IL-12 Fc fusion protein

Abstract

The present invention relates to IL-12 Fc fusion proteins and their use in medicine, pharmaceutical compositions containing same, and methods of using same as medicaments for the treatment and / or prevention of cancer.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to IL-12 Fc fusion proteins and their use in medicine, pharmaceutical compositions containing same, and methods of using same as medicaments for the treatment and / or prevention of cancer.

[0002] Background of the Invention Interleukin-12 (IL-12) is a cytokine with proven antitumor potential and has shown promising preclinical efficacy in mouse tumor models. However, drug-related toxicity has been observed in clinical trials, leading to suboptimal IL-12 dosing regimens and a lack of efficacy in patients.

[0003] To overcome drug-related toxicity, masking of IL-12 has been proposed to prevent systemic activity and toxicity and create a therapeutic window. Masking of IL-12 activity can be achieved, for example, by fusing the IL-12 receptor domain to IL-12 via a protease-cleavable linker and subsequent local activation by protease-mediated removal of the IL-12 receptor at tumor sites in cancer patients.

[0004] Currently, there are no approved IL-12-based therapies. Therefore, there remains a high unmet need to provide new therapeutic IL-12-based biological molecules that can be used for the treatment of cancer.

[0005] Summary of the Invention In a first aspect, the present invention relates to an interleukin-12 (IL-12) Fc fusion protein comprising a first polypeptide chain and a second polypeptide chain, wherein a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain; and a second Fc domain, wherein the IL-12p35 subunit or IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, and the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and the first or second polypeptide chain further comprises a binding moiety selected from the group consisting of a collagen-binding moiety, a heparin-binding moiety, and a fibronectin-binding moiety.

[0006] In further embodiments relating to an IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the binding moiety is linked to the C-terminus of the IL-12p35 subunit, or to the C-terminus of the IL-12p40 subunit, or the binding moiety is linked to the C-terminus of the masking moiety, in each case optionally via a third polypeptide linker.

[0007] In further embodiments of the IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the binding moiety is positioned between the IL-12p35 subunit and the IL-12p40 subunit, or the binding moiety is positioned between the C-terminus of the first Fc domain and the N-terminus of the IL-12p35 subunit or the N-terminus of the IL-12p40 subunit, in either case optionally flanked on one or both sides by a linker or linkers, preferably a peptide linker.

[0008] In a further embodiment relating to an IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the binding moiety is a collagen binding moiety.

[0009] In a further embodiment of the IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the collagen binding moiety binds to collagen I.

[0010] In a further embodiment relating to an IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the collagen binding moiety binds to collagen I and has the sequence LxxLxLxxN (SEQ ID NO: 41), wherein L is leucine, N is asparagine, and x is any amino acid.

[0011] In further embodiments of the IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the collagen binding moiety is 20 amino acids (aa), 19 aa, 18 aa, 17 aa, 16 aa, 15 aa, 14 aa, 13 aa, 12 aa, 11 aa, 10 a, or 9 aa in length.

[0012] In a further embodiment of the IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the collagen binding moiety comprises or consists of any one of the amino acid sequences of SEQ ID NOs: 40-47.

[0013] In a further embodiment relating to an IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the binding moiety is a heparin binding moiety.

[0014] In a further embodiment of the IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the heparin binding moiety has the sequence VRIQRKKEKMKET (SEQ ID NO: 50).

[0015] In a further embodiment relating to an IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the collagen binding moiety binds to collagen IV.

[0016] In a further embodiment of the IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the collagen binding moiety has the sequence KLWVLPK (SEQ ID NO: 40).

[0017] In a further embodiment relating to an IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the binding moiety is a fibronectin binding moiety.

[0018] In a further embodiment of the IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the fibronectin binding moiety has the sequence GGWSHW (SEQ ID NO: 49).

[0019] In a further embodiment relating to an IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the IL-12p35 subunit and the IL-12p40 subunit are human.

[0020] In further embodiments relating to an IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the IL-12p35 subunit comprises a polypeptide having at least 95% identity to SEQ ID NO:1 and the IL-12p40 subunit comprises a polypeptide having at least 95% identity to SEQ ID NO:2, preferably the IL-12p35 subunit comprises the polypeptide of SEQ ID NO:1 and the IL-12p40 subunit comprises the polypeptide of SEQ ID NO:2.

[0021] In further embodiments of the IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the IL-12p40 subunit and the IL-12p35 subunit are linked in a single chain having the structure (written N-terminus to C-terminus) IL-12p40-IL-12p35 or IL-12p35-IL-12p40.

[0022] In a further embodiment of the IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the single chain IL-12p40-IL-12p35 is linked to the C-terminus of the first Fc domain via its IL-12p40 subunit, or the single chain IL-12p35-IL-12p40 is linked to the first Fc domain via its IL-12p35 subunit, in both cases via a first peptide linker, which is protease-cleavable.

[0023] In a further embodiment of the IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the IL-12p40 subunit and the IL-12p35 subunit are linked to each other via a linker that is rich in the amino acid residues glycine and serine, preferably having a length of 5 to 20 amino acids and comprising only the amino acids glycine and serine, more preferably a glycine and serine linker having the amino acid sequence of SEQ ID NO: 22.

[0024] In further embodiments relating to an IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the single chain IL-12p40-IL-12p35 comprises a polypeptide having at least 95% identity to SEQ ID NO:8, or the single chain IL-12p35-IL-12p40 comprises a polypeptide having at least 95% identity to SEQ ID NO:9.

[0025] In a further embodiment relating to the IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the second peptide linker is not protease cleavable.

[0026] In a further embodiment relating to an IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the masking moiety binds to the IL-12p40 subunit and is selected from the group consisting of an IL-12 receptor or an IL-12p40-binding fragment thereof, an scFv, or an immunoglobulin single variable domain, preferably a VHH.

[0027] In a further embodiment relating to the IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the first and second Fc domains each comprise one or more mutations that promote heterodimerization of the Fc domains.

[0028] In further embodiments relating to the IL-12 Fc fusion protein according to the first aspect or any of its embodiments, (a) the first Fc domain is a human IgG1 Fc domain comprising the mutation T366W, and the second Fc domain is a human IgG1 Fc domain comprising the mutations T366S, L368A, and Y407V, or (b) the first Fc domain is a human IgG1 Fc domain comprising the mutations T366S, L368A, and Y407V, and the second Fc domain is a human IgG1 Fc domain comprising the mutation T366W.

[0029] In a further embodiment relating to an IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the first and second Fc domains are human IgG1 Fc domains, and one of the first or second Fc domains comprises the mutations H435R and Y436F.

[0030] In a further embodiment relating to an IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the first and second Fc domains are human IgG1 Fc domains, and either the first Fc domain, or the second Fc domain, or both Fc domains, comprise the mutations L234A and L235A.

[0031] In further embodiments relating to the IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the first Fc domain comprises the amino acid sequence of SEQ ID NO: 15, and the second Fc domain comprises the amino acid sequence of SEQ ID NO: 16, or the first Fc domain comprises the amino acid sequence of SEQ ID NO: 17, and the second Fc domain comprises the amino acid sequence of SEQ ID NO: 18, or the first Fc domain comprises the amino acid sequence of SEQ ID NO: 16, and the second Fc domain comprises the amino acid sequence of SEQ ID NO: 15, or the first Fc domain comprises the amino acid sequence of SEQ ID NO: 18, and the second Fc domain comprises the amino acid sequence of SEQ ID NO: 17.

[0032] In a further embodiment relating to the IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the protease-cleavable linker is cleavable by a matrix metalloprotease (MMP), preferably MMP-2, MMP-9, or MMP-13.

[0033] In a further embodiment of the IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the protease-cleavable linker comprises or consists of any one of the amino acid sequences of SEQ ID NOs: 232-241.

[0034] In a second aspect, the present invention provides an IL-12 polypeptide comprising a first polypeptide chain and a second polypeptide chain. For Fc fusion proteins, a) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:208, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:209; b) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:210, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:211; c) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:212, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:213; d) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:214, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:215; e) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:216, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:217; , comprising or consisting of the amino acid sequence of SEQ ID NO: 219; g) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 220 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 221; h) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 222 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 223; i) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 224 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 225; j) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 226 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 227; k) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 228 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 229; l) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 230;and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 231, or m) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 242 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 243.

[0035] In a further embodiment of the IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the masking moiety comprises an IL-12-binding immunoglobulin single variable domain comprising three CDRs comprised within any one of the sequences of SEQ ID NOs: 61-109.

[0036] In a further embodiment of the IL-12 Fc fusion protein according to the first aspect or any of its embodiments, the masking moiety comprises an IL-12-binding immunoglobulin single variable domain comprising any one of the amino acid sequences of SEQ ID NOs: 61-109.

