Multifunctional molecule containing shielding interleukin 12 and method of use

Abstract

This disclosure relates to a multifunctional molecule containing a shielding interleukin 12, and to methods for fabricating and using the same. In certain embodiments, the multifunctional molecule disclosed herein includes a cleavable linker.

Description

[Technical Field]

[0001] This disclosure relates to a multifunctional molecule containing shielding interleukin 12, and to methods for producing and using the same. [Background technology]

[0002] Interleukin-12 (IL12) is a 74 kDa heterodimer molecule containing P35 and P40 subunits covalently linked by a disulfide bond. IL12 is known as an activator of natural killer (NK) cells, as well as an inducer of IFN-γ from NK and T cells. Studies have shown that IL12 can enhance tumor cell killing mediated by therapeutic antibodies and immune cells. However, systemic administration of IL12 can cause serious side effects, including fever, gastrointestinal reactions, lymphopenia, cytokine release syndrome, and even death. To address these issues, IL12 prodrugs engineered with shielding domains such as IL12 receptor beta have been used. However, these prodrugs often face challenges in terms of stability and developability due to their complex structure. Because IL12 plays a crucial role in immunomodulation and oncology, there is still a need in this field for the development of therapeutic molecules of IL12 for immunotherapy and cancer treatment that have reduced side effects and improved stability and developability. [Overview of the project]

[0003] This disclosure provides a multifunctional shielding IL12 molecule and methods of use. Furthermore, this disclosure provides the multifunctional molecule disclosed herein, as well as methods for producing and using pharmaceutical compositions containing it, for example, for treating diseases and disorders, such as cancer. The present invention is in part based on the discovery of a novel multifunctional shielding IL12 molecule having a novel structure and shielding moiety that exhibits excellent shielding effect and enhanced molecular development potential.

[0004] In certain embodiments, the multifunctional molecule disclosed herein comprises: a) a first chain comprising a first shielding portion, a first cleavable linker, a P35 molecule, and a first Fc region; and b) a second chain comprising a second shielding portion, a second cleavable linker, a P40 molecule, and a second Fc region.

[0005] In certain embodiments, the first shielding portion includes the IL12Rβ2 portion. In certain embodiments, the first shielding portion includes the amino acid sequence shown in SEQ ID NO: 4 or 5. In certain embodiments, the first shielding portion includes the amino acid sequence shown in SEQ ID NO: 4. In certain embodiments, the first shielding portion includes the amino acid sequence shown in SEQ ID NO: 5. In certain embodiments, the second shielding portion includes the IL12Rβ1 portion. In certain embodiments, the second shielding portion includes the amino acid sequence shown in SEQ ID NO: 3.

[0006] In certain embodiments, each of the first and second cleavable linkers can be recognized and hydrolyzed by a proteolytic enzyme specifically expressed in the tumor microenvironment. In certain embodiments, the proteolytic enzyme is a matrix metalloproteinase. In certain embodiments, the matrix metalloproteinase is matrix metalloproteinase 14 (MMP14). In certain embodiments, each of the first and second cleavable linkers contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 67-112. In certain embodiments, the first cleavable linker contains the amino acid sequence shown in SEQ ID NO: 6 or 7. In certain embodiments, the first cleavable linker contains the amino acid sequence shown in SEQ ID NO: 6. In certain embodiments, the first cleavable linker contains the amino acid sequence shown in SEQ ID NO: 7. In certain embodiments, the second cleavable linker contains the amino acid sequence shown in SEQ ID NO: 8.

[0007] In certain embodiments, the first Fc region and the second Fc region form a dimerized Fc region. In certain embodiments, the dimerized Fc region includes a human Fc region. In certain embodiments, the dimerized Fc region includes an Fc region selected from the group consisting of IgG, IgA, IgD, IgE, and IgM Fc regions. In certain embodiments, the dimerized Fc region includes an Fc region selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 Fc regions. In certain embodiments, the dimerized Fc region includes an IgG1 Fc region. In certain embodiments, the first Fc region includes a knob chain and the second Fc region includes a hole chain. In certain embodiments, the first Fc region includes a hole chain and the second Fc region includes a knob chain. In certain embodiments, the knob chain includes the mutations S354C, T366W, and K408A. In certain embodiments, the hole chain includes mutations Y349C, T366S, L368A, F405K, and Y406V. In certain embodiments, each of the first and second Fc regions includes one or more mutations that reduce FcγR and C1q binding to the Fc region. In certain embodiments, each of the first and second Fc regions includes mutations L234A and L235A. In certain embodiments, each of the first and second Fc regions includes mutations L234A, L235A, and P329G.

[0008] In certain embodiments, the P35 and P40 portions are linked to the Fc region via a linker. In certain embodiments, the linker is a peptide linker. In certain embodiments, the peptide linker contains about 4 to about 30 amino acids. In certain embodiments, the peptide linker contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 49 to 66.

[0009] In certain embodiments, the first chain comprises the amino acid sequence set forth in SEQ ID NO: 9 or 10. In certain embodiments, the first chain comprises the amino acid sequence set forth in SEQ ID NO: 9. In certain embodiments, the first chain comprises the amino acid sequence set forth in SEQ ID NO: 10. In certain embodiments, the second chain comprises the amino acid sequence set forth in SEQ ID NO: 11.

[0010] In certain embodiments, the second shielding portion comprises an IL12Rβ1 portion comprising one or more mutations of C6S, C55S, Y85S, or Q108L. In certain embodiments, the second shielding portion comprises an IL12Rβ1 portion comprising the mutations of C6S, C55S, Y85S, and Q108L. In certain embodiments, the second chain comprises the amino acid sequence set forth in SEQ ID NO: 34.

[0011] In certain embodiments, the first chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-29. In certain embodiments, the first chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-29. In certain embodiments, the second chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 30-40. In certain embodiments, the second chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-40.

[0012] In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 20, and the second chain includes the amino acid sequence shown in SEQ ID NO: 31. In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 20, and the second chain includes the amino acid sequence shown in SEQ ID NO: 34. In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 20, and the second chain includes the amino acid sequence shown in SEQ ID NO: 36. In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 21, and the second chain includes the amino acid sequence shown in SEQ ID NO: 30. In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 21, and the second chain includes the amino acid sequence shown in SEQ ID NO: 31. In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 21, and the second chain includes the amino acid sequence shown in SEQ ID NO: 34. In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 21, and the second chain includes the amino acid sequence shown in SEQ ID NO: 36. In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 25, and the second chain includes the amino acid sequence shown in SEQ ID NO: 30. In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 25, and the second chain includes the amino acid sequence shown in SEQ ID NO: 31. In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 25, and the second chain includes the amino acid sequence shown in SEQ ID NO: 34. In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 25, and the second chain includes the amino acid sequence shown in SEQ ID NO: 36. In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 28, and the second chain includes the amino acid sequence shown in SEQ ID NO: 30. In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 28, and the second chain includes the amino acid sequence shown in SEQ ID NO: 31. In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 28, and the second chain includes the amino acid sequence shown in SEQ ID NO: 34.In a particular embodiment, the first chain comprises the amino acid sequence shown in SEQ ID NO: 28, and the second chain comprises the amino acid sequence shown in SEQ ID NO: 36.

[0013] In certain embodiments, the multifunctional molecule further comprises an antibody moiety. In certain embodiments, the antibody moiety comprises a full-length immunoglobulin, a single-chain Fv(scFv) fragment, a Fab fragment, a Fab' fragment, F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, VHH, a VHH-Fc fusion, an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a tribody, a tetrabody, or any combination thereof. In certain embodiments, the antibody moiety comprises VHH. In certain embodiments, the antibody moiety comprises scFv. In certain embodiments, the antibody moiety is linked to the C-terminus of a first chain. In certain embodiments, the antibody moiety is linked to the C-terminus of a second chain. In certain embodiments, the antibody moiety is linked to the C-terminus of the first chain and / or the second chain via a second linker. In certain embodiments, the second linker is a peptide linker. In certain embodiments, the second linker contains about 4 to about 30 amino acids. In certain embodiments, the second linker contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 49 to 66.

[0014] In certain embodiments, the antibody portion binds to a tumor-associated antigen. In certain embodiments, tumor-associated antigens include PDL1, CD10, CD19, CD20, CD21, CD22, CD25, CD30, CD33, CD34, CD37, CD44v6, CD45, CDw52, Fms-like tyrosine kinase 3 (FLT-3, CD135), c-Kit (CD117), CSF1R (CD115), CD133, PDGFR-alpha (CD140a), PDGFR-beta (CD140b), chondroitin sulfate proteoglycan 4 (CSPG4), Muc-1, EGFR, de2-7-EGFR, EGFRvIII, folate-binding protein, Her2neu, Her3, PSMA, PSCA, PSA, TAG-72, HLA-DR, IGFR, IL3R, fibroblast-activating protein (FAP), and carboanhydrase IX (MN / CA). IX) is selected from the group consisting of carcinoembryonic antigen (CEA), EpCAM, CDCP1, Delrin 1, Tenascin, frizzled1-10, vascular antigens VEGFR2 (KDR / FLK1), VEGFR3 (FLT4, CD309), Endoglin, CLEC14, Tem1-8, and Tie2, as well as any combination thereof. In certain embodiments, the tumor-associated antigen is PDL1.

[0015] In certain embodiments, the antibody portion includes a VHH containing a heavy chain variable region CDR1 containing the amino acid sequence shown in SEQ ID NO: 12, a heavy chain variable region CDR2 containing the amino acid sequence shown in SEQ ID NO: 13, and a heavy chain variable region CDR3 containing the amino acid sequence shown in SEQ ID NO: 14. In certain embodiments, the antibody portion includes a VHH containing the amino acid sequence shown in SEQ ID NO: 15. In certain embodiments, the first chain contains the amino acid sequence shown in SEQ ID NO: 41. In certain embodiments, the first chain contains the amino acid sequence shown in SEQ ID NO: 43. In certain embodiments, the second chain contains the amino acid sequence shown in SEQ ID NO: 42. In certain embodiments, the second chain contains the amino acid sequence shown in SEQ ID NO: 44. In certain embodiments, the first chain contains the amino acid sequence shown in SEQ ID NO: 41, and the second chain contains the amino acid sequence shown in SEQ ID NO: 42. In certain embodiments, the first chain contains the amino acid sequence shown in SEQ ID NO: 43, and the second chain contains the amino acid sequence shown in SEQ ID NO: 44.

[0016] In certain embodiments, the multifunctional molecule further comprises a second cytokine moiety. In certain embodiments, the second cytokine moiety comprises TNFα, IFN-γ, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL9, IL13, IL15, IL17, IL18, IL21, IL22, IL15α, TGFβ, G-CSF, GM-CSF, or any combination thereof. In certain embodiments, the second cytokine moiety comprises an IL7 molecule. In certain embodiments, the multifunctional molecule comprises a first chain comprising the amino acid sequence shown in SEQ ID NO: 21 and a second chain comprising the amino acid sequence shown in SEQ ID NO: 45. In certain embodiments, the second cytokine moiety comprises an IL15 molecule. In certain embodiments, the multifunctional molecule comprises a first chain comprising the amino acid sequence shown in SEQ ID NO: 21 and a second chain comprising the amino acid sequence shown in SEQ ID NO: 46. In certain embodiments, the second cytokine moiety comprises an IL18 molecule. In certain embodiments, the multifunctional molecule comprises a first chain containing the amino acid sequence shown in SEQ ID NO: 21 and a second chain containing the amino acid sequence shown in SEQ ID NO: 47. In certain embodiments, the second cytokine moiety contains the IL15 molecule and IL15α sushi. In certain embodiments, the multifunctional molecule comprises a first chain containing the amino acid sequence shown in SEQ ID NO: 48 and a second chain containing the amino acid sequence shown in SEQ ID NO: 46.

[0017] Furthermore, this disclosure provides pharmaceutical compositions. In certain embodiments, the pharmaceutical composition comprises a) a multifunctional molecule disclosed herein, and b) a pharmaceutically acceptable carrier.

[0018] Furthermore, this disclosure provides one or more nucleic acids encoding the multifunctional molecules disclosed herein, one or more vectors comprising the nucleic acids disclosed herein, and host cells comprising the nucleic acids or vectors disclosed herein. In certain embodiments, the host cells have no or reduced endogenous expression of matrix metalloproteinases. In certain embodiments, the host cells have no or reduced endogenous expression of MMP14. In certain embodiments, the host cells are HEK293 cells, their mutants, or derivatives thereof. In certain embodiments, the host cells are Expi293 cells, their mutants, or derivatives thereof. In certain embodiments, the host cells are Expi293 cells. In certain embodiments, the host cells are Expi293F cells. In certain embodiments, the host cells are CHO cells, their mutants, or derivatives thereof in which the MMP14 gene is knocked out or knocked down.

[0019] Furthermore, this disclosure provides methods for preparing the multifunctional molecules disclosed herein. In certain embodiments, the method includes expressing the multifunctional molecules in the host cells disclosed herein and isolating the multifunctional molecules from the host cells. In certain embodiments, the host cells are Expi293 cells that do not endogenously express MMP14. In certain embodiments, the host cells are CHO cells, mutants thereof, or derivatives thereof in which the MMP14 gene is knocked out or knocked down.

[0020] Furthermore, this disclosure provides methods for treating and / or preventing cancer. In certain embodiments, the method involves administering an effective amount of a multifunctional molecule or a pharmaceutical composition disclosed herein to a target. In certain embodiments, cancer exhibits high microsatellite instability (MSI).

[0021] Furthermore, this disclosure provides any multifunctional molecules disclosed herein for use as pharmaceuticals. Furthermore, this disclosure provides any multifunctional molecules disclosed herein for use in the treatment of cancer. Furthermore, this disclosure provides pharmaceutical compositions disclosed herein for use as pharmaceuticals. Furthermore, this disclosure provides pharmaceutical compositions disclosed herein for use in the treatment of cancer.

[0022] In certain embodiments, the cancer is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial cancer, gastric cancer, bile duct cancer, head and neck cancer, hematological cancer, and combinations thereof.

[0023] Furthermore, this disclosure provides kits comprising multifunctional molecules, immunoconjugates, pharmaceutical compositions, nucleic acids, vectors, or host cells disclosed herein. In certain embodiments, the kit includes written instructions for use in treating and / or preventing neoplasms.

[0024] Furthermore, this disclosure provides a method for preparing a multifunctional molecule comprising a cleavable linker that can be recognized and hydrolyzed by MMP14. In certain embodiments, the method comprises expressing the multifunctional molecule in a host cell containing the nucleic acid of the multifunctional molecule, and isolating the multifunctional molecule from the host cell. In certain embodiments, the host cell has no or reduced endogenous expression of MMP14. In certain embodiments, the host cell is HEK293 cell, a mutant thereof, or a derivative thereof. In certain embodiments, the host cell is Expi293 cell, a mutant thereof, or a derivative thereof. In certain embodiments, the host cell is Expi293 cell. In certain embodiments, the host cell is Expi293F cell. In certain embodiments, the host cell is CHO cell, a mutant thereof, or a derivative thereof in which the MMP14 gene is knocked out or knocked down.

[0025] Furthermore, this disclosure provides a method for treating a target cancer, comprising administering an effective amount of the multifunctional molecule and anti-PD-L1 antibody disclosed herein to the target. In certain embodiments, the cancer exhibits high microsatellite instability (MSI). In certain embodiments, the cancer is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial cancer, gastric cancer, bile duct cancer, head and neck cancer, hematological cancer, and combinations thereof. In certain embodiments, the multifunctional molecule and anti-PD-L1 antibody are administered simultaneously or sequentially. In certain embodiments, the multifunctional molecule and anti-PD-L1 antibody are administered simultaneously. In certain embodiments, one or more doses of the anti-PD-L1 antibody are administered before the administration of the multifunctional molecule. In certain embodiments, the target underwent the entire course of anti-PD-L1 antibody therapy before the administration of the multifunctional molecule. In certain embodiments, the multifunctional molecule is administered during a second course of anti-PD-L1 antibody therapy. In certain embodiments, the subject receives at least one, at least two, at least three, or at least four doses of anti-PD-L1 antibody prior to the administration of the multifunctional molecule. In certain embodiments, at least one dose of anti-PD-L1 antibody is administered concurrently with the multifunctional molecule. In certain embodiments, one or more doses of the multifunctional molecule are administered before the administration of anti-PD-L1 antibody. In certain embodiments, the subject receives at least two, at least three, at least three, or at least four doses of the multifunctional molecule prior to the administration of anti-PD-L1 antibody. In certain embodiments, at least one dose of the multifunctional molecule is administered concurrently with anti-PD-L1 antibody. In certain embodiments, the multifunctional molecule and anti-PD-L1 antibody are administered once every 1, 2, 3, 4, or 5 weeks. In certain embodiments, the cancer is recurrent or progressive after therapy selected from the group consisting of surgery, chemotherapy, radiotherapy, and any combination thereof. [Brief explanation of the drawing]

