Treg-cell-targeting il12 fusion protein and preparation method therefor, and therapeutic method using same

Abstract

The present disclosure relates to a Treg-cell-targeting IL12 fusion protein, a corresponding nucleic acid molecule, vector, cell and pharmaceutical composition, a corresponding production method, a corresponding method for treating tumors, and the pharmaceutical use. Relative to a systemic immune system, the Treg-cell-targeting IL12 fusion protein of the present disclosure selectively activates the tumor microenvironment, especially inducing the secretion of IFNg by intratumoral Treg cells, thereby reducing or eliminating the immunosuppressive effect thereof.

Description

Treg cell-targeting IL12 fusion proteins and methods of making and treating therewith TECHNICAL FIELD

[0001] The present disclosure relates to IL12 fusion proteins, in particular, to Treg cell-targeting IL12 fusion proteins and methods of making and treating therewith. BACKGROUND

[0002] Regulatory T cells (Tregs) are a subset of CD4 + T cells that exhibit immunosuppressive effects, mainly on CD4 + CD25 + or CD4 + CD25 + FoxP3 + (References 1-2), which play a key role in maintaining homeostasis and preventing autoimmune diseases. Treg cells induce immunosuppression using a variety of mechanisms, including secretion of cytokines (IL10, IL35, TGFB), disruption of metabolism by regulating CD39 and CD73 ratios, expression of inhibitory molecules (PD1, CTLA4, LAG3, etc.), and deprivation of IL2 in the microenvironment.

[0003] Increased numbers of Treg cells have been observed in the peripheral blood and tumor tissues of various cancer patients. One study found that the proportion of Treg cells among CD4 cells in the tumor microenvironment was as high as 40% to 60%. In various cancers, including NSCLC, melanoma, ovarian cancer, breast cancer, and gastric cancer, higher Treg proportions were associated with worse disease-free survival. In the tumor microenvironment, Treg cells are in a state of suppressive hyperactivation and proliferation. Compared with peripheral Treg cells, tumor-infiltrating Treg cells express higher levels of T cell activation-associated cell surface molecules or chemokine receptors, such as CD25, CTLA4, PD1, LAG3, TIGIT, ICOS, 4-1BB, OX-40, GITR, CCR4, and CCR8 (Reference 3). Highly activated tumor-infiltrating Treg cells have strong inhibitory effects on anti-tumor immunity and are a major limiting factor for current immunotherapy strategies (References 1-2).

[0004] Currently, the main therapeutic strategy against Tregs is to deplete intratumoral Treg cells by antibodies. For example, antibodies against CD25, CTLA4, CCR8, 4-1BB, GITR and OX-40 with ADCC effect or enhanced ADCC effect, produce anti-tumor effect by depleting Treg cells. However, Treg depletion therapy not only depletes Treg cells in the tumor microenvironment, but also depletes systemic Treg cells, which is prone to cause systemic immune instability, leading to autoimmune diseases; in addition, the cell surface molecules highly expressed by tumor-infiltrating Treg cells are also expressed on activated effector T cells, so it is difficult to selectively deplete tumor-infiltrating Treg cells without affecting effector T cells (reference 3).

[0005] Recently, a new therapeutic strategy targeting Tregs has been proposed: inducing Treg cells (especially Treg cells in the tumor microenvironment) to exhibit fragility. Treg fragility is defined as maintaining Foxp3 expression but losing immunosuppressive function. Fragile Treg cells produce IFNγ and upregulate IFNγ receptor and transcription factor Tbet. Fragile Treg cells reduce the expression of inhibitory molecules such as CD73 and IL10, and have lower immunosuppressive activity in the tumor microenvironment. Currently, it is still under study in this field which molecules help to transition from traditional Treg cells to fragile Treg cells.

[0006] When FOXp3 + Treg cells are exposed to IL-12, they are converted into Th1-like Tregs (T-bet + IFNγ + FOXp3 + Tregs, Th1-like Tregs are defined in reference 4). In vitro studies have shown that T-bet + Tregs are able to produce IFNγ independently of IL-12, which indicates that T-bet expression alone is sufficient to trigger IFNγ production, and IL-12 can further enhance this effect. In addition, IL-12 has a unique property that it can induce Tregs and CD4 + Tcon cells (CD4 + FOXp3 - T cells), CD8 + T cells have different effects. IL-12 can induce upregulation of CD25 on activated CD4 + Tcon and CD8 + t cells (reference 5).

[0007] A major limitation of IL-12 therapy is the severe 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 therapy. In the clinic, severe side effects, including 2 deaths, occurred in renal cancer patients after receiving a dose of 500 ng / kg / day of rhIL12 for 5 consecutive days (Refs. 6-8). The toxicity of IL12 has been shown to be associated with the release of systemic IFNy. In the above-mentioned clinical study where deaths occurred, severe toxicity of IL12 was accompanied by high levels of systemic IFNy release, with peak IFNy concentrations in plasma exceeding 24,000 pg / mL. In contrast, a single dose of IL12 given 14 days before the administration cycle can lead to a decrease in plasma IFNy levels (peak of about 6,000 pg / ml) and reduce IL12-induced severe toxicity events. IL-12 toxicity studies based on IFNy neutralizing antibodies and IFNy knockout mice have also further confirmed that many IL-12 acute toxicities are IFNy dependent (Refs. 7-8). Reducing systemic release of IFNy helps to improve the safety of IL-12 therapy. However, a decrease in plasma IFNy concentration is usually accompanied by a decrease in the effectiveness of IL-12 therapy (Refs. 9-10). IL-12 therapy at a safe dose shows very limited clinical efficacy. SUMMARY

[0008] The present disclosure relates generally to a Treg cell-targeting IL12 fusion protein, corresponding nucleic acid molecules, vectors, cells, pharmaceutical compositions, methods of production, methods of tumor treatment, and pharmaceutical uses.

[0009] In a first aspect, the present disclosure provides a Treg cell-targeting IL12 fusion protein.

[0010] Non-limiting exemplary embodiments of the Treg cell-targeting IL12 fusion protein described in the present disclosure can include one or more of the following features.

[0011] In some embodiments, the Treg cell-targeting IL12 fusion protein comprises one or more Treg cell-targeting modules that specifically bind to a Treg cell surface molecule and IL12 directly or indirectly linked, and the IL12 comprises one or more attenuated activity mutations compared to wild-type IL12.

[0012] In some embodiments, the one or more attenuated activity mutations are located on the p35 subunit and / or the p40 subunit of the IL12.

[0013] In some embodiments, the one or more attenuated activity mutations comprise one or more mutations occurring at positions W15, E59, F60, K84, and K195 of the p40 subunit, the positions being located with reference to SEQ ID NO: 5.

[0014] In some embodiments, the one or more attenuated activity mutations comprise mutations occurring at any one of the following sets of positions of 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.

[0015] In some embodiments, the one or more attenuated activity mutations comprise one or more mutations from a wild-type amino acid to A.

[0016] In some embodiments, the one or more attenuated activity mutations comprise any one of the following sets 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 E59A / F60A / K84A / K195A.

[0017] In some embodiments, the p40 subunit comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 9-17; preferably, the p40 subunit comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 14.

[0018] In some embodiments, the p35 subunit and the p40 subunit of the IL12 are connected by a linker, preferably, the IL12 after being connected by the linker comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 18-26; more preferably, the IL12 after being connected by the linker comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 23.

[0019] In some embodiments, the Treg cell surface molecule comprises one or more selected from the group consisting of CTLA4, CCR8, CCR4, CCR10, CD25, GITR, OX-40, ICOS, and 4-1BB.

[0020] In some embodiments, the Treg cell targeting IL12 fusion protein further comprises a PD1 targeting moiety that specifically binds PD1.

[0021] In some embodiments, the Treg cell targeting moiety and / or the PD1 targeting moiety comprises a ligand or a ligand fragment, or an antibody or a fragment of an antibody; optionally, the Treg cell targeting moiety and / or the PD1 targeting moiety comprises a ligand extracellular domain (LECD), a Fab, a scFab, a Fab’, a (Fab’)2, a Fv, a scFv, or a VHH.

[0022] In some embodiments, the Treg cell targeting IL12 fusion protein further comprises a first Fc unit and a second Fc unit with a hinge region, the first Fc unit dimerizes with the second unit to form a dimer, optionally, the IL12 is located between the Treg cell targeting moiety or the PD1 targeting moiety and the hinge region of the first Fc unit or the second Fc unit.

[0023] In some embodiments, the Treg cell targeting IL12 fusion protein comprises a dimer consisting of A-[L1] n1 -B-[L2] n2 -C and A’-[L3] n3 -C’ wherein:

[0024] A and A’ represent a first targeting moiety and a second targeting moiety, respectively, at least one of the A and A’ specifically binds a Treg cell surface molecule;

[0025] B represents a p40 subunit-[L4] n4 -p35 subunit or a p35 subunit-[L4] n4IL12 consisting of a p40 subunit, at least one of the p35 subunit and the p40 subunit comprising one or more attenuating mutations that attenuate the affinity of the IL12 to bind to its receptor as compared to wild type;

[0026] C and C’ represent a first Fc unit and a second Fc unit for dimerization, respectively;

[0027] L1, L2, L3, and L4 represent linkers, n1, n2, n3, and n4 are selected from 0 or 1; and,

[0028] - represents a peptide bond.

[0029] In some embodiments, the Treg cell-targeting IL12 fusion protein comprises a dimer consisting of A-[L1] n1 -B-[L2] n2 -C and A'-[L3] n3 -B'-[L4] n4 -C’; wherein:

[0030] A and A’ represent a first targeting moiety and a second targeting moiety, respectively, at least one of the A and A’ specifically binds to a Treg cell surface molecule;

[0031] B represents a p40 subunit and B’ represents a p35 subunit, or, B represents a p35 subunit and B’ represents a p40 subunit; at least one of the p35 subunit and the p40 subunit comprises one or more attenuating mutations that attenuate the affinity of the IL12 to bind to its receptor as compared to wild type;

[0032] C and C’ represent a first Fc unit and a second Fc unit for dimerization, respectively;

[0033] L1, L2, L3, and L4 represent linkers, n1, n2, n3, and n4 are selected from 0 or 1; and,

[0034] - represents a peptide.

[0035] In some embodiments, the first targeting moiety and / or the second targeting moiety comprises a ligand, an antibody, or a fragment of the ligand or the antibody that specifically binds to a target antigen; optionally, the first targeting moiety and / or the second targeting moiety comprises a ligand extracellular domain (LECD), a Fab, a scFab, a Fab’, a (Fab’)2, a Fv, a scFv, or a VHH that specifically binds to the target antigen.

[0036] In some embodiments, the first targeting moiety and the second targeting moiety both comprise a Fab.

[0037] In some specific embodiments, the Treg cell-targeting IL12 fusion protein comprises a first peptide, a second peptide, a third peptide, and a fourth peptide, the first peptide comprising VH1-(H1-CH1)-[L1] n1 -B-[L2] n2 -C, the second peptide comprising VH2-(H2-CH1)-[L3] n3 -C', the third peptide comprising VL1-(L1-CL), and the fourth peptide comprising 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).

[0038] In some embodiments, the first targeting module comprises a Fab, and the second targeting module comprises a VHH or a LECD.

[0039] In some specific embodiments, the Treg cell-targeting IL12 fusion protein comprises a first peptide, a second peptide, and a third peptide, the first peptide comprising VH1-(H1-CH1)-[L1] n1 -B-[L2] n2 -C, the second peptide comprising VHH2-[L3] n3 -C' or LECD2-[L3] n3 -C', the third peptide comprising 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 VHH2 or LECD2 of the second peptide forms the second targeting module.

[0040] In some embodiments, the first targeting module comprises a VHH or a LECD, and the second targeting module comprises a Fab.

[0041] In some specific embodiments, the Treg cell-targeting IL12 fusion protein comprises a first peptide, a second peptide, and a third peptide, the first peptide comprising VHH1-[L1] n1 -B-[L2] n2 -C or LECD1-[L1] n1 -B-[L2] n2 -C, the second peptide comprising VH2-(H2-CH1)-[L3] n3-C, the third peptide comprising 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).

[0042] In some embodiments, the first targeting module comprises a VHH or LECD, and the second targeting module comprises a VHH or LECD. In some specific embodiments, the Treg cell targeting IL12 fusion protein comprises a first peptide and a second peptide, the first peptide comprising VHH1-[L1] n1 -B-[L2] n2 -C or LECD1-[L1] n1 -B-[L2] n2 -C, the second peptide comprising 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; VHH2 or LECD2 of the second peptide forms the second targeting module.

[0043] In some embodiments, the first targeting module specifically binds to a Treg cell surface molecule, and the second targeting module specifically binds to a Treg cell surface molecule, optionally, 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. In some specific embodiments, the first targeting module has a KD value of binding to its target antigen of ≤1E-7 M, ≤1E-8 M, ≤1E-9 M, ≤1E-10 M, ≤1E-11 M, or ≤1E-12 M, preferably, the KD value is in the range of 1E-10 M to 1E-7 M, more preferably, the KD value is in the range of 1E-9 M to 1E-8 M; the second targeting module has a KD value of binding to its target antigen of ≤1E-7 M, ≤1E-8 M, ≤1E-9 M, ≤1E-10 M, ≤1E-11 M, or ≤1E-12 M, preferably, the KD value is in the range of 1E-10 M to 1E-7 M, more preferably, the KD value is in the range of 1E-9 M to 1E-8 M.

[0044] In some embodiments, the first targeting moiety specifically binds to a Treg cell surface molecule, the second targeting moiety specifically binds to PD1, optionally, the first targeting moiety specifically binds to CTLA4 or CCR8, the second targeting moiety specifically binds to PD1. In some specific embodiments, the KD value of the first targeting moiety binding to its target antigen is <1E-7 M, <1E-8 M, <1E-9 M, <1E-10 M, <1E-11 M, or <1E-12 M, preferably the KD value is in the range of 1E-10 M to 1E-7 M, more preferably the KD value is in the range of 1E-9 M to 1E-8 M; the KD value of the second targeting moiety binding to its target antigen is <1E-6 M, <1E-7 M, <1E-8 M, <1E-9 M, <1E-10 M, <1E-11 M, or <1E-12 M, preferably the KD value is in the range of 1E-8 M to 1E-6 M, more preferably the KD value is in the range of 1E-7 M to 10E-6 M, or in the range of 1E-8 to 1E-7.

[0045] In some embodiments, the first targeting moiety specifically binds to PD1, the second targeting moiety specifically binds to a Treg cell surface molecule, optionally, the first targeting moiety specifically binds to PD1, the second targeting moiety specifically binds to CTLA4 or CCR8. In some specific embodiments, the KD value of the first targeting moiety binding to its target antigen is <1E-6 M, <1E-7 M, <1E-8 M, <1E-9 M, <1E-10 M, <1E-11 M, or <1E-12 M, preferably the KD value is in the range of 1E-8 M to 1E-6 M, more preferably the KD value is in the range of 1E-7 M to 10E-6 M, or in the range of 1E-8 to 1E-7; the KD value of the second targeting moiety binding to its target antigen is <1E-7 M, <1E-8 M, <1E-9 M, <1E-10 M, <1E-11 M, or <1E-12 M, preferably the KD value is in the range of 1E-10 M to 1E-7 M, more preferably the KD value is in the range of 1E-9 M to 1E-8 M.

[0046] In some embodiments, the KD value of the Treg cell targeting moiety binding to its target antigen is <1E-7 M, <1E-8 M, <1E-9 M, <1E-10 M, <1E-11 M, or <1E-12 M, preferably the KD value is in the range of 1E-10 M to 1E-7 M, more preferably the KD value is in the range of 1E-9 M to 1E-8 M.

[0047] In some embodiments, the PD1 targeting moiety binds to its target antigen with a KD value of <1E-6 M, <1E-7 M, <1E-8 M, <1E-9 M, <1E-10 M, <1E-11 M, or <1E-12 M, preferably the KD value is in the range of 1E-8 M to 1E-6 M, more preferably the KD value is in the range of 1E-7 M to 10E-6 M, or in the range of 1E-8 to 1E-7.

[0048] In some embodiments, the CTLA4 targeting moiety comprises a Fab, scFab, Fab', (Fab')2, Fv, scFv, or VHH that specifically binds to CTLA4;

[0049] Optionally, the Fab, scFab, Fab', (Fab')2, Fv, and scFv that specifically binds to CTLA4 comprises a VH and VL as shown below:

[0050] (1) the VH comprises a HCDR1 as set forth in SEQ ID NO: 79 or 82, a HCDR2 as set forth in SEQ ID NO: 80 or 83, and a HCDR3 as set forth in SEQ ID NO: 81 or 84; and the VL comprises a LCDR1 as set forth in SEQ ID NO: 85 or 88, a LCDR2 as set forth in SEQ ID NO: 86, and a LCDR3 as set forth in SEQ ID NO: 87;

[0051] (2) the VH comprises a HCDR1 as set forth in SEQ ID NO: 89, a HCDR2 as set forth in SEQ ID NO: 90, and a HCDR3 as set forth in SEQ ID NO: 91; and the VL comprises a LCDR1 as set forth in SEQ ID NO: 92, a LCDR2 as set forth in SEQ ID NO: 93, and a LCDR3 as set forth in SEQ ID NO: 94;

[0052] (3) the VH comprises a HCDR1 as set forth in SEQ ID NO: 95, a HCDR2 as set forth in SEQ ID NO: 96, and a HCDR3 as set forth in SEQ ID NO: 97; and the VL comprises a LCDR1 as set forth in SEQ ID NO: 98, a LCDR2 as set forth in SEQ ID NO: 99, and a LCDR3 as set forth in SEQ ID NO: 100;

[0053] (4) the VH comprises HCDR1 as set forth in SEQ ID NO: 134, HCDR2 as set forth in SEQ ID NO: 135, and HCDR3 as set forth in SEQ ID NO: 136; and the VL comprises LCDR1 as set forth in SEQ ID NO: 137, LCDR2 as set forth in SEQ ID NO: 138, and LCDR3 as set forth in SEQ ID NO: 139;

[0054] (5) the VH comprises HCDR1 as set forth in SEQ ID NO: 149, HCDR2 as set forth in SEQ ID NO: 150, and HCDR3 as set forth in SEQ ID NO: 151; and the VL comprises LCDR1 as set forth in SEQ ID NO: 152, LCDR2 as set forth in SEQ ID NO: 153, and LCDR3 as set forth in SEQ ID NO: 154;

[0055] (6) the VH comprises HCDR1 as set forth in SEQ ID NO: 155, HCDR2 as set forth in SEQ ID NO: 156, and HCDR3 as set forth in SEQ ID NO: 157; and the VL comprises LCDR1 as set forth in SEQ ID NO: 158, LCDR2 as set forth in SEQ ID NO: 159, and LCDR3 as set forth in SEQ ID NO: 160;

[0056] (7) the VH comprises HCDR1 as set forth in SEQ ID NO: 161, HCDR2 as set forth in SEQ ID NO: 162, and HCDR3 as set forth in SEQ ID NO: 163; and the VL comprises LCDR1 as set forth in SEQ ID NO: 164, LCDR2 as set forth in SEQ ID NO: 165, and LCDR3 as set forth in SEQ ID NO: 166;

[0057] (8) the VH comprises HCDR1 as set forth in SEQ ID NO: 167, HCDR2 as set forth in SEQ ID NO: 168, and HCDR3 as set forth in SEQ ID NO: 169; and the VL comprises LCDR1 as set forth in SEQ ID NO: 170, LCDR2 as set forth in SEQ ID NO: 171, and LCDR3 as set forth in SEQ ID NO: 172;

[0058] (9) the VH comprises HCDR1 as set forth in SEQ ID NO: 173, HCDR2 as set forth in SEQ ID NO: 174, and HCDR3 as set forth in SEQ ID NO: 175; and the VL comprises LCDR1 as set forth in SEQ ID NO: 176, LCDR2 as set forth in SEQ ID NO: 177, and LCDR3 as set forth in SEQ ID NO: 178;

[0059] (10) the VH comprises HCDR1 as set forth in SEQ ID NO: 179, HCDR2 as set forth in SEQ ID NO: 180, and HCDR3 as set forth in SEQ ID NO: 181; and the VL comprises LCDR1 as set forth in SEQ ID NO: 182, LCDR2 as set forth in SEQ ID NO: 183, and LCDR3 as set forth in SEQ ID NO: 184;

[0060] (11) the VH comprises HCDR1 as set forth in SEQ ID NO: 188, HCDR2 as set forth in SEQ ID NO: 189, and HCDR3 as set forth in SEQ ID NO: 190; and the VL comprises LCDR1 as set forth in SEQ ID NO: 191, LCDR2 as set forth in SEQ ID NO: 192, and LCDR3 as set forth in SEQ ID NO: 193;

[0061] (12) the VH comprises HCDR1 as set forth in SEQ ID NO: 194, HCDR2 as set forth in SEQ ID NO: 195, and HCDR3 as set forth in SEQ ID NO: 196; and the VL comprises LCDR1 as set forth in SEQ ID NO: 197, LCDR2 as set forth in SEQ ID NO: 198, and LCDR3 as set forth in SEQ ID NO: 199;

[0062] (13) the VH comprises HCDR1 as set forth in SEQ ID NO: 200, HCDR2 as set forth in SEQ ID NO: 201, and HCDR3 as set forth in SEQ ID NO: 202; and the VL comprises LCDR1 as set forth in SEQ ID NO: 203, LCDR2 as set forth in SEQ ID NO: 204, and LCDR3 as set forth in SEQ ID NO: 205;

[0063] (14) the VH comprises HCDR1 as set forth in SEQ ID NO:206, HCDR2 as set forth in SEQ ID NO:207, and HCDR3 as set forth in SEQ ID NO:208; and the VL comprises LCDR1 as set forth in SEQ ID NO:209, LCDR2 as set forth in SEQ ID NO:210, and LCDR3 as set forth in SEQ ID NO:211; or,

[0064] (15) the VH comprises HCDR1 as set forth in SEQ ID NO:212, HCDR2 as set forth in SEQ ID NO:213, and HCDR3 as set forth in SEQ ID NO:214; and the VL comprises LCDR1 as set forth in SEQ ID NO:215, LCDR2 as set forth in SEQ ID NO:216, and LCDR3 as set forth in SEQ ID NO:217;

[0065] Optionally, the VHH that specifically binds to CTLA4 comprises:

[0066] (1) HCDR1 as set forth in SEQ ID NO: 101, HCDR2 as set forth in SEQ ID NO: 102, and HCDR3 as set forth in SEQ ID NO: 103;

[0067] (2) HCDR1 as set forth in SEQ ID NO: 104, HCDR2 as set forth in SEQ ID NO: 105, and HCDR3 as set forth in SEQ ID NO: 106;

[0068] (3) HCDR1 as set forth in SEQ ID NO: 107, HCDR2 as set forth in SEQ ID NO: 108, and HCDR3 as set forth in SEQ ID NO: 109;

[0069] (4) HCDR1 as set forth in SEQ ID NO: 110, HCDR2 as set forth in SEQ ID NO: 111, and HCDR3 as set forth in SEQ ID NO: 112;

[0070] (5) HCDR1 as set forth in SEQ ID NO: 113, HCDR2 as set forth in SEQ ID NO: 114, and HCDR3 as set forth in SEQ ID NO: 115;

[0071] (6) HCDR1 as set forth in SEQ ID NO: 116, HCDR2 as set forth in SEQ ID NO: 117, and HCDR3 as set forth in SEQ ID NO: 118;

[0072] (7) HCDR1 as depicted in SEQ ID NO: 119, HCDR2 as depicted in SEQ ID NO: 120 and HCDR3 as depicted in SEQ ID NO: 121;

[0073] (8) HCDR1 as depicted in SEQ ID NO: 122, HCDR2 as depicted in SEQ ID NO: 123 and HCDR3 as depicted in SEQ ID NO: 124;

[0074] (9) HCDR1 as depicted in SEQ ID NO: 125, HCDR2 as depicted in SEQ ID NO: 126 and HCDR3 as depicted in SEQ ID NO: 127;

[0075] (10) HCDR1 as depicted in SEQ ID NO: 128, HCDR2 as depicted in SEQ ID NO: 129 and HCDR3 as depicted in SEQ ID NO: 130;

[0076] (11) HCDR1 as depicted in SEQ ID NO: 131, HCDR2 as depicted in SEQ ID NO: 132 and HCDR3 as depicted in SEQ ID NO: 133;

[0077] (12) HCDR1 as depicted in SEQ ID NO: 140, HCDR2 as depicted in SEQ ID NO: 141 and HCDR3 as depicted in SEQ ID NO: 142;

[0078] (13) HCDR1 as depicted in SEQ ID NO: 143, HCDR2 as depicted in SEQ ID NO: 144 and HCDR3 as depicted in SEQ ID NO: 145;

[0079] (14) HCDR1 as depicted in SEQ ID NO: 146, HCDR2 as depicted in SEQ ID NO: 147 and HCDR3 as depicted in SEQ ID NO: 148; or,

[0080] (15) HCDR1 as depicted in SEQ ID NO: 185, HCDR2 as depicted in SEQ ID NO: 186 and HCDR3 as depicted in SEQ ID NO: 187.

[0081] In some embodiments, the Fab, scFab, Fab', (Fab')2, Fv and scFv that specifically bind to CTLA4 comprise a VH and a VL as set forth below:

[0082] (1) the VH comprises a sequence as shown in any one of SEQ ID NOs: 31-33, and the VL comprises a sequence as shown in SEQ ID NO: 34 or 35;

[0083] (2) the VH comprises a sequence as shown in SEQ ID NO: 36, and the VL comprises a sequence as shown in SEQ ID NO: 37;

[0084] (3) the VH comprises a sequence as shown in SEQ ID NO: 38, and the VL comprises a sequence as shown in SEQ ID NO: 39;

[0085] (4) the VH comprises a sequence as shown in SEQ ID NO: 51, and the VL comprises a sequence as shown in SEQ ID NO: 52;

[0086] (5) the VH comprises a sequence as shown in SEQ ID NO: 56, and the VL comprises a sequence as shown in SEQ ID NO: 57;

[0087] (6) the VH comprises a sequence as shown in SEQ ID NO: 58, and the VL comprises a sequence as shown in SEQ ID NO: 59;

[0088] (7) the VH comprises a sequence as shown in SEQ ID NO: 60, and the VL comprises a sequence as shown in SEQ ID NO: 61;

[0089] (8) the VH comprises a sequence as shown in SEQ ID NO: 62, and the VL comprises a sequence as shown in SEQ ID NO: 63;

[0090] (9) the VH comprises a sequence as shown in SEQ ID NO: 64, and the VL comprises a sequence as shown in SEQ ID NO: 65;

[0091] (10) the VH comprises a sequence as shown in SEQ ID NO: 66, and the VL comprises a sequence as shown in SEQ ID NO: 67;

[0092] (11) the VH comprises a sequence as shown in SEQ ID NO: 69, and the VL comprises a sequence as shown in SEQ ID NO: 70;

[0093] (12) the VH comprises a sequence as shown in SEQ ID NO: 71, and the VL comprises a sequence as shown in SEQ ID NO: 72;

[0094] (13) the VH comprises a sequence as set forth in SEQ ID NO: 73, and the VL comprises a sequence as set forth in SEQ ID NO: 74;

[0095] (14) the VH comprises a sequence as set forth in SEQ ID NO: 75, and the VL comprises a sequence as set forth in SEQ ID NO: 76;

[0096] (15) the VH comprises a sequence as set forth in SEQ ID NO: 77, and the VL comprises a sequence as set forth in SEQ ID NO: 78; or,

[0097] (16) an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to the VH and / or VL as set forth in any one of groups (1)-(15).

[0098] In some embodiments, the VHH that specifically binds to CTLA4 comprises:

[0099] (1) a sequence as set forth in any one of SEQ ID NOs: 40-50, 53-55, 68;

[0100] (2) an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to the sequence as set forth in group (1).