[0037] In a third aspect, the present invention relates to a cleavage product capable of binding to the human IL-12 receptor comprising the IL-12 cytokine following proteolytic cleavage of the cleavable linker as defined in any one of the IL-12 Fc fusion proteins of the preceding aspects and related embodiments.

[0038] In a further embodiment relating to a cleavage product according to the third aspect or any of its embodiments, the cleavage product comprises an IL-12 cytokine and a binding moiety.

[0039] In further embodiments relating to the cleavage product according to the third aspect or any of its embodiments, the cleavage product comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, or 242 after proteolytic cleavage of the cleavable linker.

[0040] In a fourth aspect, the invention relates to an IL-12 binding immunoglobulin single variable domain comprising three CDRs contained within any one of the sequences SEQ ID NOs: 61-109.

[0041] In a further embodiment relating to an IL-12-binding immunoglobulin single variable domain according to the fourth aspect or any of its embodiments, the immunoglobulin single variable domain is a VHH.

[0042] In a further embodiment relating to an IL-12 binding immunoglobulin single variable domain according to the fourth aspect or any of its embodiments, the immunoglobulin single variable domain comprises the amino acid sequence of any one of SEQ ID NOs: 61-109.

[0043] In a fifth aspect, the present invention relates to a nucleic acid encoding at least one polypeptide of an IL-12 Fc fusion protein of the preceding aspects or any embodiment thereof related thereto, or a nucleic acid encoding one of the polypeptide chains of an IL-12 Fc fusion protein of the preceding aspects or any embodiment thereof related thereto, or a nucleic acid encoding an IL-12-binding immunoglobulin single variable domain of the preceding aspects or any embodiment thereof related thereto.

[0044] In a sixth aspect, the invention relates to a vector comprising a nucleic acid of the fifth aspect, optionally wherein the vector comprises nucleic acid encoding both chains of an IL-12 Fc fusion protein.

[0045] In a seventh aspect, the present invention relates to a host cell comprising the nucleic acid of the fifth aspect or the vector of the sixth aspect, optionally wherein the cell comprises one or more nucleic acids encoding both chains of an IL-12 Fc fusion protein.

[0046] In an eighth aspect, the present invention relates to a method for producing an IL-12 Fc fusion protein, comprising culturing a host cell of the seventh aspect under conditions for producing the fusion protein, and optionally purifying the IL-12 Fc fusion protein.

[0047] In a ninth aspect, the invention relates to a composition comprising an IL-12 Fc fusion protein of any of the preceding aspects or related embodiments thereof.

[0048] In a tenth aspect, the invention relates to a pharmaceutical composition comprising an IL-12 Fc fusion protein of any of the preceding aspects or related embodiments, and a pharmaceutically acceptable carrier.

[0049] In an eleventh aspect, the invention relates to a kit comprising an IL-12 Fc fusion protein of any of the preceding aspects or related embodiments, or a composition of the ninth aspect, or a pharmaceutical composition of the tenth aspect.

[0050] In a twelfth aspect, the present invention relates to an IL-12 Fc fusion protein as defined in any of the preceding aspects or embodiments related thereto, for use in medicine.

[0051] In a thirteenth aspect, the present invention relates to a cleavage product as defined in the third aspect or any embodiment related thereto, for use in medicine.

[0052] In a fourteenth aspect, the present invention relates to a method of treating or reducing the incidence of cancer in a subject, the method comprising administering to the subject an effective amount of an IL-12 Fc fusion protein according to any of the preceding aspects or embodiments related thereto.

[0053] In a fifteenth aspect, the present invention relates to an IL-12 Fc fusion protein according to any of the preceding aspects or related embodiments, for use in the treatment or prevention of cancer.

[0054] In a sixteenth aspect, the present invention relates to the use of an IL-12 Fc fusion protein according to any of the preceding aspects or embodiments related thereto for the manufacture of a medicament.

[0055] In a seventeenth aspect, the present invention relates to the use of an IL-12 Fc fusion protein according to any of the preceding aspects or related embodiments for the manufacture of a medicament for reducing the incidence of or treating cancer.

[0056] In any of the foregoing aspects and embodiments, the IL-12 Fc fusion protein, cleavage product, IL-12-binding immunoglobulin single variable domain, nucleic acid, vector, or host cell may be isolated, i.e., an isolated IL-12 Fc fusion protein, isolated cleavage product, isolated IL-12-binding immunoglobulin single variable domain, isolated nucleic acid, isolated vector, or isolated host cell. [Brief explanation of the drawings]