[0026] [Figure 1]This figure shows the endogenous MMP9 expression of Expi293 cells, Wayne293 cells, and CHO cells as evaluated by FACS. [Figure 2] This figure shows the endogenous MMP14 expression of Expi293 cells, Wayne293 cells, and CHO cells as evaluated by FACS. [Figure 3] This figure shows schematics of the IL12 prodrug and the control. [Figure 4] This figure shows the protein expression of the IL12 prodrug as evaluated by SDS-PAGE. [Figure 5] This figure shows the purity and homogeneity of the IL12 prodrug as evaluated by high-performance liquid chromatography (HPLC) analysis. [Figure 6] This figure shows the protein expression of the IL12 prodrug as evaluated by SDS-PAGE. [Figure 7] This figure shows the purity and homogeneity of the IL12 prodrug as evaluated by high-performance liquid chromatography (HPLC) analysis. [Figure 8] This figure shows the IL12 function of IL12 prodrugs without MMP, as evaluated by the HEK-Blue IL12 reporter assay. [Figure 9] This figure shows the IL12 function of IL12 prodrugs with and without MMP, as evaluated by the HEK-Blue IL12 reporter assay. [Figure 10] This figure shows the IL12 function of IL12 prodrugs with and without MMP, as evaluated by the HEK-Blue IL12 reporter assay. [Figure 11] This figure shows the IL12 function of IL12 prodrugs with and without MMP, as evaluated by the HEK-Blue IL12 reporter assay. [Figure 12] This figure shows the shielding efficiency of IL12 prodrugs as evaluated by an IL12 receptor binding assay. [Figure 13A] This figure shows the purity of L1 and L2 as evaluated by SEC-HPLC (13A) and non-reducing CE-SDS (CE-NR) (13B). [Figure 13B] This figure shows the purity of L1 and L2 as evaluated by SEC-HPLC (13A) and non-reducing CE-SDS (CE-NR) (13B). [Figure 14] This figure shows the IL12 function of L1 and L2 cells, evaluated with and without MMP, as assessed by the HEK-Blue IL12 reporter assay. [Figure 15] This figure shows the immune-activating capacity of L2 as evaluated by IFNγ induction in peripheral blood mononuclear cells (PBMCs). [Figure 16] This figure shows the SEC-HPLC profiles of L2 and A-D1-3+B-D12 under the same culture and purification conditions. [Figure 17] This figure shows the IL12 function of L2 and A-D1-3+B-D12, evaluated with and without MMP, as assessed by the HEK-Blue IL12 reporter assay. [Figure 18] This figure shows the binding of L2 to the human IL12 receptor on HEK-Blue® IL12 reporter cells. [Figure 19] This figure shows the L2 shielding efficiency and immune activation capacity measured by IFNγ induction using peripheral blood mononuclear cells (PBMCs). [Figure 20A] This figure shows the in vivo antitumor efficacy of L2 in a B16F10 melanoma mouse model. In Figure 20A, the upper panel shows the mean tumor growth curve, and the lower panel shows the individual tumor growth curves. Figure 20B shows the changes in mouse body weight. [Figure 20B] This figure shows the in vivo antitumor efficacy of L2 in a B16F10 melanoma mouse model. In Figure 20A, the upper panel shows the mean tumor growth curve, and the lower panel shows the individual tumor growth curves. Figure 20B shows the changes in mouse body weight. [Figure 21A] This figure shows the in vivo antitumor efficacy of L2 in the COLO205 colon cancer mouse model. Figure 21A shows the mean tumor growth curve. Figure 21B shows the change in mouse body weight. [Figure 21B]This figure shows the in vivo antitumor efficacy of L2 in the COLO205 colon cancer mouse model. Figure 21A shows the mean tumor growth curve. Figure 21B shows the change in mouse body weight. [Figure 22A] This figure shows the in vivo antitumor efficacy of L2 in NSG mice carrying human colon cancer HCT116 cells. Figure 22A shows the mean tumor growth curve. Figure 22B shows the mean tumor weight 19 days after treatment. Figure 22C shows the change in mouse body weight. Figure 22D shows lymphocyte tumor infiltration 19 days after treatment. [Figure 22B] This figure shows the in vivo antitumor efficacy of L2 in NSG mice carrying human colon cancer HCT116 cells. Figure 22A shows the mean tumor growth curve. Figure 22B shows the mean tumor weight 19 days after treatment. Figure 22C shows the change in mouse body weight. Figure 22D shows lymphocyte tumor infiltration 19 days after treatment. [Figure 22C] This figure shows the in vivo antitumor efficacy of L2 in NSG mice carrying human colon cancer HCT116 cells. Figure 22A shows the mean tumor growth curve. Figure 22B shows the mean tumor weight 19 days after treatment. Figure 22C shows the change in mouse body weight. Figure 22D shows lymphocyte tumor infiltration 19 days after treatment. [Figure 22D] This figure shows the in vivo antitumor efficacy of L2 in NSG mice carrying human colon cancer HCT116 cells. Figure 22A shows the mean tumor growth curve. Figure 22B shows the mean tumor weight 19 days after treatment. Figure 22C shows the change in mouse body weight. Figure 22D shows lymphocyte tumor infiltration 19 days after treatment. [Figure 23] This figure shows the serum concentrations of tumor-bearing female NSG mice after single-dose intravenous infusion of L2 at doses of 0.06 and 0.18 mg / kg. [Figure 24] This figure shows the pharmacokinetic and pharmacodynamic characteristics of L2. [Figure 25]This figure shows the in vivo antitumor efficacy of L2 in a human malignant melanoma / hPBMC xenograft mouse model. [Figure 26] This figure shows the in vivo antitumor efficacy of a combination of L2 and in-house anti-PD-L1 antibodies in a human colorectal cancer / hPBMC xenograft mouse model. [Figure 27] This figure shows the in vivo antitumor efficacy of a combination of L2 and commercially available anti-PD-L1 antibodies in a human colorectal cancer / hPBMC xenograft mouse model. [Figure 28] This figure shows the final tumor volume (30 days after inoculation) of mice treated with KGX101, KGX101+KN035, and KGX101+atezolizumab in the presence of hPBMCs. [Figure 29] This figure shows the percentage of human CD45-PD-L1+ cells and T cell tumor infiltration in tumor sites of mice treated with KGX101, KGX101+KN035, and KGX101+atezolizumab in the presence of hPBMCs. [Figure 30] This figure shows the serum IFNγ concentrations in mice treated with KGX101, KGX101+KN035, and KGX101+atezolizumab in the presence of hPBMCs. [Figure 31] This figure shows additional in vivo studies of L2 and commercially available anti-PD-L1 antibody combinations in a human colorectal cancer / hPBMC xenograft mouse model. [Figure 32] This figure shows the percentage of circulating T cells (CD3+%) and T cell infiltration (CD3+) in the tumor microenvironment. [Figure 33] This figure shows the stability of L2 and a+b in cynomolgus monkey serum. [Figure 34] This figure shows the stability of L2 and a+b in human serum. [Figure 35] This figure shows the ex vivo cleavage efficiency of L2 by tumor tissue and normal mouse tissue. [Figure 36] This figure shows schematics of multifunctional IL12 prodrugs and controls. [Figure 37] This figure shows the protein expression of L2-αPDL1 and the control prodrug as evaluated by SDS-PAGE. [Figure 38] This figure shows the purity and homogeneity of L2-αPDL1 and the control prodrug as evaluated by high-performance liquid chromatography (HPLC) analysis. [Figure 39] This figure shows the PDL1 coupling dynamics of L2-αPDL1 as evaluated by biolayer interferometry (BLI). [Figure 40] This figure shows the purity and homogeneity of L2-IL7, L2-IL15α, L2-mut, and control proteins as evaluated by high-performance liquid chromatography (HPLC) analysis. [Figure 41] This figure shows the IL12 function of multifunctional IL12 prodrugs with and without MMP, as evaluated by the HEK-Blue IL12 reporter assay. [Figure 42] This figure shows the IL12 function of multifunctional IL12 prodrugs with and without MMP, as evaluated by the HEK-Blue IL12 reporter assay. [Figure 43] This figure shows the immune-activating ability of multifunctional IL12 prodrugs in the absence of MMPs, as evaluated by IFNγ induction in PBMCs. [Figure 44] This figure shows the immunoactivating ability of multifunctional IL12 prodrugs, evaluated by IFNγ induction in PBMCs, with and without MMPs. [Figure 45] This figure shows the in vivo antitumor efficacy of the bispecific molecule L2-IL15α in human colon cancer HCT116 / hPBMC xenografted NSG mice. [Figure 46] This figure shows L1 expression detected by SDS-PAGE in MMP knockout CHO cells. [Modes for carrying out the invention]

[0027] This disclosure provides a multifunctional shielding IL12 molecule and methods of use. In certain embodiments, the multifunctional molecule disclosed herein comprises a shielding IL12 molecule and a cleavable linker. Furthermore, this disclosure provides the multifunctional molecule disclosed herein, as well as methods for producing and using pharmaceutical compositions containing it, for example, to treat diseases and disorders, such as cancer. The present invention is in part based on the discovery of a novel multifunctional shielding IL12 molecule having a novel structure and shielding moiety that exhibits excellent shielding effect and enhanced molecular development potential.

[0028] 1.Definition Where used herein, the terms “about” or “approximately” mean within the tolerance range of a particular value as determined by those skilled in the art, which will depend in part on how that value is measured or determined, i.e., on the limits of the measurement system. In certain embodiments, “about” may mean within or greater than three standard deviations, according to convention in the art. In certain embodiments, “about” may mean a range of up to 20% of a given value, e.g., up to 10%, up to 5%, or up to 1%. In certain embodiments, particularly with respect to biological systems or processes, this term may mean within that order of magnitude, e.g., within five times or two times the value. Where used herein, the term “about X ~ Y” has the same meaning as “about X ~ about Y”.

[0029] Where used herein, the term “antibody” includes full-length antibodies and any antigen-binding fragments thereof (i.e., antibody fragments). “Antibody” may be a standalone molecule or a portion of an antibody derivative, such as a multispecific antibody (e.g., a bispecific antibody) or another multifunctional molecule containing an antibody portion.

[0030] "Full-length antibody," "intact antibody," and "whole antibody" refer to antibodies that are similar in structure to natural antibodies or antibodies having a heavy chain containing an Fc region as defined herein. In certain embodiments, a full-length antibody comprises two heavy chains and two light chains. In certain embodiments, the variable regions of the light and heavy chains are responsible for antigen binding. The variable regions of the heavy and light chains may be referred to as "VH" and "VL," respectively. The variable regions of both chains generally contain three highly variable loops called complementarity-determining regions (CDRs) (light chain (LC)CDRs including LC-CDR1, LC-CDR2, and LC-CDR3; heavy chain (HC)CDRs including HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein may be defined or specified by well-known conventions, such as those of Kabat, Chothia, MacCallum, IMGT, and AHo, as described below. Three CDRs of the heavy or light chain are sandwiched between flanking intervals known as framework regions (FRs), which are more conserved than the CDRs and form a scaffold supporting the hypervariable loop. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of the heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Some of the major antibody classes are classified into subclasses such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain). In certain embodiments, the full-length antibody is glycosylated. In certain embodiments, the full-length antibody contains a glycan linked to its Fc region. In certain embodiments, the full-length antibody contains a branched glycan.

[0031] The terms “antigen-binding portion,” “antibody fragment,” and “antibody portion” of an antibody, as used herein, refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be exerted by fragments of a full-length antibody. Examples of antibody fragments, but not limited to, include Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv and scFv-Fc), single-domain antibodies, VHH, VHH-Fc, nanobodies, domain antibodies, bivalent domain antibodies, or any other fragments or combinations thereof of an antibody that binds to an antigen. “VHH” refers to a single-domain antibody isolated from a camelid animal. In certain embodiments, VHH includes a variable region of the heavy chain of a camelid heavy-chain antibody. In certain embodiments, VHH has a size of approximately 25 kDa or less. In certain embodiments, VHH has a size of approximately 20 kDa or less. In a particular embodiment, VHH has a size of approximately 15 kDa or less.

[0032] A "reference antibody and a cross-competing antibody" refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competitive assay. An example of a competitive assay is described in "Antibodies, Harlow and Lane" (Cold Spring Harbor Press, Cold Spring Harbor, New York).

[0033] "Fv" is the minimal antibody fragment containing the complete antigen recognition and binding sites. This fragment consists of a dimer of one heavy-chain variable region and one light-chain variable region, tightly coupled non-covalently. When these two domains fold, they create six hypervariable loops (three loops in each of the heavy and light chains) that lead to antigen binding of amino acid residues, conferring antigen-binding specificity to the antibody. However, a single variable domain (or half of the Fv containing only three antigen-specific CDRs) can also recognize and bind to the antigen, but may have lower affinity than the entire binding site.

[0034] "Single-chain Fv" is also abbreviated as "sFv" or "scFv," and refers to a single polypeptide chain with attached V. H and V L It is an antibody fragment containing an antibody domain. In some embodiments, the scFv polypeptide allows the scFv to form a desired structure for antigen binding. H Domain and V L It further includes polypeptide linkers between domains. For a review of scFv, see The Pharmacology of Monoclonal Antibodies, Pluckthun, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York City, pp. 269–315 (1994).

[0035] "Affinity" refers to the sum of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity between molecule X and its partner Y can generally be expressed by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including the methods described herein. Specific descriptive and exemplary embodiments for measuring binding affinity are described below.

[0036] As used herein, the terms “CDR” or “complementarity-determining region” are intended to mean discontinuous antigen-binding sites within the variable regions of the heavy and / or light chains. These specific areas include: Kabat et al., J. Biol. Chem. Vol. 252: pp. 6609-6616 (1977); Kabat et al., US Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. Vol. 196: pp. 901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., Vol. 273: pp. 927-948 (1997); MacCallum et al., J. Mol. Biol. Vol. 262: pp. 732-745 (1996); Abhinandan and Martin, Mol. Immunol., Vol. 45: pp. 3832-3839 (2008); Lefranc As described by MP et al., Dev. Comp. Immunol., Vol. 27: pp. 55-77 (2003); and Honegger and Pluckthun, J. Mol. Biol., Vol. 309: pp. 657-670 (2001), these definitions may include overlaps or subsets of amino acid residues when compared to one another. However, the application of any one of the definitions for referring to a CDR of an antibody or transplanted antibody or its variants is intended to be within the scope of the terms defined and used herein. For comparison, the amino acid residues encompassing the CDRs defined by each of the above references are shown in Table 1 below. CDR prediction algorithms and interfaces are publicly known in the art, for example, Abhinandan and Martin, Mol. Immunol., Vol. 45: pp. 3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., Vol. 38: pp. D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., Vol. 43: pp. D432-D438 (2015).The contents of the references cited in this paragraph are incorporated herein by reference in their entirety for use in this application and for possible inclusion in one or more claims herein.

[0037] [Table 1]

[0038] The expressions "Kabat variable region residue numbering" or "Kabat amino acid position numbering" and their variations refer to the numbering scheme used in the heavy chain or light chain variable regions of a series of antibodies by Kabat et al. (cited above). When using this numbering scheme, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the shortening or insertion of FR or CDR in the variable domain. For example, the heavy chain variable region may contain a single amino acid insertion after H2 residue 52 (Kabat residue 52a) and an inserted residue after heavy chain FR residue 82 (e.g., Kabat residues 82a, 82b, and 82c). The Kabat numbering of residues in a given antibody can be determined by aligning the homology regions of the antibody sequence with a "standard" Kabat numbering sequence.

[0039] In certain embodiments, the amino acid residues comprising the CDR of a single-domain antibody are defined according to the IMGT nomenclature of Lefranc et al. (cited above). In certain embodiments, the amino acid residues comprising the CDR of a full-length antibody are defined according to the Kabat nomenclature of Kabat et al. (cited above). In certain embodiments, the numbering of residues in the immunoglobulin heavy chain, such as the Fc region, is the same EU index numbering as that of Kabat et al. (cited above). "Kabat-like EU indexing" refers to the residue numbering of human IgG1 EU antibodies.

[0040] In this specification, “amino acid sequence identity percentage (%)” or “homology” for polypeptides and antibody sequences is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of the polypeptide being compared, after aligning the sequences, taking into account any conservative substitutions as part of sequence identity. Alignment for the purpose of determining amino acid sequence identity percentage can be achieved in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve maximum alignment over the entire length of the sequences being compared. However, for the purposes of this specification, amino acid sequence identity percentage values ​​are generated using the sequence comparison computer program MUSCLE (Edgar, RC, Nucleic Acids Research, Vol. 32 (No. 5): pp. 1792-1797, 2004; Edgar, RC, BMC Bioinformatics, Vol. 5 (No. 1): p. 113, 2004).

[0041] "Homologie" refers to the sequence similarity or identity between two polypeptides or two nucleic acid molecules. If the positions in both sequences being compared are occupied by the same base or amino acid monomer subunit—for example, if the positions in each of two DNA molecules are occupied by adenine—then these molecules are homologous in their positions. The percentage of homology between two sequences is a function of the number of matching or homologous positions common to both sequences, divided by the number of positions compared, and multiplied by 100. For example, if 6 out of 10 positions in two sequences are matching or homologous, the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, comparisons are performed by aligning the two sequences to obtain maximum homology.

[0042] In this specification, the terms “Fc region” or “fragmentary crystalline region” are used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions, or dimers thereof. In certain embodiments, the human IgG Fc region extends from Cys226 to its carboxyl terminus. In certain embodiments, the human IgG Fc region extends from Pro231 to its carboxyl terminus. In certain embodiments, the human IgG Fc region includes a CH2 domain and a CH3 domain. In certain embodiments, the C-terminal lysine of the Fc region (residue 447 according to the EU numbering scheme) can be removed, for example, during antibody production or purification, or by recombination of the nucleic acid encoding the antibody heavy chain. In certain embodiments, the composition of an intact antibody may include an antibody population in which all K447 residues have been removed, an antibody population in which K447 residues have not been removed, or an antibody population having a mixture of antibodies with and without K447 residues. Suitable natural sequence Fc regions for use in the antibodies described herein include the Fc regions of human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0043] The term "regulatory sequence" refers to a DNA sequence necessary to express a operably linked coding sequence in a particular host organism. Suitable regulatory sequences for prokaryotes include, for example, promoters, optional operator sequences, and ribosome binding sites. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers.

[0044] Nucleic acids are "operably linked" when they are positioned in a functional relationship with another nucleic acid sequence. For example, a pre-sequence or secretion leader DNA is operably linked to the polypeptide DNA if it is expressed as a preprotein involved in polypeptide secretion; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are contiguous, and in the case of a secretion leader, contiguous and in the reading frame. Enhancers, however, do not need to be contiguous. Linking is achieved by ligation at a convenient restriction site. If such a site does not exist, synthetic oligonucleotide adapters or linkers are used according to conventional practice.

[0045] As used herein, the term “vector” refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is ligated. This term includes not only vectors as self-replicating nucleic acid structures, but also vectors that are incorporated into the genome of the host cell to which they are introduced. Certain vectors are capable of inducing the expression of nucleic acids to which they are operably ligated. Such vectors are referred to herein as “expression vectors.”

[0046] The terms “transfected,” “transformed,” or “transduced,” as used herein, refer to the process by which an exogenous nucleic acid is transfected or introduced into a host cell. A “transfected,” “transformed,” or “transduced” cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid, and the cell includes the primary target cell and its offspring.

[0047] The terms “host cell,” “host cell line,” and “host cell culture” are used synonymously and refer to cells into which exogenous nucleic acids have been introduced, including the offspring of such cells. Host cells include “transformed cells” and “transformed cells,” including primary transformed cells and their offspring, regardless of passage number. Offspring may not have nucleic acid contents that are exactly identical to those of the parent cells and may contain mutations. This specification includes mutant offspring that have the same function or biological activity as those screened or selected in the initially transformed cells.

[0048] In this specification, the terms “subject,” “individual,” and “patient” are used synonymously and are not limited to these, but refer to mammals, including humans, cattle, horses, cats, dogs, rodents, or primates. In some embodiments, the subject is human.

[0049] The “effective dose” of an agonist refers to the effective amount in terms of dosage and duration required to achieve the desired therapeutic or prophylactic effect. The specific dose may vary depending on the timing of administration, the tissue to be imaged, and one or more physical delivery systems carrying the agonist, regardless of whether it is administered in combination with other compounds.

[0050] The “therapeutic effective dose” of the substance / molecule, agonist, or antagonist of this application may vary depending on factors such as the individual’s medical condition, age, sex, and weight, as well as the ability of the substance / molecule, agonist, or antagonist to elicit a desired response in the individual. The therapeutic effective dose is also the amount in which any toxic or adverse effects of the substance / molecule, agonist, or antagonist outweigh the therapeutic beneficial effects. The therapeutic effective dose may be delivered in one or more doses.