[0101] In some embodiments, the CCR8 targeting moiety comprises a Fab, scFab, Fab', (Fab')2, Fv, scFv, or VHH that specifically binds to CCR8;

[0102] Optionally, the Fab, scFab, Fab', (Fab')2, Fv, and scFv that specifically binds to CCR8 comprises a VH and a VL as set forth below:

[0103] (1) HCDR1 as set forth in SEQ ID NO: 265, HCDR2 as set forth in SEQ ID NO: 266, and HCDR3 as set forth in SEQ ID NO: 267; and the VL comprises LCDR1 as set forth in SEQ ID NO: 268, LCDR2 as set forth in SEQ ID NO: 269, and LCDR3 as set forth in SEQ ID NO: 270; or,

[0104] (2) the VH comprises HCDR1 as set forth in SEQ ID NO: 271, HCDR2 as set forth in SEQ ID NO: 272, and HCDR3 as set forth in SEQ ID NO: 273; and the VL comprises LCDR1 as set forth in SEQ ID NO: 274, LCDR2 as set forth in SEQ ID NO: 275, and LCDR3 as set forth in SEQ ID NO: 276;

[0105] Optionally, the VHH that specifically binds to CCR8 comprises:

[0106] (1) HCDR1 as set forth in SEQ ID NO: 259, HCDR2 as set forth in SEQ ID NO: 260, and HCDR3 as set forth in SEQ ID NO: 261

[0107] (2) HCDR1 as set forth in SEQ ID NO: 262, HCDR2 as set forth in SEQ ID NO: 263, and HCDR3 as set forth in SEQ ID NO: 264; or,

[0108] (3) HCDR1 as set forth in SEQ ID NO: 277, HCDR2 as set forth in SEQ ID NO: 278, and HCDR3 as set forth in SEQ ID NO: 279.

[0109] In some embodiments, the Fab, scFab, Fab’, (Fab’)2, Fv, and scFv that specifically binds to CCR8 comprises a VH and a VL, which are as set forth:

[0110] (1) the VH comprises a sequence as set forth in SEQ ID NO: 254, and the VL comprises a sequence as set forth in SEQ ID NO: 255;

[0111] (2) the VH comprises a sequence as set forth in SEQ ID NO: 256, and the VL comprises a sequence as set forth in SEQ ID NO: 257; or,

[0112] (3) an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to the VH and / or VL as set forth in any one of groups (1)-(2).

[0113] In some embodiments, the VHH that specifically binds to CCR8 comprises:

[0114] (1) a sequence as set forth in any one of SEQ ID NOs: 252-253 or 258; or,

[0115] (2) an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to the sequence set forth in Group (1).

[0116] In some embodiments, the PD1 targeting moiety comprises a Fab, scFab, Fab', (Fab')2, Fv, scFv, or VHH that specifically binds PD1;

[0117] Optionally, the Fab, scFab, Fab', (Fab')2, Fv, and scFv that specifically binds PD1 comprises a VH and VL as set forth below:

[0118] (1) the VH comprises a HCDR1 as set forth in SEQ ID NO: 234, a HCDR2 as set forth in SEQ ID NO: 235, and a HCDR3 as set forth in SEQ ID NO: 236, and the VL comprises a LCDR1 as set forth in SEQ ID NO: 237, a LCDR2 as set forth in SEQ ID NO: 238, and a LCDR3 as set forth in SEQ ID NO: 239;

[0119] (2) the VH comprises a HCDR1 as set forth in SEQ ID NO: 240, a HCDR2 as set forth in SEQ ID NO: 241, and a HCDR3 as set forth in SEQ ID NO: 242, and the VL comprises a LCDR1 as set forth in SEQ ID NO: 243, a LCDR2 as set forth in SEQ ID NO: 244, and a LCDR3 as set forth in SEQ ID NO: 245;

[0120] (3) the VH comprises a HCDR1 as set forth in SEQ ID NO: 246, a HCDR2 as set forth in SEQ ID NO: 247, and a HCDR3 as set forth in SEQ ID NO: 248, and the VL comprises a LCDR1 as set forth in SEQ ID NO: 249, a LCDR2 as set forth in SEQ ID NO: 250, and a LCDR3 as set forth in SEQ ID NO: 251; or,

[0121] (4) the VH comprises a HCDR1 as set forth in SEQ ID NO: 293, a HCDR2 as set forth in SEQ ID NO: 294, and a HCDR3 as set forth in SEQ ID NO: 295, and the VL comprises a LCDR1 as set forth in SEQ ID NO: 296, a LCDR2 as set forth in SEQ ID NO: 297, and a LCDR3 as set forth in SEQ ID NO: 298;

[0122] Optionally, the VHH that specifically binds PD1 comprises:

[0123] (1) 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,

[0124] (2) 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.

[0125] In some embodiments, the Fab, scFab, Fab', (Fab')2, Fv, and scFv that specifically binds PD1 comprises a VH and a VL as shown below:

[0126] (1) the VH comprises a sequence as shown in SEQ ID NO: 218, and the VL comprises a sequence as shown in SEQ ID NO: 219;

[0127] (2) the VH comprises a sequence as shown in SEQ ID NO: 222, and the VL comprises a sequence as shown in SEQ ID NO: 223;

[0128] (3) the VH comprises a sequence as shown in SEQ ID NO: 224, and the VL comprises a sequence as shown in SEQ ID NO: 225;

[0129] (4) the VH comprises a sequence as shown in SEQ ID NO: 226, and the VL comprises a sequence as shown in SEQ ID NO: 227; or,

[0130] (5) an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to the VH and / or VL as shown in any one of groups (1)-(4).

[0131] In some embodiments, the VHH that specifically binds PD1 comprises:

[0132] (1) a sequence as shown in SEQ ID NO: 220 or 221; or,

[0133] (2) an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to the sequence as shown in group (1).

[0134] In some embodiments, wherein the PD1 targeting moiety comprises a PDL1 extracellular domain and / or a PDL2 extracellular domain;

[0135] Optionally, the PD1 targeting moiety comprises:

[0136] (1) a sequence as set forth in any one of SEQ ID NOs: 27-29; or

[0137] (2) an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to a sequence as set forth in group (1).

[0138] In some embodiments, the first Fc unit and the second Fc unit comprise a Knob mutation and a Hole mutation to form a Knob-in-Hole structure.

[0139] In some embodiments, the Knob mutation is selected from S354C, T366W, preferably S354C / T366W, and the Hole mutation is selected from Y349C, T366S, L368A, and Y349C / T366S / L368A / Y349C, preferably Y349C / T366S / L368A / Y349C.

[0140] In some embodiments, the first Fc unit and the second Fc unit comprise a mutation that reduces or abrogates effector function.

[0141] In some embodiments, the mutation that reduces or abrogates effector function comprises a L234A / L235A mutation.

[0142] In some embodiments, the first Fc unit comprises:

[0143] (1) a sequence as set forth in SEQ ID NO: 299 or 300; or

[0144] (2) an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to a sequence as set forth in group (1).

[0145] In some embodiments, the second Fc unit comprises:

[0146] (1) a sequence as set forth in any one of SEQ ID NOs: 301-303; or

[0147] (2) an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to a sequence as set forth in group (1).

[0148] In some embodiments, the linker is selected from a sequence as set forth in any one of SEQ ID NOs: 280-292.

[0149] In some embodiments, the Treg-targeting IL12 fusion protein comprises:

[0150] (1) a first peptide comprising a sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 307 or 310, a second peptide comprising a sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 308, a third peptide comprising a sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 309, and a fourth peptide comprising a sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 309;

[0151] (2) a first peptide comprising a sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 307 or 310, a second peptide comprising a sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 311, and a third peptide comprising a sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 309;

[0152] (3) a first peptide comprising a sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 312 or 313, a second peptide comprising a sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 308, and a third peptide comprising a sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 309.

[0153] In a second aspect, the present disclosure provides an isolated nucleic acid molecule comprising a nucleic acid segment encoding one or more peptide chains of the Treg cell-targeting IL12 fusion protein of the first aspect of the present disclosure. In some embodiments, the nucleic acid is DNA or RNA.

[0154] In a third aspect, the present disclosure provides a vector comprising the isolated nucleic acid molecule of the second aspect of the present disclosure.

[0155] In a fourth aspect, the present disclosure provides a cell comprising the isolated nucleic acid molecule of the second aspect or the vector of the third aspect. In some embodiments, 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.

[0156] In a fifth aspect, the present disclosure provides a production method, comprising culturing the cell of the fourth aspect to express the Treg cell-targeting IL12 fusion protein of the first aspect, and isolating and purifying the Treg cell-targeting IL12 fusion protein.

[0157] In a sixth aspect, the present disclosure provides a pharmaceutical composition, comprising: (1) the Treg cell-targeting IL12 fusion protein of the first aspect, the nucleic acid of the second aspect, the vector of the third aspect, the cell of the fourth aspect, or the product of the method of the fifth aspect; and (2) a pharmaceutically acceptable carrier.

[0158] In a seventh aspect, the present disclosure provides a method of treating a tumor, 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 vector of the third aspect, the cell of the fourth aspect, the product of the method of the fifth aspect, or the pharmaceutical composition of the sixth aspect, which induces intratumoral Treg cells to secrete IFNy, and attenuates or abrogates the immunosuppressive effect of the intratumoral Treg cells.

[0159] In some embodiments, the Treg cell induces intratumoral Treg cells to transform into Th1-like Treg cells or fragile Treg cells.

[0160] In some embodiments, the Treg cell-targeting IL12 fusion protein further induces tumor microenvironment CD8 + T cells or CD4 + Tcon cells: (1) secrete IFNy; (2) upregulate the expression level of T-bet; and / or, upregulate the expression of CD25.

[0161] In some embodiments, wherein the Treg-targeting IL12 fusion protein is administered systemically, such as intravenously or subcutaneously.

[0162] In some embodiments, it is a method of monotherapy or in combination with other therapies.

[0163] In some embodiments, the other therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, toxin therapy, and surgery.

[0164] In some embodiments, the Treg cell-targeting IL12 fusion protein, the isolated nucleic acid molecule, the vector, the cell, the product of the method, or the pharmaceutical composition is administered prior to, after, or concurrently with the other therapy.

[0165] In some embodiments, the subject is an immune checkpoint inhibitor-resistant patient, preferably, the immune checkpoint inhibitor is a PD1 antibody.

[0166] In some embodiments, 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 brain glioma.

[0167] In an eighth aspect, the present disclosure provides use of the Treg cell-targeting IL12 fusion protein of the first aspect, the isolated nucleic acid molecule of the second aspect, the vector of the third aspect, the cell of the fourth aspect, the product of the method of the fifth aspect, or the pharmaceutical composition of the sixth aspect in the preparation of a medicament for treating a tumor, wherein the Treg cell-targeting IL12 fusion protein induces intratumoral Treg cells to secrete IFNy, and attenuates or abrogates the immunosuppressive effect of the Treg cells.

[0168] In some embodiments, the Treg cell induces intratumoral Treg cells to transform into Th1-like Treg cells or fragile Treg cells.

[0169] In some embodiments, the Treg cell-targeting IL12 fusion protein further induces tumor microenvironment CD8 + T cells or CD4 + Tcon cells: (1) secrete IFNy; (2) upregulate the expression level of T-bet; and / or, upregulate the expression of CD25.

[0170] In some embodiments, the Treg-targeting IL12 fusion protein is administered systemically, such as intravenously or subcutaneously.

[0171] In some embodiments, the Treg-targeting IL12 fusion protein is administered as a monotherapy or in combination with other therapies.

[0172] In some embodiments, the other therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, toxin therapy, and surgery.

[0173] In some embodiments, the Treg cell-targeting IL12 fusion protein, the isolated nucleic acid molecule, the vector, the cell, the product of the method, or the pharmaceutical composition is administered prior to, after, or concurrently with the other therapy.

[0174] In some embodiments, the subject is an immune checkpoint inhibitor resistant patient, preferably the immune checkpoint inhibitor is a PD1 antibody.

[0175] In some embodiments, 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 brain glioma. BRIEF DESCRIPTION OF DRAWINGS

[0176] In order to more clearly illustrate the technical solutions in the specific embodiments of the present application or the prior art, the drawings required to be used in the description of the specific embodiments or the prior art will be briefly introduced as follows. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0177] 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).

[0178] 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).

[0179] Figure 2A-2B: IL12Rβ1 + Cells and PD1 + Cell proportion in spleen and tumor-derived lymphocyte subsets; lymphocyte subset typing: 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 + ).

[0180] Figure 3A-Figure 3B: CTLA4 + cells or CCR8 + cells in spleen and tumor-derived T lymphocyte subsets; lymphocyte subset typing: CD3 + T cells (CD45 + CD3 + ), Tcon cells (CD45 + CD3 + CD4 + CD25 - ), Treg cells (CD45 + CD3 + CD4 + CD25 + ), CD8 + T cells (CD45 + CD3 + CD8 + ).

[0181] Figure 4: PD1 + cells, CTLA4 + cells and CCR8 + cells in tumor infiltrating lymphocyte subsets; lymphocyte subset typing: CD3 + T cells (CD45 + CD3 + ); Tcon cells (CD45 + CD3 + CD4 + CD25 - ); Treg cells (CD45 + CD3 + CD4 + CD25 + ); CD8 + T cells (CD45 + CD3 + CD8 + ).

[0182] Figure 5A: Schematic of Treg cell-targeted IL12 fusion protein, wherein ABD represents an antigen binding domain, the p35 and p40 subunits of IL12 are not shown separately, and the linker is not shown.

[0183] Figure 5B: Schematic of Treg cell-targeted IL12 fusion proteins, where ABD represents an antigen binding domain, the p35 and p40 subunits of IL12 are shown, and linkers are not shown.

[0184] Figure 6: Specific example of the schematic shown in Figure 5A (targeting CTLA4).

[0185] Figure 7: Specific example of the schematic shown in Figure 5A (targeting CCR8).

[0186] Figures 8A-8C: IL12 activity detection results for hIL12(WT)-Fc / / Fc, hIL12(4A)-Fc / / Fc, and Ipi-hIL12(4A) / / hPDL2.

[0187] Figure 9: Activity detection results for CTLA4-targeted IL12 fusion proteins (Ipi).

[0188] Figures 10A-10C: Activity detection results for CTLA4-targeted IL12 fusion proteins (VH146, HL32, Ipi).

[0189] Figures 11A-11E: Activity detection results for CCR8-targeted IL12 fusion proteins.

[0190] Figures 12A-12B: Growth inhibition of B16 melanoma by Treg cell-targeted IL12 fusion proteins shown in Table 7 (Modeling and dosing: hCTLA4 / hPD1 humanized mice (purchased from Janvier), cancer cell inoculation amount: 1E5 cells / mouse, grouping: 4 mice / group, intraperitoneal injection for dosing, dose: 5 mg / kg, dosing time: 4, 7, 11, 15 days after inoculation).

[0191] Figures 13A-13B: Growth inhibition of B16 melanoma by Treg cell-targeted IL12 fusion proteins shown in Table 8 (Modeling and dosing: hCTLA4 humanized mice (purchased from Envigo), cancer cell inoculation amount: 2.5E5 cells / mouse, grouping: 7 mice / group, tail vein injection for dosing, dose: 5 mpk, dosing time: 5, 8, 11, 14 days after inoculation).

[0192] Figures 14A-14B: Growth inhibition of RM1 prostate tumor by Treg cell-targeted IL12 fusion proteins shown in Table 9 (Modeling and dosing: hCTLA4 / hPD1 humanized mice (purchased from Janvier), cancer cell inoculation amount: 2E5 cells / mouse, grouping: 5 mice / group, intraperitoneal injection for dosing, dose: 5 mpk, dosing time: 6, 9, 12, 15 days after inoculation).

[0193] Figure 15A-15D: Inhibition of RM1 prostate tumor growth by Treg cell-targeted IL12 fusion proteins shown in Table 10 (Modeling and dosing: hCTLA4 humanized mice (purchased from BioLegend), cancer cell inoculation amount: 2E5 cells per mouse, grouping: 5 mice per group, intraperitoneal injection for dosing, dose: 5 mpk, dosing time: 6, 9, 12, 15 days after inoculation).

[0194] Figure 16A: IL12 activity detection results of 9D9-mIL12(4A) / / mPDL2.

[0195] Figure 16B: IL12 activity detection results of Ipi-hIL12(4A) / / hPDL2.

[0196] Figure 17: Inhibition of MC38 colorectal cancer growth by test drugs shown in Table 12 (Modeling and dosing: wild type C57BL / 6 mice, cancer cell inoculation amount: 5E6 cells per mouse, grouping: 20 mice per group, intraperitoneal injection for dosing, dose: 5 mpk, dosing time: 9, 13, 16, 20 days after inoculation).

[0197] Figure 18: Inhibition of EMT6 breast tumor growth by test drugs shown in Table 12 (Modeling and dosing: wild type Balb / c mice, cancer cell inoculation amount: 2E6 cells per mouse, grouping: 20 mice per group, intraperitoneal injection for dosing, dose: 5 mpk, dosing time: 6, 9, 12, 15 days after inoculation).

[0198] Figure 19: Inhibition of B16 melanoma growth by test drugs shown in Table 12 (Modeling and dosing: wild type Balb / c mice, cancer cell inoculation amount: 2E5 cells per mouse, grouping: 7 mice per group, intraperitoneal injection for dosing, dose: 5 mpk for mIpi, 10 mpk for others, dosing time: 4, 8, 11, 14 days after inoculation).

[0199] Figure 20: Inhibition of MC38 colorectal tumor growth by test drugs shown in Table 13 (Modeling and dosing: wild type C57BL / 6 mice, cancer cell inoculation amount: 5E5 cells per mouse, grouping: 6 mice per group, intraperitoneal injection for dosing, dose: 1.5 mpk, dosing time: 7, 12, 17, 21 days after inoculation).

[0200] Figure 21: Inhibition of B16 melanoma growth by test drugs shown in Table 14 (Modeling and dosing: wild type C57BL / 6 mice, cancer cell inoculation amount: 2E5 cells per mouse, grouping: 7 mice per group, tail vein injection for dosing, dose: 5 mpk, dosing time: 5, 8, 12, 15 days after inoculation).

[0201] Figure 22A-22C: Inhibition of CT26 colorectal tumor growth by the test drugs shown in Table 15, Table 16 (modeling and administration: wild-type Balb / c mice, cancer cell inoculation amount: 2.5E5 per mouse, grouping: 6 mice per group, intraperitoneal injection administration, dose: 5 mpk, administration time: 4, 7, 11, 14 days after inoculation).

[0202] Figure 23A-23B: Results of detection of serum IFNy content in mice after administration.

[0203] Figure 24A-24B: Changes in body weight of mice after administration.

[0204] Figure 25: Results of detection of serum IFNy content in cynomolgus monkeys after administration.

[0205] Figure 26: Cell count of TDLN in mice after administration.

[0206] Figure 27: Proportion of IFNy+ cells in Treg cells derived from the spleen, TDLN and tumor of mice after administration.

[0207] Figure 28: Proportion of IFNy+ cells in Tcon cells derived from the spleen, TDLN and tumor of mice after administration.

[0208] Figure 29: Proportion of IFNy+ cells in CD8 T cells derived from the spleen, TDLN and tumor of mice after administration.

[0209] Figure 30: Proportion of T-bet+ cells in Treg cells derived from the spleen, TDLN and tumor of mice after administration.

[0210] Figure 31: Proportion of T-bet+ cells in Tcon cells derived from the spleen, TDLN and tumor of mice after administration.

[0211] Figure 32: Proportion of T-bet+ cells in CD8 T cells derived from the spleen, TDLN and tumor of mice after administration.

[0212] Figure 33: Proportion of CD25+ cells in CD8 T cells derived from the spleen, TDLN and tumor of mice after administration.

[0213] Figure 34: Proportion of CD25+ cells in Tcon cells derived from the spleen, TDLN and tumor of mice after administration.

[0214] Figure 35: Summary of information of 9D9-mIL12(4A) / / mPDL2 administration group in Figures 27-34.

[0215] Figure 36: Results of detection of IFNy content in culture supernatant after 72 hours of drug treatment.

[0216] Figure 37A: The percentage of IFNy+ cells in PBMC-derived Treg cells after 72h drug treatment.

[0217] Figure 37B: The percentage of IFNy+ cells in PBMC-derived Tcon cells after 72h drug treatment.

[0218] Figure 37C: The percentage of IFNy+ cells in PBMC-derived CD8 T cells after 72h drug treatment. DETAILED DESCRIPTION

[0219] I. SUMMARY

[0220] Current Treg depletion therapy depletes Treg cells in the tumor microenvironment, but also depletes systemic Treg cells, leading to immune-related adverse events (irAEs) and autoimmune-related toxicities, and depletes activated effector T cells in the tumor microenvironment, suppressing specific anti-tumor responses. Current IL12 therapy also has significant deficiencies, as its high systemic toxicity and narrow therapeutic window limit the clinical application of IL12 therapy.

[0221] To address the deficiencies of current Treg depletion therapy and / or IL12 therapy, the present disclosure provides a novel Treg cell-targeting IL12 fusion protein, which optionally comprises one or more Treg cell-targeting moieties that specifically bind to a Treg cell surface molecule and IL12 comprising one or more attenuated mutations compared to wild-type IL12, directly or indirectly linked. The present disclosure also provides a novel tumor treatment method, which comprises administering to a subject an effective amount of a Treg cell-targeting IL12 fusion protein, which induces intratumoral Treg cells to secrete IFNy, attenuating or reversing the immunosuppressive effect of the intratumoral Treg cells.

[0222] The Treg cell-targeting IL12 fusion protein and tumor treatment method of the present disclosure have at least one of the following advantages:

[0223] (1) The present disclosure employs a targeted delivery of IL12 strategy, which is different from the current Treg depletion strategy, and does not cause the depletion of systemic Treg cells and the depletion of effector T cells in the tumor microenvironment.

[0224] (2) The strategy of the present disclosure targets the delivery of IL12 to Treg cells (especially intratumoral Treg cells), which can induce Treg cells in the tumor microenvironment to secrete IFNy, increasing their fragility, thereby attenuating or reversing their immunosuppressive effect in the tumor microenvironment.

[0225] (3) In the present disclosure, the Treg cell-targeting IL12 fusion protein has target antigen-dependent IL12 activity, which selectively acts in the tumor microenvironment and less (minimally) acts on the peripheral immune system, reducing the toxic side effects of IL12 and expanding the therapeutic window.

[0226] (4) In some cases, the Treg cell-targeting IL12 fusion protein of the present disclosure not only binds to Treg cells (e.g., through CTLA4 and CCR8), but also binds to tumor microenvironmental effector T cells (e.g., through PD1), which can not only induce Treg cells in the tumor microenvironment to secrete IFNγ, but also to a certain extent activate intratumoral effector T cells (e.g., CD8 + T cells and Tcon).

[0227] (5) In some cases, certain preferred mutations or certain preferred structural features can further strengthen the target antigen dependence of the Treg cell-targeting IL12 fusion protein, helping to achieve a balance between therapeutic effect and safety.

[0228] II Definitions

[0229] Unless otherwise defined, all technical terms, symbols, and other scientific or other terminology used in the present disclosure are intended to have the meanings commonly understood by those of ordinary skill in the art to which the present disclosure belongs. In some cases, terms with commonly understood meanings are defined in this disclosure for clarity and / or for ready reference, and inclusion of such definitions herein does not necessarily preclude an otherwise commonly understood term from being used by those of ordinary skill in the art. Numerous techniques and procedures are described or referenced in the present disclosure, and these techniques and procedures are well understood and commonly employed by those of ordinary skill in the art.

[0230] The singular forms "a," "an," and "the" include plural referents unless otherwise specified. For example, the term "a cell" includes one or more cells, including mixtures thereof.

[0231] The present disclosure uses "A and / or B" to include all of the following alternatives: "A," "B," "A or B," and "A and B" unless otherwise specified.

[0232] Unless otherwise specified, the present disclosure uses numerical or letter designations of headings (e.g., (1), (2), (3), etc., or (a), (b), (c), etc.) are merely for ease of reading and should not be interpreted as a limitation on the order of elements or method steps.

[0233] Unless otherwise indicated, the disclosure in the use of expressions such as "first", "second", "third" and "fourth", or like A and A', B and B', C and C', when used in the description of modules, units or peptides, etc., only serves to distinguish between more than one module, unit or peptide, etc., and is not to be construed as implying a specific order or direction, nor does it imply that the more than one module, unit or peptide is the same or different.

[0234] The term "about" has its ordinary meaning, i.e. approximately. If the degree of approximation is not clear from the context, "about" means within plus or minus 10% of the value provided, or rounded to the nearest significant figure, including the value provided in all cases. In the case of a range being provided, the range includes the boundary values.

[0235] The term "comprise", "comprising", "contain", "containing", "have", "having" are synonymous with "include", "including", "comprise", "comprising" and are inclusive or open-ended and do not exclude additional, unrecited elements, ingredients, or steps. The term "consisting of" is a closed term, which excludes any element, ingredient or step not recited in "consisting of". The term "consisting essentially of" does not exclude an unrecited element, ingredient or step that does not materially affect the characteristics of the subject matter.

[0236] The term "fusion protein" refers to a protein comprising at least one hybrid peptide comprising protein domains from at least two different proteins. The protein domains from at least two different proteins can be directly connected by a peptide bond, or indirectly connected by a linker or other domain. Fusion proteins can be produced by any method known in the art. For example, fusion proteins provided by the disclosure can be produced by recombinant protein expression and purification, which is particularly suitable for fusion proteins comprising 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)), which is incorporated by reference herein in its entirety.

[0237] In the present disclosure, when referring to a "amino acid mutation" of a protein, polypeptide, or fragment thereof, it means a substitution, deletion, or insertion of an amino acid residue that is typically found at that position. The "wild-type amino acid residue" typically refers to the amino acid residue that the typical native form of the protein, polypeptide should have at that position. Typically, such typical native form is one that is agreed upon in the art. In some aspects, the "amino acid mutation" can be generated using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like. Methods of altering amino acid side chain groups by means other than genetic engineering, such as chemical modification, are also contemplated to be useful.

[0238] In some aspects, a "substitution mutation" includes a substitution with an amino acid selected from the twenty standard amino acids, and also includes a substitution with a non-naturally occurring amino acid or a naturally occurring amino acid derivative of the foregoing twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). In some embodiments, the substitution mutation can be a conservative amino acid residue substitution or a non-conservative amino acid residue substitution.

[0239] A "conservative amino acid residue substitution" refers to the replacement of an amino acid residue by another amino acid residue of the same class, where the same class is defined by common physico-chemical amino acid side chain properties and high frequencies of substitution in homologous proteins found in nature, e.g., as determined by a standard Dayhoff frequency exchange matrix or a BLOSUM matrix. Illustratively, amino acids can be grouped into six major classes according to their different side chain groups, including: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gin, Glu); Class IV (His, Arg, Lys); Class V (Ile, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For example, replacing Asp with another Class III residue such as Asn, Gin, or Glu is a conservative substitution.

[0240] The term "non-conservative amino acid residue substitution" refers to the replacement of an amino acid residue in 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 Gin.

[0241] " / " is used in the present disclosure to connect mutations, and means "and". For example, "E59 / F60" means that the polypeptide and / or protein has a mutation at the position corresponding to position 59 and position 60 of the reference sequence. For example, "E59A / F60A" means that the polypeptide and / or protein has a mutation at the position corresponding to position 59 of the reference sequence from E to A, and at the position corresponding to position 60 of the reference sequence from F to A.

[0242] “Mutant,” “mutated,” and “wild type” are a set of relative concepts in the present disclosure. A mutant is understood to be a protein or polypeptide that contains an amino acid mutation. While a wild type is identical to its corresponding mutant in all other aspects, except that the wild type form has the wild type amino acid at each amino acid position of the mutant. For example, if the mutant is a full length form with a signal peptide, then its wild type is a full length form. If the mutant is a processed mature form without a signal peptide, then its corresponding wild type is also a processed mature form. If its mutant is fused to another polypeptide, or conjugated to another substance, then its wild type is also the form fused to another polypeptide or conjugated to another substance.

[0243] The term "sequence identity" refers to the extent (percentage) of amino acid / nucleic acid identity between two sequences at equivalent positions when the two sequences are optimally aligned. During alignment, gaps can be introduced to maximize the percentage of sequence identity, if necessary, but any conservative substitutions are not considered part of the sequence identity. To determine the percentage of sequence identity, alignment can be achieved by techniques known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters to be used in measuring alignment, including any algorithms needed to achieve maximal alignment throughout the length of the sequences being compared.