[0057] [Figures 1A-1H] Figures 1A-1H: Exemplary formats scouted for the assembly of IL-12 Fc fusion proteins, with cleavable IL-12 (p35 as a kidney shape, p40 as three individual spheres), an antibody fragment masking domain, a cleavable linker (light grey with an asterisk) connecting IL-12 to Fc, and either knobs-in-holes or wild-type Fc. [Figure 2] Figure 2: Functional characterization of 47 VHH-Fc discovered via llama immunization followed by subsequent phage panning. Only one of the constructs shows >90% inhibition of IL-12 relative to the control molecule in the Promega Bioassay. [Figure 3A-3B] Figures 3A-3B: Single-chain chimeric IL-12 (BI-066) (Figure 3A) or MMP9-truncated chimeric IL-12 Fc fusion protein (BI-057) (Figure 3B) was serially diluted and added to Promega IL-12 bioassay cells. After incubation, Bio-Glo™ reagent was added and luminescence was measured. Data were analyzed in GraphPad Prims, and EC50 values ​​were calculated. [Figure 4]Figure 4: Chimeric IL-12 Fc fusion protein (BI-057) was proteolytically cleaved with activated MMP9 (cleaved) or incubated without enzyme (uncleaved) for 24 hours. Both samples were then serially diluted and added to Promega IL-12 bioassay cells. After incubation, Bio-Glo™ reagent was added and luminescence was measured. Data were analyzed in GraphPad Prims, and EC50 values ​​were calculated. [Figures 5A-5D] Figures 5A-5D: C57B1 / 6 mice were injected with B16.F10 melanoma cells. Treatment began when tumors reached a volume of 70-100 mm. Animals were treated with vehicle or chimeric IL-12 Fc fusion protein at the doses indicated in the figure legend. The treatment schedule is depicted by the dotted lines. Tumor growth (Figures 5A and 5C) and body weight change (Figures 5B and 5D) were monitored twice weekly and are presented as spaghetti plots depicting individual mice. [Figures 6A-6B] Figures 6A-6B: C57B1 / 6 mice were injected with B16.F10 melanoma cells. Treatment began when tumors reached a volume of 70-100 mm. Animals were treated with vehicle or unmasked chimeric IL-12 Fc fusion protein at the doses indicated in the figure legends (Figure 6A: 0.08 mg / kg, Figure 6B: 1.6 mg / kg). The treatment schedule is depicted by the dotted line. Body weight changes were monitored twice weekly and are presented as spaghetti plots depicting individual mice. [Figures 7A-7B] Figures 7A-7B: C57B1 / 6 mice were injected with MC38 colon cancer cells. Treatment began when tumors reached a volume of 70-100 mm. Animals were treated with vehicle or chimeric IL-12 Fc fusion protein at the doses indicated in the figure legend. The treatment schedule is depicted by the dotted lines. Tumor growth (Figure 7A) and body weight change (Figure 7B) were monitored twice weekly and are presented as group means. [Figure 8A-8B]Figures 8A-8B: C57B1 / 6 mice were injected with B16.F10 melanoma cells. Treatment began when tumors reached a volume of 70-100 mm. Animals were treated with vehicle or chimeric IL-12 Fc fusion protein at the doses indicated in the figure legend. The treatment schedule is depicted by the dotted line. Tumor growth (Figure 8A) and body weight change (Figure 8B) were monitored twice weekly and are presented as spaghetti plots depicting individual mice. [Figures 9A-9C] Figures 9A-9C: Cynomolgus monkeys were injected with three different doses of human IL-12 Fc fusion protein on day 1. Blood was collected on days 1 (pre-dose), 8, and 15. The values ​​of ALT (Figure 9A), bilirubin (Figure 9B), and creatinine (Figure 9C) for each animal are shown on the graphs. The dotted line depicts the reference value. [Figures 10A-10F] Figures 10A-10F: Gene expression of MMP2 (Figure 10A), MMP9 (Figure 10B), MMP13 (Figure 10C), TIMP1 (Figure 10D), TIMP2 (Figure 10E), and TIMP3 (Figure 10F) in normal and cancer tissues. White, thin line, GTEX normal tissue corresponding to TCGA cancer tissue; white, thick line, TCGA adjacent normal tissue; gray, thick line, TCGA cancer tissue. [Figure 11] Figure 11: Five variants show efficient cleavage of the parent molecule into the IL-12 component and the Fc mask domain with the addition of MMP9. [Figure 12] Figure 12: Treatment with MMP9 using an anti-p40 Western antibody demonstrates the release of single-chain IL-12 from the intact prodrug, with the released IL-12 migrating to a ∼62 kD band in a reducing environment. The detection antibodies used in this assay were anti-IL-12 p40 (R&D Systems, AF309) 1:2500; anti-goat IgG HRP (R&D Systems, HAF017) 1:1000. [Figures 13A-13E]Figures 13A-13E: Human IL-12 Fc fusion proteins BI-050 (Figure 13A), BI-051 (Figure 13B), BI-052 (Figure 13C), BI-054 (Figure 13D), and BI-055 (Figure 13E) were proteolytically cleaved with activated MMP9 (cleaved) or incubated without enzyme (uncleaved) for 24 hours. All samples were then serially diluted and added to a Promega IL-12 bioassay cell. After incubation, Bio-Glo™ reagent was added and luminescence was measured. Data were analyzed in GraphPad Prims, and EC50 values ​​were calculated (see Table 15). [Figures 14A-14B] Figures 14A-14B: BI-059 at 2.5 μM was incubated with either buffer control, 6.5 nM (0.025 μg) activated recombinant human MMP9, or 5 μg of human colorectal cancer tumor lysate for 2 hours at 37°C. Tumor lysate was also incubated with buffer alone as a control. SDS-PAGE and Western blotting were performed using antibodies against human Fc (Figure 14A) and human IL12p40 (Figure 14B), which showed size shifts corresponding to the released masking domain / Fc fragment and free IL-12, respectively, after cleavage of the full-length IL-12 Fc fusion protein by MMPs. [Figure 15] Figure 15: C57B1 / 6 mice were injected with MC38 colon cancer cells. Treatment began when tumors reached a volume of 70-100 mm. Mice were treated twice on days 1 and 4 with vehicle or chimeric IL-12 Fc fusion protein at the doses indicated in the figure legend. Mice were sacrificed on day 5 and tumors were harvested. Tissues were digested and followed by IFNγ assessment. [Figures 16A-16F]Figures 16A-16F: C57B1 / 6 mice were injected with MC38 colon cancer cells. Treatment began when tumors reached a volume of 70-100 mm. Animals were treated twice on days 1 and 4 with vehicle or chimeric IL-12 Fc fusion protein at the doses indicated in the figure legends. Animals were sacrificed on day 5 and tumors were harvested. Tissues were digested followed by flow cytometric assessment of tumor-infiltrating leukocytes for marker expression as indicated. [Figures 17A-17I] Figures 17A-17I: Gene expression of collagen I A1 (Figure 17A), collagen I A2 (Figure 17B), fibronectin (Figure 17C), collagen IV A1 (Figure 17D), collagen IV A2 (Figure 17E), collagen IV A3 (Figure 17F), collagen IV A4 (Figure 17G), collagen IV A5 (Figure 17H), and collagen IV A6 (Figure 17I) in normal and cancer tissues. White, thin line, GTEX normal tissue corresponding to TCGA cancer tissue; white, thick line, TCGA adjacent normal tissue; gray, thick line, TCGA cancer tissue. [Figures 18A-18B] Figures 18A-18B: Human IL-12 Fc fusion protein BI-051 was serially diluted and added to collagen I-coated plates for 10 minutes. After a washing step, bound proteins were detected using a biotinylated anti-human Fc antibody. SA-HRP, followed by substrate, was added to the wells, and the OD was measured in a Tecan plate reader (Figure 18A). OD values ​​are presented. Interaction analysis was performed using a Biacore T200 (Figure 18B) equipped with a CM5 chip, in which human collagen type 1 (Merck, CC050) was amine-coupled (3000 RU) onto the active surface, and reference cells were amine-coupled without any ligand. BI-051 and mAb IgG1 (negative control) were diluted to a final concentration of 5 μM in Biacore running buffer (phosphate-buffered saline, pH 7.4, containing 0.05% Tween-20). Biacore measurements were performed using the reference subtraction method. The interaction occurred only on the activated surface on which human type 1 collagen was immobilized. [Figure 19]Figure 19: The separate chains (knob and hole chains) comprising the human IL-12 Fc fusion protein BI-051 were serially diluted and added to collagen I-coated plates for 10 minutes. After a washing step, bound protein was detected using a biotinylated anti-human Fc antibody. SA-HRP followed by substrate was added to the wells and the OD was measured in a Tecan plate reader. The OD values ​​are presented. [Figures 20A-20D] Figures 20A-20D: Plates coated with fibroblast-produced collagen I were prepared as described in the Materials and Methods section. After decellularization, 5 μg of fluorescently labeled human IL-12 Fc fusion protein (BI-051) was added to the wells (Figure 20B). Wells incubated with PBS (Figure 20D) served as controls. After a washing step, the wells were stained with anti-collagen I antibody (Figures 20A and 20C) and visualized in the Opera Phenix system. [Figures 21A-21B] Figures 21A-21B: C57B1 / 6 mice were injected with MC38 colon cancer cells. Treatment began when tumors reached a volume of 70-100 mm. Animals were treated with vehicle, chimeric IL-12 Fc fusion protein (BI-065), or chimeric IL-12 Fc fusion protein containing a fibronectin TME linker (BI-059) at a dose of 0.5 mg / kg. The treatment schedule is depicted by the dotted lines. Tumor growth was monitored twice weekly and is presented as a spaghetti plot depicting individual mice (Figure 21A). The percentage of surviving mice is presented (Figure 21B). [Figure 22]Figure 22: C57B1 / 6 mice were injected with B16.F10 melanoma cells. Treatment began when tumors reached a volume of 70-100 mm. Mice were treated with vehicle, chimeric IL-12 Fc fusion protein (BI-065), or chimeric IL-12 Fc fusion protein containing a fibronectin TME linker (BI-059) at a dose of 1.5 mg / kg. The treatment schedule is depicted by the dotted line. Tumor growth was monitored twice weekly and is presented as a spaghetti plot depicting individual mice. [Figures 23A-23B] Figures 23A-23B: C57B1 / 6 mice were injected with MC38 colon cancer cells. Treatment began when tumors reached a volume of 70-100 mm. Animals were treated with vehicle, chimeric IL-12 Fc fusion protein (BI-065), or chimeric IL-12 Fc fusion protein containing a collagen I TME linker (BI-057) at a dose of 0.5 mg / kg. The treatment schedule is depicted by the dotted lines. Tumor growth (Figure 23A) and changes in body weight (Figure 23B) were monitored twice weekly and are presented as spaghetti plots depicting individual mice. [Figures 24A-24B] Figures 24A-24B: C57B1 / 6 mice were injected with MC38 colon cancer cells. Treatment began when tumors reached a volume of 70-100 mm. Animals were treated with vehicle, chimeric IL-12 Fc fusion protein (BI-065), or chimeric IL-12 Fc fusion protein containing a collagen I TME linker (BI-057) at a dose of 1.5 mg / kg. The treatment schedule is depicted by the dotted lines. Tumor growth (Figure 24A) and changes in body weight (Figure 24B) were monitored twice weekly and are presented as spaghetti plots depicting individual mice. [Figures 25A-25B]Figures 25A-25B: Chimeric IL-12 Fc fusion proteins BI-057 or BI-065 were serially diluted and added to collagen I-coated plates for 120 minutes. Rat collagen (Corning) (Figure 25A) or human collagen (Millipore / R&D) (Figure 25B) were tested. After a washing step, bound proteins were detected using a biotinylated anti-human Fc antibody. SA-HRP followed by substrate was added to the wells and the OD was measured in a Tecan plate reader. The OD values ​​are presented. [Figures 26A-26B] Figures 26A-26B: Precision-cut liver slices were prepared from the livers of fibrotic rats. IL-12 Fc fusion protein (BI-057 or BI-065; amounts indicated in the figure legends) was added to the slices and cultured for 24 hours (Figure 26A) or 2 hours, followed by a 22-hour recovery period (Figure 26B). After the 2-hour incubation, the fusion protein was thoroughly washed and incubated in culture medium for another 22 hours before collection. After 24 hours, the slices were collected, lysed, and homogenized. The amount of IL-12 Fc fusion protein recovered from the slices was measured using the MSD U-PLEX biomarker assay, using IL-12 Fc fusion protein as a standard. [Figures 27A-27D] Figures 27A-27D: C57B1 / 6 albino mice were subcutaneously injected with KPCY pancreatic cancer cells. Animals were intravenously injected with a 30 μg dose of Dylight 650-labeled chimeric IL-12 Fc fusion protein (BI-201) or a chimeric IL-12 Fc fusion protein containing a collagen I TME linker (BI-202). To assess the kinetics of protein retention, mice were imaged using an IVIS under autoexposure epifluorescence settings at the indicated time points (Figure 27A). Image analysis to determine total radiant efficiency was performed using Living Image. Statistical analysis on the final day of the experiment was performed using the Mann-Whitney test (Figure 27B). Fluorescence data were used to calculate half-life (Figure 27C) and clearance (Figure 27D). [Figures 28A-28B]Figures 28A-28B: Balb / c mice were subcutaneously injected with EMT6 breast cancer cells. Animals were intravenously injected with a 50 μg dose of Dylight 650-labeled chimeric IL-12 Fc fusion protein (BI-200) or a chimeric IL-12 Fc fusion protein containing a collagen I TME linker (BI-051). To assess the kinetics of protein retention, mice were imaged using an IVIS under autoexposure epifluorescence settings at the indicated time points (Figure 28A). Image analysis to determine total radiant efficiency was performed using Living Image. Statistical analysis at 30 hours was performed using the Mann-Whitney test (Figure 27B). [Figures 29A-29B] Figures 29A-29B: C57B1 / 6 mice were subcutaneously injected with PDA30364 pancreatic cancer cells. Animals were injected intratumorally with a chimeric IL-12 Fc fusion protein (BI-065) or a chimeric IL-12 Fc fusion protein containing a collagen I TME linker (BI-057) at a dose of 150 pmol. Blood was collected 48 hours post-injection, and IFNγ (Figure 29A) and CXCL10 (Figure 29B) levels were determined by LegendPlex. Statistical analysis was performed using the Mann-Whitney test. [Figure 30A-30B] Figures 30A-30B: C57B1 / 6 mice were orthotopically injected with EMT6 breast cancer cells. Treatment began when tumors reached a volume of 50-120 mm3. Animals were treated with vehicle or chimeric IL-12 Fc fusion protein (BI-065), or chimeric IL-12 Fc fusion protein containing a collagen I TME linker (BI-057) at the doses indicated in the figure legends. Blood was collected 24 hours (Figure 30A) or 72 hours (Figure 30B) post-injection, and IFNγ levels were determined by LegendPlex. Statistical analysis was performed using the Mann-Whitney test.