[0051] The "prophylactic effective dose" refers to the effective amount of medication needed for the duration required to achieve the desired preventive effect. Typically, but not always, the prophylactic effective dose will be less than the therapeutic effective dose, as prophylactic doses are used for pre-disease or earlier-stage disease.

[0052] As used herein, “treatment” or “to treat” refers to a method for obtaining a beneficial or desired outcome, including clinical outcomes. For the purposes of this application, beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: relief of one or more symptoms caused by the disease; reduction of the severity of the disease; stabilization of the disease (e.g., prevention or delay of disease progression); prevention or delay of disease spread (e.g., metastasis); prevention or delay of disease recurrence; delay or slowing of disease progression; improvement of the disease state; provision of disease remission (partial or complete); reduction of the dose of one or more other drugs required to treat the disease; delay of disease progression; increase or improvement of quality of life; increase in weight gain; and / or extension of survival. “Treatment” also includes reduction of the pathological outcomes of cancer (e.g., tumor volume). The methods of this application are intended to address any one or more of these aspects of treatment. “Treatment” does not necessarily mean that the condition being treated will be cured.

[0053] The embodiments of this application described herein are understood to include embodiments "consisting of" and / or "essentially consisting of".

[0054] As used herein, the term “modulate” means a positive or negative change. Illustrative modulations include changes of approximately 1%, 2%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0055] As used herein, the term “increase” means a change of at least about 5%. The change may be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100%, or greater.

[0056] As used herein, the term “reduce” means a change of at least about 5%. The change may be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or even about 100%.

[0057] As used herein and in the appended claims, the singular forms "a," "or," and "the" refer to multiple subjects unless the circumstances clearly indicate otherwise.

[0058] "Effector function" refers to the biological activity resulting from the Fc region of an antibody, and varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cell-mediated cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0059] The term "pharmaceutical preparation" refers to a preparation in which the biological activity of the active ingredient contained therein is acceptable, and which does not contain additional ingredients that are unacceptably toxic to the target population to which the preparation will be administered.

[0060] As used herein, "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical preparation other than the active ingredient that is non-toxic to the subject. Examples of pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0061] 2. Multifunctional shielding IL12 molecules and their parts This disclosure provides a multifunctional shielding IL12 molecule and methods of use. In certain embodiments, the multifunctional molecule disclosed herein includes a shielding IL12 molecule and a cleavable linker. In certain embodiments, the multifunctional molecule includes a P35 molecule and a P40 molecule. In certain embodiments, the multifunctional molecule includes a shielding moiety comprising an IL12Rβ1 moiety and an IL12Rβ2 moiety. The present invention is in part based on the discovery of a multifunctional shielding IL12 molecule having a novel structure and shielding moiety that exhibits superior shielding effect and enhanced molecular stability compared to a reference shielding IL12 molecule, for example, the shielding IL12 molecule disclosed in International Publication No. 2019209965. In certain embodiments, the multifunctional shielding IL12 molecule of this disclosure may incorporate, individually or in combination, any of the features described in this application, for example, as detailed in sections 2.1 to 2.9 of this specification.

[0062] The multifunctional shielding IL12 molecules of this disclosure are useful, for example, as pharmaceuticals for treating neoplasms or cancers. In certain embodiments, neoplasms and cancers whose growth can be inhibited using the multifunctional shielding IL12 molecules of this disclosure are typically neoplasms and cancers that are responsive to immunotherapy. In certain embodiments, neoplasms and cancers include breast cancer (e.g., mammary gland cell carcinoma), ovarian cancer (e.g., ovarian cell carcinoma), and renal cell carcinoma (RCC). Other cancers that can be treated using the methods of this disclosure include: melanoma (e.g., metastatic melanoma), prostate cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, brain tumors, chronic or acute leukemia including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, and chronic lymphocytic leukemia, lymphoma (e.g., Hodgkin lymphoma and non-Hodgkin lymphoma, lymphocytic lymphoma, primary CNS lymphoma, T-cell lymphoma), and nasopharyngeal cancer (nasopharange). Carcinomas), head and neck cancers, cutaneous or intraocular malignant melanomas, uterine cancers, rectal cancers, anal cancers, gastric cancers, testicular cancers, uterine cancers, fallopian tube cancers, endometrial cancers, cervical cancers, vaginal cancers, vulvar cancers, esophageal cancers, small intestine cancers, endocrine cancers, thyroid cancers, parathyroid cancers, mammary gland cancers, soft tissue sarcomas, urethral cancers, penile cancers, pediatric solid tumors, bladder cancers, kidney or ureteral cancers, mammary gland and pelvic cancers, central nervous system (CNS) neoplasms, tumor angiogenesis, spinal axial tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermal carcinomas, squamous cell carcinomas, asbestos-induced cancers, such as mesothelioma, and combinations of the above cancers.

[0063] 2.1 Exemplary Shielding IL12 Molecules This disclosure provides a multifunctional shielding IL12 molecule and methods of use. In certain embodiments, the multifunctional molecule disclosed herein comprises a shielding IL12 molecule and a cleavable linker. In certain embodiments, the IL12 molecule is a human IL12 molecule. In certain embodiments, the IL12 molecule is a mouse IL12 molecule. In certain embodiments, the IL12 molecule is a physiologically active form of cytokine after secretion from a cell (i.e., the signal peptide has been cleaved). In certain embodiments, secreted IL12 refers to a polypeptide or protein whose cytokine portion has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the cytokine portion of an immunoactive protein product or fragment thereof. In certain embodiments, the multifunctional molecule comprises a P35 molecule and a P40 molecule. In certain embodiments, the P35 molecule comprises the amino acid sequence shown in SEQ ID NO: 1. In certain embodiments, the P40 molecule comprises the amino acid sequence shown in SEQ ID NO: 2.

[0064] In certain embodiments, the IL12 molecule can activate endogenous immune cells. In certain embodiments, the endogenous immune cells are selected from the group consisting of NK cells, NK-T cells, dendritic cells, and T cells. In certain embodiments, the endogenous immune cells are endogenous CD8 T cells, macrophages with the M1 phenotype, or dendritic cells with mature and activated phenotypes. In certain embodiments, the second cytokine portion can increase the endogenous immune cell population by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1000%, or more. In certain embodiments, the second cytokine portion can recruit endogenous immune cells to the tumor site.

[0065] In certain embodiments, the multifunctional molecule includes a shielding moiety. In certain embodiments, the shielding moiety includes an IL12 receptor, e.g., IL12Rβ1 or IL12Rβ2, or a portion or variant thereof that can bind to IL12. In certain embodiments, the IL12 receptor is a physiologically active form of the IL12 receptor. In certain embodiments, the IL12 receptor, e.g., IL12Rβ1 or IL12Rβ2, refers to a polypeptide or protein having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the extracellular portion of the IL12 receptor or a fragment thereof that can bind to IL12. In certain embodiments, the shielding moiety includes an IL12Rβ1 portion or a portion or variant thereof. In certain embodiments, the shielding moiety includes an IL12Rβ1 portion containing domain 1 of IL12Rβ1. In certain embodiments, the shielding moiety includes an IL12Rβ1 portion containing the amino acid sequence shown in SEQ ID NO: 3. In certain embodiments, the shielding portion includes the IL12Rβ2 portion or a portion or variant thereof. In certain embodiments, the shielding portion includes the IL12Rβ2 portion containing domains 1 and 2 of IL12Rβ2. In certain embodiments, the shielding portion includes the IL12Rβ2 portion containing the amino acid sequence shown in SEQ ID NO: 4. In certain embodiments, the shielding portion includes the IL12Rβ2 portion containing domain 1 of IL12Rβ2. In certain embodiments, the shielding portion includes the IL12Rβ2 portion containing the amino acid sequence shown in SEQ ID NO: 5.

[0066] In certain embodiments, the shielding portion includes mutations to increase the stability and / or shielding efficiency of the shielding portion. In certain embodiments, the shielding portion includes an IL12Rβ1 portion containing mutations of C6S, C55S, or a combination thereof. In certain embodiments, the shielding portion includes an IL12Rβ1 portion containing mutations of Y85S, Q108L, or a combination thereof. In certain embodiments, the shielding portion includes an IL12Rβ1 portion containing mutations of C6S, C55S, Y85S, and Q108L.

[0067] In certain embodiments, the multifunctional molecule includes a first chain and a second chain. In certain embodiments, the first chain includes a first shielding portion, a first cleavable linker, a P35 molecule, and a first Fc region. In certain embodiments, the second chain includes a second shielding portion, a second cleavable linker, a P40 molecule, and a second Fc region. In certain embodiments, the first shielding portion includes an IL12Rβ2 portion. In certain embodiments, the first shielding portion includes the amino acid sequence shown in SEQ ID NO: 4 or 5. In certain embodiments, the first shielding portion includes the amino acid sequence shown in SEQ ID NO: 4. In certain embodiments, the first shielding portion includes the amino acid sequence shown in SEQ ID NO: 5. In certain embodiments, the second shielding portion includes an IL12Rβ1 portion. In certain embodiments, the second shielding portion includes the amino acid sequence shown in SEQ ID NO: 3.

[0068] In certain embodiments, each of the first and second cleavable linkers can be recognized and hydrolyzed by a protease. In certain embodiments, the protease is specifically expressed in the tumor microenvironment. In certain embodiments, the protease is a matrix metalloproteinase (MMP). In certain embodiments, the matrix metalloproteinase is MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, MMP28, or any combination thereof. In certain embodiments, the matrix metalloproteinase is MMP14.

[0069] In certain embodiments, each of the first and second cleavable linkers includes an arbitrary amino acid sequence that can be recognized and hydrolyzed by a proteolytic enzyme, such as MMP, such as MMP14. In certain embodiments, each of the first and second cleavable linkers includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 67 to 112. In certain embodiments, the first cleavable linker includes the amino acid sequence shown in SEQ ID NO: 6 or 7. In certain embodiments, the first cleavable linker includes the amino acid sequence shown in SEQ ID NO: 6. In certain embodiments, the first cleavable linker includes the amino acid sequence shown in SEQ ID NO: 7. In certain embodiments, the second cleavable linker molecule includes the amino acid sequence shown in SEQ ID NO: 8.

[0070] In certain embodiments, the first Fc region and the second Fc region form a dimerized Fc region. In certain embodiments, the dimerized Fc region includes a human Fc region. In certain embodiments, the dimerized Fc region includes an Fc region selected from the group consisting of IgG, IgA, IgD, IgE, and IgM Fc regions. In certain embodiments, the dimerized Fc region includes an Fc region selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 Fc regions. In certain embodiments, the dimerized Fc region includes an IgG1 Fc region. In certain embodiments, the dimerized Fc region includes an IgG4 Fc region.

[0071] In certain embodiments, the Fc region includes one or more mutations due to EU numbering of residues. In certain embodiments, the Fc region includes one or more mutations that reduce FcγR and C1q binding to the Fc region. In certain embodiments, the Fc region is an IgG1 Fc region. In certain embodiments, the IgG1 Fc region includes an L234A mutation and / or an L235A mutation. In certain embodiments, the Fc region is an IgG2 or IgG4 Fc region. In certain embodiments, the Fc region is an IgG4 Fc region including an F234A and / or L235A mutation. In certain embodiments, the Fc region further includes a P329G mutation.

[0072] In certain embodiments, the first Fc region includes a knob chain and the second Fc region includes a hole chain. In certain embodiments, the first Fc region includes a hole chain and the second Fc region includes a knob chain. In certain embodiments, the knob chain includes mutations of S354C, T366W, K408A, or any combination thereof. In certain embodiments, the knob chain includes mutations of S354C, T366W, and K408A. In certain embodiments, the hole chain includes mutations of Y349C, T366S, L368A, F405K, Y406V, or any combination thereof. In certain embodiments, the hole chain includes mutations of Y349C, T366S, L368A, F405K, and Y406V.

[0073] In certain embodiments, the P35 and P40 portions are linked to the Fc region via a linker. In certain embodiments, the linker is a peptide linker. In certain embodiments, the peptide linker contains about 4 to about 30 amino acids. In certain embodiments, the peptide linker contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 49 to 66.

[0074] In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 9 or 10. In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 9. In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 10. In certain embodiments, the second chain includes the amino acid sequence shown in SEQ ID NO: 11.

[0075] In certain embodiments, the first chain includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 20-29 and any combination thereof. In certain embodiments, the first chain includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-29. In certain embodiments, the second chain includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 30-40 and any combination thereof. In certain embodiments, the second chain includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-40. In certain embodiments, the multifunctional molecule includes any molecule listed in Table 6.

[0076] In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 20, and the second chain includes the amino acid sequence shown in SEQ ID NO: 31. In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 20, and the second chain includes the amino acid sequence shown in SEQ ID NO: 34. In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 20, and the second chain includes the amino acid sequence shown in SEQ ID NO: 36. In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 21, and the second chain includes the amino acid sequence shown in SEQ ID NO: 30. In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 21, and the second chain includes the amino acid sequence shown in SEQ ID NO: 31. In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 21, and the second chain includes the amino acid sequence shown in SEQ ID NO: 34. In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 21, and the second chain includes the amino acid sequence shown in SEQ ID NO: 36. In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 25, and the second chain includes the amino acid sequence shown in SEQ ID NO: 30. In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 25, and the second chain includes the amino acid sequence shown in SEQ ID NO: 31. In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 25, and the second chain includes the amino acid sequence shown in SEQ ID NO: 34. In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 25, and the second chain includes the amino acid sequence shown in SEQ ID NO: 36. In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 28, and the second chain includes the amino acid sequence shown in SEQ ID NO: 30. In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 28, and the second chain includes the amino acid sequence shown in SEQ ID NO: 31. In certain embodiments, the first chain includes the amino acid sequence shown in SEQ ID NO: 28, and the second chain includes the amino acid sequence shown in SEQ ID NO: 34.In a particular embodiment, the first chain comprises the amino acid sequence shown in SEQ ID NO: 28, and the second chain comprises the amino acid sequence shown in SEQ ID NO: 36.

[0077] 2.2 Exemplary shielding IL12 molecule containing additional cytokine moiety In certain embodiments, the multifunctional molecule further comprises a second cytokine moiety. In certain embodiments, the second cytokine moiety comprises human cytokines. In certain embodiments, the second cytokine moiety comprises mouse cytokines. In certain embodiments, the term "cytokine" refers to the physiologically active form of cytokine after it has been secreted from a cell (i.e., the signal peptide has been cleaved). In certain embodiments, secreted cytokine refers to a polypeptide or protein whose cytokine moiety has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homology to the cytokine moiety of an immunoactive protein product or fragment thereof. In certain embodiments, the second cytokine moiety comprises TNFα, IFN-γ, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL9, IL13, IL15, IL17, IL18, IL21, IL22, IL15α, TGFβ, G-CSF, GM-CSF, or any variant thereof or any combination thereof. In a particular embodiment, the second cytokine moiety includes an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with secreted cytokines such as TNFα, IFN-γ, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL9, IL13, IL15, IL17, IL18, IL21, IL22, IL15α, TGFβ, G-CSF, or GM-CSF.

[0078] In certain embodiments, the second cytokine portion activates endogenous immune cells. In certain embodiments, the endogenous immune cells are selected from a group consisting of NK cells, NK-T cells, dendritic cells, and T cells. In certain embodiments, the endogenous immune cells are endogenous CD8 T cells, macrophages with the M1 phenotype, or dendritic cells with mature and activated phenotypes. In certain embodiments, the second cytokine portion can increase the endogenous immune cell population by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1000%, or more. In certain embodiments, the second cytokine portion can recruit endogenous immune cells to the tumor site.

[0079] In certain embodiments, the second cytokine moiety is linked to the C-terminus of the first chain of the multifunctional molecule. In certain embodiments, the second cytokine moiety is linked to the C-terminus of the second chain of the multifunctional molecule. In certain embodiments, the second cytokine moiety is linked to the Fc region via a linker. In certain embodiments, the linker is a peptide linker. In certain embodiments, the peptide linker contains about 4 to about 30 amino acids. In certain embodiments, the peptide linker contains about 4 to about 15 amino acids. In certain embodiments, the peptide linker contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 49 to 66.

[0080] In certain embodiments, the second cytokine moiety includes an IL7 molecule. In certain embodiments, the multifunctional molecule includes a first chain containing the amino acid sequence shown in SEQ ID NO: 21 and a second chain containing the amino acid sequence shown in SEQ ID NO: 45.

[0081] In certain embodiments, the second cytokine moiety includes an IL15 molecule. In certain embodiments, the multifunctional molecule includes a first chain containing the amino acid sequence shown in SEQ ID NO: 21 and a second chain containing the amino acid sequence shown in SEQ ID NO: 45.

[0082] In certain embodiments, the second cytokine portion includes an IL18 molecule. In certain embodiments, the multifunctional molecule includes a first chain containing the amino acid sequence shown in SEQ ID NO: 21 and a second chain containing the amino acid sequence shown in SEQ ID NO: 47.

[0083] In certain embodiments, the second cytokine moiety includes an IL15α molecule. In certain embodiments, the multifunctional molecule includes a first chain containing the amino acid sequence shown in SEQ ID NO: 48, and a second chain containing the amino acid sequence shown in SEQ ID NO: 46.

[0084] 2.3 Exemplary shielding IL12 molecule containing antibody moiety In certain embodiments, the multifunctional molecule further comprises an antibody moiety. In certain embodiments, the antibody moiety of the Disclosure may be or may comprise a chimeric antibody, a humanized antibody, or a monoclonal antibody containing a human antibody. In certain embodiments, the antibody comprises a human antibody. In certain embodiments, the antibody comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework. In certain embodiments, the antibody of the Disclosure may be an antibody fragment, e.g., Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In certain embodiments, the antibody is a full-length antibody, e.g., an intact IgG1 antibody, or another antibody class or isotype as defined herein. In certain embodiments, the antibody comprises a single-domain antibody. In certain embodiments, the single-domain antibody comprises a VHH. In certain embodiments, the single-domain antibody comprises a heavy chain variable region (VH). In certain embodiments, the single-domain antibody is ligated to an Fc region. In certain embodiments, the single-domain antibody is not ligated to an Fc region. In certain embodiments, the antibody may incorporate any of the features described in this application, either individually or in combination.

[0085] In certain embodiments, the antibody comprises full-length immunoglobulin, single-chain Fv(scFv) fragments, Fab fragments, Fab' fragments, F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, VHH, Fv-Fc fusions, scFv-Fc fusions, VHH-Fv fusions, diabodies, tribodies, tetrabodies, or any combination thereof.

[0086] In certain embodiments, the antibody moiety is linked to the C-terminus of the first chain of the multifunctional molecule. In certain embodiments, the antibody moiety is linked to the C-terminus of the second chain of the multifunctional molecule. In certain embodiments, the antibody moiety is linked to the Fc region via a linker. In certain embodiments, the linker is a peptide linker. In certain embodiments, the peptide linker contains about 4 to about 30 amino acids. In certain embodiments, the peptide linker contains about 4 to about 15 amino acids. In certain embodiments, the peptide linker contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 49 to 66.