[0244] In the present disclosure, “specifically binds” means a KD value of no more than 1E-6 M, 1E-7 M, 1E-8 M, 1E-9 M, 1E-10 M, 1E-11 M, or 1E-12 M to the target antigen. In the present disclosure, KD = Koff / Kon, where Koff represents the dissociation rate constant and Kon represents the association rate constant. KD values can be determined using methods known in the art. For example, measuring affinity in solution using a biosensor system such as a Biacore system to measure surface plasmon resonance, or by solution equilibrium titration (SET). In some aspects, a ligand (especially a ligand extracellular domain), an antibody, or fragment thereof specifically binds to a target antigen.

[0245] In the present disclosure, the term “antibody” includes, but is not limited to, a typical four-chain antibody, a heavy chain antibody (HCAb), and an immunoglobulin new antigen receptor (IgNAR).

[0246] A typical "four-chain antibody" refers to an immunoglobulin consisting of two heavy chains (HC) and two light chains (LC); a heavy chain refers to a polypeptide chain consisting of, in the direction of the N-terminal to C-terminal, a heavy chain variable region, a heavy chain constant region CH1 domain, a hinge region, a heavy chain constant region CH2 domain, a heavy chain constant region CH3 domain; and, when the full-length antibody is of an IgE isotype, optionally further comprising a heavy chain constant region CH4 domain; a light chain is a polypeptide chain consisting of, in the direction of the N-terminal to C-terminal, a light chain variable region and a light chain constant region; the heavy chains are connected to each other, and the heavy chains are connected to the light chains by disulfide bonds, forming a "Y" shape structure.

[0247] A "heavy-chain antibody" refers to an antibody consisting only of heavy chains, and lacking light chains, produced by Camelidae family species, which includes camels, llamas and alpacas. Typically, its heavy chain consists of, in the direction of the N-terminal to C-terminal, a heavy chain variable region, a hinge region, a heavy chain constant region CH2 domain and a heavy chain constant region CH3 domain. Among them, the heavy chain variable region of the heavy-chain antibody is called VHH (variable domain of heavy chain of heavy-chain antibody).

[0248] An "immunoglobulin new antigen receptor" refers to an antibody class from the immune spectrum of sharks, which consists of a homodimer of one variable new antigen receptor (VNAR) domain and five constant new antigen receptor (CNAR) domains.

[0249] In the present disclosure, an "antibody fragment" refers to a fragment of an antibody that has an antigen-binding function. In some aspects, 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 papain digestion of a full length antibody. The heavy chain fragment of an Fab fragment (VH-CH1) and the light chain fragment of an Fab fragment (VL-CL) are connected by a linker for it to be produced as a single protein chain, forming a single chain Fab (scFab). Furthermore, F(ab')2, which is a dimer of Fab', is a bivalent antibody fragment, produced by pepsin digestion of a complete antibody under the disulfide linkages of the hinge region. F(ab')2 can be reduced under neutral conditions to break disulfide linkages in the hinge region, thereby converting the F(ab')2 dimer into an Fab' monomer. Fab' monomers are essentially Fab fragments with the hinge region. Fv fragments consist of the VL and VH domains of a single arm of an antibody. The two domains of the Fv fragment, VL and VH, can be coded by separate genes, or can be joined, using a linker, to create a single protein chain that pairs the VL and VH domains to form a single chain Fv (scFv) in which the VL and VH regions pair to form an antigen binding site.

[0250] A "variable region" refers to the domain of the heavy or light chain of an antibody that is at the amino terminus that recognizes and binds to an antigen, the makeup and arrangement of the amino acids in this segment determine the specificity of the antibody for an antigen. The heavy chain variable domain can be referred to as "VH" and the light chain variable domain can be referred to as "VL". The variable regions of the heavy and light chains each consist of three complementarity-determining regions (CDRs) connected by four framework regions (FRs), also known as hypervariable regions.

[0251] A "complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is hypervariable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. CDRs located within the variable domain of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while CDRs located within the variable domain of the light chain are referred to as LCDR1, LCDR2, and LCDR3. The precise amino acid sequence boundaries of each CDR in a given light chain variable region or heavy chain variable region amino acid sequence can be determined using any of a number of well-known antibody CDR assignment schemes, or combinations thereof, including, for example: Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (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)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering with a large number of crystal structures. Unless otherwise specified, in the present application, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways.

[0252] In some aspects, the CDR regions of the antibody can be defined by the manner described in Table A.

[0253] Table A. CDR Description

[0254] In some aspects, the CDR regions of an antibody can include "extended CDRs" as follows: 24-36 or 24-34 in the VL (LCDR1), 46-56 or 50-56 (LCDR2), and 89-97 or 89-96 (LCDR3), and 26-35 in the VH (HCDR1), 50-65 or 49-65 (HCDR2), and 93-102, 94-102, or 95-102 (HCDR3). For each of these definitions, the variable domain residues are numbered according to Kabat et al. supra.

[0255] The expression "variable domain residue numbering in Kabat" or "amino acid position numbering in Kabat" and variants thereof, refers to the numbering system used in the antibody compilation of Kabat et al. supra for heavy chain variable domains or light chain variable domains. Using this numbering system, the actual linear amino acid sequence can include fewer or additional amino acids corresponding to a shortening of, or insertion into, the FR or HVR of the variable domain. For example, a heavy chain variable domain can include a single amino acid inserted at residue 52 of H2 (residue 52a according to Kabat) and residues inserted at residue 82 of a heavy chain FR (e.g., residues 82a, 82b, and 82c according to Kabat, etc.). The Kabat residue numbering for a given antibody can be determined by alignment of the antibody sequence with the "standard" Kabat numbered sequence through homology regions.

[0256] Unless otherwise specified, the numbering of residues in an antibody heavy chain is that of the EU index, as in Kabat et al. supra. "EU index in Kabat" refers to the residue numbering of the human IgGl EU antibody.

[0257] In some aspects, the CDR regions of an antibody can be defined using online tools. The online tools include, but are not limited to:

[0258] abYsis:

[0259] http: / / www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi)

[0260] IMGT / DomainGapAlign:

[0261] https: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi)

[0262] The term "hinge region" denotes the portion of an immunoglobulin heavy chain polypeptide that serves to connect the CH1 domain and the CH2 domain. Hinge domains are structurally diverse, varying in sequence and length between immunoglobulin classes and subclasses. Heavy chains are disulfide-bonded to one another via the hinge region. According to crystallographic studies, the immunoglobulin hinge region can be further subdivided, both structurally and functionally, into three regions: upper hinge, core, and lower hinge. See Shin et al., Immunological Reviews 130:87 (1992). The upper hinge includes those amino acids from the carboxy terminus of CH1 to the first residue in the hinge that restricts movement, which is usually the first cysteine residue that forms an interchain disulfide bond between two heavy chains. The length of the upper hinge region correlates with the segmental flexibility of the antibody. The core hinge region contains the inter-heavy chain disulfide bonds. The lower hinge region connects the amino-terminal end of the CH2 domain and includes residues therein.

[0263] The "Knob-into-Hole" technology is described, for example, in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Generally, this approach involves introducing a protuberance ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote formation of a heterodimer and impede formation of a homodimer. The protuberance is constructed by substituting a larger side chain (e.g., tyrosine or tryptophan) for a small amino acid side chain from the interface of the first polypeptide. A compensating cavity of the same or similar size as the protuberance is created in the interface of the second polypeptide by substituting a smaller amino acid side chain (e.g., alanine or threonine) for a large amino acid side chain. The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g., by site-specific mutagenesis or by peptide synthesis. In a particular embodiment, the knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole modification comprises the amino acid substitutions T366S, L368A, and Y407V in the other of the two subunits of the Fc domain. In another particular embodiment, the subunit of the Fc domain comprising the knob modification additionally comprises the amino acid substitution S354C, and the subunit of the Fc domain comprising the hole modification additionally comprises the amino acid substitution Y349C. Introduction of these two cysteine residues results in the formation of a disulfide bond between the two subunits of the Fc region, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0264] The term "EC50" refers to the concentration of an active substance at which 50% of the maximal response is induced (i.e., halfway between baseline and maximal response).

[0265] The term "effector T cell (Teff)" refers to a non-regulatory T cell and includes T helper cells and cytotoxic T cells. In some embodiments, Teff includes Tcon and CD8 + T cells. In the present disclosure, Tcon is synonymous with CD4 + Tcon is used interchangeably, and in some embodiments, Tcon is defined as CD45 + CD3 + CD4 + FOXp3 - or CD45 + CD3 + CD4 + CD25 - . In some embodiments, CD8 + T cells are defined as CD45 + CD3 + CD8 + .

[0266] The term "regulatory T cell (Treg)" or "Tregs" refers to a CD4 + T cell that is capable of suppressing the response of other T cells, including but not limited to effector T cells (Teff). In some embodiments, Treg cells can be defined as CD4 + CD25 + T cells, CD4 + FoxP3 + T cells or CD4 + CD25 + FoxP3 + T cells.

[0267] The term "Thl-like Treg cell" refers to a T-bet + IFNγ + FoxP3 + Treg cell. Further description of Thl-like Tregs can be found in reference 4. Reference 4 is incorporated by reference in its entirety.

[0268] The term "fragile Treg cells" is defined as Treg cells that maintain Foxp3 expression but lose immunosuppressive function. More description about fragile Treg cells can be found in reference 11. Reference 11 is incorporated by reference in its entirety.

[0269] The term "effector Treg cells" or "eTreg cells" refers to Treg cells differentiated from nTregs (naive Treg cells) upon stimulation. In some embodiments, effector Treg cells are CD45RA - FOXP3 hi CD25 hi CD4 + More description about eTreg cells can be found in reference 7. Reference 7 is incorporated by reference in its entirety.

[0270] III. Treg cell-targeting IL12 fusion proteins

[0271] In one aspect, the present disclosure provides Treg cell-targeting IL12 fusion proteins. In some embodiments, the Treg cell-targeting IL12 fusion protein comprises one or more Treg cell-targeting moieties that specifically bind to a Treg cell surface molecule and IL12 linked directly or indirectly, and the IL12 comprises one or more attenuated activity mutations compared to wild-type IL12.

[0272] Targeting moieties

[0273] A moiety should be understood in the present disclosure as a unit or element with certain function, and should not be limited to a certain specific structure. In the present disclosure, a targeting moiety should be understood as a moiety that specifically binds to a target antigen.

[0274] In some aspects, the targeting moiety comprises, but is not limited to, a ligand or fragment thereof that specifically binds to a target antigen, an antibody or fragment thereof. In some embodiments, the targeting moiety comprises an antibody fragment that specifically binds to a target antigen, including but not limited to Fab, Fab', F(ab')2, scFab, Fv, scFv, and VHH. In some embodiments, the targeting moiety comprises a ligand fragment that specifically binds to a target antigen, including a ligand extracellular domain.

[0275] Treg cell-targeting moieties

[0276] A Treg cell-targeting moiety refers to a moiety that specifically binds to a Treg cell surface molecule.

[0277] In some embodiments, the Treg cell surface molecule includes, but is not limited to, a target antigen selected from the group consisting of CTLA4, CCR8, CCR4, CCR10, CD25, GITR, OX-40, ICOS and 4-1BB.

[0278] In some aspects, 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, preferably, 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.

[0279] CTLA4 targeting module

[0280] In the present disclosure, the CTLA4 targeting module includes, but is not limited to, its ligand CD80, CD86 or ligand fragment (e.g. CD80 extracellular domain or CD86 extracellular domain), CTLA4 antibody or antibody fragment.

[0281] In some aspects, the CTLA4 binding module includes an anti-CTLA4 antibody or fragment thereof. In some specific embodiments, the CTLA4 antibody or 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 an antibody fragment of the foregoing antibodies, or an antibody or antibody fragment derived therefrom. In some specific embodiments, the CTLA4 antibody is selected from the CTLA4 antibodies and fragments thereof disclosed in patents CN116535506A, CN116478289A, CN111153999B, CN106188297B, WO2019152413A1 (the patents are incorporated by reference in their entirety into the present disclosure), or antibodies and fragments thereof derived therefrom.

[0282] In some embodiments, the antibody fragment that specifically binds to CTLA-4 includes Fab, scFab, Fab', (Fab')2, Fv, scFv or VHH.

[0283] In some embodiments, the Fab, scFab, Fab', (Fab')2, Fv and scFv that specifically bind to CTLA4 include VH and VL as shown below:

[0284] (1) the VH comprises a HCDR1 as set forth in SEQ ID NO: 79, a HCDR2 as set forth in SEQ ID NO: 80, and a HCDR3 as set forth in SEQ ID NO: 81; the VL comprises a LCDR1 as set forth in SEQ ID NO: 85, a LCDR2 as set forth in SEQ ID NO: 86, and a LCDR3 as set forth in SEQ ID NO: 87; or,

[0285] the VH comprises a HCDR1 as set forth in SEQ ID NO: 82, a HCDR2 as set forth in SEQ ID NO: 83, and a HCDR3 as set forth in SEQ ID NO: 81; the VL comprises a LCDR1 as set forth in SEQ ID NO: 88, a LCDR2 as set forth in SEQ ID NO: 86, and a LCDR3 as set forth in SEQ ID NO: 87; or,

[0286] the VH comprises a HCDR1 as set forth in SEQ ID NO: 82, a HCDR2 as set forth in SEQ ID NO: 83, and a HCDR3 as set forth in SEQ ID NO: 84; the VL comprises a LCDR1 as set forth in SEQ ID NO: 88, a LCDR2 as set forth in SEQ ID NO: 86, and a LCDR3 as set forth in SEQ ID NO: 87;

[0287] (2) the VH comprises a HCDR1 as set forth in SEQ ID NO: 89, a HCDR2 as set forth in SEQ ID NO: 90, and a HCDR3 as set forth in SEQ ID NO: 91; the VL comprises a LCDR1 as set forth in SEQ ID NO: 92, a LCDR2 as set forth in SEQ ID NO: 93, and a LCDR3 as set forth in SEQ ID NO: 94;

[0288] (3) the VH comprises a HCDR1 as set forth in SEQ ID NO: 95, a HCDR2 as set forth in SEQ ID NO: 96, and a HCDR3 as set forth in SEQ ID NO: 97; the VL comprises a LCDR1 as set forth in SEQ ID NO: 98, a LCDR2 as set forth in SEQ ID NO: 99, and a LCDR3 as set forth in SEQ ID NO: 100;

[0289] (4) the VH comprises HCDR1 as set forth in SEQ ID NO: 134, HCDR2 as set forth in SEQ ID NO: 135, and HCDR3 as set forth in SEQ ID NO: 136; and the VL comprises LCDR1 as set forth in SEQ ID NO: 137, LCDR2 as set forth in SEQ ID NO: 138, and LCDR3 as set forth in SEQ ID NO: 139;

[0290] (5) the VH comprises HCDR1 as set forth in SEQ ID NO: 149, HCDR2 as set forth in SEQ ID NO: 150, and HCDR3 as set forth in SEQ ID NO: 151; and the VL comprises LCDR1 as set forth in SEQ ID NO: 152, LCDR2 as set forth in SEQ ID NO: 153, and LCDR3 as set forth in SEQ ID NO: 154;

[0291] (6) the VH comprises HCDR1 as set forth in SEQ ID NO: 155, HCDR2 as set forth in SEQ ID NO: 156, and HCDR3 as set forth in SEQ ID NO: 157; and the VL comprises LCDR1 as set forth in SEQ ID NO: 158, LCDR2 as set forth in SEQ ID NO: 159, and LCDR3 as set forth in SEQ ID NO: 160;

[0292] (7) the VH comprises HCDR1 as set forth in SEQ ID NO: 161, HCDR2 as set forth in SEQ ID NO: 162, and HCDR3 as set forth in SEQ ID NO: 163; and the VL comprises LCDR1 as set forth in SEQ ID NO: 164, LCDR2 as set forth in SEQ ID NO: 165, and LCDR3 as set forth in SEQ ID NO: 166;

[0293] (8) the VH comprises HCDR1 as set forth in SEQ ID NO: 167, HCDR2 as set forth in SEQ ID NO: 168, and HCDR3 as set forth in SEQ ID NO: 169; and the VL comprises LCDR1 as set forth in SEQ ID NO: 170, LCDR2 as set forth in SEQ ID NO: 171, and LCDR3 as set forth in SEQ ID NO: 172;

[0294] (9) the VH comprises HCDR1 as set forth in SEQ ID NO: 173, HCDR2 as set forth in SEQ ID NO: 174, and HCDR3 as set forth in SEQ ID NO: 175; and the VL comprises LCDR1 as set forth in SEQ ID NO: 176, LCDR2 as set forth in SEQ ID NO: 177, and LCDR3 as set forth in SEQ ID NO: 178;

[0295] (10) the VH comprises HCDR1 as set forth in SEQ ID NO: 179, HCDR2 as set forth in SEQ ID NO: 180, and HCDR3 as set forth in SEQ ID NO: 181; and the VL comprises LCDR1 as set forth in SEQ ID NO: 182, LCDR2 as set forth in SEQ ID NO: 183, and LCDR3 as set forth in SEQ ID NO: 184;

[0296] (11) the VH comprises HCDR1 as set forth in SEQ ID NO: 188, HCDR2 as set forth in SEQ ID NO: 189, and HCDR3 as set forth in SEQ ID NO: 190; and the VL comprises LCDR1 as set forth in SEQ ID NO: 191, LCDR2 as set forth in SEQ ID NO: 192, and LCDR3 as set forth in SEQ ID NO: 193;

[0297] (12) the VH comprises HCDR1 as set forth in SEQ ID NO: 194, HCDR2 as set forth in SEQ ID NO: 195, and HCDR3 as set forth in SEQ ID NO: 196; and the VL comprises LCDR1 as set forth in SEQ ID NO: 197, LCDR2 as set forth in SEQ ID NO: 198, and LCDR3 as set forth in SEQ ID NO: 199;

[0298] (13) the VH comprises HCDR1 as set forth in SEQ ID NO: 200, HCDR2 as set forth in SEQ ID NO: 201, and HCDR3 as set forth in SEQ ID NO: 202; and the VL comprises LCDR1 as set forth in SEQ ID NO: 203, LCDR2 as set forth in SEQ ID NO: 204, and LCDR3 as set forth in SEQ ID NO: 205;

[0299] (14) the VH comprises HCDR1 as set forth in SEQ ID NO:206, HCDR2 as set forth in SEQ ID NO:207, and HCDR3 as set forth in SEQ ID NO:208; and the VL comprises LCDR1 as set forth in SEQ ID NO:209, LCDR2 as set forth in SEQ ID NO:210, and LCDR3 as set forth in SEQ ID NO:211; or,

[0300] (15) the VH comprises HCDR1 as set forth in SEQ ID NO:212, HCDR2 as set forth in SEQ ID NO:213, and HCDR3 as set forth in SEQ ID NO:214; and the VL comprises LCDR1 as set forth in SEQ ID NO:215, LCDR2 as set forth in SEQ ID NO:216, and LCDR3 as set forth in SEQ ID NO:217.

[0301] In some embodiments, the VHH that specifically binds to CTLA4 comprises:

[0302] (1) HCDR1 as set forth in SEQ ID NO: 101, HCDR2 as set forth in SEQ ID NO: 102, and HCDR3 as set forth in SEQ ID NO: 103;

[0303] (2) HCDR1 as set forth in SEQ ID NO: 104, HCDR2 as set forth in SEQ ID NO: 105, and HCDR3 as set forth in SEQ ID NO: 106;

[0304] (3) HCDR1 as set forth in SEQ ID NO: 107, HCDR2 as set forth in SEQ ID NO: 108, and HCDR3 as set forth in SEQ ID NO: 109;

[0305] (4) HCDR1 as set forth in SEQ ID NO: 110, HCDR2 as set forth in SEQ ID NO: 111, and HCDR3 as set forth in SEQ ID NO: 112;

[0306] (5) HCDR1 as set forth in SEQ ID NO: 113, HCDR2 as set forth in SEQ ID NO: 114, and HCDR3 as set forth in SEQ ID NO: 115;

[0307] (6) HCDR1 as set forth in SEQ ID NO: 116, HCDR2 as set forth in SEQ ID NO: 117, and HCDR3 as set forth in SEQ ID NO: 118;

[0308] (7) HCDR1 as depicted in SEQ ID NO: 119, HCDR2 as depicted in SEQ ID NO: 120 and HCDR3 as depicted in SEQ ID NO: 121 ;

[0309] (8) HCDR1 as depicted in SEQ ID NO: 122, HCDR2 as depicted in SEQ ID NO: 123 and HCDR3 as depicted in SEQ ID NO: 124;

[0310] (9) HCDR1 as depicted in SEQ ID NO: 125, HCDR2 as depicted in SEQ ID NO: 126 and HCDR3 as depicted in SEQ ID NO: 127;

[0311] (10) HCDR1 as depicted in SEQ ID NO: 128, HCDR2 as depicted in SEQ ID NO: 129 and HCDR3 as depicted in SEQ ID NO: 130;

[0312] (11) HCDR1 as depicted in SEQ ID NO: 131, HCDR2 as depicted in SEQ ID NO: 132 and HCDR3 as depicted in SEQ ID NO: 133;

[0313] (12) HCDR1 as depicted in SEQ ID NO: 140, HCDR2 as depicted in SEQ ID NO: 141 and HCDR3 as depicted in SEQ ID NO: 142;

[0314] (13) HCDR1 as depicted in SEQ ID NO: 143, HCDR2 as depicted in SEQ ID NO: 144 and HCDR3 as depicted in SEQ ID NO: 145;

[0315] (14) HCDR1 as depicted in SEQ ID NO: 146, HCDR2 as depicted in SEQ ID NO: 147 and HCDR3 as depicted in SEQ ID NO: 148; or,

[0316] (15) HCDR1 as depicted in SEQ ID NO: 185, HCDR2 as depicted in SEQ ID NO: 186 and HCDR3 as depicted in SEQ ID NO: 187.

[0317] In some embodiments, the Fab, scFab, Fab', (Fab')2, Fv and scFv that specifically bind to CTLA4 comprise a VH and a VL as set forth below:

[0318] (1) the VH comprises a sequence as set forth in SEQ ID NO: 31, and the VL comprises a sequence as set forth in SEQ ID NO: 34; or,

[0319] the VH comprises a sequence as set forth in SEQ ID NO: 32, and the VL comprises a sequence as set forth in SEQ ID NO: 35; or,

[0320] the VH comprises a sequence as set forth in SEQ ID NO: 33, and the VL comprises a sequence as set forth in SEQ ID NO: 35;

[0321] (2) the VH comprises a sequence as set forth in SEQ ID NO: 36, and the VL comprises a sequence as set forth in SEQ ID NO: 37;

[0322] (3) the VH comprises a sequence as set forth in SEQ ID NO: 38, and the VL comprises a sequence as set forth in SEQ ID NO: 39;

[0323] (4) the VH comprises a sequence as set forth in SEQ ID NO: 51, and the VL comprises a sequence as set forth in SEQ ID NO: 52;

[0324] (5) the VH comprises a sequence as set forth in SEQ ID NO: 56, and the VL comprises a sequence as set forth in SEQ ID NO: 57;

[0325] (6) the VH comprises a sequence as set forth in SEQ ID NO: 58, and the VL comprises a sequence as set forth in SEQ ID NO: 59;

[0326] (7) the VH comprises a sequence as set forth in SEQ ID NO: 60, and the VL comprises a sequence as set forth in SEQ ID NO: 61;

[0327] (8) the VH comprises a sequence as set forth in SEQ ID NO: 62, and the VL comprises a sequence as set forth in SEQ ID NO: 63;

[0328] (9) the VH comprises a sequence as set forth in SEQ ID NO: 64, and the VL comprises a sequence as set forth in SEQ ID NO: 65;

[0329] (10) the VH comprises a sequence as set forth in SEQ ID NO: 66, and the VL comprises a sequence as set forth in SEQ ID NO: 67;

[0330] (11) the VH comprises a sequence as set forth in SEQ ID NO: 69, and the VL comprises a sequence as set forth in SEQ ID NO: 70;

[0331] (12) the VH comprises a sequence as set forth in SEQ ID NO: 71, and the VL comprises a sequence as set forth in SEQ ID NO: 72;

[0332] (13) the VH comprises a sequence as set forth in SEQ ID NO: 73, and the VL comprises a sequence as set forth in SEQ ID NO: 74;

[0333] (14) the VH comprises a sequence as set forth in SEQ ID NO: 75, and the VL comprises a sequence as set forth in SEQ ID NO: 76;

[0334] (15) the VH comprises a sequence as set forth in SEQ ID NO: 77, and the VL comprises a sequence as set forth in SEQ ID NO: 78; or,

[0335] (16) an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to the VH and / or VL as set forth in any one of groups (1)-(15).

[0336] In some embodiments, the VHH that specifically binds to CTLA4 comprises:

[0337] (1) a sequence as set forth in any one of SEQ ID NOs: 40-50, 53-55, 68;

[0338] (2) an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to the sequence as set forth in group (1).

[0339] CCR8 targeting moiety

[0340] In the present disclosure, the CCR8 targeting moiety includes, but is not limited to, CCR8 ligand CCL1, CCL8, CCL16, CCL18, or a fragment of the ligand (e.g., CCL1 extracellular domain, CCL8 extracellular domain, CCL16 extracellular domain, CCL18 extracellular domain), an anti-CCR8 antibody, or a fragment thereof.

[0341] In some aspects, the CCR8 targeting moiety comprises an anti-CCR8 antibody or fragment thereof. In some embodiments, 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, GB2101, 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 an antibody fragment of the foregoing, or an antibody or antibody fragment derived therefrom. In some embodiments, the CCR8 antibody or fragment thereof is selected from the following patent published CCR8 antibodies and fragments thereof, or antibodies and fragments derived therefrom (the patents are incorporated by reference in their entirety into the present disclosure):

[0342] CN117777291A, WO2024088346A1, WO2024086684A2, WO2024077239A1, WO2024076514A1, WO2024062082A1, WO2024059909A1, WO2024062072A2, WO2024062019A1, WO2024062076A1, WO2024052517A2, CN117285627A, WO2024040216A2, WO2024027823A1, WO2024008110A1, WO2023230473A1, WO2023219147A1, WO2023208182A1, WO2023208203A1, WO2023193732A1, WO2023137466A2, WO2023116880A1, WO2023098888A1, WO2023020621A1, CN117425677A, CN117693527A, CN117616046A, CN117858899A, CN117597364A, WO2022216965A1, CN116888156A, CN116917320A, CN116964091A, WO2022117569A1, CN116589583A, CN117098561A, WO2022003156A1, WO2022004760A1, CN117653725A, CN115768792A, CN115551895A, CN117964757A, CN114929278A, CN113260381A, CN110835371A, CN110573180B, WO2018112032A1, WO2007044756A2.

[0343] In some embodiments, the antibody fragment that specifically binds CCR8 comprises a Fab, scFab, Fab', (Fab')2, Fv, scFv, or VHH.

[0344] In some embodiments, the Fab, scFab, Fab', (Fab')2, Fv, and scFv that specifically binds CCR8 comprises a VH and VL as set forth below:

[0345] (1) HCDR1 as depicted in SEQ ID NO: 265, HCDR2 as depicted in SEQ ID NO: 266, and HCDR3 as depicted in SEQ ID NO: 267; the VL comprises LCDR1 as depicted in SEQ ID NO: 268, LCDR2 as depicted in SEQ ID NO: 269, and LCDR3 as depicted in SEQ ID NO: 270; or,

[0346] (2) the VH comprises HCDR1 as depicted in SEQ ID NO: 271, HCDR2 as depicted in SEQ ID NO: 272, and HCDR3 as depicted in SEQ ID NO: 273; the VL comprises LCDR1 as depicted in SEQ ID NO: 274, LCDR2 as depicted in SEQ ID NO: 275, and LCDR3 as depicted in SEQ ID NO: 276.

[0347] In some embodiments, the VHH that specifically binds CCR8 comprises:

[0348] (1) HCDR1 as depicted in SEQ ID NO: 259, HCDR2 as depicted in SEQ ID NO: 260, and HCDR3 as depicted in SEQ ID NO: 261

[0349] (2) HCDR1 as depicted in SEQ ID NO: 262, HCDR2 as depicted in SEQ ID NO: 263, and HCDR3 as depicted in SEQ ID NO: 264; or,

[0350] (3) HCDR1 as depicted in SEQ ID NO: 277, HCDR2 as depicted in SEQ ID NO: 278, and HCDR3 as depicted in SEQ ID NO: 279.