[0058] Detailed Description of the Invention The inventors set out to design a conditionally active IL-12 fusion protein that would allow for the systemic administration of IL-12 (which is known to be toxic) to patients for the treatment of tumors. Many challenges had to be overcome along the way, including selecting a suitable molecular design, finding a suitable method for blocking the activity of IL-12 to allow for systemic administration, tailoring chemistry, manufacturing, and control (CMC) properties and molecular function, and ensuring that IL-12 reaches the tumor and then becomes active again within or near the tumor.

[0059] The present invention provides an interleukin-12 (IL-12) Fc fusion protein comprising a first polypeptide chain and a second polypeptide chain, wherein a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain; and the first and second polypeptide chains are coupled to the first Fc domain and the second Fc domain. The present invention is based on the concept of providing an IL-12 Fc fusion protein in which an IL-12p35 subunit or an IL-12p40 subunit is linked to the C-terminus of a first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, and in which a masking moiety is linked to the C-terminus of a second Fc domain via a second peptide linker, and the first or second polypeptide chain further comprises a binding moiety selected from the group consisting of a collagen-binding moiety, a heparin-binding moiety, and a fibronectin-binding moiety.

[0060] An Fc-based fusion protein approach was chosen to increase the half-life of the fusion protein after systemic administration. This Fc fusion protein comprises two polypeptide chains. The first polypeptide chain comprises a first Fc domain and both IL-12 subunits, IL-12p35 and IL-12p40, which together form the active IL-12 cytokine, and is linked to the C-terminus of the first Fc domain via a protease-cleavable linker. The second polypeptide chain comprises a second Fc domain and a masking moiety linked to the C-terminus of the second Fc domain. Both polypeptide chains dimerize together via their respective Fc domains to form a dimeric Fc fusion protein, i.e., the polypeptide chains are linked via the bond of the two Fc domains that together form the Fc portion of the fusion protein. In this dimeric Fc fusion protein, the masking moiety of the second chain binds to the IL-12 cytokine on the first chain, thereby blocking, inhibiting, or attenuating the activity of the IL-12 cytokine. In addition, the Fc-based fusion protein comprises a binding moiety selected from the group consisting of a collagen-binding moiety, a heparin-binding moiety, and a fibronectin-binding moiety.

[0061] After systemic administration of the IL-12 Fc fusion protein, the activity of IL-12 on the first polypeptide chain is still blocked, inhibited, or attenuated by the masking moiety. It is then proposed that tumor-specific activation is achieved via a dual mechanism involving a protease-cleavable linker and a binding moiety. The protease-cleavable linker is preferably cleaved by a protease that is tumor-specific or upregulated in the tumor microenvironment (TME). In addition, the binding moiety binds to its respective structure in the TME, such as the extracellular matrix (ECM), and together with the protease-cleavable linker, provides a therapeutic window that allows optimal biological activity within the TME without dose-limiting systemic toxicity.

[0062] Retaining the IL-12 Fc fusion protein within the TME may have additional advantages, such as longer exposure of the fusion protein to an environment of upregulated protease activity in the TME, which may increase cleavage efficiency and ultimately the levels of cleavage products released. Also, once cleaved, the cleavage products may be retained within the tumor, which may increase the efficacy of the response and reduce systemic toxicity by preventing, reducing, or delaying the entry of cleavage products into the circulation.

[0063] In one aspect, unmasked IL-12 provides a potent Th1 polarizing stimulus to T cells at the tumor site and improves their effector function.

[0064] In another aspect, the IL-12 Fc fusion protein has improved pharmacokinetic and / or toxicity properties compared to unmasked IL-12. In another aspect, the IL-12 Fc fusion protein has improved pharmacokinetic and / or toxicity properties compared to other masked IL-12 fusion proteins.

[0065] In another embodiment, the IL-12 Fc fusion protein can be produced as a stable molecule with high processability and productivity.

[0066] In another embodiment, the IL-12 Fc fusion protein may have tumor-independent properties, i.e., may be used for the treatment of multiple cancers. In a related embodiment, the IL-12 Fc fusion protein may be useful for the immunomodulatory treatment of cancer or tumors.

[0067] In one aspect, the cleavage products of the IL-12 Fc fusion protein are prevented, reduced, or delayed from entering the circulation after cleavage in the TME.

[0068] In another aspect, the cleavage products of the IL-12 Fc fusion protein exhibit increased retention within the tumor or TME. In a related aspect, the cleavage products of the IL-12 Fc fusion protein exhibit increased efficacy responses. In a related aspect, the cleavage products of the IL-12 Fc fusion protein exhibit reduced systemic toxicity.

[0069] In another embodiment, the IL-12 activity of the uncleaved IL-12 Fc fusion protein is at least 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, 175-fold, 200-fold, 225-fold, 250-fold, 275-fold, 300-fold, 325-fold, 350-fold, 375-fold, 400-fold, 425-fold, 450-fold, 475-fold, 500-fold, 525-fold, 550-fold, 575-fold, or 600-fold less than the IL-12 activity of the IL-12 Fc fusion protein after cleavage of the cleavable linker. In other words, the delta EC of the uncleaved IL-12 Fc fusion protein and the cleaved IL-12 Fc fusion protein as measured in an IL-12 bioassay 50 (EC 50 Uncleaved IL-12 Fc fusion protein:EC 50 The cleaved IL-12 Fc fusion protein) is at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 400, 425, 450, 475, 500, 525, 550, 575, or 600, e.g., as in the Promega IL-12 bioassay described in the Examples.

[0070] definition Terms not specifically defined herein should be given the meaning that would be given them by one of ordinary skill in the art in light of the disclosure and context. However, as used herein, terms have the meaning indicated unless specified to the contrary, and the following rules are observed:

[0071] As used herein, the term "comprise" and variations thereof, such as "comprises" and "comprise," may be replaced with the terms "containing" or "including" or "having."

[0072] The term "sequence" as used herein (e.g., in terms such as "heavy chain / light chain sequence," "antibody sequence," "variable domain sequence," "constant domain sequence," or "protein sequence") should generally be understood to include both the related amino acid sequence as well as the nucleic acid or nucleotide sequence encoding same, unless the context requires a more restrictive interpretation.

[0073] The "Fc domain" of an antibody is not directly involved in binding of the antibody to an antigen, but exerts various effector functions. The term "Fc domain of an antibody" is well known to those skilled in the art and is defined based on papain digestion of an antibody. Depending on the amino acid sequence of the constant region of their heavy chain, antibodies or immunoglobulins are classified into classes: IgA, IgD, IgE, IgG, and IgM. According to the heavy chain constant region, the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. Some of these can be further classified into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The Fc portion of an antibody is directly involved in ADCC (antibody-dependent cellular cytotoxicity) and CDC (complement-dependent cytotoxicity) based on complement activation, Clq binding, and Fc receptor binding. Complement activation (CDC) is initiated by the binding of complement factor Clq to the Fc portion of most IgG antibody subclasses. While the effect of antibodies on the complement system is condition-dependent, binding to Clq occurs via defined binding sites in the Fc portion. Such binding sites include, for example, L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbered according to EU numbering (Edelman et al., Proc Natl Acad Sci US A. 1969 May;63(1):78-85)). Among these residues, L234 and L235 are most important in mediating IgG1 C1q and Fc gamma receptor binding (Hezareh et al., J. Virology 75 (2001) 12161-12168; Shields et al. (2001) JBC, 276 (9):6591-6604). Antibodies of the subclasses IgG1 and IgG3 typically exhibit complement activation and Clq and C3 binding, whereas IgG2 and IgG4 do not activate the complement system and do not bind Clq and C3.