[0087] In certain embodiments, the antibody binds to a tumor-associated antigen. In certain embodiments, tumor-associated antigens include PDL1, CD10, CD19, CD20, CD21, CD22, CD25, CD30, CD33, CD34, CD37, CD44v6, CD45, CDw52, Fms-like tyrosine kinase 3 (FLT-3, CD135), c-Kit (CD117), CSF1R (CD115), CD133, PDGFR-alpha (CD140a), PDGFR-beta (CD140b), chondroitin sulfate proteoglycan 4 (CSPG4), Muc-1, EGFR, de2-7-EGFR, EGFRvIII, folate-binding protein, Her2neu, Her3, PSMA, PSCA, PSA, TAG-72, HLA-DR, IGFR, IL3R, fibroblast-activating protein (FAP), and carboanhydrase IX (MN / CA). IX) Selected from the group consisting of carcinoembryonic antigen (CEA), EpCAM, CDCP1, Delrin 1, Tenascin, frizzled1-10, vascular antigens VEGFR2 (KDR / FLK1), VEGFR3 (FLT4, CD309), Endoglin, CLEC14, Tem1-8, and Tie2, as well as any combination thereof.

[0088] In certain embodiments, the multifunctional molecule comprises an antibody portion that binds to PDL1. In certain embodiments, the anti-PDL1 antibody of the present disclosure binds to the extracellular domain of PDL1. In certain embodiments, the anti-PDL1 antibody binds to the same epitope as the anti-PDL1 antibodies described herein. In certain embodiments, the anti-PDL1 antibodies disclosed herein can function as antagonists of the PDL1-based signaling pathway. In certain embodiments, the anti-PDL1 antibody can block or reduce a signal pathway that depends on the PDL1 protein. In certain embodiments, the anti-PDL1 antibody can reduce the activity of the signal pathway by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 99%, or about 99.9%.

[0089] In certain embodiments, the antibody portion comprises a single-domain antibody that binds to PDL1. In certain embodiments, the single-domain antibody binds to PDL1 with a KD of about 1×10 -7 M or lower. In certain embodiments, the single-domain antibody binds to PDL1 with a KD of about 1×10 -8 M or lower. In certain embodiments, the single-domain antibody binds to PDL1 with a KD of about 1×10 -9 M or lower. In certain embodiments, the single-domain antibody binds to PDL1 with a KD of about 1×10 -10 M or lower. In certain embodiments, the single-domain antibody binds to PDL1 with a KD of about 1×10 -11 M to about 1×10 -7 M. In certain embodiments, the single-domain antibody binds to PDL1 with a KD of about 1×10 -10 M to about 1×10 -7 M. In certain embodiments, the single-domain antibody binds to PDL1 with a KD of about 1×10​​​​​​​It binds to PDL1 at M's KD. In certain embodiments, the single-domain antibody is approximately 1 × 10⁶ -10 M ~ approx. 1×10 -9 M is linked with PDL1 in KD.

[0090] In a particular embodiment, a single-domain antibody cross-competes for binding to PDL1 with a reference anti-PDL1 single-domain antibody that includes heavy chain variable regions CDR1 containing the amino acid sequence shown in SEQ ID NO: 12, heavy chain variable region CDR2 containing the amino acid sequence shown in SEQ ID NO: 13, and heavy chain variable region CDR3 containing the amino acid sequence shown in SEQ ID NO: 14.

[0091] In a particular embodiment, the single-domain antibody comprises a heavy chain variable region including a CDR1 domain, a CDR2 domain, and a CDR3 domain, wherein the CDR1 domain, CDR2 domain, and CDR3 domain each comprise a reference heavy chain variable region containing the amino acid sequence of SEQ ID NO: 15.

[0092] In a particular embodiment, the single-domain antibody comprises a heavy chain variable region CDR1 containing the amino acid sequence shown in SEQ ID NO: 12, a heavy chain variable region CDR2 containing the amino acid sequence shown in SEQ ID NO: 13, and a heavy chain variable region CDR3 containing the amino acid sequence shown in SEQ ID NO: 14.

[0093] In certain embodiments, the single-domain antibody includes a heavy chain variable region containing an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the single-domain antibody includes a heavy chain variable region containing an amino acid sequence having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the single-domain antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 15.

[0094] In certain embodiments, the multifunctional molecule includes a first chain containing the amino acid sequence shown in SEQ ID NO: 41. In certain embodiments, the multifunctional molecule includes a first chain containing the amino acid sequence shown in SEQ ID NO: 43. In certain embodiments, the multifunctional molecule includes a second chain containing the amino acid sequence shown in SEQ ID NO: 42. In certain embodiments, the multifunctional molecule includes a second chain containing the amino acid sequence shown in SEQ ID NO: 44. In certain embodiments, the multifunctional molecule includes a first chain containing the amino acid sequence shown in SEQ ID NO: 41 and a second chain containing the amino acid sequence shown in SEQ ID NO: 42. In certain embodiments, the multifunctional molecule includes a first chain containing the amino acid sequence shown in SEQ ID NO: 43 and a second chain containing the amino acid sequence shown in SEQ ID NO: 44.

[0095] In certain embodiments, any one of the amino acid sequences contained in the heavy chain variable region may contain up to about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 amino acid substitutions, deletions, and / or additions. In certain embodiments, the amino acid substitutions are conservative substitutions.

[0096] In certain embodiments, the anti-PDL1 antibody portion is conjugated with a therapeutic agent or label. In certain embodiments, the label is selected from the group consisting of radioisotopes, fluorescent dyes, and enzymes.

[0097] 2.4 Substitution, Insertion, and Deletion Variants In certain embodiments, multifunctional shielding IL12 molecular variants having one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include HVR (or CDR) and FR. Conservative substitutions are shown in Table 2 under the heading "Preferred Substitutions." More substantial modifications are provided in Table 2 under the heading "Exemplary Substitutions" and are further described below in relation to amino acid side chain classes. Amino acid substitutions can be introduced into the target protein, and the product can be screened for desired activity.

[0098] [Table 2]

[0099] Amino acids can be grouped according to common side-chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. In certain embodiments, non-conservative substitutions may involve exchanging one of these classes for another.

[0100] In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs (or CDRs), provided that such modifications do not substantially reduce the antibody-antigen binding ability. For example, conservative modifications (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made to the HVRs (or CDRs). Such modifications may be made in the HVR (or CDR) "hotspot" or outside the CDR. In certain embodiments of the variant VHH sequences provided above, each HVR (or CDR) is either unchanged or contains one, two, or three or fewer amino acid substitutions.

[0101] Amino acid sequence insertions include amino-terminus and / or carboxyl-terminus fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is a multifunctional shielding IL12 molecule with an N-terminal methionyl residue. Other insertion variants include the fusion of a polypeptide that increases the serum half-life of an enzyme (e.g., ADEPT) or molecule to the N-terminus or C-terminus of a multifunctional shielding IL12 molecule.

[0102] 2.5 Glycosylated Variants In certain embodiments, the multifunctional shielding IL12 molecule is modified to increase or decrease the degree to which the construct is glycosylated. The addition or deletion of glycosylation sites to the multifunctional shielding IL12 molecule can be conveniently achieved by modifying the amino acid sequence to create or remove one or more glycosylation sites.

[0103] If a multifunctional shielding IL12 molecule contains an Fc region (e.g., scFv-Fc), the carbohydrate attached to it may be modified. The native Fc region of antibodies produced by mammalian cells is typically the C of the Fc region. H The molecule contains branched or bifurcated oligosaccharides generally attached to Asn297 of the two domains via N-linking. See, for example, Wright et al., TIBTECH Vol. 15: pp. 26-32 (1997). The oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc of the "trunk" of the bifurcated oligosaccharide structure. In certain embodiments, the oligosaccharide in the Fc region may be modified to create variants having certain improved properties.

[0104] In certain embodiments, the multifunctional shielding IL12 molecule has a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such an Fc region may be 1%–80%, 1%–65%, 5%–65%, or 20%–40%. The amount of fucose is determined by calculating the average amount of fucose in the Asn297 glycan relative to the total amount of all sugar structures attached to Asn297 (e.g., complex, hybrid, and high-mannose structures), measured by MALDI-TOF mass spectrometry, as described, for example, in International Publication No. 2008 / 077546. Asn297 refers to the asparagine residue located approximately 297th position in the Fc region (EU numbering of the Fc region residue), although Asn297 may be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to slight sequence variations. Such fucosylated variants can enhance ADCC function. See, for example, U.S. Patent Application Publication No. 2003 / 0157108 (Presta, L.) and U.S. Patent Application Publication No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.).Examples of publications relating to “defucosylated” or “fucose-deficient” variants include: U.S. Patent Application Publication No. 2003 / 0157108; International Publication No. 2000 / 61739; International Publication No. 2001 / 29246; U.S. Patent Application Publication No. 2003 / 0115614; U.S. Patent Application Publication No. 2002 / 0164328; U.S. Patent Application Publication No. 2004 / 0093621; U.S. Patent Application Publication No. 2004 / 0132140; U.S. Patent Application Publication No. 2004 / 0110704; U.S. Patent Application Publication No. 2004 / 01 Specification No. 10282; U.S. Patent Application Publication No. 2004 / 0109865; International Publication No. 2003 / 085119; International Publication No. 2003 / 084570; International Publication No. 2005 / 035586; International Publication No. 2005 / 035778; International Publication No. 2005 / 053742; International Publication No. 2002 / 031140; Okazaki et al., J.Mol.Biol. 336: pp. 1239-1249 (2004); Yamane-Ohnuki et al., Biotech.Bioeng. 87: pp. 614 (2004). Examples of cell lines capable of producing defucosylated Fc regions include Lec13 CHO cells lacking protein fucosylation (Ripka et al., Arch. Biochem. Biophys. Vol. 249: pp. 533-545 (1986); U.S. Patent Application Publication No. 2003 / 0157108, Presta, L.; and International Publication No. 2004 / 056312, Adams et al.), and knockout cell lines such as the alpha-1,6-fucosyltransferase gene, FUT8, and knockout CHO cells (see, for example, Yamane-Ohnuki et al., Biotech. Bioeng. Vol. 87: pp. 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng. Vol. 94 (No. 4): pp. 680-688 (2006); and International Publication No. 2003 / 085107).

[0105] In certain embodiments, the multifunctional shielding IL12 molecule has a bifid oligosaccharide in which a branched oligosaccharide attached to the Fc region is bifid by GlcNAc. Such variants may have reduced fucosylation and / or enhanced ADCC function. Examples of such variants are described, for example, in International Publication No. 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.). Variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such variants may have enhanced CDC function. Such variants are described, for example, in International Publication No. 1997 / 30087 (Patel et al.); International Publication No. 1998 / 58964 (Raju, S.); and International Publication No. 1999 / 22764 (Raju, S.).

[0106] 2.6 Fc region variant In certain embodiments, the Fc region of the multifunctional shielding IL12 molecule of this disclosure may include a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that includes amino acid modifications (e.g., substitutions) at one or more amino acid positions. In certain embodiments, one or more amino acid modifications can be introduced into the Fc region (e.g., scFv-Fc or VHH-Fc) to generate an Fc region variant.

[0107] In certain embodiments, the Fc region possesses some, but not all, effector functions, making it a desirable candidate for applications where the molecule's in vivo half-life is important, but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to confirm that the Fc region retains FcRn binding ability, even though it lacks FcγR binding (and therefore is likely to lack ADCC activity). NK cells, the main cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression in hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol. 9: pp. 457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of a target molecule are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA, Vol. 83: pp. 7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA, Vol. 82: pp. 1499-1502 (1985); and U.S. Patent No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med., Vol. 166: pp. 1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (see, for example, the ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, California) and the CytoTox96® non-radioactive cytotoxicity assay (Promega, Madison, Wisconsin)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.Alternatively, or in addition, the ADCC activity of the molecule of interest can be evaluated in vivo in animal models, such as those disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA, Vol. 95: pp. 652-656 (1998). A C1q binding assay can also be performed to confirm that the Fc region cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in International Publication No. 2006 / 029879 and International Publication No. 2005 / 100402. To evaluate complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J.Immunol.Methods vol. 202: p. 163 (1996); Cragg, MS et al., Blood vol. 101: pp. 1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood vol. 103: pp. 2738-2743 (2004)). Furthermore, the determination of FcRn binding and in vivo clearance / half-life can be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l.Immunol. vol. 18 (No. 12): pp. 1759-1769 (2006)).

[0108] Examples of Fc regions with reduced effector function include those having one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include the so-called "DANA" Fc mutant (U.S. Patent No. 7,332,581), in which residues 265 and 297 are substituted with alanine, and Fc mutants having two or more substitutions at amino acid positions 265, 269, 270, 297, and 327.

[0109] Certain variants exhibiting improved or reduced binding with FcR are described. (See, for example, U.S. Patent No. 6,737,056, International Publication No. 2004 / 056312, and Shields et al., J. Biol. Chem. Vol. 9 (No. 2): pp. 6591-6604 (2001)).

[0110] In certain embodiments, the Fc region includes one or more mutations due to EU numbering of residues. In certain embodiments, the Fc region includes one or more mutations that reduce FcγR and C1q binding to the Fc region. In certain embodiments, the Fc region is an IgG1 Fc region. In certain embodiments, the IgG1 Fc region includes an L234A mutation and / or an L235A mutation. In certain embodiments, the Fc region is an IgG2 or IgG4 Fc region. In certain embodiments, the Fc region is an IgG4 Fc region including an F234A and / or L235A mutation. In certain embodiments, the Fc region further includes a P329G mutation.

[0111] In certain embodiments, the Fc region includes one or more knob-in-hole mutations to facilitate pairing of the knob and hole strands. In certain embodiments, the Fc region includes a knob strand containing mutations of S354C, T366W, K408A, or any combination thereof. In certain embodiments, the Fc region includes a hole strand containing mutations of Y349C, T366S, L368A, F405K, Y406V, or any combination thereof.

[0112] In certain embodiments, the Fc region includes an IgG4 Fc region. In certain embodiments, the IgG4 Fc region includes an S228P mutation.

[0113] In certain embodiments, modifications are made to the Fc region that result in alterations (i.e., enhancements or reductions) to C1q binding and / or complement-dependent cell-mediated cytotoxicity (CDC), as described, for example, in U.S. Patent No. 6,194,551, International Publication No. 99 / 51642, and Idusogie et al., J.Immunol. 164:4178-4184 (2000).

[0114] In certain embodiments, a multifunctional shielding IL12 molecular variant includes a variant Fc region containing one or more amino acid substitutions that alter the half-life and / or alter the binding to the neonatal Fc receptor (FcRn). Fc regions with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which plays a role in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) are described in U.S. Patent Application Publication 2005 / 0014934 (Hinton et al.). Such Fc regions have one or more substitutions therein that alter the binding between the Fc region and FcRn. Such Fc variants include those having substitutions in one or more Fc region residues, for example, those having a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0115] For other examples of Fc region variants, see Duncan & Winter, Nature, Vol. 322, pp. 738–740 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and International Publication No. 94 / 29351.

[0116] 2.7 Production Method The multifunctional shielding IL12 molecules disclosed herein can be produced using any technique available or known in the art. For example, but not limited to, the multifunctional shielding IL12 molecules can be produced using recombinant methods and compositions such as those described in U.S. Patent No. 4,816,567. Detailed procedures for producing the multifunctional shielding IL12 molecules are described in the examples below.

[0117] Furthermore, the subject matter of this disclosure provides isolated nucleic acids encoding the multifunctional shielding IL12 molecule disclosed herein. For example, the isolated nucleic acid may encode an amino acid sequence comprising the first and / or second strands of the multifunctional shielding IL12 molecule.

[0118] In certain embodiments, the nucleic acid may reside in one or more vectors, such as an expression vector. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop to which additional DNA segments can be ligated. Another type of vector is a viral vector to which additional DNA segments can be ligated into a viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors), upon introduction into a host cell, are integrated into the host cell's genome and thereby replicate together with the host genome. Furthermore, certain vectors, expression vectors, can direct the expression of a gene to which they are operably ligated. Generally, expression vectors useful in recombinant DNA techniques are often in the form of plasmids (vectors). However, the subject matter of this disclosure is intended to include other forms of expression vectors that perform equivalent functions, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses).

[0119] Different parts of the multifunctional shielding IL12 molecule disclosed herein can be constructed into a single multicistronic expression cassette, multiple expression cassettes in a single vector, or multiple vectors. Examples of elements that create a polycistronic expression cassette include, but are not limited to, various viral and nonviral internal ribosome entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-κB IRES, RUNX1 IRES, p53 IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, aftvirus IRES, picornavirus IRES, poliovirus IRES, and encephalomyocarditis virus IRES), and cleavable linkers (e.g., 2A peptides, e.g., P2A, T2A, E2A, and F2A peptides). A combination of retroviral vectors and appropriate packaging strains is also preferred, in which case the capsid protein will function to infect human cells. Various amphotropic virus-producing cell lines are known, but are not limited to these, including PA12 (Miller et al. (1985) Mol. Cell. Biol. Vol. 5: pp. 431-437); PA317 (Miller et al. (1986) Mol. Cell. Biol. Vol. 6: pp. 2895-2902); and CRIP (Danos et al. (1988) Proc. Natl. Acad. Sci. USA Vol. 85: pp. 6460-6464). Non-amphotropic particles are also preferred, such as pseudotyped particles with VSVG, RD114, or GALV envelopes, and any other known in the art.

[0120] In certain embodiments, a nucleic acid encoding the multifunctional shielding IL12 molecule of this disclosure and / or one or more vectors containing such nucleic acids can be introduced into a host cell. In certain embodiments, the introduction of nucleic acids into cells can be carried out by any method known in the Art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequence, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, and the like. In certain embodiments, the host cell may contain, for example, a vector containing a nucleic acid encoding an amino acid sequence containing one or both strands of the multifunctional shielding IL12 molecule, and is transformed therein, for example. In certain embodiments, the host cell is a eukaryotic cell, for example, a Chinese hamster ovary (CHO) cell or a lymphocyte (e.g., YO, NSO, Sp20 cells).

[0121] In certain embodiments, a method for producing the multifunctional shielding IL12 molecule disclosed herein may include culturing host cells into which the nucleic acid encoding the multifunctional shielding IL12 molecule has been introduced under conditions suitable for the expression of the multifunctional shielding IL12 molecule, and optionally recovering the multifunctional shielding IL12 molecule from the host cells and / or host cell culture medium. In certain embodiments, the multifunctional shielding IL12 molecule is recovered from the host cells by a chromatographic technique.