[0351] In some embodiments, the Fab, scFab, Fab', (Fab')2, Fv, and scFv that specifically binds CCR8 comprises a VH and a VL as set forth below:

[0352] (1) the VH comprises a sequence as set forth in SEQ ID NO: 254, and the VL comprises a sequence as set forth in SEQ ID NO: 255;

[0353] (2) the VH comprises a sequence as set forth in SEQ ID NO: 256, and the VL comprises a sequence as set forth in SEQ ID NO: 257; or,

[0354] (3) an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to the VH and / or VL of any one of groups (1)-(2).

[0355] In some embodiments, the VHH that specifically binds to CCR8 comprises:

[0356] (1) the sequence of any one of SEQ ID NOs: 252-253 or 258; or,

[0357] (2) an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to the sequence of group (1).

[0358] CCR4 targeting module

[0359] In the present disclosure, the CCR4 targeting module includes, but is not limited to, CCR4 ligand CCL2, CCL4, CCL5, CCL17, CCL22, or a ligand fragment (e.g., CCL2 extracellular domain, CCL4 extracellular domain, CCL5 extracellular domain, CCL17 extracellular domain, and CCL22 extracellular domain), an anti-CCR4 antibody, or an antibody fragment.

[0360] In some aspects, the CCR4 targeting module comprises an anti-CCR4 antibody or fragment thereof. In some specific embodiments, the anti-CCR4 antibody or fragment thereof is selected from Mogamulizumab, KM 2760, TQB-2619, or GNR-015, or an antibody fragment of the foregoing antibodies, or an antibody or antibody fragment derived therefrom.

[0361] CCR10 targeting module

[0362] In the present disclosure, the CCR10 targeting module includes, but is not limited to, CCR10 ligand CCL27, CCL28, or a ligand fragment (e.g., CCL27 extracellular domain, CCL28 extracellular domain), an anti-CCR10 antibody, or an antibody fragment.

[0363] CD25 targeting module

[0364] In the present disclosure, the CD25 targeting moiety comprises an anti-CD25 antibody or fragment thereof. In some specific embodiments, the anti-CD25 antibody or fragment thereof is selected from dacliximab, Basiliximab, Daclizumab, Inolimomab, Camidanlumab, daclizumab, BA 1106, RO-7296682, vopikitug, 9MW3911, ALD2510, D02, H11E11-2V1, H3F14V2, IBIO-101, INV-1013, INV 321, INV 322, TST 010, 33B3.1, RM-1995, or an antibody fragment of the foregoing, or an antibody or antibody fragment derived therefrom.

[0365] GITR targeting moiety

[0366] In the present disclosure, the GITR targeting moiety comprises, but is not limited to, the GITR ligand GITRL or a ligand fragment (e.g., the GITRL extracellular domain), an anti-GITR antibody or antibody fragment.

[0367] In some aspects, the GITR targeting moiety comprises an anti-GITR antibody or fragment thereof. In some specific embodiments, the anti-GITR antibody or 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 an antibody fragment of the foregoing, or an antibody or antibody fragment derived therefrom.

[0368] OX 40 targeting moiety

[0369] In the present disclosure, the OX40 targeting moiety comprises, but is not limited to, the OX40 ligand OX40L or a ligand fragment (e.g., the OX40L extracellular domain), an anti-OX40 antibody or antibody fragment.

[0370] In some aspects, the OX40-targeting moiety comprises an anti-OX40 antibody or fragment thereof. In some embodiments, the anti-OX40 antibody or fragment thereof is selected from the group consisting of Rocatinlimab, Revdofilimab, Ivuxolimab, Tavolixizumab, Vonlerolizumab, ES-102, INCAGN-1949, MEDI-6469, YH-002, BAT-6026, GEN1055, IMG-007, BMS-986178, EMB09, FS-120, HFB-3010, HLX-51, IBI-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, and antibody fragments of the foregoing, or antibodies or antibody fragments derived therefrom.

[0371] ICOS-targeting moiety

[0372] In the present disclosure, the ICOS-targeting moiety comprises, but is not limited to, the ICOS ligand ICOSL or a ligand fragment (e.g., the ICOSL extracellular domain), an anti-ICOS antibody or antibody fragment.

[0373] In some aspects, the ICOS-targeting moiety comprises an anti-ICOS antibody or fragment thereof. In some embodiments, the anti-ICOS antibody or fragment thereof is selected from the group consisting of Acazicolcept, Izuralimab, Vopratelimab, Alomfilimab, BMS-986226, Feladilimab, KY1055, MEDI-570, and antibody fragments of the foregoing, or antibodies or antibody fragments derived therefrom.

[0374] 4-1BB-targeting moiety

[0375] In the present disclosure, the 4-1BB-targeting moiety comprises, but is not limited to, the 4-1BB ligand 4-1BBL or a ligand fragment (e.g., the 4-1BB extracellular domain), an anti-4-1BB antibody or antibody fragment.

[0376] In some aspects, the 4-1BB targeting moiety comprises an anti-4-1BB antibody or fragment thereof. In some specific embodiments, the anti-4-1BB antibody or 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 an antibody fragment of the foregoing, or an antibody or antibody fragment derived therefrom.

[0377] PD1 targeting moiety

[0378] The Treg cell-targeting IL12 fusion proteins described herein can further comprise a PD1 targeting moiety that specifically binds PD1. The PD1 targeting moieties described herein include, but are not limited to, PD1 ligands PDL1, PDL2, or fragments thereof (e.g., PDL1 ectodomain, PDL2 ectodomain), and anti-PD1 antibodies or fragments thereof.

[0379] In some aspects, the PD1 targeting moiety comprises its ligand PDL1 or PDL2 and fragments thereof. In some specific embodiments, the PD1 targeting moiety comprises a PDL1 ectodomain. More specifically, the PDL1 ectodomain comprises a sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 27. In some specific embodiments, the PDL1 ectodomain comprises the sequence set forth in SEQ ID NO: 27.

[0380] In some specific embodiments, the PD1 targeting moiety is a PDL2 ectodomain. More specifically, the PDL2 ectodomain comprises a sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28. In some specific embodiments, the PDL2 ectodomain comprises the sequence set forth in SEQ ID NO: 28.

[0381] In some embodiments, the PD1 -targeting moiety is a PDL2 extracellular domain. More specifically, the PDL2 extracellular domain comprises a sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 29. In some embodiments, the PDL2 extracellular domain comprises the sequence set forth in SEQ ID NO: 29.

[0382] In some embodiments, the PD1 antibody or fragment thereof is selected from the group consisting of Retifanlimab, Pucotenlimab, Cadonilimab, Serplulimab, Zimberelimab, Penpulimab, Dostarlimab-gxly, Prolgolimab, Tislelizumab, Camrelizumab, Sintilimab, Toripalimab, Cemiplimab-RWLC, Pembrolizumab, Nivolumab, Balstilimab, Finotonlimab, Iparomlimab, Ivonescimab, Enlanxubab (SG-001), Cetrelimab, Ezabenlimab, Genolimzumab, Nofazinlimab, Rilvegostomig, Sasanlimab, Spartalizumab, Tebotelimab, Volrustomig, Budigalimab, ABBV-1882, BAT-1306, Danvilostomig, HX-009, IBI363, Izuralimab, Lomvastomig, Lorigerlimab, LZM-009, Peramprizumab, Peresolimab, Pidilizumab, Rosnilimab, SSGJ-707, Tombestomig, TQB-2868, Vudalimab, Acrixolimab, EMB-02, Fidasimtamab, IAP-0971, JS-201, JS-207, LBL-015, OSE-279, Sabestomig, SAR-445877, TY-101, ZG-005, AK-129, AK-131, ASKG-915, AWT-020, BC008-1A, CC-90006, CTX-8371, Eciskafuspalfa, EMB-09, GNR-051, GS-0151, IBI-321, IBI319, INCA-33890, INCA32459, JNJ-67484703, KY-0118, Latikafusp, LVGN-3616, ONO-4538HSC, ONO-4685, Pimivalimab, RB-0004, RC-148, REMD-532, Reozalimab, SHR-1901, SOT201, SSGJ-705, STW204, Sym-021, SYN-125, VT1093, YH-008, Yinkang001, Zeluvalimab, enlonstobart, Lipustobart or Pradusinstobart, Tobemstomig, or an antibody fragment of the foregoing, or an antibody or antibody fragment derived therefrom.

[0383] In some embodiments, the PD1 antibody or fragment is selected from the PD1 antibodies and fragments thereof disclosed in patent CN111699200B, WO2023205754A (the patents are incorporated by reference in their entirety into the present disclosure), or antibodies and fragments derived therefrom.

[0384] In some embodiments, the antibody fragment that specifically binds to PD1 comprises Fab, scFab, Fab', (Fab')2, Fv, scFv, or VHH.

[0385] In some embodiments, the Fab, scFab, Fab', (Fab')2, Fv, and scFv that specifically bind to PD1 comprise VH and VL as shown below:

[0386] (1) the VH comprises 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 comprises 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;

[0387] (2) the VH comprises 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 comprises 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;

[0388] (3) the VH comprises a HCDR1 as set forth in SEQ ID NO: 246, a HCDR2 as set forth in SEQ ID NO: 247, and a HCDR3 as set forth in SEQ ID NO: 248, and the VL comprises a LCDR1 as set forth in SEQ ID NO: 249, a LCDR2 as set forth in SEQ ID NO: 250, and a LCDR3 as set forth in SEQ ID NO: 251; or,

[0389] (4) the VH comprises a HCDR1 as set forth in SEQ ID NO: 293, a HCDR2 as set forth in SEQ ID NO: 294, and a HCDR3 as set forth in SEQ ID NO: 295, and the VL comprises a LCDR1 as set forth in SEQ ID NO: 296, a LCDR2 as set forth in SEQ ID NO: 297, and a LCDR3 as set forth in SEQ ID NO: 298.

[0390] In some embodiments, the VHH that specifically binds to PD1 comprises:

[0391] (1) a HCDR1 as set forth in SEQ ID NO: 228, a HCDR2 as set forth in SEQ ID NO: 229, and a HCDR3 as set forth in SEQ ID NO: 230; or,

[0392] (2) a HCDR1 as set forth in SEQ ID NO: 231, a HCDR2 as set forth in SEQ ID NO: 232, and a HCDR3 as set forth in SEQ ID NO: 233.

[0393] In some embodiments, the Fab, scFab, Fab', (Fab')2, Fv, and scFv that specifically binds to PD1 comprises a VH and a VL as set forth below:

[0394] (1) the VH comprises a sequence as set forth in SEQ ID NO: 218, and the VL comprises a sequence as set forth in SEQ ID NO: 219;

[0395] (2) the VH comprises a sequence as set forth in SEQ ID NO: 222, and the VL comprises a sequence as set forth in SEQ ID NO: 223;

[0396] (3) the VH comprises a sequence as set forth in SEQ ID NO: 224, and the VL comprises a sequence as set forth in SEQ ID NO: 225;

[0397] (4) the VH comprises a sequence as set forth in SEQ ID NO: 226, and the VL comprises a sequence as set forth in SEQ ID NO: 227; or,

[0398] (5) an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to the VH and / or VL as set forth in any one of groups (1)-(2).

[0399] In some embodiments, the VHH that specifically binds to PD1 comprises:

[0400] (1) a sequence as set forth in SEQ ID NO: 220 or 221; or,

[0401] (2) an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to the sequence as set forth in group (1).

[0402] IL12

[0403] IL12 comprises a p35 subunit and a p40 subunit. The p35 subunit and the p40 subunit can be connected in a manner known in the art, including but not limited to: the p35 subunit and the p40 subunit are connected by forming an interchain disulfide bond, as in native IL12; or, the p35 subunit and the p40 subunit are connected by a linker; or, the p35 subunit and the p40 subunit are respectively connected to other functional modules, for example, the spatial proximity and dimerization of p35 and p40 are achieved by dimerization of Fc. The connection modes of the p35 subunit and the p40 subunit disclosed in WO2022140797A1, WO2022192898A2, WO2021189139A1, WO2021216916A1, WO2020086758A1, WO2020072821A, WO2021212083A2, WO2023070038A2, WO2023070056A2, WO2021236676A1, WO2022156773A1, CN108250303B, WO2022094046A1, WO2023114775A2, WO2023115033A2, and WO2022129313A1 are incorporated herein by reference in their entirety.

[0404] In some specific embodiments, the connection mode is as follows from N-terminus to C-terminus: p40 subunit-linker-p35 subunit.

[0405] In some specific embodiments, the connection mode is as follows from N-terminus to C-terminus: p35 subunit-linker-p40 subunit.

[0406] p35 subunit

[0407] The p35 subunit is the alpha subunit shared by IL-12 and IL-35. The p35 subunit dimerizes with the p40 subunit to form IL-12, and it dimerizes with Ebi3 to form IL-35.

[0408] The p35 subunit described herein can vary in length, including but not limited to: full-length p35 subunit with uncleaved signal peptide, mature p35 after cleavage of the signal peptide, isoforms, and fragments thereof.

[0409] The p35 subunit described herein includes p35 from different species. In some aspects, the p35 subunit is from a vertebrate, 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, cows, sheep). The p35 subunit from vertebrates includes but is 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 caballus (Horse).

[0410] The p35 subunit described herein can be wild type. In some aspects, the sequences of typical wild type human, monkey, mouse, and dog p35 subunits are shown in SEQ ID NOs: 1-4, and the sequence alignment shows their conservation (see FIG. 1A).

[0411] The p35 subunit described in the present disclosure can also be a mutant, which comprises 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. The mutations can be obtained by methods known in the art. Mutations occurring on the p35 subunit and methods for obtaining the mutations are disclosed in patents WO2020072821A2, WO2021067863A2, WO2022094046A1, WO2023004282A2, WO2023043978A2, WO2023279085A1, WO2022155263A2, US20230051304A1, WO2023133540A1, which are incorporated herein by reference in their entirety. The positions of the mutations of the human p35 subunit mutants and the non-human p35 subunit mutants described in the present disclosure are determined according to the mature human wild-type IL12 p35 subunit (SEQ ID NO: 1). For example, when the present disclosure refers to a mutation occurring at position 60, it means that the human p35 subunit mutants and the non-human p35 subunit mutants described in the present disclosure have a mutation occurring at the position corresponding to position 60 of the mature human wild-type IL12 p35 subunit (SEQ ID NO: 1), rather than requiring the mutation to occur at the 60th amino acid residue position of the mutant (thus excluding the confusion of positions that can be caused by species differences, signal peptides, truncations, etc.). The correspondence of positions can be determined by sequence alignment, for example, as shown in FIG. 1A.

[0412] In some aspects, the one or more mutations decrease the affinity of the p35 subunit or the corresponding IL12 to bind to its receptor as compared to the wild-type amino acid residue.

[0413] In some aspects, the one or more p35 subunit mutations occur at a position selected from the group consisting of: 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, L123, L124, M125, D126, P127, K128, R129, Q130, I131, Q135, N136, E143, Q146, N151, E153, K158, E162, E163, P164, D165, F166, Y167, K168, T169, K170, I171, K172, L173, I175, R181, I182, R183, V185, T186, D188, R189, V190, S192, Y193, N195, A196, S197, according to the numbering of SEQ ID NO: 1.

[0414] In some aspects, the one or more p35 subunit mutations are mutated from wild type to A.

[0415] In some aspects, the one or more p35 subunit mutations are selected from the group consisting of (the mutation positions 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, E38S, E38T, E38V, E38W, F39A, F39D, F39E, F39G, F39H, F39I, F39K, F39L, F39M, F39N, F39P, F39Q, F39R, F39S, F39T, F39V, F39W, F39Y, Y40A, Y40G, Y40S, P41A, P41D, P41E, P41F, P41G, P41H, P41I, P41K, P41L, P41M, P41N, P41Q, P41R, P41S, P41T, P41V, P41W, P41Y, T43A, S44G, E45A, E46A, D48A, H49A, E50A, D55Q, D55K, N71D, N71Q, C74A, C74S, L75A, N76D, E79A, E79Q, T80A, F82A, N85D, N85Q, L89A, F96A, M97A, L124A, M125A, K128A, K128D, K128E, K128F, K128G, K128H, K128I, K128L, K128M, K128N, K128P, K128Q, K128R, K128S, K128T, K128V, K128W, K128Y, Q130E, Q135E, N136D, E143Q, Q146E, N151D, N151K, E153K, E153Q, K158A, K158D, K158E, K158H, K158R, E162A, E162D, E162H, E162K, E162R, E162Q, E163A, E163D, E163Q, E163H, E163R, E163K, D165A, D165H, D165E, D165R, D165K, D165N, F166A, F166D, F166E, F166G, F166H, F166I, F166K, F166L, F166M, F166N, F166P, F166Q, F166R, F166S, F166T, F166V, F166W, F166Y, Y167A, Y167D, Y167E, Y167F, Y167G, Y167H, Y167I, Y167K, Y167L, Y167N, Y167Q, Y167S, Y167T, Y167V, Y167R, K168A, K168H,K168D, K168E, K168R, K170A, K170H, K170D, K170E, K170R, I171A, I171V, I171E, K172A, K172H, K172D, K172E, K172R, I175A, D188A, R189A, R189K, N195D, N195Q, S197A.

[0416] In some aspects, the p35 subunit of the present disclosure is a p35 subunit of a human or a non-human animal (e.g., a monkey, a dog, or a mouse); the p35 subunit can be a wild-type or a human p35 subunit mutant or a non-human p35 subunit mutant comprising the aforementioned mutations; alternatively, the p35 subunit of the present disclosure has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 1-4.

[0417] p40 subunit

[0418] “IL12 p40”, “p40”, or “IL12B” are used interchangeably in the present disclosure. p40 is the beta subunit shared by IL-12 and IL-23, the p40 subunit dimerizes with the p35 subunit to form the cytokine IL-12, and the p40 subunit dimerizes with the p19 subunit to form the cytokine IL-23.

[0419] The p40 subunit of the present disclosure has diversity in length, which includes but is not limited to: full-length p40 subunit without cleavage of signal peptide, mature p40 after cleavage of signal peptide, isoforms, and fragments thereof.

[0420] The p40 subunit described herein includes p40 from different species of origin. In some aspects, the p40 subunit described herein is 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, cows, sheep). The p40 subunit of vertebrate origin includes, but is 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).

[0421] The p40 subunit described herein can be wild type. In some aspects, the sequences of typical wild type human, monkey, mouse, and dog p40 subunits are shown in SEQ ID NOs: 5-8, and sequence alignment results show that they are conserved (see FIG. IB).

[0422] The p40 subunit described in the present disclosure can also be a mutant, which comprises 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. The mutations can be obtained according to methods known in the art. Mutations occurring on the p40 subunit and methods for obtaining mutations are disclosed in patents WO2022140797A1, WO2022192898A2, WO2020072821A2, WO2021067863A2, WO2021212083A2, WO2022094046A1, WO2023004282A2, WO2023023503A1, WO2023043978A2, WO2023050006A1, WO2023070038A2, WO2023070056A2, US20230051304A1, WO2023133540A1, which are incorporated herein by reference in their entirety. The mutation positions of the human p40 subunit mutants and the non-human p40 subunit mutants described in the present disclosure are determined according to the mature human wild-type IL12 p40 subunit (SEQ ID NO: 5). For example, when the present disclosure refers to a mutation occurring at position 60, it means that the human p40 subunit mutants and the non-human p40 subunit mutants described in the present disclosure 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 that the mutation occurs at the 60th amino acid residue position of the mutant (thus excluding the confusion of positions that can be caused by species differences, signal peptides, truncations, etc.). The correspondence of positions can be determined by sequence alignment, for example, as shown in FIG. IB.

[0423] In some aspects, the one or more mutations decrease the affinity of the p40 subunit or the corresponding IL12 to bind to its receptor as compared to the wild-type amino acid residue.

[0424] In some aspects, the one or more mutations in the p40 subunit occur at a position selected from the group consisting of (the positions of the mutations are 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, L47, T54, 155, Q56, K58, E59, F60, G61, D62, Q65, Y66, E73, K84, E86, D87, G88, I89, W90, D93, K96, D97, K99, E100, K102, N103, K104, F106, E110, N113, Y114, D129, D142, Q144, E156, R159, D161, N162, K163, D166, D170, Q172, D174, A176, C177, P178, A179, A180, E181, S183, P185, E187, H194, K195, L196, K197, N200, S204, F206, R208, D209, D214, N218, N220, N226, Q229, E231, E235, T242, P243, S245, Y246, F247, S248, C252, Q256, K258, S259, K260, R261, E262, K264, D265, D270, N281, Q289, D290, R291, Y292, Y293, E299.

[0425] In some aspects, the one or more mutations in the p40 subunit occur at a position selected from (the positions of the mutations are 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.

[0426] In some aspects, the one or more mutations in the p40 subunit are mutated from wild type to A.

[0427] In some aspects, the one or more p40 subunit mutations are selected from the group consisting of: K6A, W15A, W15H, W15K, W15R, P17A, D18A, D18G, D18N, D18K, E32Q, E33Q, D34N, D34K, Q42E, S43E, S43K, E45K, E45Q, Q56E, K58H, K58W, E59A, E59F, E59H, E59K, E59L, E59Q, E59S, E59D, E59G, E59R, F60A, F60D, F60E, F60K, F60R, F60V, D62N, D62H, D62I, D62N, E73Q, K84A, K84N, K84Q, K84T, K84R, K84E, K84I, K84L, K84V, K84W, K84Y, E86A, E86L, E86R, E86S, E86W, D87A, D87N, D93A, D93E, D93H, D93R, D93W, K96A, K99E, K99Y, E100Q, N103D, N103Q, K104A, N113D, N113Q, Q144E, D161N, D161R, D161S, R159E, K163E, C177A, E187Q, H194A, K195A, K197A, K197D, K197E, K197Q, K197T, K197W, N200D, N200Q, N218Q, Q229E, E235Q, Y246V, Y246F, C252S, Q256N, K258E, K258Q, S259D, K260E, K260Q, R261D, E262Q, K264E, N281D, N281Q, D290A, Y292F, E299Q, according to the numbering of SEQ ID NO: 5.

[0428] In some aspects, the one or more p40 subunit mutations 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 / K84A / K195A; (17) E59A / F60A / K84A / E86A / D93A; (18) E59F / F60A; (19) E59K / F60A; (20) E59L / F60A; (21) E59H / F60A; (22) E59S / F60A; (23) E59A / F60A / K84N; (24) E59A / F60A / K84Q; (25) E59A / F60A / K84T; (26) E59A / F60A / K84R; (27) P17A / D18A / E59A / F60A.

[0429] In some aspects, the p40 subunit is a human or non-human animal (e.g., monkey, dog, or mouse) p40 subunit; the p40 subunit can be wild-type or a human p40 subunit mutant or non-human p40 subunit mutant comprising the foregoing mutations; alternatively, the p40 subunit has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 5-8.

[0430] Fc unit

[0431] In one aspect, the targeted IL12 fusion protein of the present disclosure further comprises a first Fc unit and a second Fc unit, wherein the first Fc unit and the second Fc unit are dimerized.

[0432] In the present disclosure, "Fc unit" or "Fc region" is used to define the carboxy-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In some embodiments, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxy-terminus of the heavy chain. However, an antibody produced by a host cell can undergo post-translational cleavage of one or more, particularly one or two, amino acids from the carboxy-terminus of the heavy chain. Thus, an antibody produced by a host cell via expression of a particular nucleic acid molecule encoding a full-length heavy chain can include a full-length heavy chain, or the antibody can include a cleaved variant of the full-length heavy chain. This can be the case where the last two carboxy-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU numbering). Thus, the carboxy-terminal lysine (Lys447) or the carboxy-terminal glycine (Gly446) and lysine (Lys447) of an Fc region can or can not be present. Unless otherwise indicated herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as set forth by Kabat et al. (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0433] In some aspects, the Fc unit or Fc region described herein comprises a hinge region.

[0434] In some embodiments, the hinge region is an intact hinge region or a fragment thereof.

[0435] In some embodiments, the hinge region of a human IgGl antibody corresponds to amino acid positions 216-230, or positions 226-230, according to the EU numbering as specified in Kabat. The core hinge region of human IgGl contains the sequence Cys-Pro-Pro-Cys, which when dimerized by disulfide bond formation, creates a cyclic octapeptide, which is believed to act as a pivot, conferring flexibility. Conformational changes permitted by the structure and flexibility of immunoglobulin hinge region polypeptide sequences can influence the effector functions of the Fc portion of an antibody. The hinge region of other IgG subclasses can be determined by alignment of the hinge region cysteine residues with the IgGl subclass sequence.

[0436] In some aspects, the Fc unit or Fc region comprises a mutation design that facilitates heterodimer formation. Representative are the "Knob-into-Hole" format proposed by Cater et al.; the Electrostatic Steering format used by Amgen to form Fc-containing heterodimeric formats (US20100286374 Al); the SEEDbody format proposed by Jonathan H. Davis et al. by IgG / Ig chain exchange; the DuoBody platform technology of Genmab to form bispecific molecules; the structural computation and Fc amino acid mutation synthesis of Xencor to form heterodimeric protein formats (mAbs 3:6, 546-557; November / December 2011); the Fc modification method based on charge network of Suzhou Conjugate to form heterodimeric protein formats (CN201110459100.7); and other Fc amino acid change or functional modification means to achieve gene engineering methods to form heterodimeric functional proteins.

[0437] In some aspects, the Fc unit or Fc region comprises a mutation that reduces or eliminates effector function (e.g., ADCC function or ADCP function).

[0438] Reduction or ablation of effector function can be obtained by mutation of the antibody Fc region and is 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., supra); and DAPA (D265A and P329A) (Shields RL., J Biol Chem. 2001, 276(9): 6591-604; US2015 / 0320880). Examples of mutations that reduce or ablate effector function include LALA mutants comprising L234A and L235A mutations in the IgGl Fc amino acid sequence, DAPA (D265A, P329A) (see, e.g., US 6,737,056), N297A, DANAPA (D265A, N297A and P329A), and / or LALADANAPS (L234A, L235A, D265A, N297A and P331S). Additionally, non-limiting exemplary embodiments that reduce or ablate 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.

[0439] In some embodiments, the first Fc unit comprises a Knob mutation and the second Fc unit comprises a Hole mutation.

[0440] In some embodiments, the first Fc unit comprises a Hole mutation and the second Fc unit comprises a Knob mutation.

[0441] In some embodiments, the first Fc unit comprises S354C / T366W mutations and the second Fc unit comprises Y349C / T366S / L368A / Y349C mutations.

[0442] In some embodiments, the first Fc unit comprises L234A / L235A mutations and the second Fc unit comprises L234A / L235A mutations.

[0443] In some embodiments, the first Fc unit comprises the sequence set forth in SEQ ID NO: 299, and the second Fc unit comprises the sequence set forth in 301.

[0444] In some embodiments, the first Fc unit comprises the sequence set forth in SEQ ID NO: 299, and the second Fc unit comprises the sequence set forth in 302.

[0445] In some embodiments, the first Fc unit comprises the sequence set forth in SEQ ID NO: 299, and the second Fc unit comprises the sequence set forth in 303.

[0446] In some embodiments, the first Fc unit comprises the sequence set forth in SEQ ID NO: 300, and the second Fc unit comprises the sequence set forth in 301.

[0447] In some embodiments, the first Fc unit comprises the sequence set forth in SEQ ID NO: 300, and the second Fc unit comprises the sequence set forth in 302.

[0448] In some embodiments, the first Fc unit comprises the sequence set forth in SEQ ID NO: 300, and the second Fc unit comprises the sequence set forth in 303.

[0449] Linker

[0450] In the present disclosure, "linker" is synonymous with "linker peptide", "peptide linker", and refers to a linking unit used to connect two domains. The linker usually has a certain flexibility, and the use of the linker helps the domains to maintain their original spatial conformation and function.

[0451] In some embodiments, the linker is rich in glycine and / or serine. For example, the linker can be selected from (G4S)x, wherein x is an integer of 1 or greater than 1.

[0452] In some embodiments, the linker comprises 1 or more amino acids, typically, about 1-30, 2-24, or 3-15 amino acids.

[0453] Exemplary linkers include, but are not limited to: GGGGSGGGSGGGG, GGPGGGGSGGGSGGGGSG, SGGGGS, GGSG, SGGSG, PGGGSG, SGGGGSGGGGS, GGGGS, GGSGGS, GGGSG, SGGGSG, TGGSG.