[0074] "Single-chain Fv" or "scFv" antibody fragments are single-chain Fv variants comprising the VH and VL domains of an antibody, wherein the domains are present in a single polypeptide chain. Single-chain Fvs are capable of recognizing and binding to antigen. The scFv polypeptide may also optionally comprise a polypeptide linker disposed between the VH and VL domains to facilitate the formation of the desired three-dimensional structure for antigen binding by the scFv (see, e.g., Pluckthun, 1994, In The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315).

[0075] As used herein, a "non-cleavable linker" or "non-protease-cleavable linker" refers to a peptide linker that does not contain a peptide sequence or mimetic thereof that is a target for a protease. Exemplary non-cleavable linkers are described in the "Linkers" section.

[0076] An antigen-binding molecule / protein (such as an immunoglobulin, antibody, antigen-binding unit, or fragment of such an antigen-binding molecule / protein) that "binds," "binds to," "specifically binds," or is capable of "specifically binding to," "has affinity for," "is specific for," and / or "has specificity for" a particular epitope, antigen, or protein (or at least one portion, fragment, or epitope thereof) is said to be "against" or "directed against" that epitope, antigen, or protein, or is a "binding" molecule / protein with respect to such epitope, antigen, or protein.

[0077] As used herein, the terms "binding" and "specific binding" refer to the binding of an antibody or antigen-binding portion (e.g., an immunoglobulin, antibody, antigen-binding unit, or fragment of such an antigen-binding molecule / protein) to an epitope of an antigen in an in vitro assay, preferably a plasmon resonance assay, using purified wild-type antigen (Malmqvist M., "Surface plasmon resonance for detection and measurement of antibody-antigen affinity and kinetics." Curr Opin Immunol. 1993 Apr;5(2):282-6.). Antibody affinity can also be measured using equilibrium exclusion assay (Kinetic Exclusion Assay) technology (Darling, RJ, and Brault PA., "Kinetic Exclusion Assay Technology: Characterization of Molecular Interactions." ASSAY and Drug Development Technologies. 2004, Dec 2(6):647-657).

[0078] Generally, the term "specificity" refers to the number of different antigens or epitopes that a particular antigen-binding molecule / protein (e.g., an immunoglobulin, an antibody, an antigen-binding unit, or a fragment of such an antigen-binding molecule / protein) can bind to. The specificity of an antigen-binding molecule / protein can be determined based on its affinity and / or avidity. Affinity is determined by the equilibrium constant (K D ) and is a measure of the binding strength between an epitope and an antigen-binding site on an antigen-binding molecule / protein: K D The smaller the value of K, the stronger the binding strength between the epitope and the antigen-binding molecule / protein (alternatively, affinity can also be expressed as the affinity constant (K A ), which is 1 / K D(wherein the avidity is the affinity of the antigen-binding molecule / protein (e.g., an immunoglobulin, an antibody, an antigen-binding unit, or a fragment of such an antigen-binding molecule / protein) to the antigen. The avidity is related to both the affinity between the epitope and its antigen-binding site on the antigen-binding molecule / protein and the number of relevant binding sites present on the antigen-binding molecule / protein. As will be apparent to those skilled in the art (e.g., based on the further disclosure herein), affinity can be determined in a manner known per se depending on the particular antigen of interest. Avidity is a measure of the strength of binding between an antigen-binding molecule / protein (e.g., an immunoglobulin, an antibody, an antigen-binding unit, or a fragment of such an antigen-binding molecule / protein) and the antigen. Avidity is related to both the affinity between an epitope and its antigen-binding site on the antigen-binding molecule / protein and the number of relevant binding sites present on the antigen-binding molecule / protein.

[0079] For human applications, it is often desirable to reduce the immunogenicity of therapeutic molecules, such as antibodies or binding proteins comprising the antigen-binding units described herein, originally derived from other species, such as mice. This can be achieved by constructing chimeric antibodies / binding proteins, or by a process called "humanization." In this context, a "chimeric antibody" or "chimeric antigen-binding unit" is understood to be an antibody or antigen-binding unit that comprises a sequence portion (e.g., a variable domain) derived from one species (e.g., a mouse) fused to a sequence portion (e.g., a constant domain) derived from a different species (e.g., a human). In this context, a "humanized antibody," "humanized binding protein," or "humanized antigen-binding unit" is an antibody, protein, or antigen-binding unit that comprises a variable domain originally derived from a non-human species, in which certain amino acids have been mutated so that the overall sequence of the variable domain more closely resembles the sequence of a human variable domain. Methods for humanizing antibodies are well known in the art (Billetta R, Lobuglio AF. "Chimeric antibodies". Int Rev Immunol. 1993;10(2-3):165-76; Riechmann L, Clark M, Waldmann H, Winter G (1988). "Reshaping human antibodies for therapy". Nature: 332:323).

[0080] An "optimized antibody" or "optimized antigen-binding unit or protein" is a specific type of humanized antibody or humanized antigen-binding unit / protein that has the ability to bind to a predetermined antigen and comprises an immunoglobulin amino acid sequence variant, or fragment thereof, that comprises one or more FRs having substantially the amino acid sequence of a human immunoglobulin and one or more CDRs having substantially the amino acid sequence of a non-human immunoglobulin. This non-human amino acid sequence is often referred to as an "import" sequence and is typically taken from an "import" antibody domain, particularly the variable domain. Generally, optimized antibodies contain at least a CDR (or HVL) of or derived from a non-human antibody inserted between the FRs of a human heavy or light chain variable domain. It will be understood that certain murine FR residues may be important for the function of an optimized antibody, and therefore some of the heavy and light chain variable domain residues of the human germline sequences have been altered to be identical to those of the corresponding murine sequences. During this process, undesired amino acids may also be removed or changed, for example, to avoid deamidation, undesired charge or lipophilicity, or nonspecific binding. An "optimized antibody," "optimized antibody fragment," or "optimized" may sometimes be referred to as a "humanized antibody," "humanized antibody fragment," or "humanized," or as "sequence-optimized."

[0081] Furthermore, technologies have been developed to generate antibodies or VH / VL domains based on sequences derived from the human genome, for example, by the use of phage display or transgenic animals (WWW.Alexis.com / technology-alivamab.php; WO 90 / 05144; D. Marks, HR Hoogenboom, TP Bonnert, J. McCafferty, AD Griffiths and G. Winter (1991) "Bypassing immunization. Human antibodies from V-gene libraries displayed on phage." J. Mol. Biol., 222, 581-597; Knappik et al., J. Mol. Biol. 296: 57-86, 2000; S. Carmen and L. Jermutus, "Concepts in antibody phage display." Briefings in Functional Genomics and Proteomics 2002 1(2):189-203; Lonberg N, Huszar D. "Human antibodies from transgenic mice". Int Rev Immunol. 1995;13(1):65-93.; Bruggemann M, Taussig MJ. "Production of human antibody repertoires in transgenic mice". Curr Opin Biotechnol. 1997 Aug;8(4):455-8.) Such antibodies or antigen-binding units or VH / VL domains are, in the context of the present invention, "human antibodies", "human antigen-binding units", or "human VH / VL domains".

[0082] As used herein, the terms "identical" or "percent identity," in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of the same nucleotides or amino acid residues when compared and aligned for maximum correspondence. To determine percent identity, the sequences are aligned for optimal comparison (e.g., gaps can be introduced into the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # identical positions / total # positions (e.g., overlapping positions) × 100). In some embodiments, the two sequences being compared are the same length after introducing gaps, if appropriate, into the sequence (e.g., excluding additional sequence that extends beyond the sequence being compared). For example, when comparing variable region sequences, leader and / or constant domain sequences are not considered. For sequence comparison between two sequences, a "corresponding" CDR refers to the CDR at the same position in both sequences (e.g., CDR-H1 of each sequence).