[0122] To recombinantly produce the multifunctional shielding IL12 molecule of this disclosure, for example, nucleic acids encoding the multifunctional shielding IL12 molecule as described above can be isolated, further cloned, and / or inserted into one or more vectors for expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes capable of specifically binding to the gene encoding the multifunctional shielding IL12 molecule). Suitable host cells for cloning or expression of the vector include prokaryotic or eukaryotic cells known in the art.

[0123] In certain embodiments, vertebrate cells can also be used as hosts. For example, but not limited to, mammalian cell lines adapted to grow in suspensions may be useful. Non-limiting examples of useful mammalian host cell lines include: SY40(COS-7) transformed monkey kidney CV1 cell line; human fetal kidney cell line (HEK293, or 293 cells described, e.g., Graham et al., J Gen Viral. Vol. 36: p. 59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described, Mather, Biol. Reprod. Vol. 23: pp. 243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL3A); human lung cells (W138); human hepatocytes (Hep02); mouse mammary tumor cells (MMT060562); TRI cells, e.g., Mather et al., Annals These include TRI cells; MRC5 cells; and FS4 cells, as described in NYAcad.Sci. Vol. 383: pp. 44-68 (1982). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFK CHO cells (Urlaub et al., Proc. Natl. Acad.Sci. USA Vol. 77: 42 I6 (1980)); and myeloma cell lines such as YO, NSO, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo ed., Humana Press, Totowa, New Jersey), pp. 255-268 (2003).

[0124] In certain embodiments, the host cells have no or reduced endogenous expression of a matrix metalloproteinase capable of recognizing and hydrolyzing the cleavable linker contained in the multifunctional shielding IL12 molecule of this disclosure. In certain embodiments, the host cells have no or reduced endogenous expression of MMP14. In certain embodiments, the host cells are HEK293 cells, a variant thereof, or a derivative thereof. In certain embodiments, the host cells are Expi293 cells, Wayne293 cells, a variant thereof, or a derivative thereof. In certain embodiments, the host cells are Expi293 cells. In certain embodiments, the host cells are Expi293F cells. In certain embodiments, the host cells are Wayne293 cells. In certain embodiments, the host cells are CHO cells, a variant thereof, or a derivative thereof in which the MMP14 gene is knocked out or knocked down. Endogenous gene expression of MMP14 can be eliminated or reduced using any gene knockout or knockdown method, including, but not limited to, mutation, deletion, or knockout of part or all of the MMP14 gene, epigenetic silencing of the MMP14 gene, or RNAi-induced gene silencing. In certain embodiments, the host cells are, for example, CHO cells, mutants thereof, or derivatives thereof, in which the MMP14 gene has been knocked out using a CRISPR / Cas9 system. In certain embodiments, the host cells are, for example, CHO cells, mutants thereof, or derivatives thereof, in which the MMP14 gene has been knocked down using an RNAi system. In certain embodiments, the host cells are CHO-K cells, mutants thereof, or derivatives thereof.

[0125] Furthermore, this disclosure provides a method for preparing a multifunctional molecule comprising a cleavable linker that can be recognized and hydrolyzed by MMP14. In certain embodiments, this method includes expressing the multifunctional molecule in a host cell containing the nucleic acid of the multifunctional molecule, and isolating the multifunctional molecule from the host cell. Furthermore, this disclosure provides a method for preparing the multifunctional shielding IL12 molecule of this disclosure. In certain embodiments, this method includes expressing the multifunctional molecule in a host cell disclosed herein, and isolating the multifunctional molecule from the host cell. In certain embodiments, the host cell has no or reduced endogenous expression of MMP14. In certain embodiments, the host cell is HEK293 cell, a variant thereof, or a derivative thereof. In certain embodiments, the host cell is Expi293 cell, Wayne293 cell, a variant thereof, or a derivative thereof. In certain embodiments, the host cell is Expi293 cell. In certain embodiments, the host cell is Expi293F cell. In certain embodiments, the host cell is Wayne293 cell. In certain embodiments, the host cell is a CHO cell, a mutant thereof, or a derivative thereof in which the MMP14 gene is knocked out or knocked down. In certain embodiments, the host cell is a CHO cell, a mutant thereof, or a derivative thereof in which the MMP14 gene is knocked out using, for example, a CRISPR / Cas9 system. In certain embodiments, the host cell is a CHO cell, a mutant thereof, or a derivative thereof in which the MMP14 gene is knocked down using, for example, an RNAi system. In certain embodiments, the host cell is a CHO-K cell, a mutant thereof, or a derivative thereof.

[0126] 3. How to use Furthermore, the subject matter of this disclosure provides methods for using the multifunctional shielding IL12 molecules of this disclosure. In certain embodiments, such methods relate to the therapeutic use of the multifunctional shielding IL12 molecules of this disclosure.

[0127] 3.1 Treatment method This disclosure provides methods and uses of the multifunctional shielding IL12 molecules disclosed herein for treating diseases and disorders or for increasing immune responses. In certain embodiments, the multifunctional shielding IL12 molecules disclosed herein or pharmaceutical compositions comprising them may be administered to subjects (e.g., mammals such as humans) to treat diseases and disorders or to increase immune responses. In certain embodiments, the diseases and disorders involve immune checkpoint inhibition and / or abnormal IL12 activity. In certain embodiments, diseases and disorders that can be treated with the multifunctional shielding IL12 molecules disclosed herein include, but are not limited to, tumors, such as cancer.

[0128] In certain embodiments, the Disclosure provides multifunctional shielding IL12 molecules (or fragments thereof) as described herein for use in the manufacture of a pharmaceutical. In certain embodiments, the Disclosure provides multifunctional shielding IL12 molecules (or fragments thereof) as described herein for use in the manufacture of a pharmaceutical for treating cancer. In certain embodiments, the Disclosure provides multifunctional shielding IL12 molecules (or fragments thereof) as described herein for use in the treatment of a cancer of interest. In certain embodiments, the Disclosure provides a pharmaceutical composition comprising the multifunctional shielding IL12 molecules (or fragments thereof) provided herein for use in the treatment of a cancer of interest. In certain embodiments, cancer may be hematological cancers (e.g., leukemia, lymphoma, and myeloma), ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, glioblastoma, pharyngeal cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma, and various carcinomas (including prostate cancer and small cell lung cancer). Suitable carcinomas include, but are not limited to, all carcinomas known in the field of oncology, such as astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primary neuroectodermal tumor (PNET), chondrosarcoma, osteosarcoma, pancreatic ductal adenocarcinoma, small cell lung adenocarcinoma and large cell lung adenocarcinoma, chordoma, angiosarcoma, endosarcoma, squamous cell carcinoma, bronchoalveolar carcinoma, epithelial adenocarcinoma and its liver metastases, lymphangiosarcoma, intralymphatic sarcoma, hepatocellular carcinoma, cholangiocarcinoma, synoviomas, mesothelioma, Ewing's tumor, rhabdomyosarcoma, colon cancer, basal cell carcinoma, sweat gland carcinoma, papillary carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, and medullary carcinoma. Cancer, bronchial cancer, renal cell carcinoma, cholangiocarcinoma, choriocarcinoma, seminomas, embryonal carcinoma, Wilms' tumor, testicular tumor, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma, Waldenström macroglobulinemia, mammary gland tumors, e.g., ductal carcinoma and lobular adenocarcinoma, squamous cell carcinoma and adenocarcinoma of the cervix, uterine and ovarian epithelial carcinoma, prostate adenocarcinoma, transitional squamous cell carcinoma of the bladder, B-cell and T-cell lymphomas (nodular and diffuse), plasmacytoma, acute and chronic leukemia, malignant melanoma, soft tissue sarcoma, and leiomyosarcoma.

[0129] In certain embodiments, cancer may be melanoma, NSCLC, head and neck cancer, urothelial carcinoma, breast cancer (e.g., triple-negative breast cancer, TNBC), gastric cancer, cholangiocarcinoma, classical Hodgkin lymphoma (cHL), non-Hodgkin lymphoma primary mediastinal B-cell lymphoma (NHL PMBCL), mesothelioma, ovarian cancer, lung cancer (e.g., small cell lung cancer), esophageal cancer, nasopharyngeal cancer (NPC), biliary tract cancer, colorectal cancer, cervical cancer, or thyroid cancer.

[0130] In certain embodiments, the subject to be treated is a mammal (e.g., human, non-human primate, rat, mouse, cattle, horse, pig, sheep, goat, dog, cat, etc.). In certain embodiments, the subject is human. In certain embodiments, the subject is suspected of having cancer, at risk of having cancer, or has been diagnosed with cancer or any other disease that has abnormal IL12 expression or activity.

[0131] Numerous diagnostic methods and clinical descriptions of cancer or any other disease exhibiting abnormal IL12 activity are known in the art. Such methods include, but are not limited to, immunohistochemistry, PCR, and fluorescence in situ hybridization (FISH). Additional details on diagnostic methods for abnormal IL12 activity or expression can be found, for example, in Gupta et al. (2009) Mod Pathol. Vol. 22 (No. 1): pp. 128-133; Lopez-Rios et al. (2013) J Clin Pathol. Vol. 66 (No. 5): pp. 381-385; Ellison et al. (2013) J Clin Pathol. Vol. 66 (No. 2): pp. 79-89; and Guha et al. (2013) PLoS ONE Vol. 8 (No. 6): e67782.

[0132] Administration may be by any preferred route, including, for example, intravenous, intramuscular, or subcutaneous. In some embodiments, the multifunctional shielding IL12 molecules (or fragments thereof) and / or compositions provided herein are administered in combination with a second, third, or fourth activator (e.g., an antineoplastic agent, a growth inhibitor, a cytotoxic agent, or a chemotherapeutic agent) to treat a disease or disorder involving abnormal IL12 activity. Examples of such agonists include docetaxel, gefitinib, FOLFIRI (irinotecan, 5-fluorouracil, and leucovorin), irinotecan, cisplatin, carboplatin, paclitaxel, bevacizumab, FOLFOX-4, fluorouracil injection, leucovorin, and oxaliplatin, afatinib, gemcitabine, capecitabine, pemetrexed, tivantinib, everolimus, CpG-ODN, rapamycin, lenalidomide, vemurafenib, endostatin, lapatinib, PX-866, imprime PGG, and irlotinibm. In some embodiments, the multifunctional shielding IL12 molecule (or fragment thereof) is conjugated to an additional agonist.

[0133] In certain embodiments, the multifunctional shielding IL12 molecules (or fragments thereof) and / or compositions provided herein are administered in combination with one or more additional therapies such as radiotherapy, surgery, chemotherapy, and / or targeted therapy. In certain embodiments, the multifunctional shielding IL12 molecules (or fragments thereof) provided herein are administered in combination with radiotherapy. In certain embodiments, the combination of the multifunctional shielding IL12 molecules (or fragments thereof) and / or compositions provided herein with radiotherapy is used to treat neoplasms or cancers disclosed herein.

[0134] In certain embodiments, the multifunctional shielding IL12 molecules (or fragments thereof) and / or compositions provided herein are administered in combination with an anti-PD-L1 antibody, such as celpurimab. In certain embodiments, the multifunctional shielding IL12 molecules (or fragments thereof) and / or compositions and the anti-PD-L1 antibody are administered simultaneously or sequentially. In certain embodiments, the multifunctional shielding IL12 molecules (or fragments thereof) and / or compositions and the anti-PD-L1 antibody are administered simultaneously. In certain embodiments, one or more doses of the anti-PD-L1 antibody are administered before the administration of the multifunctional shielding IL12 molecules (or fragments thereof) and / or compositions. In certain embodiments, the subject underwent the entire course of anti-PD-L1 antibody therapy before the administration of the multifunctional shielding IL12 molecules (or fragments thereof) and / or compositions. In certain embodiments, the multifunctional shielding IL12 molecules (or fragments thereof) and / or compositions are administered during a second course of anti-PD-L1 antibody therapy. In certain embodiments, subjects received at least one, at least two, at least three, or at least four doses of anti-PD-L1 antibody prior to administration of multifunctional shielding IL12 molecules (or fragments thereof) and / or compositions. In certain embodiments, at least one dose of anti-PD-L1 antibody is administered concurrently with multifunctional shielding IL12 molecules (or fragments thereof) and / or compositions. In certain embodiments, one or more doses of multifunctional shielding IL12 molecules (or fragments thereof) and / or compositions are administered before administration of anti-PD-L1 antibody. In certain embodiments, subjects received at least two, at least three, at least three, or at least four doses of multifunctional shielding IL12 molecules (or fragments thereof) and / or compositions prior to administration of anti-PD-L1 antibody. In certain embodiments, at least one dose of multifunctional shielding IL12 molecules (or fragments thereof) and / or compositions is administered concurrently with anti-PD-L1 antibody. In certain embodiments, the multifunctional shielding IL12 molecule (or fragment thereof) and / or composition, as well as the anti-PD-L1 antibody, are administered once every 1, 2, 3, 4, or 5 weeks.In certain embodiments, the cancer is recurrent or progressive after a therapy selected from the group consisting of surgery, chemotherapy, radiotherapy, and any combination thereof.

[0135] Depending on the indication to be treated and factors related to dosage that are expected to be familiar to experienced physicians in the field, the multifunctional shielding IL12 molecules provided herein will be administered in a dosage effective to treat the indication while minimizing toxicity and side effects. When treating cancer, a typical dose may be in the range of, for example, 0.001 to 1000 μg, but doses below or above this exemplary range are within the scope of the invention. The daily dose may be about 0.1 μg / kg to about 100 mg / kg of total body weight, about 0.1 μg / kg to about 100 μg / kg of total body weight, or about 1 μg / kg to about 100 μg / kg of total body weight. As described above, the therapeutic or preventive effect can be monitored by regularly evaluating the treated patient. When repeated administration is performed over several days or longer, the treatment is repeated, depending on the condition, until the desired suppression of disease symptoms occurs. However, other drug regimens may be useful and are within the scope of the invention. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.

[0136] Pharmaceutical compositions comprising the multifunctional shielding IL12 molecules disclosed herein may be administered once, twice, three times, or four times daily. The compositions may also be administered less frequently than once daily, for example, six times a week, five times a week, four times a week, three times a week, twice a week, once a week, once every two weeks, once every three weeks, once a month, once every two months, once every three months, or once every six months. The compositions may also be administered in sustained-release formulations such as implants, which gradually release the composition over a period of time, allowing for administration at even less frequent doses, such as once a month, once every two to six months, once a year, or even a single dose. Sustained-release devices (such as pellets, nanoparticles, microparticles, nanospheres, and microspheres) may be administered by injection or surgically implanted at various locations.

[0137] Cancer treatment can be evaluated by, for example, but not limited to, tumor regression, reduction in tumor weight or size, progression-free survival, overall survival, overall response rate, duration of response, quality of life, and protein expression and / or activity. Techniques can be used to determine the efficacy of therapy, including, for example, measuring the response by radiographic imaging.

[0138] In certain embodiments, the efficacy of the treatment is measured by the tumor growth inhibition percentage (TGI%) calculated using the formula: 100 - (T / C × 100), where T is the mean relative tumor volume of the treated tumor and C is the mean relative tumor volume of the untreated tumor. In certain embodiments, the TGI% is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, or greater than 95%.

[0139] 4. Pharmaceutical preparations Furthermore, the subject matter of this disclosure provides pharmaceutical formulations comprising the multifunctional shielding IL12 molecules disclosed herein, together with a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition may comprise a combination of multiple (e.g., two or more) multifunctional shielding IL12 molecules of the subject matter of this disclosure.

[0140] In certain embodiments, the pharmaceutical formulations of the present disclosure can be prepared in the form of lyophilized formulations or aqueous solutions by combining a multifunctional shielding IL12 molecule of desired purity with one or more optionally selected pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). For example, lyophilized formulations are described in U.S. Patent No. 6,267,958, but are not limited thereto. In certain embodiments, aqueous formulations are described in U.S. Patent No. 6,171,586 and International Publication No. 2006 / 044908, the latter formulation comprising a histidine acetate buffer. In certain embodiments, the multifunctional shielding IL12 molecule may have a purity of more than about 80%, more than about 90%, more than about 91%, more than about 92%, more than about 93%, more than about 94%, more than about 95%, more than about 96%, more than about 97%, more than about 98%, more than about 99%, more than about 99.1%, more than about 99.2%, more than about 99.3%, more than about 99.4%, more than about 99.5%, more than about 99.6%, more than about 99.7%, more than about 99.8%, or more than about 99.9%.

[0141] Pharmacochemically acceptable carriers are generally non-toxic to the recipient at the dosage and concentration used, and include, but are not limited to, the following: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol) Examples of pharmaceutically acceptable carriers herein include low molecular weight (less than approximately 10 residues) polypeptides, proteins such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or nonionic surfactants such as polyethylene glycol (PEG). Further examples of pharmaceutically acceptable carriers herein include interstitial drug dispersants such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), such as human soluble PH-20 hyaluronidase glycoproteins such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Application Publications 2005 / 0260186 and 2006 / 0104968. In certain embodiments, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

[0142] The carrier may be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, for example, the multifunctional shielding IL12 molecule, may be coated with a substance that protects the compound from the action of acids and other natural conditions that may inactivate the compound.

[0143] The pharmaceutical compositions disclosed herein can also be administered in combination therapy, that is, in combination with other agents. Furthermore, in certain embodiments, the pharmaceutical compositions disclosed herein may contain more than one active ingredient, for example, those having complementary activities that do not adversely affect each other, depending on the needs of the specific indication being treated. In certain embodiments, the pharmaceutical formulation may contain a second active ingredient for treating the same disease treated by the first therapeutic agent. Such active ingredients are preferably present in a combination of amounts effective for the intended purpose. Furthermore, for example, but not limited to, the formulations disclosed herein may contain more than one active ingredient, preferably those having complementary activities that do not adversely affect each other, depending on the needs of the specific indication being treated. For example, it may be desirable to further provide a second therapeutic agent useful for treating the same disease. Such active ingredients are preferably present in a combination of amounts effective for the intended purpose.

[0144] The compositions of this disclosure can be administered by various methods known in the art. The route and / or method of administration will vary depending on the desired outcome. The active compound can be prepared using a carrier that prevents the compound from being rapidly released, such as controlled-release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyacid anhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Numerous methods for preparing such formulations are described, for example, in *Sustained and Controlled Release Drug Delivery Systems*, edited by JR Robinson, Marcel Dekker, Inc., New York City, 1978. In certain embodiments, the pharmaceutical composition is manufactured under Good Manufacturing Practice (GMP) conditions of the U.S. Food and Drug Administration.

[0145] Furthermore, sustained-release formulations containing the multifunctional shielding IL12 molecules disclosed herein can be prepared. A preferred example of a sustained-release formulation is a semipermeable matrix of a solid hydrophobic polymer containing the multifunctional shielding IL12 molecules, such matrix in the form of a molded article, e.g., a film or microcapsules. In certain embodiments, the active ingredient may be encapsulated in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, in, for example, hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, edited by Osol, A. (1980).