[0454] Format

[0455] The IL2 fusion proteins targeting Treg cells described in the present disclosure can adopt any reasonable conformation. The conformation shown in the embodiments of the present disclosure should not be understood as a limitation of the present disclosure.

[0456] The Treg cell targeting module of the Treg cell targeting IL12 fusion proteins described in the present disclosure and IL12 can be connected in any possible way in the art, including but not limited to directly connected by a peptide bond, connected by a linker, facilitated non-covalent connection between peptide chains, etc.

[0457] Figures 5A and 5B disclose possible ways of the IL12 fusion proteins described in the present disclosure. Figure 5A: [ABD1]-[linker]-[p40 subunit]-[linker]-[p35 subunit]-[linker]-[Fc1 with hinge region] and [ABD2]-[linker]-[Fc2 with hinge region] form a heterodimer through Fc1 and Fc2. Figure 5B: [ABD1]-[linker]-[p35 subunit]-[linker]-[Fc1 with hinge region] and [ABD2]-[linker]-[p40 subunit]-[Fc2 with hinge region] form a heterodimer through Fc1 and Fc2. In these two possible ways, IL12 is placed between the Treg cell targeting module and the Fc unit, in the hinge region, and the activity of IL12 can be shielded, which can reduce the non-specific activation of non-target cells.

[0458] Figures 6 and 7 are further illustrations of the structure shown in Figure 5A, which respectively illustrate possible forms of the Treg cell targeting IL12 fusion proteins for the CTLA target and the CCR8 target, but are not exhaustive, and Figures 6 and 7 should not be understood as a limitation of the present disclosure.

[0459] In some embodiments, the Treg cell targeting IL12 fusion protein further comprises a first Fc unit with a hinge region and a second Fc unit, the first Fc unit dimerizes with the second unit to form a dimer, and optionally, the IL12 is located between the Treg cell targeting module or PD1 targeting module and the hinge region of the first Fc unit or the second Fc unit.

[0460] In some embodiments, the Treg cell targeting IL12 fusion protein comprises a dimer consisting of A-[L1] n1 -B-[L2] n2 -C and A'-[L3] n3 -C' wherein:

[0461] A and A' respectively represent a first targeting module and a second targeting module, at least one of A and A' specifically binds to a Treg cell surface molecule;

[0462] B represents IL12 consisting of a p40 subunit - [L4] n4 - a p35 subunit or a p35 subunit - [L4] n4 - a p40 subunit, at least one of said p35 and p40 subunits comprising one or more attenuating mutations that attenuate the affinity of said IL12 to bind to its receptor as compared to wild type;

[0463] C and C’ represent a first and a second Fc unit for dimerization, respectively;

[0464] L1, L2, L3 and L4 represent linkers, n1, n2, n3 and n4 are selected from 0 or 1; and,

[0465] - represents a peptide bond.

[0466] In some embodiments, the Treg cell-targeting IL12 fusion protein comprises a dimer consisting of A - [L1] n1 - B - [L2] n2 - C and A’ - [L3] n3 - B’ - [L4] n4 - C’ wherein:

[0467] A and A’ represent a first and a second targeting moiety, respectively, at least one of said A and A’ specifically binds to a Treg cell surface molecule;

[0468] B represents a p40 subunit and B’ represents a p35 subunit, or, B represents a p35 subunit and B’ represents a p40 subunit; at least one of said p35 and p40 subunits comprising one or more attenuating mutations that attenuate the affinity of said IL12 to bind to its receptor as compared to wild type;

[0469] C and C’ represent a first and a second Fc unit for dimerization, respectively;

[0470] L1, L2, L3 and L4 represent linkers, n1, n2, n3 and n4 are selected from 0 or 1; and,

[0471] - represents a peptide.

[0472] In some embodiments, the first and / or the second targeting moiety comprises a ligand, an antibody or a fragment of said ligand or antibody that specifically binds to said target antigen; optionally, the first and / or the second targeting moiety comprises a ligand extracellular domain (LECD), a Fab, a scFab, a Fab’, a (Fab’)2, a Fv, a scFv or a VHH that specifically binds to said target antigen.

[0473] In some embodiments, the first targeting module and the second targeting module each comprise a Fab.

[0474] In some specific embodiments, the Treg cell-targeting IL12 fusion protein comprises a first peptide, a second peptide, a third peptide, and a fourth peptide, the first peptide comprising VH1-(H1-CH1)-[L1] n1 -B-[L2] n2 -C, the second peptide comprising VH2-(H2-CH1)-[L3] n3 -C', the third peptide comprising VL1-(L1-CL), and the fourth peptide comprising 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).

[0475] In some embodiments, the first targeting module comprises a Fab, and the second targeting module comprises a VHH or a LECD.

[0476] In some specific embodiments, the Treg cell-targeting IL12 fusion protein comprises a first peptide, a second peptide, and a third peptide, the first peptide comprising VH1-(H1-CH1)-[L1] n1 -B-[L2] n2 -C, the second peptide comprising VHH2-[L3] n3 -C' or LECD2-[L3] n3 -C', and the third peptide comprising 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 VHH2 or LECD2 of the second peptide forms the second targeting module.

[0477] In some embodiments, the first targeting module comprises a VHH or a LECD, and the second targeting module comprises a Fab.

[0478] In some specific embodiments, the Treg cell-targeting IL12 fusion protein comprises a first peptide, a second peptide, and a third peptide, the first peptide comprising VHH1-[L1] n1 -B-[L2] n2 -C or LECD1-[L1] n1 -B-[L2] n2 -C, the second peptide comprising VH2-(H2-CH1)-[L3]n3 -C', the third peptide comprises 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).

[0479] In some embodiments, the first targeting module comprises VHH or LECD, and the second targeting module comprises VHH or LECD. In some specific embodiments, the Treg cell-targeting IL12 fusion protein comprises a first peptide and a second peptide, the first peptide comprises VHH1-[L1] n1 -B-[L2] n2 -C or LECD1-[L1] n1 -B-[L2] n2 -C, the second peptide comprises 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; VHH2 or LECD2 of the second peptide forms the second targeting module.

[0480] In the present application, VH1 and H1-CH1 represent the corresponding VH (heavy chain variable region) and CH1 (heavy chain first constant region domain) of the first targeting module, respectively, and VL1 and L1-CL represent the corresponding VL (light chain variable region) and CL (light chain constant region domain) of the first targeting module, respectively.

[0481] In the present application, VH2 and H2-CH1 represent the corresponding VH (heavy chain variable region) and CH1 (heavy chain first constant region domain) of the second targeting module, respectively, and VL2 and L2-CL represent the corresponding VL (light chain variable region) and CL (light chain constant region domain) of the second targeting module, respectively.

[0482] In the present application, VHH1 represents the corresponding VHH (heavy chain variable region of heavy chain antibody) of the first targeting module.

[0483] In the present application, VHH2 represents the corresponding VHH (heavy chain variable region of heavy chain antibody) of the second targeting module.

[0484] In the present application, LECD1 represents the corresponding LECD (ligand extracellular domain) of the first targeting module.

[0485] In the present application, LECD2 represents the corresponding LECD (ligand extracellular domain) of the second targeting module.

[0486] In some embodiments, the first targeting moiety specifically binds to a Treg cell surface molecule, the second targeting moiety specifically binds to a Treg cell surface molecule, optionally, the first targeting moiety specifically binds to CTLA4 and the second targeting moiety specifically binds to CTLA4, or the first targeting moiety specifically binds to CCR8 and the second targeting moiety specifically binds to CCR8, or the first targeting moiety specifically binds to CTLA4 and the second targeting moiety specifically binds to CCR8, or the first targeting moiety specifically binds to CCR8 and the second targeting moiety specifically binds to CTLA4.

[0487] In some embodiments, the first targeting moiety specifically binds to a Treg cell surface molecule, the second targeting moiety specifically binds to PD1, optionally, the first targeting moiety specifically binds to CTLA4 or CCR8, and the second targeting moiety specifically binds to PD1.

[0488] In some embodiments, the first targeting moiety specifically binds to PD1, the second targeting moiety specifically binds to a Treg cell surface molecule, optionally, the first targeting moiety specifically binds to PD1, the second targeting moiety specifically binds to CTLA4 or CCR8.

[0489] In some embodiments, the Treg cell-targeting IL12 fusion protein comprises:

[0490] (1) a first peptide, a second peptide, a third peptide, and a fourth peptide, the first peptide comprising a sequence set forth in SEQ ID NO: 307 or 310, the second peptide comprising a sequence set forth in SEQ ID NO: 308, the third peptide comprising a sequence set forth in SEQ ID NO: 309, and the fourth peptide comprising a sequence set forth in SEQ ID NO: 309;

[0491] (2) a first peptide, a second peptide, and a third peptide, the first peptide comprising a sequence set forth in SEQ ID NO: 307 or 310, the second peptide comprising a sequence set forth in SEQ ID NO: 311, and the third peptide comprising a sequence set forth in SEQ ID NO: 309;

[0492] (3) a first peptide, a second peptide, and a third peptide, the first peptide comprising a sequence set forth in SEQ ID NO: 312 or 313, the second peptide comprising a sequence set forth in SEQ ID NO: 308, and the third peptide comprising a sequence set forth in SEQ ID NO: 309.

[0493] IV Nucleic acid molecules and vectors

[0494] In one aspect, 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.

[0495] The terms "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.

[0496] In some embodiments, the nucleotide sequence is incorporated into an expression cassette or expression vector. It should be understood that 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. Typically, the expression cassette can be inserted into a vector and / or individual for targeting a desired host cell. In some embodiments, 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.

[0497] In some embodiments, the nucleotide sequence is incorporated into an expression vector. Those skilled in the art will understand that the term "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. In some embodiments, the expression vector may be an integration vector.

[0498] In some embodiments, the expression vector can be a viral vector. As will be appreciated by those skilled in the art, the term “viral vector” is used broadly to refer to a nucleic acid molecule (e.g., a transfer plasmid) that includes nucleic acid elements of viral origin that typically facilitate transfer or integration of the nucleic acid molecule into the genome of a cell, or to a viral particle that mediates the transfer of nucleic acid. Viral particles will typically include various viral components, and sometimes also include host cell components in addition to the nucleic acid(s). The term viral vector can refer to a virus or viral particle that is 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 that are derived primarily from viruses. In some embodiments, the viral vector is a bacculorival vector, a retroviral vector, or a lentiviral vector. The term “retroviral vector” refers to a viral vector or plasmid that contains structural and functional genetic elements derived primarily from a retrovirus, or portions thereof. The term “lentiviral vector” refers to a viral vector or plasmid that contains structural and functional genetic elements derived primarily from a lentivirus, which is a genus of retroviruses, or portions thereof, including LTRs.

[0499] Accordingly, the present disclosure also provides vectors, plasmids, or viruses containing one or more nucleic acid molecules encoding the Treg cell-targeting IL12 fusion proteins of the present disclosure. The nucleic acid molecules can be contained within a vector that is capable of directing the expression of the nucleic acid molecule in, for example, a cell that has been transformed / transduced with the vector. Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available or readily prepared by the skilled artisan.

[0500] DNA vectors can be introduced into eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting cells can be found in Sambrook et al. (2012, supra) and other standard molecular biology laboratory manuals, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, nuclear injection, hydrodynamic shock, and infection.

[0501] Viral vectors that can be used in the present disclosure include, for example, baculoviral vectors, retroviral vectors, adenoviral vectors and adeno-associated viral vectors, lentiviral vectors, herpes viruses, simian virus 40 (SV40), and bovine papilloma virus vectors (see, e.g., Gluzman (ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.).

[0502] The nucleic acid molecules provided by the present disclosure can contain sequences that occur naturally, or sequences that are different from those that occur naturally, but encode the same polypeptide (e.g., an antibody) due to the degeneracy of the genetic code. These nucleic acid molecules can be composed of RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA such as DNA produced by phosphoramidite-based synthesis), or combinations or modifications of nucleotides within these types of nucleic acids. Furthermore, the nucleic acid molecules can be double-stranded or single-stranded (e.g., a sense strand or an antisense strand).

[0503] In some embodiments, the expression cassette or expression vector can be one or more. In some specific embodiments, the expression cassette or expression vector is one, in which all the peptide chains of the targeting IL12 fusion protein are encoded in the one expression cassette or expression vector, connected by self-cleaving peptides (e.g., P2A, T2A) between the peptide chains, or connected by IRES between the open reading frames (ORFs) encoding the peptide chains. In some specific embodiments, the expression cassette or expression vector is more than one, each expression cassette or expression vector independently expresses each peptide chain of the targeting IL12 fusion protein, respectively. In some embodiments, the expression cassette or expression vector is more than one, which includes expression cassettes or expression vectors expressing a single peptide chain of the targeting IL12 fusion protein, and expression cassettes or expression vectors expressing multiple peptide chains of the targeting IL12 fusion protein, connected by self-cleaving peptides (e.g., P2A, T2A) between the peptide chains, or connected by IRES between the open reading frames (ORFs) encoding the peptide chains.

[0504] V Recombinant cells and cell cultures

[0505] The present disclosure also provides a cell comprising the aforementioned isolated nucleic acid molecule encoding the nucleotide fragment sequence of one or more polypeptides of the aforementioned Treg cell-targeting IL12 fusion protein.

[0506] In the present disclosure, “cell,” “cell culture,” “cell line” refers not only to the particular subject cell, cell culture, or cell line, but also to the progeny or potential progeny of such a cell, cell culture, or cell line, without regard to the number of transfers or passages. It is also understood that all progeny can not be identical to the parental cell since there can be, for example, mutations that occur during replication. However, such progeny are still included within the scope of the term as used herein, as long as such progeny retain the functional equivalent of the original cell, cell culture, or cell line.

[0507] Introduction of the nucleic acid molecules of the present disclosure into a cell can be performed by methods known to those of skill in the art, for example, viral infection, transfection, conjugation, protoplast fusion, liposome transfection, electroporation, nucleofection, calcium phosphate precipitation, polyethylenimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

[0508] In some embodiments, the nucleic acid molecules can be delivered by viral or non-viral delivery vehicles known in the art. For example, the nucleic acid molecules can be stably integrated in the genome of the recombinant cell, or can replicate as an episome, or be present in the recombinant cell as a minicircle expression vector for transient expression.

[0509] Thus, in some embodiments, the nucleic acid molecules are maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the nucleic acid molecules are stably integrated into the genome of the recombinant cell. Stable integration can be achieved using classical random genome recombination techniques or using more precise techniques such as guide RNA guided CRISPR / Cas9 genome editing, or DNA guided endonuclease genome editing with NgAgo (Natronobacterium gregoryi Argonaute), or TALEN genome editing (transcription activator-like effector nucleases). In some embodiments, the nucleic acid molecules are present in the recombinant cell as a minicircle expression vector for transient expression.

[0510] Thus, in some embodiments, the nucleic acid molecules can be encapsulated in a viral capsid or a lipid nanoparticle, or can be delivered by viral or non-viral delivery means and methods known in the art, such as electroporation. For example, introduction of the nucleic acid into a cell can be achieved by viral transduction. In one non-limiting example, a baculovirus or an adeno-associated virus (AAV) can be engineered to deliver the nucleic acid to the target cell by viral transduction. Several AAV serotypes have been described, and all known serotypes can infect cells from a variety of different tissue types. AAV is capable of transducing a broad range of species and tissues in vivo without signs of toxicity, and it produces a relatively mild innate and adaptive immune response.

[0511] Thus, in some embodiments, a host cell can be genetically engineered (e.g., transduced or transformed or transfected) with, for example, a vector construct of the present application, which can be, for example, a viral vector or a vector for homologous recombination (including a nucleic acid sequence homologous to a portion of the host cell genome), or can be an expression vector for expression of a polypeptide of interest. The host cell can be an untransformed cell or a cell that has been transfected with at least one nucleic acid molecule.

[0512] In some embodiments, the cell is a prokaryotic or eukaryotic cell, such as a bacterium (E. coli), a fungus (yeast), an insect cell, a mammalian cell (e.g., CHO cell line, HEK293 cell line).

[0513] In another aspect, the present disclosure provides a cell culture comprising at least one recombinant cell as described herein and a culture medium. Generally, the culture medium can be any suitable culture medium 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. Accordingly, a cell culture comprising at least one recombinant cell as disclosed herein is also within the scope of the present disclosure. Methods and systems suitable for producing and maintaining cell cultures are known in the art.

[0514] VI Compositions

[0515] The Treg cell-targeting IL12 fusion proteins, nucleic acids, recombinant cells, and / or cell cultures of the present disclosure can be incorporated into compositions, including pharmaceutical compositions. Such compositions generally comprise one or more of the Treg cell-targeting IL12 fusion proteins, nucleic acids, recombinant cells, and / or cell cultures as provided and described herein, and a pharmaceutically acceptable excipient (e.g., carrier). In some embodiments, the pharmaceutical compositions of the present disclosure are formulated for treating, preventing, ameliorating a disease, such as cancer, or for reducing or delaying the onset of a disease.

[0516] Accordingly, one aspect of the present disclosure relates to a pharmaceutical composition comprising: (a) a Treg cell-targeting IL12 fusion protein, a recombinant nucleic acid, a recombinant cell, or a cell culture as disclosed herein; and (b) a pharmaceutically acceptable carrier. “Pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, and the like that are physiologically compatible.

[0517] VII Production Methods

[0518] In another aspect, the present disclosure relates to various methods for producing the Treg cell-targeting IL12 fusion proteins of the present disclosure, the methods comprising: (a) providing one or more recombinant cells as disclosed herein; and culturing the one or more recombinant cells in a culture medium, such that the cells produce a Treg cell-targeting IL12 fusion protein encoded by a recombinant nucleic acid molecule. Accordingly, the Treg cell-targeting IL12 fusion proteins produced by the methods disclosed herein are also within the scope of the present disclosure.

[0519] In some embodiments, the method further comprises isolating and / or purifying the produced Treg cell-targeting IL12 fusion protein. In some embodiments, the method further comprises structurally modifying (e.g., PEGylating) the produced Treg cell-targeting IL12 fusion protein to increase half-life.

[0520] VIII Therapeutic methods

[0521] In one aspect, the present disclosure relates to a method of treating a tumor, the method comprising administering to a subject an effective amount of a Treg cell-targeting IL12 fusion protein provided by the present disclosure, the IL12 fusion protein inducing intratumoral Treg cells to secrete IFNy thereby attenuating or abrogating their immunosuppressive effect.

[0522] In the present disclosure, “treatment” refers to clinical intervention with the intent to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or reoccurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies disclosed herein are used to delay development of a disease or to slow the progression of a disease.

[0523] In the present disclosure, “administering” refers to delivering a biologically active composition or formulation by administration routes including, but not limited to, intravenous, intraarterial, intramuscular, intraperitoneal, subcutaneous, intramuscular, and topical administration, or a combination thereof. It includes, but is not limited to, administration by a medical professional and self-administration.

[0524] In the present disclosure, “subject” includes animals, such as humans and non-human animals. In some embodiments, a “subject” is a patient under the care of a physician. Thus, the subject can be a human patient or individual who has, is at risk for, or is suspected of having a disease of interest (e.g., cancer) and / or one or more symptoms of the disease. A subject can also be an individual who is at risk of being diagnosed with a condition of interest at the time of diagnosis or thereafter. The term “non-human animal” includes all vertebrates, e.g., mammals, e.g., rodents (e.g., mice) and other mammals, e.g., non-human primates, and other mammals, such as, for example, sheep, dogs, cows, chickens, and non-mammals, such as, for example, amphibians, reptiles, etc.

[0525] In the present disclosure, an "effective amount" refers to the amount of a composition sufficient to accomplish a stated purpose (e.g., achieve an effect for which it is administered, treat a disease, reduce a signaling pathway, or alleviate one or more symptoms of a disease or health condition) relative to the absence of the composition. An example of an "effective amount" is an amount sufficient to contribute to treatment, prevention, or reduction of one or more symptoms of a disease, which can also be referred to as a "therapeutically effective amount." "Alleviation" of a symptom means a decrease in severity or frequency of the symptom or elimination of the symptom. The exact amount of a composition (including a "therapeutically effective amount") will depend on the purpose of the treatment, and will be

[0526] In some embodiments, the Treg cell induces intratumoral Treg cell conversion to Thl-like Treg cells or fragile Treg cells.

[0527] In some embodiments, the Treg cell-targeting IL12 fusion protein also induces tumor microenvironment CD8 + T cells or CD4 + Tcon cells: (1) secrete IFNy; (2) upregulate expression levels of T-bet; and / or, upregulate expression of CD25.

[0528] In some embodiments, the Treg cell-targeting IL12 fusion protein is administered systemically, such as intravenously or subcutaneously.

[0529] In some embodiments, the method is a monotherapy or is administered in combination with other therapies. In some aspects, the other therapy can be selected from the group consisting of chemotherapy, radiation therapy, immunotherapy, toxin therapy, and surgery. In some aspects, the Treg cell-targeting IL12 fusion protein is administered prior to, after, or concurrently with the other therapy.

[0530] In some embodiments, the subject is an immune checkpoint inhibitor resistant patient, e.g., a PD1 antibody resistant patient.

[0531] In some embodiments, 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 brain glioma.

[0532] IX Applications

[0533] In another aspect, the present disclosure relates to 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 produced by the aforementioned production method, or the aforementioned pharmaceutical composition in preparation of a tumor treatment drug, wherein the Treg cell-targeting IL12 fusion protein induces intratumoral Treg cells to secrete IFNy, and attenuates or eliminates the immunosuppressive effect of the Treg cells.

[0534] In some embodiments, the Treg cells induce intratumoral Treg cells to transform into Th1-like Treg cells or fragile Treg cells.

[0535] In some embodiments, the Treg cell-targeting IL12 fusion protein further induces tumor microenvironment CD8 + T cells or CD4 + Tcon cells: (1) secrete IFNy; (2) upregulate the expression level of T-bet; and / or, upregulate the expression of CD25.

[0536] In some embodiments, the Treg-targeting IL12 fusion protein is administered systemically, e.g., intravenously or subcutaneously.

[0537] In some embodiments, the Treg-targeting IL12 fusion protein is administered as a monotherapy or in combination with other therapies.

[0538] In some embodiments, the other therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, toxin therapy, and surgery.

[0539] In some embodiments, the Treg cell-targeting IL12 fusion protein is administered prior to, after, or concurrently with the other therapy.

[0540] In some embodiments, 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 brain glioma.

[0541] The embodiments of the present application will be described in detail below with examples, but those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. The specific conditions not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not specified by the manufacturer, which are conventional products that can be obtained by commercial purchase.

[0542] Example 1 IL12Rβ1, PD1, CTLA4 and CCR8 are differentially expressed in tumor infiltrating lymphocyte subsets or spleen lymphocyte subsets

[0543] The tumor volume is taken to be 200-400 mm 3 or 300-500 mm 3 in size of MC38 colorectal cancer tumor model mice (C57BL / 6), and the spleen and tumor tissues are isolated to obtain spleen lymphocytes and tumor infiltrating lymphocytes (TILs). The isolated lymphocytes are immunotyped by fluorescence-activated cell sorting (FACS), and the expression levels of IL12Rβ1, PD1, CTLA-4 and / or CCR8 on different lymphocyte subsets are detected. The positive and negative gates are determined by fluorescence minus one (FMO) staining. The results are shown in Figures 2-4.

[0544] IL12Rβ1 + cells and PD1 + cells account for

[0545] As shown in Figure 2A, the proportion of IL12Rβ1 + cells in the spleen lymphocyte subsets is extremely low, except for NK cells and NKT cells; unlike this, the proportion of IL12Rβ1 + in the tumor infiltrating lymphocyte subsets is generally significantly higher than that in the corresponding spleen immune cell subsets.

[0546] As shown in Figure 2B, the proportion of PD1 + cells in the tumor infiltrating T cell subsets is significantly higher than that in the corresponding spleen T cell subsets.

[0547] Therefore, compared with the spleen, the expression of IL12Rβ1 and PD1 in the immune cell subsets derived from the tumor is significantly increased, which implies that the tumor microenvironment is more inflammatory than the peripheral immune system.

[0548] CTLA4 +Cell, CCR8 + Cell and PD1 + Cell ratio

[0549] CTLA4 + Cell and CCR8 + The ratio of the cell in tumor-infiltrating Treg cells is much higher than that in spleen-derived Treg cells or other tumor-infiltrating lymphocytes other than Treg cells, showing the specificity of target antigens CTLA4 and CCR8 in tumor-infiltrating Treg cells.

[0550] Comparing PD1 in tumor-infiltrating T lymphocyte subgroups + Cell, CTLA4 + Cell and CCR8 + Cell ratio. As shown in Figure 4, PD1 is highly expressed in all intratumoral T cell subgroups, while CTLA4 and CCR8 are only highly expressed in intratumoral Treg cell subgroups, indicating that PD1 is specific to tumor-infiltrating T lymphocytes, while CTLA4 and CCR8 are specific to tumor-infiltrating Treg cells.

[0551] Example 2 Design of Treg cell-targeting IL12 fusion protein

[0552] Design of Treg cell-targeting IL12 fusion protein, which can have the structure shown in Figure 5A or Figure 5B.

[0553] Figure 5A: [ABD1]-[linker]-[p40 subunit]-[linker]-[p35 subunit]-[linker]-[Fc1 with hinge region]; and, [ABD2]-[linker]-[Fc2 with hinge region], and form a heterodimer through Fc1 and Fc2.

[0554] Figure 5B: [ABD1]-[linker]-[p35 subunit]-[linker]-[Fc1 with hinge region]; and, [ABD2]-[linker]-[p40 subunit]-[Fc2 with hinge region], and form a heterodimer through Fc1 and Fc2.

[0555] Target antigen binding domain (ABD) and target point

[0556] ABD represents Antigen Binding Domain, which can be selected from ligands or fragments thereof (e.g., ligand extracellular domain) that specifically bind to target antigens, antibodies or fragments thereof (e.g., Fab, scFab, Fab', (Fab')2, Fv, scFv or VHH).

[0557] The IL12 fusion protein can be single target specific, with ABD1 and ABD2 binding to the same target antigen. The target antigen can be a Treg cell specific target antigen as shown in Example 1: CTLA4 or CCR8. The target antigen can also be other homologous target antigens known in the art, for example, CCR4, CCR10, CD25, GITR, OX-40, ICOS, and 4-1BB.

[0558] The IL12 fusion protein can also be dual target specific, with ABD1 and ABD2 binding to different target antigens. The dual targets can both be selected from Treg cell specific target antigens, for example, CTLA4, CCR8, CCR4, CCR10, CD25, GITR, OX-40, ICOS, and 4-1BB; or one of the dual targets is selected from the aforementioned Treg cell specific target antigens, and the other target is selected from tumor infiltrating T lymphocyte specific target antigens, for example, PD1. As an example, the dual targets can be selected from the group consisting of: (1) CTLA4 and CCR8, (2) CTLA4 and PD1, (3) CCR8 and PD1.

[0559] The specific structure of the IL12 fusion protein shown in FIG. 5A can be as shown in FIG. 6 or 7.

[0560] For CTLA4 / PD1 dual target or CCR8 / PD1 dual target Treg cell targeting IL12 fusion proteins, there are mainly two types: (1) opposite structure, i.e., IL12 is located opposite to the PD1 binding domain, which includes FIG. 6B, 6E, 6H, 6J, 7B, 7E, 7H, 7J, 7M; (2) same side structure, i.e., IL12 is located on the same side of the PD1 binding domain, which includes FIG. 6C, 6F, 6I, 6K, 7C, 7F, 7I, 7K, 7L.

[0561] IL12 and mutations thereof

[0562] To reduce toxicity, the IL12 of the IL12 fusion protein can be subjected to a weakening mutation. Compared with the wild-type amino acid residue, the weakening mutation results in a decrease in the ability of IL12 to bind and activate its receptor. The weakening mutation can occur in the p35 subunit and / or the p40 subunit. As an example, the weakening mutation occurs in the p40 subunit. Illustratively, the weakening mutation can be selected from the group consisting of: (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.