[0083] The determination of percent identity or percent similarity between two sequences can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed using the NBLAST program, score=100, word length=12, to obtain nucleotide sequences homologous to a nucleic acid encoding a protein of interest. BLAST protein searches can be performed using the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein of interest. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules (ibid.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. Another preferred, non-limiting example of a mathematical algorithm utilized for sequence comparison is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package.When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art, including ADVANCE and ADAM, described in Torellis and Robotti, 1994, Comput. Appl. Biosci. 10:3-5; and FASTA, described in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search. When ktup=2, similar regions in the two sequences being compared are found by looking at pairs of aligned residues; when ktup=1, single aligned amino acids are examined. ktup can be set to 2 or 1 for protein sequences, or 1 to 6 for DNA sequences. If ktup is not specified, the default is 2 for proteins and 6 for DNA. Alternatively, protein sequence alignments may be performed using the CLUSTAL W algorithm as described by Higgins et al., 1996, Methods Enzymol. 266:383-402.

[0084] As used herein, the term "linkage" refers to the association of different components of an IL-12 Fc fusion protein and includes (i) any means by which the different components can be linked, such as a linker, or (ii) any chemical association for linking the different components of the IL-12 Fc fusion protein, including both covalent and non-covalent interactions, preferably covalent interactions. A covalent interaction may be, for example, a direct covalent bond between residues, such as a peptide bond or a disulfide bond. The linker may be a peptide linker or a non-peptide linker, preferably a peptide linker. When the linker is a peptide linker, it may be composed of one or more amino acids.

[0085] "Immunoglobulin single variable domains" (ISVDs) are antibody fragments consisting of a single variable antibody domain. Like whole antibodies, they can selectively bind to a specific antigen. With a molecular weight of only 12-18 kDa, they are significantly smaller than conventional antibodies (150-160 kDa) composed of two heavy and two light protein chains, and even smaller than Fab fragments (approximately 50 kDa, one light chain and half a heavy chain) and single-chain variable fragments (approximately 25 kDa, two variable domains, one from the light chain and one from the heavy chain). Generally, immunoglobulin single variable domains have an amino acid sequence comprising four framework regions (FR1-FR4) and three complementarity-determining regions (CDR1-CDR3), preferably according to the following formula: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The term immunoglobulin single variable domain, as used herein, includes, but is not limited to, variable domains of camelid heavy chain antibodies (VHH), referred to as Nanobodies™, domain antibodies (dAbs), and immunoglobulin single variable domains derived from sharks (IgNAR domains).

[0086] "VHH domain" also refers to VHH, V H Known as H domains, VHH antibody fragments, and VHH antibodies, they were originally described as antigen-binding immunoglobulin (variable) domains of "heavy chain antibodies" (i.e., "light chain-free antibodies"; Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). The term "VHH domain" refers to the separation of these variable domains from the heavy chain variable domains present in conventional four-chain antibodies (which are referred to herein as "VH domains" or "VH domains") and from the light chain variable domains present in conventional four-chain antibodies (which are referred to herein as "VH domains" or "VH domains"). LVHH domains are chosen to distinguish them from VHH domains (referred to as "VH domains" or "VL domains"). VHH domains can specifically bind to epitopes without an additional antigen-binding domain (in contrast to the VH or VL domains in conventional four-chain antibodies, where the epitope is recognized by the VL domain together with the VH domain). VHH domains are small, robust, and efficient antigen recognition units formed by a single immunoglobulin domain.

[0087] In the context of the present invention, the terms VHH domain, VHH, V H H domain, VHH antibody fragment, VHH antibody, as well as "Nanobody®" and "Nanobody® domain" ("Nanobody" is a trademark of Ablynx NV, Ghent, Belgium) are used interchangeably and represent an ISVD (having the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and specifically binding to an epitope without requiring the presence of a second immunoglobulin variable domain), which can also be distinguished from a VH domain by the so-called "hallmark residues", as defined, for example, in WO2009 / 109635, Figure 1.

[0088] Methods for obtaining VHH domains that bind to specific antigens or epitopes have been previously described, for example, in WO2006 / 040153 and WO2006 / 122786. VHH domains derived from Camelidae can be "humanized" by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues that occur at the corresponding positions in a VH domain from a conventional four-chain antibody from humans. Humanized VHH domains can comprise one or more fully human framework region sequences; and in even more specific embodiments, they can comprise human framework region sequences derived from DP-29, DP-47, DP-51, or portions thereof, optionally in combination with a JH sequence, such as JH5.

[0089] IL-12 Interleukin-12 (IL-12) is a heterodimeric molecule composed of an α chain (IL-12p35 subunit) and a β chain (IL-12p40 subunit), covalently linked by disulfide bridges to form a biologically active 70 kDa dimer. It is produced by antigen-presenting cells, such as dendritic cells and macrophages, and is critical for the recruitment and effector function of CD8+ T cells and NK cells. IL-12 is therefore a major contributor to effective antitumor immune responses. IL-12 signals through the IL-12Rβ1 and IL-12Rβ2 receptors expressed on target cells, enabling downstream Jak2 and Tyk2 to promote STAT4 phosphorylation and homodimerization. Further studies have demonstrated that IL-12 is not only required for the activation of effector antitumor immune responses, but can also directly inhibit immunosuppression. Thus, the use of IL-12 as a cancer immunotherapy may be beneficial in controlling tumor growth by activating antitumor cytotoxic immune responses. Overall, IL-12 targets and regulates T cells, NK cells, and antigen-presenting cells (APCs), which regulate the fate of the antitumor immune response against cancer cells.

[0090] In certain embodiments, the IL-12 cytokine comprises the IL-12p35 amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the IL-12 cytokine comprises the IL-12p40 amino acid sequence set forth in SEQ ID NO: 2. In certain embodiments, the IL-12 cytokine comprises the IL-12p35 amino acid sequence set forth in SEQ ID NO: 1 and the IL-12p40 amino acid sequence set forth in SEQ ID NO: 2.

[0091] In another embodiment, as opposed to maintaining IL-12 as a native heterodimer, the IL-12 cytokine is comprised of a single chain IL-12 having the configuration (written from N-terminus to C-terminus): IL-12p40-IL-12p35 or IL-12p35-IL-12p40. In a specific embodiment, IL-12p40-IL-12p35 comprises the amino acid sequence set forth in SEQ ID NO: 3. In a specific embodiment, IL-12p35-IL-12p40 comprises the amino acid sequence set forth in SEQ ID NO: 4.

[0092] In another embodiment, the IL-12 cytokine may comprise subunits from different species, i.e., a chimeric IL-12 cytokine. In a related embodiment, the IL-12p35 subunit is derived from a mouse and the IL-12p40 subunit is derived from a human. In a specific embodiment, the IL-12 cytokine comprises the IL-12p35 amino acid sequence set forth in SEQ ID NO:5. In a specific embodiment, the IL-12 cytokine comprises the IL-12p35 amino acid sequence set forth in SEQ ID NO:5 and the IL-12p40 amino acid sequence set forth in SEQ ID NO:2.

[0093] Such chimeric molecules have been found to be valuable for generating in vitro and in vivo data in mice without the need for an alternative masking moiety. The masking moiety is specific for the human IL-12p40 subunit, thereby blocking the activity of the IL-12 cytokine. The IL-12p35 subunit derived from mouse nevertheless forms a functional IL-12 cytokine with the human IL-12p40 subunit and is active in mouse models. For use in humans, the IL-12p35 subunit from mouse is replaced with the IL-12p35 subunit from human, but the masking moiety and all other components of the IL-12 Fc fusion protein remain the same.

[0094] In certain embodiments, the chimeric IL-12p40-IL-12p35 comprises the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the chimeric IL-12p35-IL-12p40 comprises the amino acid sequence set forth in SEQ ID NO: 7.

[0095] In related embodiments, the subunits within the single-chain IL-12 cytokine may be linked to each other via a linker, for example, IL-12p40(linker)IL-12p35 or IL-12p35(linker)IL-12p40. The linker may be a peptide linker, particularly any of the peptide linkers disclosed herein, preferably a GS linker. Thus, in related embodiments, the subunits in a single-chain IL-12 cytokine comprising the amino acid sequence set forth in any one of SEQ ID NOs: 3, 4, 6, or 7 are linked to each other via a linker disclosed herein, preferably a GS linker. In related embodiments, the GS linker has the following amino acid sequence: GGGGSGGGSGGGGS (SEQ ID NO: 22). In a preferred embodiment, a single-chain IL-12 cytokine is provided in the construct IL-12p40-15GS-IL-12p35 (SEQ ID NO: 8). In another embodiment, the single chain IL-12 cytokine is provided in the construct IL-12p35-15GS-IL-12p40 (SEQ ID NO: 9).

[0096] In a related embodiment, the single chain IL-12 cytokine is provided in the configuration IL-12p40-15GS-IL-12p35 (SEQ ID NO: 10). In another embodiment, the single chain IL-12 cytokine is provided in the configuration IL-12p35-15GS-IL-12p40 (SEQ ID NO: 11).