[0146] To administer the multifunctional shielding IL12 molecule of this disclosure via a specific route of administration, it may be necessary to coat the compound with a substance to prevent its inactivation, or to co-administer the compound with such a substance. For example, the compound may be administered to the subject in a suitable carrier, such as a liposome or diluent. Pharmaceutically acceptable diluents include physiological saline and aqueous buffer solutions. In addition to conventional liposomes, water-in-oil-in-water CGF emulsions are also used as liposomes (Strejan et al. (1984) J Neuroimmunol. 7: p. 27).

[0147] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. The use of such media and activators with pharmaceutically active substances is well known in the art.

[0148] Any conventional medium or activator is considered for use in the pharmaceutical compositions of this disclosure unless it is incompatible with the active compound. Auxiliary active compounds may also be incorporated into the compositions.

[0149] Therapeutic compositions are typically sterile, substantially isotonic, and stable under manufacturing and storage conditions. Compositions can be formulated into solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. In many cases, it is preferable to include isotonic agents, such as sugars, polyhydric alcohols such as mannitol and sorbitol, or sodium chloride in the composition. Sustained absorption of injectable compositions can be achieved by including absorption-delaying agents, such as monostearate and gelatin, in the composition.

[0150] A sterile injection solution can be prepared by incorporating one or more multifunctional shielding IL12 molecules disclosed herein in the required amount into a suitable solvent having, if necessary, one or a combination of the components listed above, and then performing sterile microfiltration, for example, by filtration through a sterile filter membrane. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a base dispersion medium and other necessary components of those listed above. In the case of sterile powders for preparing sterile injection solutions, preferred preparation methods are vacuum drying and freeze-drying, which yield a powder of the active ingredient and any additional desired components derived from its pre-sterilized filtered solution.

[0151] The therapeutic compositions may also be administered using medical devices known in the art. For example, the therapeutic compositions of the present disclosure may be administered using needle-free subcutaneous injection devices such as those disclosed in U.S. Patent No. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of implants and modules useful to this disclosure include U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for administering drugs at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering drugs through the skin; U.S. Patent No. 4,447,233, which discloses a drug infusion pump for delivering drugs at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow rate implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system with multiple chambers; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. Numerous other such implants, delivery systems, and modules are known.

[0152] For therapeutic compositions, formulations of the present disclosure include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration. The formulations can conveniently exist in unit dosage forms and can be prepared by any method known in the pharmaceutical field. The amount of multifunctional shielding IL12 molecules that can be combined with a carrier to produce a single dosage form varies depending on the target being treated and the specific method of administration. The amount of multifunctional shielding IL12 molecules that can be combined with a carrier to produce a single dosage form is generally the amount of the composition that produces the therapeutic effect. Generally, this amount is in the range of about 0.01 percent to about 99 percent, about 0.1 percent to about 70 percent, or about 1 percent to about 30 percent of the active ingredient out of 100 percent.

[0153] Dosage forms for topical or transdermal administration of the compositions of this disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0154] The terms "parenteral administration" and "administered parenterally" refer to methods of administration other than enteral and local administration, which are usually by injection and include, but are not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.

[0155] Furthermore, these pharmaceutical compositions may contain auxiliary agents such as preservatives, humectants, emulsifiers, and dispersants. Prevention of the presence of microorganisms can be ensured both by the sterilization procedures described above and by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, and phenolsorbic acid. It may also be desirable to include isotonic agents such as sugars and sodium chloride in the composition. In addition, sustained absorption of the injectable pharmaceutical form can be achieved by including absorption-delaying agents such as aluminum monostearate and gelatin.

[0156] In certain embodiments, when administered to humans and animals as a pharmaceutical, the multifunctional shielding IL12 molecules of this disclosure may be administered alone or, for example, in combination with a pharmaceutically acceptable carrier as a pharmaceutical composition containing about 0.01% to about 99.5% (or about 0.1% to about 90%) of the multifunctional shielding IL12 molecules.

[0157] 5. Manufactured products Furthermore, the subject matter of this disclosure provides products comprising substances useful for the treatment, prevention, and / or diagnosis of the disorders described above.

[0158] In certain embodiments, the product includes a container and labels or accompanying documents on or attached to the container. Non-limiting examples of suitable containers include bottles, vials, syringes, and IV solution bags. Containers can be formed from a variety of materials, such as glass or plastic. Containers may hold the compound itself or the compound in combination with another composition effective for the treatment, prevention, and / or diagnosis of a condition, and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be punctured with a subcutaneous needle).

[0159] In certain embodiments, at least one activator in the composition is the multifunctional shielding IL12 molecule of this disclosure. The label or accompanying information may indicate that the composition is used to treat a selected condition.

[0160] In certain embodiments, the product may include (a) a first container containing a composition comprising the multifunctional shielding IL12 molecule of the Disclosure, and (b) a second container containing a composition comprising a further cytotoxic or otherwise therapeutic agent. In certain embodiments, the product may further include a document indicating that the composition can be used to treat a particular condition.

[0161] Alternatively or in addition, the product may further include additional containers, such as a second or third container, containing pharmaceutically acceptable buffers such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution, but not limited to these. The product may also include other substances desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0162] The following examples are merely illustrative of the subject matter of this disclosure and should not be considered limiting in any way. [Examples]

[0163] Example 1. Generation and screening of novel IL12 prodrugs with improved stability and development potential. International application PCT / US2019 / 028933, published as international publication brochure 2019209965, discloses various IL12 prodrugs. One prodrug contains IL12 P35 and P40 subunits linked to IL12 receptors β1 and β2 (IL12Rβ1 and IL12Rβ2) by a cleavable linker, as a shielding domain and an Fc domain (Figure 1, A-D1-3+B-D12). This construct has the advantage of reducing peripheral cell damage while exerting therapeutic effects in tumor sites with high levels of MMPs that allow the shielding domain to be removed from the construct. However, A-D1-3+B-D12 faces numerous challenges regarding stability and development potential. For example, initial protein production resulted in reduced purity due to homodimerization of the B chain. Furthermore, the prodrug was poorly stable due to its high hydrophobicity and rapid degradation. Furthermore, although CHO cells are widely used for mammalian proteins due to their high productivity, the expression of intact prodrugs was low in typical CHO cells due to protein cleavage by proteases expressed in CHO cells and complex glycosylation.

[0164] To enhance protein production, endogenous MMP9 expression was tested in various cell lines. Expi293, Wayne293, and CHO cells were collected, and 2E5 cells per 100 µl were dispensed into each well of a U-bottom 96-well plate. Cells were fixed with 4% formaldehyde and then permeabilized with 0.1% PBS-Triton X-100 for 15 minutes. Cells were stained with AF488-MMP9 antibody (1:500) at 4°C for 30 minutes, washed twice with FACS buffer, and analyzed by flow cytometry. As shown in Figure 1, compared to unstained controls, all cell lines showed that the majority of the cell population expressed MMP9.

[0165] Cell lines were further tested for endogenous MMP14 expression. Expi293, Wayne293, and CHO cells were collected, and 2E5 cells per 100 μl were dispensed into each well of a U-bottom 96-well plate. Cells were stained with PE-MMP14 antibody (1:100) for 30 minutes at 4°C. For CHO cells, anti-MMP14 antibody (1:200) and AF488 goat anti-rabbit antibody (1:2000) were added and stained for 1 hour at 4°C. Cells were washed twice with FACS buffer and analyzed by flow cytometry. As shown in Figure 2, compared to unstained controls, Expi293 and Wayne293 cells did not express MMP14, but a fairly large population of CHO cells expressed high levels of MMP14.

[0166] Based on these results, we selected Expi293 cells as a production platform and used a cleavable linker that can be cleaved by MMP14 for various novel constructs of the IL12 prodrug.

[0167] To further improve the stability and development potential of the IL12 prodrug, various constructs of the IL12 prodrug were fabricated as shown in Figure 3. In Figure 3, A represents the IL12 P35 subunit, B represents the IL12 P40 subunit, R1 or M1 represents the IL12Rβ1 peptide, R2 or M2 represents the IL12Rβ2 peptide, D1-3, D12, and D1 represent the various domains contained in the IL12Rβ1 or IL12Rβ2 peptide, Fck represents the Fc knob chain, Fch represents the Fc hole chain, and asterisks represent cleavable linkers. The first round of operations included exchanging the Fc knob chain and hole chain between the A and B chains to improve chain heterodimerization and modifying the domains of the shielding portion to reduce the hydrophobicity of the prodrug.

[0168] IL-12 prodrug protein was produced by transient transfection in Expi293 cells and purified by one-step affinity chromatography (Cytiva). The purified protein was characterized by non-reducible and reduced SDS-PAGE for purity assessment. Representative SDS-PAGE results are shown in Figures 4 and 6. The purified protein was also characterized by high-performance liquid chromatography (HPLC) for homogeneity assessment. HPLC analysis was performed using BioResolve SEC mAb (Waters) at 100 mM PB, pH 6.8, and 150 mM NaCl. Representative HPLC results, along with the percentage of the listed major peaks, are shown in Figures 5 and 7. The HPLC results were consistent with the SDS-PAGE results. As shown in Figures 4 and 6, shorter shielding domains used in the molecule were associated with higher expression levels and better stability. As shown in Figure 5 and Table 3, A-D1+B-D1 and A-D1+B-D12 had the highest SEC-HPLC purity compared to the previously disclosed IL12 prodrug (A-D1-3+B-D12), with main peak % values ​​of 75.3% and 70.9%, respectively. As shown in Figure 7 and Table 4, A-D1+B-D1 and A-D12+B-D1 also had the highest SEC-HPLC purity, with main peak % values ​​of 73.8% and 71.7%, respectively. These results indicate that the novel prodrugs exhibited improved stability and development potential compared to A-D1-3+B-D12.

[0169] [Table 3]

[0170] [Table 4]

[0171] The prodrug IL12 function with and without MMP was tested using the HEK-Blue IL12 reporter assay. HEK-Blue IL12 reporter cells were purchased from InvivoGen and cultured according to the manufacturer's instructions for use. To detect IL12 activity, HEK-Blue IL12 cell suspensions were placed in test medium without Normocin and a select antibiotic, according to the instructions for use, in a 5 × 10⁶ mixture. 5 Cells / mL were prepared. 100 μL of cells were dispensed into each well of a flat-bottomed 96-well plate. Prodrug proteins or MMP14 digested proteins were prepared by sequentially diluting them 5-fold in test medium from 60 nM to 3.8 pM. 100 μL of the protein dilution was added to 100 μL of cells, and the plate was incubated at 37°C in 5% CO2 for 24 hours. The following day, QuantiBlue solution, a SEAP detection reagent, was prepared according to the manufacturer's instructions. 180 μL of QuantiBlue solution was mixed with 20 μL of induced HEK-Blue IL-12 cell supernatant in a flat-bottomed 96-well plate and incubated at 37°C for 1–3 hours. SEAP levels were measured at 620 nm using a spectrophotometer. Results were analyzed and calculated using nonlinear regression fitting in GraphPad Prism 8. a+b are IL12-Fc constructs without any shielding domains, and serve as a control.

[0172] As shown in Figure 8, the prodrug construct containing B-D1 (IL12Rβ1 peptide having only domain 1) showed higher shielding efficacy (higher EC50 values) than the construct containing the same A chain and B-D12 (IL12Rβ1 peptide having both domain 1 and domain 2). As shown in Figure 9, the shielding effect can be removed by MMP14 activity. Furthermore, as shown by the multiplicative change in IL12 activity between EC50 without MMP and EC50 with MMP, all new prodrugs showed improved shielding efficiency compared to A-D1-3 + B-D12. As shown in Figures 10 and 11, as shown by the multiplicative change in IL12 activity between EC50 without MMP and EC50 with MMP, A-D12 + B-D1 and A-D1 + B-D1 showed the highest shielding efficiency.

[0173] The shielding efficiency of the prodrug was further tested using an IL12 receptor binding assay. His-tagged human IL12Rβ1 protein was immobilized overnight at 4°C in a 96-well plate. Fusion proteins were prepared by serial 3-fold dilution from 3000 ng / mL to 12.3 ng / mL using Superblock reagent (Thermo Fisher, 37516). The IL12Rβ1-coated plates were washed and blocked at room temperature for 2 hours using Superblock. The diluted fusion proteins were added to the plates and incubated at room temperature for 1-2 hours. The bound fusion proteins were detected with HRP-conjugated anti-human IgG Fc antibody (Jackson ImmunoResearch). Exemplary ELISA results are shown in Figure 12. A-D12+B-D1 and A-D1+B-D1 showed higher EC50 binding to RB1 than A-D12+b and A-D1+b, indicating better shielding efficacy.

[0174] Based on the above results, A-D12+B-D1 and A-D1+B-D1 were selected as Lead 1 (L1) and Lead 2 (L2), respectively, for further testing, as they exhibited the best stability, developability, and shielding efficiency among the novel prodrug constructs. As shown in Figure 13A, after SEC (second column) purification, the SEC-HPLC purity of the L1 and L2 main peaks increased from approximately 65% ​​(after ProA) to 90% and 80%, respectively. As shown in Figure 13B, the CE-NR results also showed high purity for L1 and L2. As shown in Figure 14, the shielding efficacy of L1 and L2 was 20-fold and approximately 40-fold compared to the MMP digested sample. The efficacy of both lead molecules can be restored by MMP activity.

[0175] The thermal stability (Tm and Tonset) of L1 at phosphate pH 7.0 and pH 7.5 was high, there was no apparent aggregation (turbidity) at pH 7.0 and pH 7.5, and there was no apparent change in protein size from 25 to 95°C, indicating a narrow size distribution. After five freeze-thaw cycles at 25°C o / n and 8°C o / n, the SEC-HPLC purity of L1 decreased by 2.5%, 6%, and 4%, respectively. For formulations of 2 mg / ml, 20 mM phosphate, 50 mg / ml sucrose, and 0.02% PS20, after 18 hours at 37°C, the SEC-HPLC purity of L1 at pH 6.9, 7.2, and 7.5 decreased by 4.1%, 3.09%, and 2.82%, respectively, while the SEC-HPLC purity of L2 decreased by 3.08%, 2.84%, and 3.29%, respectively. These results demonstrate that L1 and L2 possess high stability and development potential as IL12 prodrugs.

[0176] The shielding efficacy and immunoactivating capacity of L2 were further investigated by measuring IFNγ induction using peripheral blood mononuclear cells (PBMCs). Human PBMCs were isolated and 1 × 10⁶ cells were used per 100 μL. 5Cells were dispensed into each well of a U-bottom 96-well plate and incubated overnight at 37°C. The following day, different fusion proteins or MMP14 digested proteins were added to PBMCs in 10-fold serial dilutions from 160 nM to 1.6 pM, along with different concentrations of anti-hCD3 antibody (clone OKT3, Biolegend 317301). PBMCs were incubated at 37°C for 48–72 hours. The supernatant was collected, and cytokine-dependent IFNγ secretion was quantified by ELISA (Mabtech 3420-1H). Dose-response curves were fitted using GraphPad Prism 8. As shown in Figure 15, in the absence of MMP cleavage, the L2 shielding effect reduced IL-12 potency by approximately 50-fold compared to unshielded IL-12 protein measured by activation of IFNγ secretion in PBMCs.

[0177] The developmental potential and efficacy of L2 were further compared with A-D1-3+B-D12, an IL12 prodrug from international patent application PCT / US2019 / 028933. Both A-D1-3+B-D12 and L2 were expressed in 100 mL of Expi-293 cells under the same conditions, the supernatant was collected, and purified using protein A chromatography (Cytiva). The titers of both recovered products were tested using Octet (Sartorius). As shown in Table 5, the titer of L2 was approximately 2.7 times higher than that of A-D1-3+B-D12. As shown in Figure 16, the SEC-HPLC profile also showed that L2 had higher purity (70.5%) after one-step purification compared to A-D1-3+B-D12 (55.7%).

[0178] [Table 5]

[0179] The efficacy of L2 and A-D1-3+B-D12 was compared using the HEK-Blue IL12 reporter assay. HEK-Blue IL12 reporter cells were purchased from InvivoGen and cultured according to the manufacturer's instructions for use. To detect IL12 activity, HEK-Blue IL12 cell suspensions were cultured in test medium without Normocin and a select antibiotic, according to the instructions for use, in a 5 × 10⁶ cubic meter. 5 Cells / mL were prepared. 100 μL of cells were dispensed into each well of a flat-bottomed 96-well plate. Fusion proteins or MMP14 digested proteins were prepared by serially diluting them 5-fold from 60 nM to 3.8 pM in test medium. 100 μL of the protein dilution was added to 100 μL of cells, and the plate was incubated at 37°C in 5% CO2 for 24 hours. The following day, QuantiBlue solution, a SEAP detection reagent, was prepared according to the manufacturer's instructions. 180 μL of QuantiBlue solution was mixed with 20 μL of induced HEK-Blue IL-12 cell supernatant in a flat-bottomed 96-well plate and incubated at 37°C for 1–3 hours. SEAP levels were measured at 620 nm using a spectrophotometer. Results were analyzed and calculated using nonlinear regression fitting in GraphPad Prism 8. As shown in Figure 17, without MMP14, the IL12 function of both L2 and A-D1-3+B-D12 was shielded by the shielding portion, and with the addition of MMP14, the IL12 function of both L2 and A-D1-3+B-D12 was restored. Furthermore, L2 showed a higher magnification change (20.09 times) in IL12 function recovery compared to A-D1-3+B-D12 (17.63 times). This indicates that the modified shielding portion of L2 improved shielding efficiency compared to A-D1-3+B-D12.

