il-12 heterodimeric fc-fusion proteins

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112638938A (zh) * 2018-04-25 2021-04-09 免疫靶向有限公司 白介素12融合蛋白及其组合物和治疗方法
US20210355185A1 (en) * 2019-10-03 2021-11-18 Xencor, Inc. Targeted il-12 heterodimeric fc-fusion proteins

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