[0097] In another embodiment, the IL-12p35 subunit of the IL-12 Fc fusion protein comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:1, and the IL-12p40 subunit of the IL-12 Fc fusion protein comprises a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:2, preferably, the IL-12p35 subunit comprises or consists of the polypeptide of SEQ ID NO:1, and the IL-12p40 subunit comprises or consists of the polypeptide of SEQ ID NO:2.

[0098] In another embodiment, the single-chain IL-12p40-IL-12p35 is linked to the C-terminus of the first Fc domain via its IL-12p40 subunit. In another embodiment, the single-chain IL-12p35-IL-12p40 is linked to the first Fc domain via its IL-12p35 subunit. In both cases, the single-chain IL-12p40-IL-12p35 or IL-12p35-IL-12p40 is linked to the C-terminus via a first peptide linker, which is protease-cleavable.

[0099] In another embodiment, the IL-12p40 subunit and the IL-12p35 subunit are linked to each other via a linker that is rich in the amino acid residues glycine and serine. In a related embodiment, the linker is 5-20 amino acids in length and contains only the amino acids glycine and serine. In a preferred embodiment, the linker has the amino acid sequence of SEQ ID NO: 22.

[0100] In a preferred embodiment, the IL-12 Fc fusion protein comprises a single chain IL-12p40-IL-12p35 polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 8. In another embodiment, the IL-12 Fc fusion protein comprises a single chain IL-12p35-IL-12p40 polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 9.

[0101] The IL-12 cytokine may include a variant of the IL-12p35 and / or IL-12p40 sequence. The variant encodes a protein that retains the functional activity of IL-12 compared to wild-type IL-12. The variant may encode an IL-12 subunit or any single-chain IL-12 disclosed herein. In one embodiment, the variant encodes an IL-12 subunit or any single-chain IL-12 set forth in any of SEQ ID NOS: 1-11 and further has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid mutations, deletions, substitutions, and / or additions compared to the amino acid sequence set forth in any of SEQ ID NOS: 1-11.

[0102] The functional activity of IL-12 can be measured in the assay shown in Example 5.

[0103] [Table 1] TIFF2026504906000002.tif249170 TIFF2026504906000003.tif212170

[0104] F C According to the present invention, the first and second polypeptide chains of the IL-12 Fc fusion protein are linked to each other via their respective Fc domains, i.e., both polypeptide chains dimerize via their Fc domains.

[0105] In the context of the present invention, an Fc domain is derived from the heavy chain of, for example, an IgG, such as IgG1, IgG2, or IgG4. For example, an Fc domain of the present invention is the Fc domain of the heavy chain of IgG1, which comprises a hinge region and two constant domains (C H2 and C H3 An example of an Fc domain (including the hinge region) is shown in SEQ ID NO: 14.

[0106] For all constant region (CL, CH1, hinge, CH2, and CH3) positions discussed in this invention, numbering is according to the EU numbering scheme (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda), unless otherwise specified, which refers to EU antibody numbering (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85). This means that the amino acid numbers indicated herein correspond to positions in the heavy chain of the corresponding subtype (e.g., IgG1 or IgG4) according to the EU numbering system, unless otherwise specified. For all variable region (VL and VH) and J segment (JH and JL) positions considered in this invention, numbering follows the Kabat numbering scheme (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda). Exceptions to these numbering schemes are noted where they occur. Those skilled in the art of antibodies will understand that these conventions consist of non-contiguous numbering in certain regions of an immunoglobulin sequence, allowing for standardized reference to conserved positions within an immunoglobulin family. Thus, the positions of any given immunoglobulin defined by EU numbering or Kabat numbering will not necessarily correspond to its contiguous sequence.

[0107] In some embodiments, the first Fc domain and the second Fc domain in the fusion proteins of the invention each contain one or more amino acid substitutions that reduce the formation of homodimers of the first or second polypeptide chains instead of heterodimers of the first and second polypeptide chains. Through these changes, a "protrusion" is created in one of the Fc domains by replacing one or more small amino acid side chains from the interface of one of the heavy chains with larger side chains (e.g., tyrosine or tryptophan). A compensatory "cavity" of identical or similar size is created on the interface of the other Fc domain by replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers, particularly Fc domain homodimers with "bumps" (see, e.g., Ridgway et al. Protein Eng, 1996. 9(7): p. 617-21; Atwell et al, JMB, 1997, 270, 26-35).

[0108] In some embodiments, such amino acid mutations are tyrosine (Y) at position 366 of the first Fc domain [T366Y] and threonine (T) at position 407 of the second Fc domain [Y407T]. In some embodiments, the first Fc domain comprises serine (S) at position 366 [T366S], and the second Fc domain comprises tryptophan (W) at position 366 [T366W], alanine (A) at position 368 [L368A], and valine (V) at position 407 [Y407V]. In a preferred embodiment, the first Fc domain comprises tryptophan (W) at position 366 [T366W], and the second Fc domain comprises serine (S) at position 366 [T366S], alanine (A) at position 368 [L368A], and valine (V) at position 407 [Y407V]. For example, position 366 of the Fc domain according to EU numbering corresponds to amino acid position 151 in the human IgG1 Fc sequence of SEQ ID NO: 14 and is changed from T at position 151 in SEQ ID NO: 14 to W at position 151 in SEQ ID NO: 15; and positions 366, 368, and 407 according to EU numbering correspond to amino acids 151, 153, and 192, respectively, in SEQ ID NO: 14 and are changed from T, L, and Y at these positions in SEQ ID NO: 14 to S, A, and V at these positions in SEQ ID NO: 16. In any of these embodiments, the amino acid changes described for the first Fc domain may also be located in the second Fc domain, and the respective amino acid changes for the second Fc domain may also be located in the first Fc domain. In other words, the terms "first" and "second" can be interchanged in these embodiments. In some embodiments, such an Fc domain is an Fc domain derived from the heavy chain of IgG1.

[0109] In some embodiments, the first Fc domain comprises a cysteine ​​(C) at position 354 [S354C] in addition to a tryptophan (W) at position 366 [T366W], and the second Fc domain comprises a cysteine ​​(C) at position 349 [Y349C] in addition to a serine (S) at position 66 [T366S], an alanine (A) at position 368 [L368A], and a valine (V) at position 407 [Y407V]. In one aspect, such an Fc domain is an Fc domain derived from the heavy chain of an IgG1.

[0110] The first and / or second Fc domains of the present invention derived from IgG1 also comprise "KO" mutations (L234A, L235A).

[0111] In some embodiments, the first Fc domain or the second Fc domain in the Fc fusion proteins of the invention further comprises one or more amino acid mutations that reduce binding of the Fc domain to Protein A. In some embodiments, such amino acid mutations are an arginine at position 435 [H435R] and a phenylalanine at position 436 [Y436F] in one of the Fc domains. Both changes are derived from the sequence of human IgG3 (IgG3 does not bind to Protein A). These two mutations are located in the CH3 domain and are incorporated into one of the Fc domains to reduce binding to Protein A (see, e.g., Jendeberg et al. J Immunol Methods, 1997. 201(1): pp. 25-34). These two changes facilitate removal of heavy chain homodimers containing these changes during protein purification.

[0112] In some embodiments, in a fusion protein of the invention, the Fc domain comprises a threonine (T) at position 407 [Y407T], and further comprises an arginine at position 435 [H435R] and a phenylalanine at position 436 [Y436F]. In this case, the other heavy chain comprises a tyrosine (Y) at position 366 [T366Y], but does not comprise the two changes at positions 435 and 436. Alternatively, in some embodiments, in a fusion protein of the invention, the Fc domain comprises a serine (S) at position 366 [T366S], an alanine (A) at position 368 [L368A], and a valine (V) at position 407 [Y407V], and further comprises an arginine at position 435 [H435R] and a phenylalanine at position 436 [Y436F]. In this case, the other Fc domain contains a tryptophan (W) at position 366 [T366W], but does not contain the two changes at positions 435 and 436. Thus, the Fc domain containing the amino acid changes that result in the "cavity" described above also contains amino acid changes that reduce binding to Protein A. Homodimers containing this Fc domain are eliminated through reduced binding to Protein A. The production of homodimers of other Fc domains that contain the "protuberance" is reduced by the presence of the "protuberance."

[0113] In a preferred embodiment, the first Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14. In a preferred embodiment, the first Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15. In a preferred embodiment, the first Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16. In a preferred embodiment, the first Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17. In another preferred embodiment, the second Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 18. In a related preferred embodiment, the IL-12 Fc fusion protein comprises a first Fc domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17 and a second Fc domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18.

[0114] In any of the foregoing embodiments, the serine at position 5 in the amino acid sequences of SEQ ID NOs: 14-18 may be substituted with a cysteine.

[0115] In some embodiments, the Fc domain of the fusion proteins of the invention may or may not further comprise YTE mutations (M252Y / S254T / T256E, EU numbering (Dall'Acqua, Kiener et al. 2006)), which have been shown to improve the pharmacokinetic properties of the Fc domain through preferentially increasing binding affinity for the neonatal FcRn receptor at pH 6.0.