[0180] The binding ability of L2 prodrugs to the human IL12 receptor on the surface of HEK-Blue® IL12 reporter cells was quantified by flow cytometry. HEK-Blue® IL-12 reporter cells were resuspended in staining buffer (Biolegend, 420201) and co-incubated with serially diluted L2 or a+b at 2–8°C for 1 hour. Subsequently, HEK-Blue IL-12® reporter cells were stained with PE conjugate goat anti-human IgG-Fc (Abcam, ab98596) and analyzed using an Attune NxT flow cytometer (ThermoFisher). Binding curves were fitted and analyzed using nonlinear regression fitting in GraphPad Prism 8. a+b is an IL12-Fc construct without any shielding domains and serves as a control. As shown in Figure 18, L2 was able to bind to the human IL-12 receptor in a dose-dependent manner, with an EC50 of 141.80 nM, compared to an EC50 of 2.92 nM for a+b bound to reporter cells. These results indicate that the prodrug L2 bound to the IL-12 receptor on the cell surface, reducing the EC50 to approximately 1 / 50th compared to a+b without shielding. The shielding and immunoactivating capacity of L2 were tested by measuring IFNγ induction using peripheral blood mononuclear cells (PBMCs). The donors used in the assay were W-20200034 and WEZ4117. Human PBMCs were isolated and 1 × 10⁶ per 100 μL. 5Cells were dispensed into each well of a U-bottom 96-well plate and incubated overnight at 37°C. The following day, different fusion proteins or MMP14 digested proteins were added to the PBMCs along with 1 μg / mL anti-hCD3 antibody (clone OKT3, Biolegend 317301) at 150 nM or 10-fold series dilutions. The PBMCs were incubated at 37°C for 48–72 hours. The supernatant was collected, and cytokine-dependent IFNγ secretion was quantified by ELISA (Mabtech 3420-1H). Dose-response curves were fitted using GraphPad Prism 8. As shown in Figure 19, the L2 EC50 inducing IFNγ secretion in PBMCs increased 500–2500-fold compared to IL-12, and its activation capacity could be restored by MMP14 digestion. The EC50 results are summarized in Figure 19. When PBMCs from donor W-20200034 were used, the L2 shielding effect showed a reduction of approximately 1 / 2500th of the IFNγ secretion function of the PBMCs compared to the EC50 of unshielded IL-12-Fc control. When PBMCs from WEZ4117 were used, the L2 EC50 was approximately 300 times higher than the EC50 of unshielded IL-12-Fc. This is thought to be due to the higher responsiveness of donor WEZ4117, which had higher IFNγ secretion than donor W-20200034. After MMP14 digestion, the shielding effect in both donors was almost restored, and the MMP14-digested L2 EC50s were 0.205 and 0.702 pM, respectively, which were similar to the values ​​for IL-12-Fc and IL-12. These in vitro results revealed that L2 showed reduced IL12 activity due to its shielding portion, and that function recovered after the shielding portion was removed by MMP14 digestion. EC50 can be used to support the selection of the starting dose in Phase 1 clinical trials.

[0181] Example 2. In vivo efficacy of a novel IL12 prodrug. The in vivo antitumor efficacy of L2 was tested in hIL12A / hIL12B / hIL12RB1 / hIL12RB2 transgenic mice using a B16F10 melanoma model. 2 × 10 5One mouse B16F10 melanoma cell was subcutaneously transplanted into Biocytogen's homozygous B-hIL12A / hIL12B / hIL12RB1 / hIL12RB2 transgenic mice (female, 5-8 weeks old, n=5). Tumor volume was 80-100 mm². 3 When the mice reached a certain stage, they were divided into groups and treated with an initial dose (day 0) of PBS or 0.04 mg / kg L2 via ip injection. Subsequent doses of PBS or 0.06 mg / kg L2 were administered on days 3, 6, and 9. Tumor volume and body weight were measured throughout the study.

[0182] As shown in Figure 20A, L2 demonstrated effective in vivo antitumor activity compared to the control. As shown in Figure 20B, no significant weight loss was observed in mice, indicating good tolerance to this treatment due to reduced peripheral toxicity.

[0183] The in vivo antitumor efficacy of L2 was further investigated using the COLO205 colon cancer model in PBMC humanized NOG mice. 3×10⁶ mice exhibiting PD-L1 overexpression were tested. 6 Human colon cancer COLO205 cells were subcutaneously transplanted into NOG mice (female, 6-8 weeks old, n=4). On a different day after tumor inoculation, the mice were raised to 5 × 10 6 The tumor was reconstituted with human PBMC cells. The tumor volume was 60-80 mm. 3 When the mice reached a certain stage, they were divided into groups and treated every three days with IV injections of PBS, 0.077 mg, or 0.31 mg / kg KGX101-L2. Tumor volume and body weight were measured during the study.

[0184] As shown in Figure 21A, L2 demonstrated effective in vivo antitumor activity in a dose-dependent manner. As shown in Figure 21B, no significant weight loss was observed in the mice, indicating good tolerance to this treatment due to reduced peripheral toxicity.

[0185] The in vivo antitumor efficacy of L2 was further investigated in NSG mice using the human colon cancer HCT116 cell line. 6 × 10 6 Individual human colon cancer HCT116 cells, 2 × 10 6 Human PBMCs were mixed and subcutaneously transplanted into NSG mice (female, 6-8 weeks old). The average tumor size was approximately 100 mm. 3 When the mice reached a certain stage (n=6), they were grouped and treated five times every four days with IV injections of PBS, 0.06, 0.18, and 0.54 mg / kg L2. Tumor volume and body weight were measured during the study. For PBMC and TIL analysis, transplanted flank tumors were collected and digested with 0.25% trypsin (Thermo Fisher) to obtain single-cell suspensions. Peripheral blood cells were also collected. The cells were further washed, and flow cytometry was performed using FACS Calibur (BD Biosciences) with FITC anti-human CD45 and APC anti-human CD3, APC anti-human CD4, or anti-human CD8. Data were analyzed using FlowJo software (Treestar). As shown in Figures 22A and 22B, L2 demonstrated effective in vivo antitumor activity in a dose-dependent manner in the HCT116 / PBMC xenograft NSG mouse model. The final TGI% (19 days after treatment) of KGX101 in the presence of hPBMCs was 36.99%, 42.13%, and 41.77% for doses of 0.06, 0.18, and 0.54 mg / kg, respectively. As shown in Figure 22C, no significant weight loss was observed in the mice, indicating good tolerance to this treatment due to reduced peripheral toxicity. As shown in Figure 22D, tumor-infiltrating lymphocyte (TIL) analysis further showed that L2 treatment at 0.06, 0.18, and 0.54 mg / kg increased the percentage of T cells (CD3+, CD4+, and CD8+) in the tumor environment, and although there were some outliers due to individual variability, no statistically significant increase was observed in peripheral blood cells.

[0186] Furthermore, the in vivo PK and PD profiles of L2 were studied in HCT116 / hPBMC xenografted NSG mice. 6×10 6 Individual human colon cancer HCT116 cells, 2 × 10 6 Human PBMCs were mixed and subcutaneously transplanted into NSG mice (female, 6-8 weeks old). The average tumor size was approximately 100 mm. 3 When the mice reached a certain time point (n=3), they were divided into groups. At that point, each mouse received a single dose of L2 at either 0.06 mg / kg or 0.18 mg / kg via intravenous infusion. The infusion rate was 30 mL / kg / hour, the dose volume was 10 mL / kg, and the infusion time was 0.33 hours. Blood samples (60–80 μL) were collected at various time points (0 / 2 / 8 / 24 / 48 / 72 / 120 / 168 hours) using the vena orbital posterior plexus method. After collection, the blood samples were centrifuged at 5,000 rpm for 5 minutes at 4°C, and the supernatant was collected. L2 concentrations in serum samples were measured by sandwich enzyme-linked immunosorbent assay (ELISA). Serum drug concentrations were obtained by 4-parameter fitting based on OD450 nm absorbance values. Drug concentration-time curves were fitted using GraphPad Prism8. In vivo PK data were analyzed using Phoenix 64 WinNonlin 6.1 software (version 6.1, Pharsight). Non-compartmental model (moment) analysis was performed to analyze the following parameters: half-life, AUC. 0-t , clearance, and distributed volume (V d) was obtained. The time point was automatically selected as the first option by a “best-fit” model for terminal half-life estimation. If the “best-fit” model could not adequately define the terminal stage, manual selection was applied. In addition, IFN-γ in serum samples was measured as a PD marker using the human IFN-γ ELISA set (BD, catalog number: 555142). To show the PK-PD relationship, IFN-γ levels and IL-12 concentrations were plotted on the same figure. As shown in Figure 23, L2 showed a dose-dependent increase in serum concentration after intravenous infusion of 0.06 and 0.18 mg / kg into tumor-bearing female NSG mice. max and AUC last The exposure measured by was dose-proportional, and at 0.18 mg / kg, C max The value is 6.8 times, AUC last The value increased 8.7 times (compared to the value at 0.06 mg / kg). MRT ranged from 75.83 to 81.94 hours, and t1 / 2 ranged from 52.75 to 64.87 hours. Mean PK parameters are shown in Figure 23. The pharmacokinetic characteristics of this study provide evidence for finding the L2 therapeutic dose range in humans. As shown in Figure 24, IFN-γ levels and IL-12 concentrations were plotted on the same figure to show the PK-PD relationship. Serum IFN-γ levels peaked on day 7 (168 hours), and the increase was not dose-dependent, nor was it at the time of PK Tmax. The delayed effect on PD markers may be due to the generation of human IFN-γ from tumor sites and its leakage into the circulatory system, suggesting that clinical trials should select drug cycles based on PD markers rather than L2 serum concentrations.

[0187] Furthermore, the in vivo antitumor efficacy of L2 was confirmed in a human malignant melanoma A375 (showing hPD-L1 overexpression) / hPBMC xenograft model. In this study, 8 × 10 6 2 × 10¹ PD-L1-A375 cells 6 The tumor was mixed with several hPBMCs (in a 4:1 ratio) and subcutaneously transplanted into the right flank of NSG mice (female, 6-8 weeks old). Fifteen days after tumor inoculation, the average tumor size was approximately 90-100 mm. 3At this point, tumor-bearing mice were randomized to different groups. Mice were administered PBS or L2 by IV injection at dose levels of 0.007, 0.02, and 0.06 mg / kg every two weeks (biw) for four doses. Tumor volume and body weight were measured during the study. As shown in Figure 25, L2 showed antitumor activity of 60.36%, 76.30%, and 76.68% TGI% (25 days after inoculation) at doses of 0.007 mg / kg, 0.02 mg / kg, and 0.06 mg / kg, respectively. L2 showed good antitumor activity in PD-L1-A375 xenograft in the presence of human PBMCs.

[0188] Example 3. In vivo efficacy of a combination of a novel IL12 prodrug and an anti-PDL1 antibody. While immune checkpoint blockers (ICBs), such as anti-PD-1 / PD-L1, are well-recognized for their efficacy against a wide range of tumors, many patients develop resistance even after an initial response. Overcoming resistance is clinically necessary. The human colorectal cancer cell line HCT116 exhibits low baseline expression of programmed cell death ligand 1 (PD-L1). Monotherapy with anti-PD-L1 antibodies and combinations with TKIs do not demonstrate superior efficacy in colorectal cancer. Considering L2's superior ability to enhance tumor-specific T cell responses, we evaluated the efficacy of a combination of L2 and anti-PD-L1 antibodies in an HCT116 / hPBMC xenograft model. 6 × 10 6 Individual human colon cancer HCT116 cells, 2 × 10 6 Human PBMCs were mixed and subcutaneously transplanted into NSG mice (female, 6-8 weeks old). Tumor volume was 80-120 mm. 3When the mice reached a certain stage, they were divided into groups and treated every four days with PBS, 0.18 mg / kg of L2 (iv), 4 mg / kg of αPD-L1 VHH antibody (ip), or a combination thereof. Tumor volume and PD-L1 expression in the tumors were measured. As shown in Figure 26, L2, when administered every four days at a dose of 0.18 mg / kg together with αPD-L1 VHH antibody, showed excellent synergistic antitumor efficacy in the HCT116 / PBMC xenograft NSG mouse model. hPD-L1+% was significantly increased in tumor cells (hCD45-), which is thought to be responsible for the synergistic effect in combination therapy.

[0189] The in vivo combined antitumor efficacy in an HCT116 / hPBMC xenograft model was further analyzed using additional commercially available PD-L1 antibodies. 6 × 10 6 Individual human colon cancer HCT116 cells, 2 × 10 6 Human PBMCs were mixed and subcutaneously transplanted into NSG mice (female, 6-8 weeks old). Tumor volume was 80-120 mm. 3When the mice reached a certain stage, they were divided into groups and treated every three days with PBS, L2(iv), two αPD-L1 antibodies (ip), and a combination thereof. Tumor volume, PD-L1 expression, T cell infiltration in the tumors, and serum IFNγ concentration were measured. As shown in Figure 27, L2 showed synergistic antitumor efficacy with both the αPD-L1 antibodies KN035 (Envafolimab, Alphamab Oncology) and atezolizumab (Roche) in the HCT116 / PBMC xenograft NSG mouse model when administered at a dose of 0.06 mg / kg every three days. As shown in Figure 28, when comparing the final tumor volume (30 days after inoculation) in mice treated with KGX101, KGX101+KN035, and KGX101+atezolizumab in the presence of hPBMCs, there was a significant difference (p<0.05) between the treatment group and the placebo group. The TGI% for KGX101 at 0.06 mg / kg, KGX101+KN035 at 0.06+0.3 mg / kg, and KGX101+atezolizumab at 0.06+2.5 mg / kg were 47.80%, 92.70%, and 99.48%, respectively. L2 demonstrated superior synergistic antitumor efficacy with both αPD-L1 antibody, KN035, and atezolizumab compared to monotherapy (p<0.05), suggesting a universal antitumor synergy with the combined use of IL-12 and anti-PD-L1 antibodies. As shown in Figure 29, after administration of 0.02 and 0.06 mg / kg of L2 monotherapy, hPD-L1+% in tumor cells (hCD45-) significantly increased, potentially generating an inhibitory signal in the TME. With the anti-PD-L1 combination, hPD-L1 expression in tumor cells was lower than with L2 monotherapy, and the PD-L1 inhibition generated by L2 monotherapy was relieved. This likely explains the synergistic effect of the combination therapy of IL-12 and anti-PD-L1 antibodies. This hypothesis was further supported by the synergistic increase in T cell infiltration (CD3+%) in the TME after combination therapy. In addition, serum IFNγ concentrations were measured to support synergistic immune activation (Figure 30). L2 at 0.06 mg / kg showed a significant increase in serum IFNγ.The combination of L2 and KN035, as well as the combination of L2 and atezolizumab, showed a significant increase in IFNγ concentration compared to monotherapy, suggesting synergistic immune activation through combination therapy.

[0190] The above study was further repeated. As shown in Figure 31, the tumor volume of L2 at a dose of 0.06 mg / kg and the tumor volume at a dose of 0.06 mg / kg + 1 mg / kg with KGX101 + KN035 were significantly smaller than in the placebo group. Comparing the final tumor volume (38 days after administration), the TGI% for KGX101 at 0.06 mg / kg and KGX101 + KN035 at 0.06 mg / kg + 1 mg / kg were 36.56% and 66.22%, respectively. These results indicate that KGX101 alone and in combination with KN035 significantly inhibited PD-L1-resistant HCT116 tumor growth in vivo. The combination of KGX101 and KN035 showed synergistic antitumor efficacy compared to monotherapy.

[0191] Tumor volume, PD-L1 expression, T cell infiltration in tumors, and serum IFNγ concentration were measured. As shown in Figure 32, the percentage of circulating T cells (CD3+%) and T cell infiltration in the TME (CD3+%) increased after L2 treatment. Serum IFNγ concentration also increased after L2 treatment. The combined use of L2 and KN035 showed a synergistic effect or tendency toward synergistic effect on immune activation. A significant increase in hPD-L1+% in tumor cells (hCD45-) was also observed after repeated L2 administration. The increased PD-L1 expression and the resulting inhibitory TME environment can explain the combined mechanism of action (MoA) of IL-12 and anti-PD-L1 antibodies, as well as the better antitumor efficacy of the anti-PD-L1 antibody in combination. By using the anti-PD-L1 antibody, IL-12 was able to further unleash its immune-activating capacity.

[0192] Example 4. Stability and ex vivo cleavage of IL12 prodrug The serum stability of L2 and a+b was studied in cynomolgus monkey serum and human serum. L2 and a+b were incubated in cynomolgus monkey serum or human serum at 37°C. Stability was studied by identifying intact and degraded molecules using Western blotting. As shown in Figures 33 and 34, L2 was relatively stable in cynomolgus monkey serum and human serum after 72 hours of incubation, with only slight degradation bands. Most of the molecules remained intact even after 168 hours of incubation.

[0193] To test the ex vivo cleavage efficiency of L2 by tumor tissue, xenograft tumors (A375 or HCT116) and normal mouse tissues (liver and lung) were obtained from an NSG mouse model, cleaved in PBS, and digested with collagenase. The prepared cell suspensions were filtered and counted. 1 microgram of L2 was cleaved at 2 × 10¹⁶ cells per 100 μL. 6 The suspension was added to individual cells and incubated at 37°C for different durations. After centrifugation, the suspension was analyzed by Western blotting. As shown in Figure 35, the A chain of L2 was cleaved in tumor cells after 3 hours of incubation and slowly cleaved over 16 hours in hepatocytes. The B chain of L2 was relatively stable, and the cleaved B chain could only be observed at 16 and 24 hours in tumor and hepatocytes. In lung cells, almost no cleavage of L2 was observed even after 24 hours. These results indicate that L2 was more fragile in TME than in other tissues. This is consistent with the molecular design of the MMP14-specific linker.

[0194] Example 5. Production and manufacturing of a multifunctional IL12 prodrug Multifunctional IL12 prodrugs can be fabricated based on novel IL12 prodrugs to improve tumor targeting, enhance IL12 function, and / or achieve synergistic effects with additional cytokine activity. Figure 36 shows designs of multifunctional IL12 prodrugs based on L2 and control molecules. L2-αPDL1 contains L2 and an anti-PDL1 VHH antibody ligated to the C-terminus of each L2 chain. L2-mut contains L2 and a mutation in its R1(M1) portion that reduces its shielding effect and thus increases IL12 function. L2-IL7, L2-IL15, and IL-IL18 contain L2 and an additional cytokine molecule (IL7, IL15, or IL18) ligated to the C-terminus of the B chain of L2. L2-IL15α contains L2, an IL15 molecule, and an IL15 receptor sushi domain ligated to the C-terminus of each L2 chain. These chain mutations included additional mutations, such as the substitution of SKREKKD in P40 to reduce C-terminal degradation of P40, and the C252S mutation in P40 to reduce aggregation due to incorrect disulfide bond formation.

[0195] L2-αPDL1 and control proteins were produced by transient transfection in Expi293 cells and purified by one-step affinity chromatography (Cytiva). The purified proteins were characterized by non-reducible and reduced SDS-PAGE for purity assessment. Representative SDS-PAGE results are shown in Figure 37. The purified proteins were also characterized by high-performance liquid chromatography (HPLC) for homogeneity assessment. HPLC analysis was performed using BioResolve SEC mAb (Waters) at 100 mM PB, pH 6.8, and 150 mM NaCl. Representative HPLC results are shown in Figure 38, with the percentage of the main peak indicated.

[0196] The PDL1 binding dynamics of L2-αPDL1 were analyzed by BLI. Recombinant PDL1 protein was loaded and immobilized onto the sensor. The L2-αPDL1 protein was diluted from 50 nM to 0.78 nM and loaded onto the sensor containing PDL1, kon The rate was determined at 25°C. After equilibrium, the buffer solution was changed to PBST, and k off The rate was determined. As shown in Figure 39, the KD of L2-αPD-L1 and a+b-αPD-L1 was similar to that of anti-PDL1 VHH antibody alone.