[0563] General method for preparing proteins of interest (construction of vectors, expression and purification of proteins)

[0564] After the design of the protein of interest (e.g., the Treg cell-targeting IL12 fusion protein shown in Example 2) is completed, the corresponding expression vector is constructed, and the protein of interest is expressed, isolated and purified: The DNA fragment corresponding to the protein of interest is chemically synthesized, and the DNA fragment is loaded into the expression vector PKS001 (purchased from Kangsheng Biology, item number: A13201) by means of molecular cloning techniques such as enzyme digestion, ligation and transformation, which are well known to those skilled in the art. The plasmid is extracted and sent for sequencing. After sequencing verification, the plasmid expressing the protein of interest is electroporated into the host cell CHO K1Q (purchased from Kangsheng Biology, item number: A13101) in suspension culture, and if necessary, pressure screening is performed to obtain a stable cell line. The transiently or stably transfected CHO K1Q cells are cultured, the supernatant is collected by centrifugation, or the purified protein is obtained after Protein A affinity chromatography, and is ready for use.

[0565] General method for detecting the activity of the test molecule IL12 using target cells or non-target cells

[0566] Construction of IL12 reporter cell line HEK293 / IL12 (i.e., non-target cell)

[0567] The STAT4-induced luciferase reporter gene is introduced into HEK293 cells using a lentiviral vector, and a screening agent is added to screen for a monoclonal cell line stably expressing the gene of interest. Subsequently, the IL12Rβ2-IL12Rβ1 and STAT4 genes are introduced into the monoclonal cell line in sequence, and a stable cell line is screened, i.e., the IL12 reporter cell line (non-target cell), named HEK293 / IL12R. The IL12 reporter cell line has a complete IL12 downstream signaling pathway. IL12 activates the IL12 receptors on the surface of the reporter cells and the downstream JAK2 and STAT4, induces the expression of luciferase, and reflects the activity of IL12 through the detectable luciferase activity.

[0568] Construction of target cells with IL12 reporter system

[0569] On the basis of the IL12 reporter cell, the hCTLA4, hPD1 or hCTLA4 / hPD1 genes are introduced by means of a lentiviral vector, and pressure screening is performed to obtain a monoclonal cell line stably expressing hCLTA4, hPD1 or hCTLA4 / hPD1 (i.e., target cell). Among them, to avoid the endocytosis of CTLA4, the introduced CTLA4 gene lacks a fragment encoding the intracellular signaling region.

[0570] Reference to the foregoing method, a monoclonal cell line stably expressing mCLTA4, mPD1 or mCTLA4 / mPD1 (i.e., target cells) is constructed.

[0571] Reference to the foregoing method, a monoclonal cell line stably expressing hCCR8, hPD1 or hCCR8 / hPD1 (i.e., target cells) is constructed.

[0572] Preparation of test proteins

[0573] Reference to Examples 2-3, test proteins are designed, corresponding expression vectors are constructed, and the test proteins are transiently expressed or stably expressed in CHO K1Q cells and secreted into the culture medium. The culture supernatant is collected for use, or the supernatant is purified for use.

[0574] Activation and detection of IL12 signal

[0575] The test sample or PBS control at serial dilutions is incubated with the foregoing target cells or non-target cells (150,000 cells) at 37°C, 5% CO2 for 24 hours. After incubation, the supernatant is mixed with an equal volume of luciferase substrate, incubated at room temperature for 3-5 minutes, and the chemiluminescence signal is detected by a microplate reader. The detection value of the test supernatant is taken as the signal value (Signal), and the detection value of the PBS control is taken as the background value (Background). The signal-to-noise ratio (signal value / background value) reflects the activity of luciferase and IL12.

[0576] Example 5 Activity of Treg cell-targeting IL12 fusion proteins highly dependent on expression of target antigen

[0577] IL12 fusion proteins as shown in Table 1 are designed, and the activation of IL12 receptors on target cells and non-target cells by the test proteins is detected according to Example 4. The results are shown in Figures 8A-8C and Tables 2-3.

[0578] Mutations reduce IL12 activity

[0579] Compared with wild-type hIL12 (WT)-Fc / / Fc, hIL12 (4A)-Fc / / Fc has 4A mutations in the IL12p40 subunit. As shown in Figures 8A-8B and Tables 2-3, the EC50 of hIL12 (WT)-Fc / / Fc is in the range of 0.09-0.031 nM, while the EC50 of hIL12 (4A)-Fc / / Fc is in the range of 1.16-5.28 nM, with a huge difference between the two. It can be seen that mutations greatly reduce IL12 activity.

[0580] Steric hindrance reduces IL12 activity

[0581] As shown in FIGS. 8B-8C and Tables 2-3, on non-target cell HEK293 / IL12, the EC50 of non-targeted hIL12(4A)-Fc / / Fc was 1.16 nM, while the EC50 of targeted Ipi-hIL12(4A) / hPDL2 was 20.23 nM, despite the same IL12 mutation occurred. The latter was further greatly reduced from the former. This might be due to the fact that in the Treg cell-targeted IL12 fusion protein, IL12 was placed between the targeting domains Ipi Fab, hPDL2 and Fc subunit (see FIG. 6B for details), and the steric hindrance of Ipi Fab and hPDL2 played a masking role on IL12, thus further reducing its activity.

[0582] Restoration of IL12 activity depends on the target antigen

[0583] As shown in FIGS. 8A-8C and Tables 2-3, compared with wild-type hIL12(WT)-Fc / / Fc and non-targeted hIL12(4A)-Fc / / Fc, the activity of targeted IL12(4A) fusion protein Ipi-hIL12(4A) / / hPDL2 on non-target cells was greatly reduced (EC50: 1 vs 1685.8; 96.7 vs 1685.8), while the reduced activity was restored on CTLA4 + Target cell or PD1 + CTLA4 + Target cell to the activity level close to that of wild-type IL12 fusion protein (EC50: 1 vs 15.6; 1 vs 14.4). Thus, the Treg cell-targeted IL12 fusion protein can maintain the non-specific IL12 activity at a minimum level, and preferentially activate CTLA4 + Cell or CTLA4 + PD1 + Cell.

[0584] Table 1 Molecular information of test proteins

[0585] Table 2 EC50 detection results of test proteins (unit: nM)

[0586] Table 3 EC50 detection results after homogenization

[0587] Example 6 CTLA4-targeted IL12 fusion proteins generally have target antigen-dependent activation, and the target antigen-dependent activation is better in the “opposite structure” and 4A mutation

[0588] CTLA4-targeted IL12 fusion proteins were constructed (see Tables 4-5 for details), and their ability to activate IL12 receptors on the surface of target or non-target cells was tested according to Example 4. The results are shown in Figures 9-10.

[0589] CTLA4-targeted IL12 fusion proteins generally have good target-dependent activation

[0590] The CTLA4-targeted IL12 fusion proteins shown in Tables 3-4 exhibit diversity and differentiation in terms of target combinations, formats, IL12 mutations, and CTLA4 antibodies. However, as shown in Figures 9-10, all of the CTLA4-targeted IL12 fusion proteins have good target-dependent activation of IL12 activity on CTLA4 + target cells or CTLA4 + PD1 + The response curves on target cells are all significantly left-shifted relative to non-target cells HEK293 / IL12R, and IL12 activity is restored, all showing good target-dependent activation.

[0591] Thus, CTLA4-targeted IL12 fusion proteins generally have good target-dependent activation, and are not limited to specific target combinations, formats, mutation types, or CDR regions of CTLA4 antibodies.

[0592] “Opposite configuration” has better target-dependent activation than “same configuration” (Figure 6B v. Figure 6C)

[0593] Opposite configuration (Figure 6B): Ipi-hIL12(2A) / / hPDL2, Ipi-hIL12(4A) / / hPDL2.

[0594] Same configuration (Figure 6C): hPDL2-hIL12(2A) / / Ipi, hPDL2-hIL12(4A) / / Ipi.

[0595] As shown in Figures 6B and 6C, the opposite configuration is very similar to the same configuration. However, as shown in Figure 9, in terms of the left-shift of the IL12 activity curve (left-shift from non-target cells HEK293 / IL12R to CTLA4 + or CTLA4 + / PD1 + target cells), the left-shift of the opposite configuration is significantly greater than that of the same configuration. This phenomenon is particularly evident in the 2A mutation. Thus, the opposite configuration has stronger target-dependent activation than the same configuration.

[0596] 4A mutation has stronger target-dependent activation than 2A mutation

[0597] As shown in FIG. 9, the targeted IL12 fusion protein with 4A mutations (E59A / F60A / K84A / K195A) has much lower ability to activate IL12 receptor on non-target cells than the targeted IL12 fusion protein with 2A mutations (E59A / F60A). However, on target cells, its IL12 activity can be restored to a level comparable to that of the targeted IL12 fusion protein with 2A mutations. It can be seen that the targeted Treg cell-targeted IL12 fusion protein (4A mutations) exhibits surprisingly high target antigen-dependent activity in terms of activity, suggesting the possibility of achieving a good balance between anti-tumor effect and safety.

[0598] Table 4. Molecular information of test proteins

[0599] Table 5. Molecular information of test proteins

[0600] Example 7. CCR8-targeted IL12 fusion proteins generally have target antigen-dependent activity, and "opposite structure" has stronger target antigen-dependent activity than "same structure"

[0601] CCR8-targeted IL12 fusion proteins were constructed (see Table 6 for details), and their activation effects on the surface IL12R of target cells and non-target cells were detected according to Example 4. The results are shown in FIGS. 11A-11E.

[0602] CCR8-targeted IL12 fusion proteins generally have target antigen-dependent activity

[0603] The Treg cell-targeted IL12 fusion proteins shown in Table 6 exhibit diversity and differentiation in terms of target point combination, format, and PD1 binding domain. However, as shown in FIGS. 11A-11E, the Treg cell-targeted IL12 fusion proteins shown in Table 6 all exhibit CTLA4 + target cell or CTLA4 + PD1 + target cell-dependent IL12 activity. It can be seen that the CCR8-based targeted Treg cell-targeted IL12 fusion proteins generally have target antigen-dependent activity.

[0604] "opposite structure" has stronger target antigen-dependent activity than "same structure"

[0605] Opposite structure (FIG. 7H): 17-64-mIL12(4A) / / mPDL2.

[0606] Same structure (FIG. 7I): mPDL2-mhIL12(4A) / / 17-64.

[0607] As shown in Figure 11, the contralateral structure showed lower non-specific activity on non-target cells and stronger activity on target cells CTLA4 + cells and CTLA4 + PD1 + cells (see Emax). It can be seen that IL12 on the contralateral structure has stronger target antigen-dependent activity than the ipsilateral structure.

[0608] Table 6 Molecular information of test proteins

[0609] Example 8 General method for constructing mouse tumor models and evaluating drug efficacy

[0610] Unless otherwise specified, the mouse tumor models and drug efficacy evaluation involved in the embodiments of the present disclosure are performed according to the following methods:

[0611] The tumor cells are inoculated subcutaneously on the right side of the back of the mouse. When the tumor reaches a certain volume, mice with appropriate body weight are selected, randomly grouped, and given the test drug or PBS control according to the established dosing regimen. The body weight and tumor volume of the mice are measured and recorded 3 times a week. As needed, the amount of IFNγ released in the peripheral blood of the mice 48 h after administration is detected (IFNγ Kit, purchased from Xinboseng, Catalog No.: EMC101g.96.10). At the end of the experiment, the mice are euthanized, the tumors are stripped and weighed, and the relative tumor inhibition rate (Tumor Growth Inhibition value, TGI) is calculated. The TGI calculation method is as follows:

[0612] Tumor volume calculation formula: V = 1 / 2ab 2 (a is the long diameter of the tumor, and b is the short diameter of the tumor).

[0613] RTV (Relative Tumor Volume) is the relative tumor volume. RTV = V t / V0. V0: The tumor volume at the beginning of the experiment. Each experimental group and control group has one RTV. t : The tumor volume at the end of an experimental period (usually about 30 days). V0: The tumor volume at the beginning of the experiment. Each experimental group and control group has one RTV.

[0614] TGI = [1-RTV (experimental group) / RTV (control group)] * 100%.

[0615] Example 9 Evaluation of the anti-tumor effect of CTLA4-targeted IL12 fusion protein in a humanized mouse tumor model

[0616] As shown in Example 8, the growth inhibitory effects of the targeted IL12 fusion proteins shown in Tables 7-10 on melanoma or prostate tumor were evaluated in the CTLA4 humanized mouse model or the CTLA4 / PD1 humanized mouse model established tumor model, and the specific results are shown in Figures 12-15.

[0617] The targeted IL12 fusion proteins based on Ipi or its variants have growth inhibitory effects on melanoma or prostate tumor

[0618] As shown in Figures 12A, 13A-13B and 14A, Ipi-mIL12(4A) / / hPDL2 and its corresponding Ipi-mIL12(4A) / / mPDL2 both showed significant growth inhibitory effects on B16 melanoma and RM1 prostate tumor.

[0619] However, the targeted Treg cell-targeted IL12 fusion proteins also showed significant anti-tumor effects when IL12 was other weakened mutations (2A or 3A), CTLA4 antibody was Ipi variants (Ipi.105 or Ipi.106) or the target combination was CTLA4 single target (see Figures 12B, 13B, 14B and 15A for details). The targeted Treg cell-targeted IL12 fusion proteins with 2A mutation showed stronger anti-tumor effect than 3A mutation and 4A mutation (Figure 14A), which may be due to the lowest degree of weakening of 2A mutation, retaining the strongest IL12 activity. For the description of Ipi variants, see reference 12.

[0620] The anti-tumor effect of CTLA4-targeted IL12 fusion proteins does not depend on specific CTLA4 antibodies or CDR regions

[0621] As shown in Figures 12B, 14B and 15B-15C, the targeted Treg cell-targeted IL12 fusion proteins also showed significant anti-tumor effects when using other CTLA4 antibodies other than Ipi or its variants. Thus, the CTLA4-based targeted Treg cell-targeted IL12 fusion proteins generally have anti-tumor effects, and do not depend on specific CTLA4 antibodies or CDR regions.

[0622] Changes in mouse body weight and release amount of peripheral blood IFNγ

[0623] Compared with the PBS control group, the targeted Treg cell-targeted IL12 fusion proteins shown in Tables 7-10 did not cause significant decrease in mouse body weight after administration, and no obvious toxic side effects were observed (specific data not shown). At the same time, the content of IFNγ in the peripheral blood of mice after administration of some test molecules was also detected, and the results showed that it was basically at a low level (<500 pg / mL, specific data not shown).

[0624] Table 7 Molecular information of test proteins

[0625] Table 8 Molecular information of test proteins

[0626] Table 9 Molecular information of test proteins

[0627] Table 10 Molecular information of test proteins

[0628] Example 10 Ipi-hIL12(4A) / / hPDL2 has similar target antigen-dependent activity as its surrogate molecule

[0629] Ipilimumab (Ipi) only binds to human CTLA4, but not to murine CTLA4. The 9D9 antibody is commonly used in the art as a surrogate molecule for Ipi to evaluate its performance in wild-type mouse models (Ref. 13). To further verify whether 9D9 can also be used as a surrogate molecule for Ipi in the Treg cell-targeted IL12 fusion proteins shown in the present disclosure, Treg cell-targeted IL12 fusion proteins as shown in Table 11 were constructed, and their activation effects on target cells and non-target cells were detected. The results are shown in Figures 16A-16B.

[0630] The results show that 9D9-mIL12(4A) / / mPDL2 has similar target antigen-dependent activity as Ipi-hIL12(4A) / / hPDL2. Therefore, 9D9-mIL12(4A) / / mPDL2 can be used as a surrogate molecule for Ipi-hIL12(4A) / / hPDL2 to evaluate the efficacy of wild-type mouse tumor models or explore the mechanism of drug action.

[0631] Table 11 Molecular information

[0632] Example 11 Evaluation of the anti-tumor effect of CTLA4-targeted Treg cell-targeted IL12 fusion proteins in wild-type mouse tumor models

[0633] As shown in Example 9, the growth inhibition effects of Treg cell-targeted IL12 fusion proteins shown in Tables 12-15 on colorectal cancer, breast cancer, and melanoma were evaluated in wild-type mouse tumor models established in immune-competent mice, and the specific detection results are shown in Figures 17-22A.

[0634] CTLA4-targeted IL12 fusion proteins (9D9) showed growth inhibition effect on colorectal cancer, breast cancer and melanoma, and showed good anti-tumor effect on PD1 antibody-sensitive tumor model (MC38) and PD1 antibody-resistant tumor model (B16, EMT6) compared with PD1 antibody or mIpi antibody

[0635] As shown in FIGS. 17-19, 21 and 22A, the 9D9-based Treg cell-targeted IL12 fusion proteins shown in Tables 12, 14 and 15 showed significant tumor growth inhibition effect on various tumors including colorectal cancer, breast cancer or melanoma.

[0636] As shown in FIG. 17, for the PD1 antibody-sensitive MC38 tumor model (tumor volume > 400mm 3 ), the CR rate of 9D9-mIL12 (4A) / / mPDL2 was the highest, reaching 65%. In contrast, mPD1 antibody failed to achieve any CR, but did result in 50% TGI. The CR rate of mIpi was 60%.

[0637] As shown in FIG. 18, for the PD1 antibody-resistant EMT6 tumor model (tumor volume > 400mm 3 ), the CR rate of 9D9-mIL12 (4A) / / mPDL2 reached 75%. In contrast, mPD1 antibody did not achieve any CR, while the CR rate of mIpi was 10%.

[0638] As shown in FIG. 19, in the PD1 antibody-resistant B16 tumor model, 9D9-mIL12 (4A) / / mPDL2 reached 65% TGI on day 16. In contrast, mPD1 antibody promoted tumor growth (TGI16 = -4%). mIpi reached 35% TGI.

[0639] The anti-tumor effect of CTLA4-targeted IL12 fusion proteins is not dependent on specific CTLA4 antibodies or CDR regions

[0640] As shown in FIG. 20, Treg cell-targeted IL12 fusion proteins using various CTLA4 antibodies other than 9D9 also showed varying degrees of anti-tumor effect. Thus, the anti-tumor effect of CTLA4-targeted IL12 fusion proteins is universal and not limited to specific CTLA4 antibodies or CDR regions.

[0641] “Opposite structure” showed better anti-tumor effect than “same structure”

[0642] As shown in FIGS. 21 and 22A, the Treg cell-targeting IL12 fusion proteins with "contralateral structure" (FIG. 6B, FIG. 6E) in Table 14-15 had a significantly better inhibitory effect on tumor growth than the corresponding "ipsilateral structure" (FIG. 6C, FIG. 6F), and had a stronger anti-tumor effect.

[0643] Changes in body weight of mice and release of IFNγ in peripheral blood

[0644] Compared with the PBS control group, the Treg cell-targeting IL12 fusion proteins in Table 12-15 did not cause a significant decrease in body weight of mice after administration, and no obvious toxic side effects were observed (specific data not shown). At the same time, the content of IFNγ in the serum of mice after administration of some test molecules was also detected, and the results showed that it was basically at a low level (<500 pg / mL, specific data not shown).

[0645] Molecular information of test proteins in Table 12

[0646] Molecular information of test proteins in Table 13

[0647] Molecular information of test proteins in Table 14

[0648] Molecular information of test proteins in Table 15

[0649] Example 12 Evaluation of anti-tumor effect of CCR8-targeting IL12 fusion proteins in wild-type mice

[0650] As shown in Example 9, the anti-tumor effect of Treg cell-targeting IL12 fusion proteins in Table 16 was evaluated in a tumor model established in wild-type mice with a healthy immune system. The specific detection results are shown in FIGS. 22B-22C.

[0651] As shown in FIGS. 22B-22C, the CCR8-targeting IL12 fusion proteins in Table 16 all had an inhibitory effect on CT26 colorectal cancer, and as shown in FIG. 22C, the "contralateral structure" (FIG. 7H, FIG. 7J, FIG. 7M) showed a significantly better anti-tumor effect than the corresponding "ipsilateral structure" (FIG. 7I, FIG. 7K, FIG. 7L).

[0652] In addition, compared with the PBS control, the mice in the test groups in Table 16 did not show a significant decrease in body weight or other toxic side effects after administration (relevant data not shown).

[0653] Molecular information of test proteins in Table 16

[0654] Example 13 Toxicology test of non-tumor-bearing mice

[0655] Suitable wild-type C57BL / 6 mice were selected and randomly grouped, 3 mice per group. On days 5, 8, 11, and 14 after grouping, the mice were injected with PBS control or different doses of test molecules shown in Table 17. The serum IFNγ content of the mice was detected at 24 h, 48 h, and 72 h after the first administration. The body weight of the mice was measured and recorded twice a week. At the end of the experiment, the animals were euthanized. The results are shown in Figures 23A-23B and 24A-24B.

[0656] As shown in Figures 23A-23B, mIL12(WT)-Fc showed a clear dose-dependent release of IFNγ compared with 9D9-mIL12(4A) / / mPDL2. Figures 24A-24B show the results of the body weight monitoring of the mice during the 4 administrations. mIL12(WT)-Fc showed a clear dose-dependent body weight loss compared with 9D9-mIL12(4A) / / mPDL2. More importantly, it was observed that mIL12(WT)-Fc started to show death from 0.1 mpk, while 9D9-mIL12(4A) / / mPDL2 did not show any death at the administration dose of 15 mpk.

[0657] As can be seen, 9D9-mIL12(4A) / / mPDL2 has high safety and significantly reduced toxicity compared with mIL12(WT)-Fc fusion protein in WT non-tumor-bearing mice. The mice can tolerate a dose of up to 15 mpk without significant toxicity.

[0658] Table 17 Molecular information of test proteins

[0659] Example 14 Monkey toxicity test of Treg cell-targeting IL12 fusion proteins

[0660] The test molecules shown in Table 18 were evaluated for toxicity in cynomolgus monkeys: 4 cynomolgus monkeys were intravenously infused with the test molecules shown in Table 18 once a week for 4 consecutive weeks. The administration dose of Ipi-hIL12(2A) / / hPDL2 and hPDL2-hIL12(4A) / / Ipi was 5 mpk, while the administration dose of Ipi-hIL12(4A) / / hPDL2 was 5 mpk or 15 mpk. During the experiment, the release of peripheral blood cytokine IFNγ and blood immunology changes of the cynomolgus monkeys were detected. The results showed that Ipi-hIL12(2A) / / hPDL2 was highly safe for cynomolgus monkeys, and the cynomolgus monkeys could tolerate a dose of 15 mpk without significant toxicity. The specific results are as follows:

[0661] Detection results of cynomolgus monkey serum IFNγ content after administration

[0662] The results of serum IFNγ detection in cynomolgus monkeys after the first administration are shown in Figure 25. The peripheral blood IFNγ 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.

[0663] Results of blood immunological index detection in cynomolgus monkeys after administration

[0664] On the day of administration, and on days 2, 6, 9, 13, 16, 20, 23 and 29, the hematological indexes including ALT, AST, CK, TBIL, WBC, NEU, MONO, LYM and EOS were detected. The results showed that the hematological indexes of cynomolgus monkeys were within the normal range after administration of the molecules shown in Table 18, or returned to the normal range quickly after a few time points out of the normal range (this may be caused by multiple blood sampling) (relevant data not shown).

[0665] No adverse reaction dose level

[0666] No test product related death was caused by administration, and no test product related abnormal changes in histopathology were caused. Under the test conditions of this example, the no adverse reaction dose level (NOAEL) of Ipi-hIL12(2A) / / hPDL2 and hPDL2-hIL12(4A) / / Ipi was 5 mg / kg, and the no adverse reaction dose level (NOAEL) of Ipi-hIL12(4A) / / hPDL2 was 15 mg / kg.

[0667] Table 18 Molecular information of test proteins

[0668] Examples 15-16 Analysis of the mechanism of action of Treg cell targeting IL12 fusion proteins

[0669] Example 15 Treg cell targeting IL12 fusion proteins selectively activate TDLN and tumor-derived T lymphocytes, with a different mechanism of action from PD1 antibodies, CTLA4 antibodies

[0670] Wild type C57BL / 6 mice were inoculated subcutaneously with 2.5E5 B16 melanoma cells on the right side of the back. On day 5 after inoculation, the body weight of the mice was measured, and mice with appropriate body weight were selected and randomly divided into groups, with 20 mice in each group. On day 5 and day 8 after inoculation, the mice were intraperitoneally injected with PBS control or the test molecules shown in Table 19 (dose: 5 mpk). The tumor volume and body weight of the mice were measured and recorded 3 times per week. On day 10 after inoculation, the tumor volume reached 300-500 mm 3Mice were euthanized and spleen, draining lymph nodes (TDLN) and tumor tissue were harvested. T cells were typed by FACS: CD4 Tcon cells (CD45 + CD3 + CD4 + FOXp3 - ) ; CD4 Treg cells (CD45 + CD3 + CD4 + FOXp3 + ) ; CD8 T cells (CD45 + CD3 + CD8 + ) ; IFNy secretion (4h stimulation with PMA / ionomycin / BFA prior to staining) ; T-bet and CD25 expression (no additional stimulation required for T-bet and CD25 staining). The results are shown in Figures 26-34, and a summary of the key information for the 9D9-mIL12(4A) / / mPDL2 group is shown in Figure 35.

[0671] The results show that the Treg cell-targeted IL12 fusion protein 9D9-mIL12(4A) / / mPDL2 differentially activates T cell subsets from the spleen, TDLN and tumor: it weakly or not at all activates T cells from the spleen, strongly activates intratumoral T cells (TILs), and activates TDLN-derived T cells that have a "crosstalk" with tumor immune cells to an extent intermediate between that of T cells from the spleen and TILs.

[0672] The Treg cell-targeted IL12 fusion protein stimulates intratumoral and TDLN Tregs to express T-bet and secrete IFNy, converting them into fragile Tregs that lose or reduce their immunosuppressive effect. This is likely to be a central mechanism of action of the Treg cell-targeted IL12 fusion protein.

[0673] The Treg cell-targeted IL12 fusion protein also induces intratumoral CD8 T cells and Tcon cells to express T-bet and secrete IFNy, enhancing their killing effect.

[0674] The Treg cell-targeted IL12 fusion protein also upregulates CD8 T cells and Tcon cells to express CD25, enhancing their utilization of the cytokine IL2 and promoting their proliferation.

[0675] In addition, as shown in Figure 26, 9D9-mIL12(4A) / / mPDL2 also stimulated a significant increase in the size of TDLN after administration, a phenomenon that did not occur in the mPD1 antibody group or the mIpi group.

[0676] Table 19 Molecular information of the tested Dai Bai

[0677] Example 16 Treg cell-targeting IL12 fusion protein selectively induces activated Treg cells to secrete IFNy

[0678] CD3+T cells and Treg cells were isolated from human PBMCs using a kit (EasySep TM Human CD4+CD127lowCD25+Regulatory T Cell Isolation Kit) and the resulting Treg cells were labeled with CSFE. CD3+T cells and CSFE-labeled Treg cells were mixed at 95%:5%. The mixed cells were seeded into a 96-well plate at a seeding amount of 2.5E6 / well, and were incubated with Ipi-hIL12(4A) / / hPDL2 shown in Table 20 under stimulation (1 μg / ml coated anti-CD3 antibody, 1 μg / ml soluble anti-CD28 antibody) or non-stimulation conditions. The non-stimulation condition simulates the peripheral immune system, and the stimulation condition simulates the tumor microenvironment (inflammatory). After 72 hours of incubation, the concentration of IFNy in the supernatant was detected by ELISA. The T cell subpopulations were typed (Tregs (CSFE + CD3 + CD4 + FOXp3 + ) and the intracellular IFNy expression of T cell subpopulations was detected by FACS. The specific detection results are shown in Figures 36-37. - CD3 + CD8 +) ; Tcon: (CSFE - CD3 + CD4 + FOXp3 - )) and the intracellular IFNy expression of T cell subpopulations was detected by FACS. The specific detection results are shown in Figures 36-37.

[0679] Ipi-hIL12(4A) / / hPDL2 does not induce IFNy release from unactivated PBMCs, showing high safety

[0680] As shown in FIG. 36, ELISA results show that unstimulated PBMC cells do not release IFNy even at a concentration of 100 nM, indicating that Ipi-hIL12(4A) / / hPDL2 has very low degree of activation of immune cells in systemic peripheral tissues, showing high safety; while stimulated PBMC cells release a large amount of IFNy, indicating that the environment of activated PBMC is highly inflammatory (TME).

[0681] In the activated PBMC cell subpopulation, Ipi-hIL12(4A) / / hPDL2 induced Treg cells to release IFNy

[0682] As shown in FIGS. 37A-37C, Ipi-hIL12(4A) / / hPDL2 can significantly induce activated Treg cells to release IFNy, and has a slight but measurable dose-dependent increase in IFNy release of activated CD8 and Tcon cells.

[0683] Table 20 Molecular information of proteins to be tested

[0684] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement to part or all of the technical features thereof; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

[0685] References

[0686] 1. Yosuke Togashi et al., Regulatory T cells in cancer immunosuppression- implications for anticancer therapy, Nature reviews clinical oncology, 16, 356-371 (2019).

[0687] 2. Feng Shan et al., Therapeutic targeting of regulatory T cells in cancer, Trends in Cancer, Volume 8, Issue 11, November 2022, Pages 944-961.

[0688] 3. Astushi Tanaka and Shimon Sakaguchi, Targeting Treg cells in cancer immunotherapy, European Journal of Immunology, 2019, 49: 1140-1146.

[0689] 4. Margarita Dominguez-Villar et al., Identification of T helper type 1-like, Foxp3 + regulatory T cells in human autoimmune disease. Nat Med. 2011 June; 17(6): 673-675.

[0690] 5. Jingxian Zhao et al., Differential Effects of IL-12 on Tregs and Non-Treg T Cells: Roles of IFN-c, IL-2 and IL-2R, PLOS ONE, Volume 7, Issue 9, p1-12.

[0691] 6. S Tugues et al., New insights into IL12-mediated tumor suppression, Cell Death and Differentiation, (2015) 22, 237-246.

[0692] 7. Bruce D. Car et al., The Toxicology of Interleukin-12: A Review. Toxicologic Pathology, Vol. 27, no. 1, pp. 58-63, 1999.

[0693] 8. John P. Leonard et al., Effects of Single-Dose Interleukin-12 Exposure on Interleukin-12-Associated Toxicity and Interferon-γ Production, Blood, Vol 90, No 7 (October 1), 1997: 2541-2548.

[0694] 9. Coughlin CM et al., The effect of interleukin 12 desensitization on the antitumor efficacy of recombinant interleukin 12. Cancer Res. 57:2460-2467.

[0695] 10. Ozmen L., et al., The in vivo antiviral activity of interleukin-12 is mediated by gamma interferon. J. Viro. 69(12):8147-8150.

[0696] 11. Yu Li et al., Potential anti-tumor efects of regulatory T cells in the tumor microenvironment: a review. Journal of Translational Medicine (2024) 22:293.

[0697] 12. Peter S. Lee et al., Improved therapeutic index of an acidic pH selective antibody, MABS, 2022, VOL. 14, NO. 1, e2024642 (14 pages).

[0698] 13. Keunok Jung et al., A Neuropilin-1 Antagonist Exerts Antitumor Immunity by Inhibiting the Suppressive Function of Intratumoral Regulatory T Cells. Cancer Immunol Res 2020; 8:46-56.

[0699] SEQUENCE LISTING

[0700] Human mature wild-type IL12 p35 subunit (SEQ ID NO: 1)

[0701] Monkey mature wild-type IL12 p35 subunit (SEQ ID NO: 2)

[0702] Mouse mature wild-type IL12 p35 subunit (SEQ ID NO: 3)

[0703] Dog mature wild-type IL12 p35 subunit (SEQ ID NO: 4)

[0704] Human mature wild-type IL12 p40 subunit (SEQ ID NO: 5)

[0705] Monkey mature wild-type IL12 p40 subunit (SEQ ID NO: 6)

[0706] Mouse mature wild-type IL12 p40 subunit (SEQ ID NO: 7)

[0707] Dog mature wild-type IL12 p40 subunit (SEQ ID NO: 8)

[0708] IL12 p40 subunit with F60A mutation (SEQ ID NO: 9)

[0709] IL12 p40 subunit with F60E mutation (SEQ ID NO: 10)

[0710] IL12 p40 subunit with F60D mutation (SEQ ID NO: 11)

[0711] IL12 p40 subunit with E59A / F60A mutations (SEQ ID NO: 12)

[0712] IL12 p40 subunit with E59A / F60A / K84A mutations (SEQ ID NO: 13)

[0713] IL12 p40 subunit with E59A / F60A / K84A / K195A mutations (SEQ ID NO: 14)

[0714] IL12 p40 subunit with W15 / E59A / F60A mutations (SEQ ID NO: 15)

[0715] IL12 p40 subunit with W15A / E59A / F60A / K84A mutations (SEQ ID NO: 16)

[0716] IL12 p40 subunit with W15A / E59A / F60A / K84A / K195A mutations (SEQ ID NO: 17)

[0717] Human p40 subunit (F60A)-linker-human p35 subunit (SEQ ID NO: 18)

[0718] Human p40 subunit (F60E)-linker-human p35 subunit (SEQ ID NO: 19)

[0719] Human p40 subunit (F60D)-linker-human p35 subunit (SEQ ID NO: 20)

[0720] Human p40 subunit (E59A / F60A)-linker-human p35 subunit (SEQ ID NO: 21)

[0721] Human p40 subunit (E59A / F60A / K84A)-linker-human p35 subunit (SEQ ID NO: 22)

[0722] Human p40 subunit (E59A / F60A / K84A / K195A)-linker-human p35 subunit (SEQ ID NO: 23)

[0723] Human p40 subunit (W15 / E59A / F60A)-linker-human p35 subunit (SEQ ID NO: 24)

[0724] Human p40 subunit (W15A / E59A / F60A / K84A)-linker-human p35 subunit (SEQ ID NO: 25)

[0725] Human p40 subunit (W15A / E59A / F60A / K84A / K195A)-linker-human p35 subunit (SEQ ID NO: 26)

[0726] Wild-type human PDL1 ectodomain (SEQ ID NO: 27)

[0727] Wild-type human PDL2 ectodomain (SEQ ID NO: 28)

[0728] Wild-type human PDL2 ectodomain without C-terminal "HPT" (SEQ ID NO: 29)

[0729] Wild-type murine PDL2 ectodomain (SEQ ID NO: 30)

[0730] Ipi VH (anti-CTLA4) (SEQ ID NO: 31)

[0731] Ipi.105 VH (anti-CTLA4) (SEQ ID NO: 32)

[0732] Ipi.106 VH (anti-CTLA4) (SEQ ID NO: 33)

[0733] Ipi VL (anti-CTLA4) (SEQ ID NO: 34)

[0734] Ipi.105 or Ipi.106 VL (anti-CTLA4) (SEQ ID NO: 35)

[0735] VH146 VH (anti-CTLA4) (SEQ ID NO: 36)

[0736] VH146 VL (anti-CTLA4) (SEQ ID NO: 37)

[0737] HL32 VH (anti CTLA4) (SEQ ID NO: 38)

[0738] HL32 VL (anti CTLA4) (SEQ ID NO: 39)

[0739] 311 VHH (anti-CTLA4) (SEQ ID NO: 40)

[0740] Erf VHH (anti-CTLA4) (SEQ ID NO: 41)

[0741] AU VHH (anti-CTLA4) (SEQ ID NO: 42)

[0742] 310 VHH (anti-CTLA4) (SEQ ID NO: 43)

[0743] 313 VHH (anti-CTLA4) (SEQ ID NO: 44)

[0744] 625 VHH (anti-CTLA4) (SEQ ID NO: 45)

[0745] 566 VHH (anti-CTLA4) (SEQ ID NO: 46)

[0746] 922 VHH (anti-CTLA4) (SEQ ID NO: 47)

[0747] 067 VHH (anti-CTLA4) (SEQ ID NO: 48)

[0748] 014 VHH (anti-CTLA4) (SEQ ID NO: 49)

[0749] 189 VHH (anti-CTLA4) (SEQ ID NO: 50)

[0750] 9D9 VH (anti-CTLA4) (SEQ ID NO: 51)

[0751] 9D9 VL (anti-CTLA4) (SEQ ID NO: 52)

[0752] Nb91 VHH (anti-CTLA4) (SEQ ID NO: 53)

[0753] 745 VHH (anti-CTLA4) (SEQ ID NO: 54)

[0754] 636 VHH (anti-CTLA4) (SEQ ID NO: 55)

[0755] Tremelimumab VH (anti-CTLA4) (SEQ ID NO: 56)

[0756] Tremelimumab VL (anti-CTLA4) (SEQ ID NO: 57)

[0757] anti-CTLA4 VH of Cadonilimab (SEQ ID NO: 58)

[0758] anti-CTLA4 VL of Cadonilimab (SEQ ID NO: 59)

[0759] Tuvonralimab VH (anti-CTLA4) (SEQ ID NO: 60)

[0760] Tuvonralimab VL (anti-CTLA4) (SEQ ID NO: 61)

[0761] Quavonlimab VH (anti-CTLA4) (SEQ ID NO: 62)

[0762] Quavonlimab VL (anti-CTLA4) (SEQ ID NO: 63)

[0763] Botensilimab VH (anti-CTLA4) (SEQ ID NO: 64)

[0764] Botensilimab VL (anti-CTLA4) (SEQ ID NO: 65)

[0765] anti-CTLA4 VH of Lorigerlimab (SEQ ID NO: 66)

[0766] anti-CTLA4 VL of Lorigerlimab (SEQ ID NO: 67)

[0767] Porustobart VHH (anti-CTLA4) (SEQ ID NO: 68)

[0768] anti-CTLA4 VH of Vudalimab (SEQ ID NO: 69):

[0769] anti-CTLA4 VL of Vudalimab (SEQ ID NO: 70)

[0770] YH-001 VH (anti-CTLA4) (SEQ ID NO: 71)

[0771] YH-001 VL (anti-CTLA4) (SEQ ID NO: 72)

[0772] JS-007 VH (anti-CTLA4) (SEQ ID NO: 73)

[0773] JS-007 VL (anti-CTLA4) (SEQ ID NO: 74)

[0774] firastotug VH (anti-CTLA4) (SEQ ID NO: 75)

[0775] firastotug VL (anti-CTLA4) (SEQ ID NO: 76)

[0776] Muzastotug VH (anti-CTLA4) (SEQ ID NO: 77)

[0777] Muzastotug VL (anti-CTLA4) (SEQ ID NO: 78)

[0778] HCDR1 of Ipi VH (SEQ ID NO: 79)

[0779] HCDR2 of Ipi VH (SEQ ID NO: 80)

[0780] HCDR3 of Ipi, Ipi.105 VH (SEQ ID NO: 81)

[0781] HCDR1 of Ipi.105, Ipi.106 VH (SEQ ID NO: 82)

[0782] HCDR2 of Ipi.105, Ipi.106 VH (SEQ ID NO: 83)

[0783] HCDR3 of Ipi.106 VH (SEQ ID NO: 84)

[0784] LCDR1 of Ipi VL (SEQ ID NO: 85)

[0785] LCDR2 of Ipi VL (SEQ ID NO: 86)

[0786] LCDR3 of Ipi VL (SEQ ID NO: 87)

[0787] LCDR1 of Ipi.105, Ipi.106 VL (SEQ ID NO: 88)

[0788] HCDR1 of 146 VH (SEQ ID NO: 89)

[0789] HCDR2 of 146 VH (SEQ ID NO: 90)

[0790] HCDR3 of 146 VH (SEQ ID NO: 91)

[0791] LCDR1 of 146 VL (SEQ ID NO: 92)

[0792] LCDR2 of 146 VL (SEQ ID NO: 93)

[0793] LCDR3 of 146 VL (SEQ ID NO: 94)

[0794] HCDR1 of HL32 VH (SEQ ID NO: 95)

[0795] HCDR2 of HL32 VH (SEQ ID NO: 96)

[0796] HCDR3 of HL32 VH (SEQ ID NO: 97)

[0797] LCDR1 of HL32 VL (SEQ ID NO: 98)

[0798] LCDR2 of HL32 VL (SEQ ID NO: 99)

[0799] LCDR3 of HL32 VL (SEQ ID NO: 100)

[0800] HCDR1 of 311 VHH (SEQ ID NO: 101)

[0801] HCDR2 of 311 VHH (SEQ ID NO: 102)

[0802] HCDR3 of 311 VHH (SEQ ID NO: 103)

[0803] HCDR1 of Erf VHH (SEQ ID NO: 104)

[0804] HCDR2 of Erf VHH (SEQ ID NO: 105)

[0805] HCDR3 of Erf VHH (SEQ ID NO: 106)

[0806] HCDR1 of AU VHH (SEQ ID NO: 107)

[0807] HCDR2 of AU VHH (SEQ ID NO: 108)

[0808] HCDR3 of AU VHH (SEQ ID NO: 109)

[0809] HCDR1 of 310 VHH (SEQ ID NO: 110)

[0810] HCDR2 of 310 VHH (SEQ ID NO: 111)

[0811] HCDR3 of 310 VHH (SEQ ID NO: 112)

[0812] HCDR1 of 313 VHH (SEQ ID NO: 113)

[0813] HCDR2 of 313 VHH (SEQ ID NO: 114)

[0814] HCDR3 of 313 VHH (SEQ ID NO: 115)

[0815] HCDR1 of 625 VHH (SEQ ID NO: 116)

[0816] HCDR2 of 625 VHH (SEQ ID NO: 117)

[0817] HCDR3 of 625 VHH (SEQ ID NO: 118)

[0818] HCDR1 of 566 VHH (SEQ ID NO: 119)

[0819] HCDR2 of 566 VHH (SEQ ID NO: 120)

[0820] HCDR3 of 566 VHH (SEQ ID NO: 121)

[0821] HCDR1 of 922 VHH (SEQ ID NO: 122)

[0822] HCDR2 of 922 VHH (SEQ ID NO: 123):

[0823] HCDR3 of 922 VHH (SEQ ID NO: 124)

[0824] HCDR1 of 067 VHH (SEQ ID NO: 125)

[0825] HCDR2 of 067 VHH (SEQ ID NO: 126)

[0826] HCDR3 of 067 VHH (SEQ ID NO: 127)

[0827] HCDR1 of 014 VHH (SEQ ID NO: 128)

[0828] HCDR2 of 014 VHH (SEQ ID NO: 129)

[0829] HCDR3 of 014 VHH (SEQ ID NO: 130)

[0830] HCDR1 of 189 VHH (SEQ ID NO: 131)

[0831] HCDR2 of 189 VHH (SEQ ID NO: 132)

[0832] HCDR3 of 189 VHH (SEQ ID NO: 133)

[0833] HCDR1 of 9D9 VH (SEQ ID NO: 134)

[0834] HCDR2 of 9D9 VH (SEQ ID NO: 135)

[0835] HCDR3 of 9D9 VH (SEQ ID NO: 136)

[0836] LCDR1 of 9D9 VH (SEQ ID NO: 137)

[0837] LCDR2 of 9D9 VH (SEQ ID NO: 138)

[0838] LCDR3 of 9D9 VH (SEQ ID NO: 139)

[0839] HCDR1 of Nb91 VHH (SEQ ID NO: 140)

[0840] HCDR2 of Nb91 VHH (SEQ ID NO: 141)

[0841] HCDR3 of Nb91 VHH (SEQ ID NO: 142)

[0842] HCDR1 of 745 VHH (SEQ ID NO: 143)

[0843] HCDR2 of 745 VHH (SEQ ID NO: 144)

[0844] HCDR3 of 745 VHH (SEQ ID NO: 145)

[0845] HCDR1 of 636 VHH (SEQ ID NO: 146)

[0846] HCDR2 of 636 VHH (SEQ ID NO: 147)

[0847] HCDR3 of 636 VHH (SEQ ID NO: 148)

[0848] HCDR1 of Tremelimumab VH (SEQ ID NO: 149)

[0849] HCDR2 of Tremelimumab VH (SEQ ID NO: 150)

[0850] HCDR3 of Tremelimumab VH (SEQ ID NO: 151)

[0851] LCDR1 of Tremelimumab VL (SEQ ID NO: 152)

[0852] LCDR2 of Tremelimumab VL (SEQ ID NO: 153)

[0853] LCDR3 of Tremelimumab VL (SEQ ID NO: 154)

[0854] HCDR1 of anti-CTLA4 VH of Cadonilimab (SEQ ID NO: 155)

[0855] HCDR2 of anti-CTLA4 VH of Cadonilimab (SEQ ID NO: 156)

[0856] HCDR3 of anti-CTLA4 VH of Cadonilimab (SEQ ID NO: 157)

[0857] LCDR1 of anti-CTLA4 VL of Cadonilimab (SEQ ID NO: 158)

[0858] LCDR2 of anti-CTLA4 VL of Cadonilimab (SEQ ID NO: 159)

[0859] LCDR3 of anti-CTLA4 VL of Cadonilimab (SEQ ID NO: 160)

[0860] HCDR1 of Tuvonralimab (SEQ ID NO: 161)

[0861] HCDR2 of Tuvonralimab (SEQ ID NO: 162)

[0862] HCDR3 of Tuvonralimab (SEQ ID NO: 163)

[0863] LCDR1 of Tuvonralimab (SEQ ID NO: 164)

[0864] LCDR2 of Tuvonralimab (SEQ ID NO: 165)

[0865] LCDR3 of Tuvonralimab (SEQ ID NO: 166)

[0866] HCDR1 of Quavonlimab (SEQ ID NO: 167)

[0867] HCDR2 of Quavonlimab (SEQ ID NO: 168)

[0868] HCDR3 of Quavonlimab (SEQ ID NO: 169)

[0869] LCDR1 of Quavonlimab (SEQ ID NO: 170)

[0870] LCDR2 of Quavonlimab (SEQ ID NO: 171)

[0871] LCDR3 of Quavonlimab (SEQ ID NO: 172)

[0872] HCDR1 of Botensilimab (SEQ ID NO: 173)

[0873] HCDR2 of Botensilimab (SEQ ID NO: 174)

[0874] HCDR3 of Botensilimab (SEQ ID NO: 175)

[0875] LCDR1 of Botensilimab (SEQ ID NO: 176)

[0876] LCDR2 of Botensilimab (SEQ ID NO: 177)

[0877] LCDR3 of Botensilimab (SEQ ID NO: 178)

[0878] HCDR1 of Lorigerlimab (SEQ ID NO: 179)

[0879] HCDR2 of Lorigerlimab (SEQ ID NO: 180)

[0880] HCDR3 of Lorigerlimab (SEQ ID NO: 181)

[0881] LCDR1 of Lorigerlimab (SEQ ID NO: 182)

[0882] LCDR2 of Lorigerlimab (SEQ ID NO: 183)

[0883] LCDR3 of Lorigerlimab (SEQ ID NO: 184)

[0884] HCDR1 of Porustobart (SEQ ID NO: 185)

[0885] HCDR2 of Porustobart (SEQ ID NO: 186)

[0886] HCDR3 of Porustobart (SEQ ID NO: 187)

[0887] HCDR1 of anti-CTLA4 VH of Vudalimab (SEQ ID NO: 188)

[0888] HCDR2 of anti-CTLA4 VH of Vudalimab (SEQ ID NO: 189)

[0889] HCDR3 of anti-CTLA4 VH of Vudalimab (SEQ ID NO: 190)

[0890] LCDR1 of anti-CTLA4 VL of Vudalimab (SEQ ID NO: 191)

[0891] LCDR2 of anti-CTLA4 VL of Vudalimab (SEQ ID NO: 192)

[0892] LCDR3 of anti-CTLA4 VL of Vudalimab (SEQ ID NO: 193)

[0893] HCDR1 of YH-001 (SEQ ID NO: 194)

[0894] HCDR2 of YH-001 (SEQ ID NO: 195)

[0895] HCDR3 of YH-001 (SEQ ID NO: 196)

[0896] LCDR1 of YH-001 (SEQ ID NO: 197)

[0897] LCDR2 of YH-001 (SEQ ID NO: 198)

[0898] LCDR3 of YH-001 (SEQ ID NO: 199)

[0899] HCDR1 of JS-007 (SEQ ID NO: 200)

[0900] HCDR2 of JS-007 (SEQ ID NO: 201)

[0901] HCDR3 of JS-007 (SEQ ID NO: 202)

[0902] LCDR1 of JS-007 (SEQ ID NO: 203)

[0903] LCDR2 of JS-007 (SEQ ID NO: 204)

[0904] LCDR3 of JS-007 (SEQ ID NO: 205)

[0905] HCDR1 of firastotug (SEQ ID NO: 206)

[0906] HCDR2 of firastotug (SEQ ID NO: 207)

[0907] HCDR3 of firastotug (SEQ ID NO: 208)

[0908] LCDR1 of firastotug (SEQ ID NO: 209)

[0909] LCDR2 of firastotug (SEQ ID NO: 210)

[0910] LCDR3 of firastotug (SEQ ID NO: 211)

[0911] HCDR1 of Muzastotug (SEQ ID NO: 212)

[0912] HCDR2 of Muzastotug (SEQ ID NO: 213)

[0913] HCDR3 of Muzastotug (SEQ ID NO: 214)

[0914] LCDR1 of Muzastotug (SEQ ID NO: 215)

[0915] LCDR2 of Muzastotug (SEQ ID NO: 216)

[0916] LCDR3 of Muzastotug (SEQ ID NO: 217)

[0917] VH of Clone D (SEQ ID NO: 218)

[0918] VL of Clone D (SEQ ID NO: 219)

[0919] 090 VHH (anti-PD1) (SEQ ID NO: 220)

[0920] 170 VHH (anti-PD1) (SEQ ID NO: 221)

[0921] 1-14 VH (anti-PD1) (SEQ ID NO: 222)

[0922] 1-14 VL (anti-PD1) (SEQ ID NO: 223)

[0923] Nivolumab VH (anti-PD1) (SEQ ID NO: 224)

[0924] Nivolumab VL (anti-PD1) (SEQ ID NO: 225)

[0925] Pembrolizumab VH (anti-PD1) (SEQ ID NO: 226)

[0926] Pembrolizumab VL (anti-PD1) (SEQ ID NO: 227)

[0927] HCDR1 of 090 VHH (SEQ ID NO: 228)

[0928] HCDR2 of 090 VHH (SEQ ID NO: 229)

[0929] HCDR3 of 090 VHH (SEQ ID NO: 230)

[0930] HCDR1 of 170 VHH (SEQ ID NO: 231)

[0931] HCDR2 of 170 VHH (SEQ ID NO: 232)

[0932] HCDR3 of 170 VHH (SEQ ID NO: 233)

[0933] HCDR1 of 1-14 VH (SEQ ID NO: 234)

[0934] HCDR2 of 1-14 VH (SEQ ID NO: 235)

[0935] HCDR3 of 1-14 VH (SEQ ID NO: 236)

[0936] LCDR1 of 1-14 VH (SEQ ID NO: 237)

[0937] LCDR2 of 1-14 VH (SEQ ID NO: 238)

[0938] LCDR3 of 1-14 VH (SEQ ID NO: 239)

[0939] (SEQ ID NO: 240): HCDR1 of Nivolumab

[0940] HCDR2 of Nivolumab (SEQ ID NO: 241)

[0941] HCDR3 of Nivolumab (SEQ ID NO: 242)

[0942] LCDR1 of Nivolumab (SEQ ID NO: 243)

[0943] LCDR2 of Nivolumab (SEQ ID NO: 244)

[0944] LCDR3 of Nivolumab (SEQ ID NO: 245)

[0945] HCDR1 of: Pembrolizumab (SEQ ID NO: 246)

[0946] HCDR2 of: Pembrolizumab (SEQ ID NO: 247)

[0947] HCDR3 of: Pembrolizumab (SEQ ID NO: 248)

[0948] LCDR1 of: Pembrolizumab (SEQ ID NO: 249)

[0949] LCDR2 of: Pembrolizumab (SEQ ID NO: 250)

[0950] LCDR3 of: Pembrolizumab (SEQ ID NO: 251)

[0951] 20-67 VHH (anti-CCR8) (SEQ ID NO: 252)

[0952] 17-64 VHH (anti-CCR8) (SEQ ID NO: 253)

[0953] BMS-986340 VH (anti-CCR8) (SEQ ID NO: 254)

[0954] BMS-986340 VL (anti-CCR8) (SEQ ID NO: 255)

[0955] LM-108 VH (anti-CCR8) (SEQ ID NO: 256)

[0956] LM-108 VL (anti-CCR8) (SEQ ID NO: 257)

[0957] 21-67 VHH (anti-CCR8) (SEQ ID NO: 258)

[0958] HCDR1 of 20-67 VHH (SEQ ID NO: 259)

[0959] HCDR2 of 20-67 VHH (SEQ ID NO: 260)

[0960] HCDR3 of 20-67 VHH (SEQ ID NO: 261)

[0961] HCDR1 of 17-64 VHH (SEQ ID NO: 262)

[0962] HCDR2 of 17-64 VHH (SEQ ID NO: 263)

[0963] HCDR3 of 17-64 VHH (SEQ ID NO: 264)

[0964] HCDR1 of BMS-986340 VH (SEQ ID NO: 265)

[0965] HCDR2 of BMS-986340 VH (SEQ ID NO: 266)

[0966] HCDR3 of BMS-986340 VH (SEQ ID NO: 267)

[0967] LCDR1 of BMS-986340 VL (SEQ ID NO: 268)

[0968] LCDR2 of BMS-986340 VL (SEQ ID NO: 269)

[0969] LCDR3 of BMS-986340 VL (SEQ ID NO: 270)

[0970] HCDR1 of LM-108 VH (SEQ ID NO: 271)

[0971] HCDR2 of LM-108 VH (SEQ ID NO: 272)

[0972] HCDR3 of LM-108 VH (SEQ ID NO: 273)

[0973] LCDR1 of LM-108 VL (SEQ ID NO: 274)

[0974] LCDR2 of LM-108 VL (SEQ ID NO: 275)

[0975] LCDR3 of LM-108 VL (SEQ ID NO: 276)

[0976] HCDR1 of 21-67 VHH (anti-CCR8) (SEQ ID NO: 277)

[0977] HCDR2 of 21-67 VHH (anti-CCR8) (SEQ ID NO: 278)

[0978] HCDR3 of 21-67 VHH (anti-CCR8) (SEQ ID NO: 279)

[0979] Linker (SEQ ID NO: 280)

[0980] Linker (SEQ ID NO: 281)

[0981] Linker (SEQ ID NO: 282)

[0982] Linker (SEQ ID NO: 283)

[0983] Linker (SEQ ID NO: 284)

[0984] Linker (SEQ ID NO: 285)

[0985] Linker (SEQ ID NO: 286)

[0986] Linker (SEQ ID NO: 287)

[0987] Linker (SEQ ID NO: 288)

[0988] Linker (SEQ ID NO: 289)

[0989] Linker (SEQ ID NO: 290)

[0990] Linker (SEQ ID NO: 291)

[0991] Linker (SEQ ID NO: 292)

[0992] HCDR1 of Clone D (anti-PD1 Ab) (SEQ ID NO: 293)

[0993] HCDR2 of Clone D (anti-PD1 Ab) (SEQ ID NO: 294)

[0994] HCDR3 of Clone D (anti-PD1 Ab) (SEQ ID NO: 295)

[0995] LCDR1 of Clone D (anti-PD1 Ab) (SEQ ID NO: 296)

[0996] LCDR2 of Clone D (anti-PD1 Ab) (SEQ ID NO: 297)

[0997] LCDR3 of Clone D (anti-PD1 Ab) (SEQ ID NO: 298)

[0998] First Fc unit (SEQ ID NO: 299)

[0999] First Fc unit (SEQ ID NO: 300)

[1000] Second Fc unit (SEQ ID NO: 301)

[1001] Second Fc unit (SEQ ID NO: 302)

[1002] Second Fc unit (SEQ ID NO: 303)

[1003] hIL12(WT)-Fc (SEQ ID NO: 304)

[1004] Fc (SEQ ID NO: 305)

[1005] hIL12(4A)-Fc (SEQ ID NO: 306)

[1006] Ipi VH-CH1-hIL12(2A)-Fc (SEQ ID NO: 307)

[1007] Ipi VH-CH1-Fc (SEQ ID NO: 308)

[1008] Ipi VL-CL (SEQ ID NO: 309)

[1009] Ipi VH-CH1-hIL12(4A)-Fc (SEQ ID NO: 310)

[1010] hPDL2-Fc (SEQ ID NO: 311)

[1011] hPDL2-hIL12(2A)-Fc (SEQ ID NO: 312)

[1012] hPDL2-hIL12(4A)-Fc (SEQ ID NO: 313)

[1013] 146 VH-CH1-mIL12(4A)-Fc (SEQ ID NO: 314)

[1014] hPDL2-Fc (SEQ ID NO: 315)