[0197] L2-IL7, L2-IL15α, L2-mut, and control proteins were produced by transient transfection in Expi293 cells and purified by one-step affinity chromatography (Cytiva). The purified proteins were characterized by high-performance liquid chromatography (HPLC) to assess homogeneity. HPLC analysis was performed using BioResolve SEC mAb (Waters) at 100 mM PB, pH 6.8, and 150 mM NaCl. Representative HPLC results are shown in Figure 40, with peak percentages indicated.

[0198] Example 6. Characterization of the multifunctional IL12 prodrug The IL12 function of multifunctional IL12 prodrugs was tested using the HEK-Blue IL12 reporter assay. HEK-Blue IL12 reporter cells were purchased from InvivoGen and cultured according to the manufacturer's instructions for use. To detect IL12 activity, HEK-Blue IL12 cell suspensions were placed in test medium without Normocin and a select antibiotic, according to the instructions for use, in a 5 × 10⁶ solution. 5Cells / mL were prepared. 100 μL of cells were dispensed into each well of a flat-bottomed 96-well plate. Prodrug proteins or MMP14 digested proteins were prepared by sequentially diluting them 5-fold in test medium from 60 nM to 3.8 pM. 100 μL of the protein dilution was added to 100 μL of cells, and the plate was incubated at 37°C in 5% CO2 for 24 hours. The following day, QuantiBlue solution, a SEAP detection reagent, was prepared according to the manufacturer's instructions. 180 μL of QuantiBlue solution was mixed with 20 μL of induced HEK-Blue IL-12 cell supernatant in a flat-bottomed 96-well plate and incubated at 37°C for 1–3 hours. SEAP levels were measured at 620 nm using a spectrophotometer. Results were analyzed and calculated using nonlinear regression fitting in GraphPad Prism 8.

[0199] As shown in Figure 41, L2-αPDL1, L2-IL7, and L2-mut exhibited IL12 activity in the presence of MMP14, but their IL12 activity was shielded in the absence of MMP14. Similarly, as shown in Figure 42, L2-IL15α exhibited IL12 activity in the presence of MMP14, but their IL12 activity was shielded in the absence of MMP14. Since all proteins in this example were prepared using only one-step purification, the L2 samples here showed lower potency and higher EC50 compared to the higher-purity samples in previous examples, but are more suitable for direct comparison with similarly purified multifunctional IL12 prodrugs.

[0200] The immune system activating capacity of multifunctional prodrugs was further investigated by measuring IFNγ and TNFα induction using peripheral blood mononuclear cells (PBMCs). Human PBMCs were isolated and 1 × 10⁶ per 100 μL. 5Cells were dispensed into each well of a U-bottom 96-well plate and incubated overnight at 37°C. The following day, different fusion proteins or MMP14 digested proteins were added to PBMCs in 10-fold serial dilutions from 160 nM to 1.6 pM, along with different concentrations of anti-hCD3 antibody (clone OKT3, Biolegend 317301). The PBMCs were incubated at 37°C for 48–72 hours. The supernatant was then collected, and cytokine-dependent secretion of IFNγ or TNFα was quantified by ELISA. Dose-response curves were fitted using GraphPad Prism 8.

[0201] As shown in the upper panel of Figure 43, L2-mut and a+B-D1-mut induced IFNγ secretion from PBMCs at a lower EC50 compared to L2. This indicates that the mutation in IL12RB1 in L2-mut and a+B-D1-mut reduced the shielding effect of IL12 compared to L2. IL12 and IL12-Fc molecules were used as positive controls. Furthermore, as shown in the lower panel of Figure 43, L2-IL15α and L2-IL7 significantly enhanced maximal IFNγ secretion from PBMCs compared to L2. This indicates that the additional cytokine molecules in these prodrugs enhanced the immunoactivating effect of the prodrugs.

[0202] As shown in the upper panel of Figure 44, L2 shielding effectively reduced the in vitro potency of IL-12 in activating IFNγ secretion in PBMCs. Digestion with MMP14 nearly restored the potency. Furthermore, as shown in the lower panel of Figure 44, L2-IL15α and L2-IL7 significantly enhanced maximal IFNγ secretion in PBMCs compared to samples with single cytokine components such as L2, L2 with MMP, and IL15α-Fc. Similar to L2, the IFNγ-stimulating activity of L2-IL7 and L2-IL15α can be enhanced after MMP14 digestion. These results demonstrate that the additional cytokine molecules L2-IL7 and L2-IL15α can enhance the immunoactivating effects of these prodrugs, with or without the presence of MMP.

[0203] Example 7. In vivo efficacy of a combination of multifunctional IL12 prodrugs. The in vivo antitumor efficacy of the bispecific molecule L2-IL15α was tested in NSG mice using the human colon cancer HCT116 cell line. 6 × 10 6 Individual human colon cancer HCT116 cells, 2 × 10 6 Human PBMCs were mixed and subcutaneously transplanted into NSG mice (female, 6-8 weeks old). Tumor volume was 80-120 mm. 3 When the mice reached a certain stage, they were divided into groups and treated every four days with IV injections of PBS, L2, L2-IL15α, and combinations of L2 and L2-IL15α. Tumor volume and body weight were measured during the study.

[0204] As shown in Figure 45, L2-IL15α at 0.21 mg / kg (equimolar to 0.18 mg / kg L2) demonstrated the best antitumor efficacy compared to L2 monotherapy and the combination of L2 and IL15α-Fc. The TGI% (19 days after treatment) for 0.18 mg / kg L2 and 0.21 mg / kg L2-IL15α in the presence of hPBMCs were 40.3% and 75.5%, respectively. These results indicate that bispecific L2-IL15α could further enhance antitumor efficacy compared to combination therapy and monotherapy.

[0205] Example 8. Additional production platform for IL12 prodrug As described in Example 1, Expi293 cells were selected as a production platform for the IL12 prodrug based on the lack of endogenous expression of MMP14. An additional production platform was developed using MMP knockout CHO cells. MMP14 and MMP2 were knocked out of CHO cells using the CRISPR / Cas9 system, and a stable cell pool of MMP14 knockout (MMP14-KO) CHO cells and MMP2 knockout (MMP2-KO) CHO cells was established. The L1 molecule was expressed by transient transfection in wild-type (WT), MMP14-KO, and MMP2-KO CHO cell pools and purified by one-step purification using affinity chromatography (Cytiva). The purified protein was characterized by non-reducing SDS-PAGE for purity assessment. As shown in Figure 46, the L1 molecule showed the highest expression levels in MMP14-KO CHO cells compared to MMP2-KO and WT CHO cells. These results indicate that, in addition to the Expi293 cell platform, MMP14 knockout CHO cells can be a platform for the production of IL12 prodrugs.

[0206] [Table 6]

[0207] [Table 7] TIFF2026514090000009.tif249170 TIFF2026514090000010.tif249170 TIFF2026514090000011.tif249170 TIFF2026514090000012.tif249170 TIFF2026514090000013.tif249170 TIFF2026514090000014.tif249170 TIFF2026514090000015.tif249170 TIFF2026514090000016.tif244170 TIFF2026514090000017.tif239170

[0208] This disclosure includes preferred embodiments described herein with reference to the drawings, where similar numbers in the drawings represent the same or similar elements. Throughout this specification, where the phrases “one embodiment,” “one embodiment,” or similar phrases are used, it means that certain features, structures, or characteristics described in relation to that embodiment are included in at least one embodiment of the present invention. Thus, throughout this specification, occurrences of the phrases “in one embodiment,” “in one embodiment,” and similar phrases do not necessarily all refer to the same embodiment.

[0209] In addition to the various embodiments illustrated and claimed, the subject matter of this disclosure also relates to other embodiments having other combinations of the features disclosed and claimed herein. Thus, certain features presented herein can be combined with one another in other ways within the scope of the subject matter of this disclosure, and the subject matter of this disclosure includes any preferred combination of the features disclosed herein. Furthermore, any features, structures, or characteristics described herein can be combined in any preferred way in one or more embodiments. The above descriptions relating to specific embodiments of the subject matter of this disclosure are presented for illustrative and explanatory purposes only. They are not intended to be exhaustive, nor are they intended to limit the subject matter of this disclosure to the embodiments of this disclosure.

[0210] However, those skilled in the art will recognize that the applicant's compositions and / or methods can be carried out without using one or more of the specific details, or using other methods, components, and substances, etc. In other cases, well-known structures, substances, or operations are not illustrated or described in detail so as not to obscure the aspects of this disclosure.

[0211] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Any methods and materials similar to or equivalent to those described herein may also be used in the implementation or testing of the disclosure, but preferred methods and materials are described herein. The methods described herein may be performed in any logically possible order, in addition to the specific order of the disclosure.

[0212] Embedding by reference This disclosure includes references and citations to other documents, such as patents, patent applications, patent publications, academic journals, books, articles, and web content. All such documents are incorporated herein by reference in their entirety for all purposes. Any document or part thereof that is incorporated herein by reference but conflicts with existing definitions, descriptions, or other disclosures expressly provided herein is incorporated only to the extent that it does not create a conflict between the incorporated document and the document in this disclosure. In the event of a conflict, the conflict shall be resolved such that this disclosure takes precedence as the preferred disclosure.

[0213] Equal portions Representative examples are intended to aid in the explanation of the present invention and are not intended to limit the scope of the invention, nor should they be construed as such. In fact, various modifications of the invention and numerous further embodiments thereof, in addition to those illustrated and described herein, will be apparent to those skilled in the art from the entirety of this document, including the examples contained herein and references to scientific and patent literature. Such examples include important additional information, illustrations, and guidance that may enable the invention to be carried out in various embodiments and their equivalents.

Claims

1. a) A first chain comprising a first shielding portion, a first cleavable linker, a P35 molecule, and a first Fc region; and b) A multifunctional molecule comprising a second shielding portion, a second cleavable linker, a P40 molecule, and a second chain containing a second Fc region.

2. The first shielding portion includes the IL12Rβ2 portion, the amino acid sequence shown in SEQ ID NO: 4, or the amino acid sequence shown in SEQ ID NO: 5, or The second shielding portion includes the IL12Rβ1 portion, or the amino acid sequence shown in Sequence ID No. 3, or The second shielding portion includes an IL12Rβ1 portion containing one or more mutations of C6S, C55S, Y85S, or Q108L, or The second shielding portion includes an IL12Rβ1 portion containing mutations of C6S, C55S, Y85S, and Q108L, or combinations thereof. The multifunctional molecule according to claim 1.

3. Each of the first cleavable linker and the second cleavable linker is recognized and hydrolyzed by a proteolytic enzyme specifically expressed in the tumor microenvironment, The proteolytic enzyme is a matrix metalloproteinase, and the matrix metalloproteinase is matrix metalloproteinase 14 (MMP14), or Each of the first cleavable linker and the second cleavable linker includes an amino acid sequence selected from the group consisting of SEQ ID NOs. 67 to 112, or a combination thereof, The first cleavable linker comprises the amino acid sequence shown in SEQ ID NO: 6 or 7, or The second cleavable linker comprises the amino acid sequence shown in Sequence ID No.

8. The multifunctional molecule according to claim 1.

4. The first Fc region and the second Fc region form a dimerized Fc region, or The aforementioned dimerized Fc region includes a human Fc region, or The dimerized Fc region includes an Fc region selected from the group consisting of Fc regions of IgG, IgA, IgD, IgE, and IgM, or The dimerized Fc region includes an Fc region selected from the group consisting of Fc regions of IgG1, IgG2, IgG3, and IgG4, or The first Fc region includes a knob chain, and the second Fc region includes a hole chain, or The first Fc region includes a hole chain, and the second Fc region includes a knob chain, or The knob strand contains mutations S354C, T366W, and K408A, and the hole strand contains mutations Y349C, T366S, L368A, F405K, and Y406V, or Each of the first Fc region and the second Fc region contains one or more mutations that reduce FcγR and C1q binding to the Fc region, or Each of the first Fc region and the second Fc region contains mutations L234A and L235A, or Each of the first Fc region and the second Fc region contains mutations L234A, L235A, and P329G. The multifunctional molecule according to claim 1.

5. Each of the P35 molecule and the P40 molecule is linked to the Fc region via a linker, The linker is a peptide linker, or The peptide linker contains 4 to 30 amino acids, or The peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 49 to 66. The multifunctional molecule according to claim 1.

6. The second chain comprises the amino acid sequence shown in SEQ ID NO: 11 or 34, The second chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 30 to 40. The multifunctional molecule according to claim 1.

7. The first chain comprises the amino acid sequence shown in SEQ ID NO: 9 or 10, The first chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 to 29. The multifunctional molecule according to claim 1.

8. The first chain comprises the amino acid sequence shown in SEQ ID NO: 20, and the second chain comprises the amino acid sequence shown in SEQ ID NO: 31, 34, or 36, or The first chain comprises the amino acid sequence shown in SEQ ID NO: 21, 25, or 28, and the second chain comprises the amino acid sequence shown in SEQ ID NO: 30, 31, 34, or 36. The multifunctional molecule according to claim 1.

9. The multifunctional molecule further comprises an antibody moiety, The antibody portion includes full-length immunoglobulin, single-chain Fv (scFv) fragment, Fab fragment, Fab' fragment, F(ab')2, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, VHH, VHH-Fc fusion, Fv-Fc fusion, scFv-Fc fusion, scFv-Fv fusion, diabody, tribody, tetrabody, or a combination thereof, or The antibody portion is linked to the C-terminus of the first chain, or to the C-terminus of the second chain, or The antibody portion is linked to the first chain and / or the C-terminus of the second chain via a second linker, wherein the second linker is a peptide linker, or The second linker contains 4 to 30 amino acids. The second linker contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 49 to 66, or The antibody portion includes a VHH comprising a heavy chain variable region CDR1 containing the amino acid sequence shown in SEQ ID NO: 12, a heavy chain variable region CDR2 containing the amino acid sequence shown in SEQ ID NO: 13, and a heavy chain variable region CDR3 containing the amino acid sequence shown in SEQ ID NO: 14, or The antibody portion includes VHH containing the amino acid sequence shown in SEQ ID NO:

15. The multifunctional molecule according to claim 1.

10. The antibody portion is bound to a tumor-associated antigen, The tumor-associated antigens are PDL1, CD10, CD19, CD20, CD21, CD22, CD25, CD30, CD33, CD34, CD37, CD44v6, CD45, CDw52, Fms-like tyrosine kinase 3 (FLT-3, CD135), c-Kit (CD117), CSF1R (CD115), CD133, PDGFR-alpha (CD140a), and PDGFR-beta. (CD140b), chondroitin sulfate proteoglycan 4 (CSPG4), Muc-1, EGFR, de2-7-EGFR, EGFRvIII, folate-binding protein, Her2neu, Her3, PSMA, PSCA, PSA, TAG-72, HLA-DR, IGFR, IL3R, fibroblast-activating protein (FAP), carboanhydrase IX (MN / CA IX), carcinoembryonic antigen (CEA), EpCAM, CDCP1, Delrin 1, tenascin, frizzled1-10, vascular antigens VEGFR2 (KDR / FLK1), VEGFR3 (FLT4, CD309), endoglin, CLEC14, Temp1-8, and Tie2, and combinations thereof selected from the group. The multifunctional molecule according to claim 9.

11. The first chain comprises the amino acid sequence shown in SEQ ID NO: 41 or 43, The second chain comprises the amino acid sequence shown in SEQ ID NO: 42 or 44. The multifunctional molecule according to claim 1.

12. Further comprising a second cytokine portion, The second cytokine portion includes TNFα, IFN-γ, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL9, IL13, IL15, IL17, IL18, IL21, IL22, IL15α, TGFβ, G-CSF, GM-CSF, or a combination thereof. The second cytokine portion includes IL7, IL15, or IL18, or The second cytokine portion includes an IL15 molecule and IL15α sushi. The multifunctional molecule according to claim 1.

13. A first chain comprising the amino acid sequence shown in SEQ ID NO: 21, and a second chain comprising the amino acid sequence shown in SEQ ID NO: 45, 46, or 47, or A first chain containing the amino acid sequence shown in SEQ ID NO: 48, and a second chain containing the amino acid sequence shown in SEQ ID NO: 45, 46, or 46, The multifunctional molecule according to claim 1.

14. a) a multifunctional molecule according to any one of claims 1 to 13, and b) a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

15. One or more nucleic acids encoding the multifunctional molecule described in Claim 1.

16. One or more nucleic acids according to claim 15, further comprising a vector.

17. A host cell comprising the nucleic acid described in claim 15 or the vector described in claim 16.

18. Endogenous expression of matrix metalloproteinase is absent or reduced, or endogenous expression of MMP14 is absent or reduced, HEK293 cells, Expi293 cells, Expi293F cells, their variants, or their derivatives, or The host cell according to claim 17, which is a CHO cell, a mutant thereof, or a derivative thereof, in which the MMP14 gene is knocked out or knocked down.

19. A method for preparing the multifunctional molecule described in Claim 1, comprising expressing the multifunctional molecule in the host cell described in Claim 17, and isolating the multifunctional molecule from the host cell.

20. A pharmaceutical composition for treating and / or preventing cancer, the pharmaceutical composition according to claim 14.

21. A kit comprising the multifunctional molecule described in Claim 1, the pharmaceutical composition described in Claim 14, the nucleic acid described in Claim 15, or the vector described in Claim 16.

22. The kit according to claim 21, further comprising a written instruction manual for use in treating and / or preventing cancer.

23. The pharmaceutical composition according to claim 20, further comprising an effective amount of anti-PD-L1 antibody.

24. For simultaneously administering the multifunctional molecule and the anti-PD-L1 antibody, For administering the multifunctional molecule during the second step of the anti-PD-L1 antibody therapy, or For administration to subjects who have received at least one, at least two, at least three, or at least four doses of the anti-PD-L1 antibody prior to the administration of the multifunctional molecule, For administering at least one dose of the anti-PD-L1 antibody simultaneously with the multifunctional molecule, or The pharmaceutical composition according to claim 23, for administering the multifunctional molecule and the anti-PD-L1 antibody once every 1, 2, 3, 4, or 5 weeks.

25. The cancer is recurrent or progressive after a therapy selected from the group consisting of surgery, chemotherapy, radiotherapy, and combinations thereof, The aforementioned cancer is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial cancer, gastric cancer, bile duct cancer, head and neck cancer, hematological cancer, and combinations thereof, or The aforementioned cancer exhibits high microsatellite instability (MSI). The pharmaceutical composition according to claim 20.

26. A cleavable linker comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 8 and SEQ ID NOs: 67 to 112.

27. ​​A method for preparing a multifunctional molecule comprising a cleavable linker that can be recognized and hydrolyzed by a matrix metalloproteinase, This includes expressing the multifunctional molecule in a host cell containing the nucleic acid of the multifunctional molecule, and isolating the multifunctional molecule from the host cell. A preparation method wherein the host cells have no or reduced endogenous expression of the matrix metalloproteinase.

28. The preparation method according to claim 27, wherein the matrix metalloproteinase is MMP14.

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