[1015] 146 VL-CL (SEQ ID NO: 316)

[1016] HL32 VH-CH1-mIL12(4A)-Fc (SEQ ID NO: 317)

[1017] hPDL2-Fc (SEQ ID NO: 318)

[1018] HL32 VL-CL1 (SEQ ID NO: 319)

[1019] Ipi VH-CH1-mIL12(4A)-Fc (SEQ ID NO: 320)

[1020] hPDL2-Fc (SEQ ID NO: 321)

[1021] 17-64 VHH-mIL12(4A)-Fc (SEQ ID NO: 322)

[1022] 17-64 VHH-Fc (SEQ ID NO: 323)

[1023] mPDL2-Fc (SEQ ID NO: 324)

[1024] mPDL2-mIL12(4A)-Fc (SEQ ID NO: 325)

[1025] Clone D VH1-CH1-mIL12(4A)-Fc (SEQ ID NO: 326)

[1026] Clone D-VL-CL (SEQ ID NO: 327)

[1027] 090 VHH-mIL2(4A)-Fc (SEQ ID NO: 328)

[1028] AU VHH-CH1-mIL12(4A)-Fc (SEQ ID NO: 329)

[1029] Ipi VH-CH1-mIL12(2A)-Fc (SEQ ID NO: 330)

[1030] mPDL2-Fc (SEQ ID NO: 331)

[1031] Ipi VH-CH1-IL12(3A)-Fc (SEQ ID NO: 332)

[1032] mPDL2-Fc (SEQ ID NO: 333)

[1033] Ipi VH-CH1-mIL12(4A)-Fc (SEQ ID NO: 334)

[1034] Ipi.105-mIL12(2A)-Fc (Knob) (SEQ ID NO: 335)

[1035] (Ipi.105 or Ipi.106) VL-CL (SEQ ID NO: 336)

[1036] Ipi.106 VH-CH1-mIL12(2A)-Fc (SEQ ID NO: 337)

[1037] Erf VHH-mIL12(4A)-Fc (SEQ ID NO: 338)

[1038] 311 VHH-mIL12(4A)-Fc (SEQ ID NO: 339)

[1039] 311 VHH-Fc (SEQ ID NO: 340)

[1040] Erf VHH-Fc (SEQ ID NO: 341)

[1041] AU-Fc (SEQ ID NO: 342)

[1042] 310 VHH-mIL12(4A)-Fc (SEQ ID NO: 343)

[1043] 310 VHH-Fc (SEQ ID NO: 344)

[1044] 313 VHH-mIL12(4A)-Fc (SEQ ID NO: 345)

[1045] 313 VHH-Fc (SEQ ID NO: 346)

[1046] 625 VHH-mIL12(4A)-Fc (SEQ ID NO: 347)

[1047] 625 VHH-Fc (SEQ ID NO: 348)

[1048] 566 VHH-mIL12(4A)-Fc (SEQ ID NO: 349)

[1049] 566 VHH-Fc (SEQ ID NO: 350)

[1050] 922 VHH-mIL12(4A)-Fc (SEQ ID NO: 351)

[1051] 922 VHH-Fc (SEQ ID NO: 352)

[1052] 067 VHH-mIL12(4A)-Fc (SEQ ID NO: 353)

[1053] 067 VHH-Fc (SEQ ID NO: 354)

[1054] 014 VHH-mIL12(4A)-Fc (SEQ ID NO: 355)

[1055] 014 VHH-Fc (SEQ ID NO: 356)

[1056] 189 VHH-mIL12(4A)-Fc (SEQ ID NO: 357)

[1057] 189 VHH-Fc (SEQ ID NO: 358)

[1058] 9D9 VH-CH1-mIL12(4A)-Fc (SEQ ID NO: 359)

[1059] 9D9 VL-CL (SEQ ID NO: 360)

[1060] mPD1 Ab-HC (SEQ ID NO: 361)

[1061] mPD1 Ab-LC (SEQ ID NO: 362)

[1062] mIpi-HC (SEQ ID NO: 363)

[1063] mIpi-LC (SEQ ID NO: 364)

[1064] Nb91 VHH-mIL12(4A)-F (SEQ ID NO: 365)

[1065] WT mIL12-Fc (SEQ ID NO: 366)

[1066] Nb91 VHH-Fc (SEQ ID NO: 367)

[1067] 189 VHH-mIL12(4A)-Fc (SEQ ID NO: 368)

[1068] 189 VHH-Fc (SEQ ID NO: 369)

[1069] 745 VHH-mIL12(4A)-Fc (SEQ ID NO: 370)

[1070] 745 VHH-Fc (SEQ ID NO: 371)

[1071] 636 VHH-mIL12(4A)-Fc (SEQ ID NO: 372)

[1072] 636 VHH-Fc (SEQ ID NO: 373)

[1073] mPDL2-mIL12(4A)-Fc (SEQ ID NO: 374)

[1074] 9D9 VH-CH1-Fc (SEQ ID NO: 375):

[1075] 090 VHH-Fc (SEQ ID NO: 376)

[1076] 9D9 VH-Fc (SEQ ID NO: 377)

[1077] 20-67 VHH-mIL12(4A)-Fc (SEQ ID NO: 378)

[1078] 20-67 VHH-Fc (SEQ ID NO: 379)

[1079] 20-67 VHH-Fc (SEQ ID NO: 380)

[1080] 20-67 VHH-mIL12(4A)-Fc (SEQ ID NO: 381)

[1081] 21-67 VHH-Fc (SEQ ID NO: 382)

[1082] 1-14 VH-CH1-Fc (SEQ ID NO: 383)

[1083] 1-14 VL-CL (SEQ ID NO: 384)

[1084] 1-14 VH-CH1-mIL12(4A)-Fc (SEQ ID NO: 385)

Claims

A method of treating a tumor, the method comprising administering to a subject an effective amount of a Treg cell-targeting IL12 fusion protein that induces intratumoral Treg cells to secrete IFNγ, attenuating or abrogating the immunosuppressive effect of the intratumoral Treg cells. The method of claim 1, wherein, The Treg cell-targeting IL12 fusion protein comprises one or more Treg cell-targeting moieties that specifically bind to a Treg cell surface molecule and IL12, and the IL12 comprises one or more attenuated-activity mutations compared to wild-type IL12. The method according to claim 1 or 2, wherein The one or more attenuated-activity mutations are located in the p35 subunit and / or the p40 subunit of the IL12. The method of claim 3, wherein, The one or more attenuated-activity mutations include one or more mutations at positions W15, E59, F60, K84, and K195 of the p40 subunit, the positions being located according to SEQ ID NO:

5. The method of claim 4, wherein, The one or more attenuated-activity mutations include mutations at any one of the following sets of positions of 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 method according to any one of claims 3-5, wherein, The one or more attenuated-activity mutations include one or more mutations from a wild-type amino acid to A. The method according to any one of claims 3-5, wherein, The one or more attenuated-activity mutations include any one of the following sets 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 E59A / F60A / K84A / K195A. The method according to any one of claims 3-7, wherein, The p40 subunit comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 9-17; preferably, the p40 subunit comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:

14. The method according to any one of claims 1 to 8, wherein The p35 and p40 subunits of the IL12 are connected by a linker, preferably the IL12 after connection by the linker comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 18-26; more preferably the IL12 after connection by the linker comprises an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:

23. The method according to any one of claims 2-9, wherein, The Treg cell surface molecule comprises one or more selected from the group consisting of CTLA4, CCR8, CCR4, CCR10, CD25, GITR, OX-40, ICOS and 4-1BB. The method according to any one of claims 2-10, wherein, The Treg cell targeting IL12 fusion protein further comprises a PD1 targeting moiety that specifically binds PD1. The method according to any one of claims 2-11, wherein, The Treg cell targeting moiety and / or the PD1 targeting moiety comprises a ligand or a ligand fragment, or an antibody or a fragment of an antibody; optionally, the Treg cell targeting moiety and / or the PD1 targeting moiety comprises a ligand extracellular domain (LECD), a Fab, a scFab, a Fab', a (Fab')2, a Fv, a scFv or a VHH. The method of any one of claims 2-12, wherein, The Treg cell targeting IL12 fusion protein further comprises a first Fc unit and a second Fc unit with a hinge region, the first Fc unit dimerizes with the second Fc unit to form a dimer, optionally the IL12 is located between the Treg cell targeting moiety or PD1 targeting moiety and the hinge region of the first Fc unit or second Fc unit. The method of claim 13, wherein, The Treg cell-targeting IL12 fusion proteins include a dimer consisting of A-[L1] n1 -B-[L2] n2 -C and A'-[L3] n3 -C' wherein: A and A' represent a first targeting moiety and a second targeting moiety, respectively, at least one of A and A' specifically binds to a Treg cell surface molecule; B represents IL12 consisting of a p40 subunit - [L4] n4 a p35 subunit or a p35 subunit - [L4] n4 at least one of the p35 and p40 subunits includes one or more attenuating mutations that attenuate the affinity of the IL12 for its receptor as compared to wild type. C and C' represent a first Fc unit and a second Fc unit for dimerization, respectively; L1, L2, L3 and L4 represent linkers, n1, n2, n3 and n4 are selected from 0 or 1; and, - represents a peptide bond. The method of claim 13, wherein, The Treg cell-targeting IL12 fusion proteins include a dimer consisting of A-[L1] n1 -B-[L2] n2 -C and A'-[L3] n3 -B'-[L4] n4 -C'. A and A' represent a first targeting moiety and a second targeting moiety, respectively, at least one of A and A' specifically binds to a Treg cell surface molecule; B represents a p40 subunit and B' represents a p35 subunit, or B represents a p35 subunit and B' represents a p40 subunit; at least one of the p35 and p40 subunits comprises one or more attenuating mutations that attenuate the affinity of the IL12 to bind to its receptor as compared to wild type; C and C' represent a first Fc unit and a second Fc unit for dimerization, respectively; L1, L2, L3 and L4 represent linkers, n1, n2, n3 and n4 are selected from 0 or 1; and, - represents a peptide bond. The method according to claim 14 or 15, wherein The first targeting module and / or the second targeting module comprises a ligand, an antibody or a fragment of the ligand or antibody that specifically binds to a target antigen; optionally, the first targeting module and / or the second targeting module comprises a ligand extracellular domain (LECD), a Fab, a scFab, a Fab', a (Fab')2, a Fv, a scFv or a VHH that specifically binds to the target antigen. The method of claim 16, wherein, The first targeting module and the second targeting module each comprise a Fab. The method of claim 17, wherein, The Treg cell-targeting IL12 fusion protein comprises a first peptide, a second peptide, a third peptide, and a fourth peptide, the first peptide comprising VH1-(H1-CH1)-[L1] n1 -B-[L2] n2 -C, the second peptide comprising VH2-(H2-CH1)-[L3] n3 -C', the third peptide comprising VL1-(L1-CL), and the fourth peptide comprising VL2-(L2-CL); The first peptide forms a dimer with the second peptide through C and C'; The first peptide forms the first targeting module with the third peptide through VH1-(H1-CH1) and VL1-(L1-CL); The second peptide forms the second targeting module with the fourth peptide through VH2-(H2-CH1) and VL2-(L2-CL). The method of claim 16, wherein, The first targeting module comprises a Fab and the second targeting module comprises a VHH or a LECD. The method of claim 19, wherein, The Treg cell-targeting IL12 fusion protein comprises a first peptide, a second peptide, and a third peptide, the first peptide comprising VH1-(H1-CH1)-[L1] n1 -B-[L2] n2 -C, the second peptide comprising VHH2-[L3] n3 -C' or LECD2-[L3] n3 -C', the third peptide comprising VL1-(L1-CL); The first peptide forms a dimer with the second peptide through C and C'; The first peptide forms the first targeting module with the third peptide through VH1-(H1-CH1) and VL1-(L1-CL); The second peptide forms the second targeting module through VHH2 or LECD2 of the second peptide. The method of claim 16, wherein, The first targeting module comprises a VHH or a LECD and the second targeting module comprises a Fab. The method of claim 21, wherein, The Treg cell-targeting IL12 fusion protein comprises a first peptide, a second peptide, and a third peptide, the first peptide comprising VHH1-[L1] n1 -B-[L2] n2 -C or LECD1-[L1] n1 -B-[L2] n2 -C, the second peptide comprising VH2-(H2-CH1)-[L3] n3 -C', the third peptide comprising VL2-(L2-CL). The first peptide forms a dimer with the second peptide through C and C'; VHH1 or LECD1 of the first peptide forms the first targeting module; The second peptide forms the second targeting module with the third peptide through VH1-(H2-CH1) and VL2-(L2-CL). The method of claim 16, wherein, The first targeting module comprises a VHH or a LECD and the second targeting module comprises a VHH or a LECD. The method of claim 23, wherein, The Treg cell-targeting IL12 fusion protein comprises a first peptide and a second peptide, the first peptide comprising VHH1-[L1] n1 -B-[L2] n2 -C or LECD1-[L1] n1 -B-[L2] n2 -C, the second peptide comprising VHH2-[L3] n3 -C’ or LECD2-[L3] n3 -C’; The first peptide forms a dimer with the second peptide through C and C'; VHH1 or LECD1 of the first peptide forms the first targeting module; VHH2 or LECD2 of the second peptide forms the second targeting module. The method of any one of claims 14-24, wherein, The first targeting module specifically binds to a Treg cell surface molecule and the second targeting module specifically binds to a Treg cell surface molecule, optionally, 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 method of any one of claims 14-24, wherein The first targeting module specifically binds to a Treg cell surface molecule and the second targeting module specifically binds to PD1, optionally, the first targeting module specifically binds to CTLA4 or CCR8 and the second targeting module specifically binds to PD1. The method of any one of claims 14-24, wherein, the first targeting moiety specifically binds to PD1 and the second targeting moiety specifically binds to CTLA4 or CCR8. The method of claim 25, wherein, the first targeting moiety binds to its target antigen with a KD value in the range of 1E-10 M to 1E-7 M and the second targeting moiety binds to its target antigen with a KD value in the range of 1E-10 M to 1E-7 M. The method of claim 26, wherein, the first targeting moiety binds to its target antigen with a KD value in the range of 1E-10 M to 1E-7 M and the second targeting moiety binds to its target antigen with a KD value in the range of 1E-8 M to 1E-6 M. The method of claim 27, wherein, the first targeting moiety binds to its target antigen with a KD value in the range of 1E-8 M to 1E-6 M and the second targeting moiety binds to its target antigen with a KD value in the range of 1E-10 M to 1E-7 M. The method of any one of claims 10-30, wherein, the CTLA4 targeting moiety comprises a Fab, scFab, Fab', (Fab')2, Fv, scFv or VHH that specifically binds to CTLA4; optionally, the Fab, scFab, Fab', (Fab')2, Fv and scFv that specifically binds to CTLA4 comprises a VH and a VL as set forth below: (1) the VH comprises a HCDR1 as set forth in SEQ ID NO: 79 or 82, a HCDR2 as set forth in SEQ ID NO: 80 or 83 and a HCDR3 as set forth in SEQ ID NO: 81 or 84; and the VL comprises a LCDR1 as set forth in SEQ ID NO: 85 or 88, a LCDR2 as set forth in SEQ ID NO: 86 and a LCDR3 as set forth in SEQ ID NO: 87; (2) the VH comprises a HCDR1 as set forth in SEQ ID NO: 89, a HCDR2 as set forth in SEQ ID NO: 90 and a HCDR3 as set forth in SEQ ID NO: 91; and the VL comprises a LCDR1 as set forth in SEQ ID NO: 92, a LCDR2 as set forth in SEQ ID NO: 93 and a LCDR3 as set forth in SEQ ID NO: 94; (3) the VH comprises a HCDR1 as set forth in SEQ ID NO: 95, a HCDR2 as set forth in SEQ ID NO: 96 and a HCDR3 as set forth in SEQ ID NO: 97; and the VL comprises a LCDR1 as set forth in SEQ ID NO: 98, a LCDR2 as set forth in SEQ ID NO: 99 and a LCDR3 as set forth in SEQ ID NO: 100; or, (4) the VH comprises HCDR1 as set forth in SEQ ID NO: 134, HCDR2 as set forth in SEQ ID NO: 135, and HCDR3 as set forth in SEQ ID NO: 136; and the VL comprises LCDR1 as set forth in SEQ ID NO: 137, LCDR2 as set forth in SEQ ID NO: 138, and LCDR3 as set forth in SEQ ID NO: 139; Optionally, the VHH that specifically binds to CTLA4 comprises: (1) HCDR1 as set forth in SEQ ID NO: 101, HCDR2 as set forth in SEQ ID NO: 102, and HCDR3 as set forth in SEQ ID NO: 103; (2) HCDR1 as set forth in SEQ ID NO: 104, HCDR2 as set forth in SEQ ID NO: 105, and HCDR3 as set forth in SEQ ID NO: 106; (3) HCDR1 as set forth in SEQ ID NO: 107, HCDR2 as set forth in SEQ ID NO: 108, and HCDR3 as set forth in SEQ ID NO: 109; (4) HCDR1 as set forth in SEQ ID NO: 110, HCDR2 as set forth in SEQ ID NO: 111, and HCDR3 as set forth in SEQ ID NO: 112; (5) HCDR1 as set forth in SEQ ID NO: 113, HCDR2 as set forth in SEQ ID NO: 114, and HCDR3 as set forth in SEQ ID NO: 115; (6) HCDR1 as set forth in SEQ ID NO: 116, HCDR2 as set forth in SEQ ID NO: 117, and HCDR3 as set forth in SEQ ID NO: 118; (7) HCDR1 as set forth in SEQ ID NO: 119, HCDR2 as set forth in SEQ ID NO: 120, and HCDR3 as set forth in SEQ ID NO: 121; (8) HCDR1 as set forth in SEQ ID NO: 122, HCDR2 as set forth in SEQ ID NO: 123, and HCDR3 as set forth in SEQ ID NO: 124; (9) HCDR1 as set forth in SEQ ID NO: 125, HCDR2 as set forth in SEQ ID NO: 126, and HCDR3 as set forth in SEQ ID NO: 127; (10) HCDR1 as set forth in SEQ ID NO: 128, HCDR2 as set forth in SEQ ID NO: 129, and HCDR3 as set forth in SEQ ID NO: 130; (11) HCDR1 as set forth in SEQ ID NO: 131, HCDR2 as set forth in SEQ ID NO: 132, and HCDR3 as set forth in SEQ ID NO: 133; (12) HCDR1 as depicted in SEQ ID NO: 140, HCDR2 as depicted in SEQ ID NO: 141, and HCDR3 as depicted in SEQ ID NO: 142; (13) HCDR1 as depicted in SEQ ID NO: 143, HCDR2 as depicted in SEQ ID NO: 144, and HCDR3 as depicted in SEQ ID NO: 145; or, (14) HCDR1 as depicted in SEQ ID NO: 146, HCDR2 as depicted in SEQ ID NO: 147, and HCDR3 as depicted in SEQ ID NO:

148. The method of claim 31, wherein, The Fab, scFab, Fab', (Fab')2, Fv, and scFv that specifically bind to CTLA4 include a VH and a VL as depicted below: (1) the VH includes a sequence as depicted in any one of SEQ ID NOs: 31-33, and the VL includes a sequence as depicted in SEQ ID NO: 34 or 35; (2) the VH includes a sequence as depicted in SEQ ID NO: 36, and the VL includes a sequence as depicted in SEQ ID NO: 37; (3) the VH includes a sequence as depicted in SEQ ID NO: 38, and the VL includes a sequence as depicted in SEQ ID NO: 39; (4) the VH includes a sequence as depicted in SEQ ID NO: 51, and the VL includes a sequence as depicted in SEQ ID NO: 52; (5) an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to the VH and / or VL depicted in any one of groups (1)-(4). The method of claim 31, wherein, The VHH that specifically binds to CTLA4 includes: (1) a sequence as depicted in any one of SEQ ID NOs: 40-50, 53-55, 68; (2) an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to the sequence depicted in group (1). The method of any one of claims 10-30, wherein, The CCR8 targeting moiety includes a Fab, scFab, Fab', (Fab')2, Fv, scFv, or VHH that specifically binds to CCR8; Optionally, the Fab, scFab, Fab', (Fab')2, Fv, and scFv that specifically binds to CCR8 include a VH and a VL as depicted below: (1) HCDR1 as depicted in SEQ ID NO: 265, HCDR2 as depicted in SEQ ID NO: 266, and HCDR3 as depicted in SEQ ID NO: 267; the VL includes LCDR1 as depicted in SEQ ID NO: 268, LCDR2 as depicted in SEQ ID NO: 269, and LCDR3 as depicted in SEQ ID NO: 270; or, (2) the VH comprises HCDR1 as set forth in SEQ ID NO: 271, HCDR2 as set forth in SEQ ID NO: 272, and HCDR3 as set forth in SEQ ID NO: 273; and the VL comprises LCDR1 as set forth in SEQ ID NO: 274, LCDR2 as set forth in SEQ ID NO: 275, and LCDR3 as set forth in SEQ ID NO: 276; Optionally, the VHH that specifically binds CCR8 comprises: (1) HCDR1 as set forth in SEQ ID NO: 259, HCDR2 as set forth in SEQ ID NO: 260, and HCDR3 as set forth in SEQ ID NO: 261 (2) HCDR1 as set forth in SEQ ID NO: 262, HCDR2 as set forth in SEQ ID NO: 263, and HCDR3 as set forth in SEQ ID NO: 264; or, (3) HCDR1 as set forth in SEQ ID NO: 277, HCDR2 as set forth in SEQ ID NO: 278, and HCDR3 as set forth in SEQ ID NO:

279. The method of claim 34, wherein, The Fab, scFab, Fab', (Fab')2, Fv, and scFv that specifically binds CCR8 comprises VH and VL as set forth: (1) the VH comprises a sequence as set forth in SEQ ID NO: 254, and the VL comprises a sequence as set forth in SEQ ID NO: 255; (2) the VH comprises a sequence as set forth in SEQ ID NO: 256, and the VL comprises a sequence as set forth in SEQ ID NO: 257; or, (3) an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to the VH and / or VL as set forth in any one of groups (1)-(2). The method of claim 34, wherein, The VHH that specifically binds CCR8 comprises: (1) a sequence as set forth in any one of SEQ ID NOs: 252-253 or 258; or, (2) an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to the sequence as set forth in group (1). The method according to any one of claims 12-24, 26-27, wherein, The PD1 targeting moiety comprises a Fab, scFab, Fab', (Fab')2, Fv, scFv, or VHH that specifically binds PD1; Optionally, the Fab, scFab, Fab', (Fab')2, Fv, and scFv comprises VH and VL as set forth: (1) the VH comprises HCDR1 as set forth in SEQ ID NO: 234, HCDR2 as set forth in SEQ ID NO: 235, and HCDR3 as set forth in SEQ ID NO: 236, and the VL comprises LCDR1 as set forth in SEQ ID NO: 237, LCDR2 as set forth in SEQ ID NO: 238, and LCDR3 as set forth in SEQ ID NO: 239; (2) the VH comprises a HCDR1 as set forth in SEQ ID NO: 240, a HCDR2 as set forth in SEQ ID NO: 241, and a HCDR3 as set forth in SEQ ID NO: 242, and the VL comprises a LCDR1 as set forth in SEQ ID NO: 243, a LCDR2 as set forth in SEQ ID NO: 244, and a LCDR3 as set forth in SEQ ID NO: 245; (3) the VH comprises a HCDR1 as set forth in SEQ ID NO: 246, a HCDR2 as set forth in SEQ ID NO: 247, and a HCDR3 as set forth in SEQ ID NO: 248, and the VL comprises a LCDR1 as set forth in SEQ ID NO: 249, a LCDR2 as set forth in SEQ ID NO: 250, and a LCDR3 as set forth in SEQ ID NO: 251; or, (4) the VH comprises a HCDR1 as set forth in SEQ ID NO: 293, a HCDR2 as set forth in SEQ ID NO: 294, and a HCDR3 as set forth in SEQ ID NO: 295, and the VL comprises a LCDR1 as set forth in SEQ ID NO: 296, a LCDR2 as set forth in SEQ ID NO: 297, and a LCDR3 as set forth in SEQ ID NO: 298; Optionally, the VHH that specifically binds PD1 comprises: (1) a HCDR1 as set forth in SEQ ID NO: 228, a HCDR2 as set forth in SEQ ID NO: 229, and a HCDR3 as set forth in SEQ ID NO: 230; or, (2) a HCDR1 as set forth in SEQ ID NO: 231, a HCDR2 as set forth in SEQ ID NO: 232, and a HCDR3 as set forth in SEQ ID NO:

233. The method of claim 37, wherein, The Fab, scFab, Fab', (Fab')2, Fv, and scFv that specifically binds PD1 comprises a VH and a VL as set forth below: (1) the VH comprises a sequence as set forth in SEQ ID NO: 218, and the VL comprises a sequence as set forth in SEQ ID NO: 219; (2) the VH comprises a sequence as set forth in SEQ ID NO: 222, and the VL comprises a sequence as set forth in SEQ ID NO: 223; (3) the VH comprises a sequence as set forth in SEQ ID NO: 224, and the VL comprises a sequence as set forth in SEQ ID NO: 225; (4) the VH comprises a sequence as set forth in SEQ ID NO: 226, and the VL comprises a sequence as set forth in SEQ ID NO: 227; or, (5) an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to the VH and / or VL set forth in any one of groups (1)-(4). The method of claim 37, wherein, The VHH that specifically binds PD1 comprises: (1) a sequence set forth in any one of SEQ ID NOs: 220 or 221; or, (2) an amino acid sequence having at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identity to a sequence set forth in group (1). The method according to any one of claims 12-24, 26-27, wherein, the PD1 targeting moiety comprises a PDL1 extracellular domain and / or a PDL2 extracellular domain; Optionally, the PD1 targeting moiety comprises: (1) a sequence set forth in any one of SEQ ID NOs: 27-29; (2) an amino acid sequence having at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identity to a sequence set forth in group (1). The method of any one of claims 13-40, wherein, the first Fc unit and the second Fc unit comprise a Knob mutation and a Hole mutation to form a Knob-in-Hole structure; Preferably, the Knob mutation is selected from S354C, T366W, preferably S354C / T366W, and the Hole mutation is selected from Y349C, T366S, L368A, and Y349C / T366S / L368A / Y349C, preferably Y349C / T366S / L368A / Y349C. The method of any one of claims 13-41, wherein, the first Fc unit and the second Fc unit comprise a mutation that reduces or abrogates effector function; Preferably, the mutation that reduces or abrogates effector function comprises L234A / L235A mutation. The method of any one of claims 13-42, wherein, the first Fc unit comprises: (1) a sequence set forth in any one of SEQ ID NOs: 299 or 300; or, (2) an amino acid sequence having at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identity to a sequence set forth in group (1). and / or, the second Fc unit comprises: (1) a sequence set forth in any one of SEQ ID NOs: 301-303; or, (2) an amino acid sequence having at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identity to a sequence set forth in group (1). The method of any one of claims 9, 14-43, wherein, the linker is selected from a sequence set forth in any one of SEQ ID NOs: 280-292. The method of claim 14, the Treg cell-targeting IL12 fusion protein comprises: (1) a first peptide, a second peptide, a third peptide, and a fourth peptide, the first peptide comprising a sequence having at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 307 or 310, the second peptide comprising a sequence having at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 308, the third peptide comprising a sequence having at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 309, and the fourth peptide comprising a sequence having at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 309; (2) a first peptide comprising a sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 307 or 310, a second peptide comprising a sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 311, and a third peptide comprising a sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 309; (3) a first peptide comprising a sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 312 or 313, a second peptide comprising a sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 308, and a third peptide comprising a sequence that is at least 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:

309. The method of any one of claims 1-45, wherein, The Treg cell-targeting IL12 fusion protein is administered systemically, e.g., intravenously or subcutaneously. The method of any one of claims 1-46, wherein, The method is a method of administering the Treg cell-targeting IL12 fusion protein as a monotherapy or in combination with another therapy. The method of claim 47, wherein, The another therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, toxin therapy, and surgery. The method of claim 45 or 46, wherein, The Treg cell-targeting IL12 fusion protein is administered before, after, or concurrently with the another therapy. The method of any one of claims 1-49, wherein The subject is an immune checkpoint inhibitor-resistant patient, preferably the immune checkpoint inhibitor is a PD1 antibody. The method of any one of claims 1-450, wherein, 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 brain glioma.

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