[0116] [Table 2]

[0117] Protease-cleavable linker

[0118] A protease-cleavable linker connects the Fc domain to the IL-12 cytokine, i.e., the protease-cleavable linker is positioned between the C-terminus of the Fc domain and the IL-12 cytokine. Once the protease-cleavable linker is cleaved by its respective protease, the IL-12 cytokine is released and is no longer attached to the Fc fusion protein.

[0119] On the Fc domain side, the protease-cleavable linker may be linked directly to the C-terminus of the Fc domain (i.e., without a linker), or it may be linked to the C-terminus of the Fc domain via a linker, such as any of the linkers described in the linker section below, preferably a peptide linker having a length of about 4 to 20 amino acids, for example. In a preferred embodiment, the protease-cleavable linker is linked to the C-terminus of the Fc domain via a linker having the sequence GGGGSGGGG (SEQ ID NO: 24).

[0120] On the IL-12 cytokine side, the protease-cleavable linker may be linked directly to the IL-12 cytokine, or it may be linked via a linker, such as any of the linkers described in the linker section below, preferably a peptide linker having a length of, for example, about 4 to 20 amino acids. Depending on the configuration of the IL-12 cytokine, the protease-cleavable linker is linked to the IL-12p40 subunit or to the IL-12p35 subunit. In a preferred embodiment, the IL-12 cytokine is provided in a single-chain configuration, and the protease-cleavable linker is linked to (a) the IL-12p40 subunit of the single-chain IL-12p40-IL-12p35, or (b) the IL-12p35 subunit of the single-chain IL-12p35-IL-12p40. In a related embodiment, the protease-cleavable linker is linked to the IL-12p35 or IL-12p40 subunit via a linker having the sequence GGGGS (SEQ ID NO: 27).

[0121] Protease-cleavable linkers typically have a short amino acid (aa) sequence of 2 aa to 20 aa, 4 aa to 15 aa, 4 aa to 12 aa, or 2 aa to 10 aa. In certain embodiments, protease-cleavable linkers can have a length of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 aa.

[0122] Preferably, the desired protease is enriched, selectively expressed, and / or more active at the desired site of cytokine activity (e.g., the TME), and thus the IL-12 Fc fusion protein is preferentially or selectively cleaved at the site of desired cytokine activity.

[0123] Proteases known to be associated with diseased cells or tissues include, but are not limited to, serine proteases, cysteine ​​proteases, aspartic acid proteases, threonine proteases, glutamic acid proteases, metalloproteases, asparagine peptide lyases, serum proteases, cathepsin, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, kallikrein, hKl, hK10, hK15, plasmin, collagenase, type IV collagenase, stromelysin, factor Xa, chymotrypsin-like proteases, trypsin-like proteases, elastase-like proteases, subtilisin-like proteases, actinidain, bromelain, calpain, caspases, These include caspase-3, Mirl-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidases, metalloendopeptidases, matrix metalloproteinases (MMPs), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, urokinase plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin-1b converting enzyme, thrombin, FAP (FAP-a), dipeptidyl peptidase, meprin, granzyme, and dipeptidyl peptidase IV (DPPIV / CD26).

[0124] The protease capable of cleaving the amino acid sequence encoded by the protease-cleavable linker sequence provided herein can be selected from the group consisting of, for example, prostate-specific antigen (PSA), matrix metalloproteinase (MMP), A distigrin and metalloproteinase (ADAM), plasminogen activator, cathepsin, caspase, tumor cell surface protease, and elastase. MMPs include, for example, matrix metalloproteinase 2 (MMP2) or matrix metalloproteinase 9 (MMP9). Preferably, the protease-cleavable linker is cleaved by MMP2, MMP9, or MMP13.

[0125] [Table 3]

[0126] In a preferred embodiment, the protease-cleavable linker sequence is GPLGVRG (SEQ ID NO: 232).

[0127] Cleavage of protease-cleavable linkers can be easily determined as shown in Examples 7-8.

[0128] Masking part As used herein, a masking moiety refers to a moiety that binds to the IL-12p35 and / or IL-12p40 subunits of the IL-12 cytokine. In one embodiment, binding of the masking moiety to the IL-12p35 and / or IL-12p40 subunits reduces the affinity of the IL-12 cytokine for its cognate receptor. In another embodiment, binding of the masking moiety to the IL-12p35 and / or IL-12p40 subunits blocks, inhibits, or attenuates the functional activity of the IL-12 cytokine.

[0129] The binding of the masking moiety to the IL-12p35 and / or IL-12p40 subunits of the IL-12 cytokine can be readily measured by methods well known in the art, see, e.g., Example 2. The strength or affinity of specific binding can be determined by the dissociation constant (K D ) and smaller K D represents a greater affinity, and a larger K Drepresents a lower affinity. Binding properties can be determined by methods such as biolayer interferometry and surface plasmon resonance-based methods, including Biacore and Octet methodologies. One such method involves measuring the association and dissociation rates of antigen-binding site / antigen or receptor / ligand complexes, where the rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rate equally in both directions. Thus, the association rate (k a ) and dissociation rate (k d ) can be determined, and k a / k d The ratio of the dissociation constant K D is equal to.

[0130] Specific binding to the IL-12p35 and / or IL-12p40 subunits may be, for example, at least about 10 -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, or at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12 M or higher K D This can be indicated by a masking portion having

[0131] In one embodiment, the masking moiety may be an IL-12 receptor or an IL-12p35 or IL-12p40 binding fragment thereof. In one embodiment, the masking moiety may be an IL-12p40 binding fragment of the IL-12 receptor. In one embodiment, the masking moiety may be an IL-12p35 binding fragment of the IL-12 receptor.

[0132] The IL-12 receptor is a type I cytokine receptor that binds to IL-12. It consists of a β1 subunit and a β2 subunit. IL-12 receptor, beta 1, or IL-12Rβ1 for short, is a subunit of the interleukin-12 receptor. IL-12RB1 is its human gene name. IL-12Rβ1 is also known as CD212 (cluster of differentiation 212). Human IL-12Rβ1 has the amino acid sequence shown in SEQ ID NO: 12. IL-12 receptor, beta 2 subunit, is a subunit of the interleukin-12 receptor. IL12RB2 is its human gene. Human IL-12Rβ2 has the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the masking moiety comprises an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to human IL-12Rβ1, having the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the masking moiety comprises an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to human IL-12Rβ2, having the amino acid sequence set forth in SEQ ID NO: 13.

[0133] In some embodiments, the masking moiety comprises the extracellular domain of IL-12Rβ1 or IL-12Rβ2, or a fragment, portion, or variant thereof that retains affinity for IL-12. The extracellular domains are shown underlined in SEQ ID NOs: 12 and 13 in Table 1. In some embodiments, the masking moiety comprises an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the extracellular domain of human IL-12Rβ1, having the underlined amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the masking moiety comprises an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the extracellular domain of human IL-12Rβ2, having the amino acid sequence shown in SEQ ID NO: 13.

[0134] The masking moiety may also be an scFv or an immunoglobulin single variable domain. Preferably, the masking moiety is a VHH, more preferably a humanized VHH.

[0135] In some embodiments, the scFv comprises the same light chain CDRs and heavy chain CDRs or the same light chain variable region (VL) and heavy chain variable region (VH) as the IL-12 antibody briakinumab or ustekinumab.

[0136] The CDRs disclosed herein and depicted in SEQ ID NOS: 61-109 are presented according to Kabat nomenclature. The underlined sequences correspond to CDR-1, CDR-2, and CDR-3, respectively, according to Kabat nomenclature. The CDRs are again individually identified in SEQ ID NOS: 333-479 in the Kabat nomenclature.

[0137] Because additional nomenclature systems are known in the art, CDR sequences based on the most commonly used of these nomenclatures are also shown, but only for instances where application of these alternative nomenclatures resulted in different amino acid sequences. These numbering systems are based on (i) CCG (Chemical Computing Group, as exemplified in Almagro et al., Proteins 2011; 79:3050-3066 and Maier et al., Proteins 2014; 82:1599-1610), (ii) Chothia (Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-917), (iii) IMGT (Lefranc MP, Dev Comp Immunol. 2003 Jan;27(1):55-77), and (iv) North (North B, J Mol Biol.(2011)406:228-56).

[0138] The amino acid residues of the VHH domains are numbered according to the general numbering system for VH domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91), and applies to VHH domains from the Camelidae family, e.g., as shown in Figure 2 of Riechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999).

[0139] According to this numbering, - FR1 comprises amino acid residues at positions 1 to 30, - CDR1 comprises the amino acid residues at positions 31 to 35, FR2 comprises the amino acids at positions 36 to 49, - CDR2 comprises amino acid residues at positions 50 to 65, FR3 comprises amino acid residues at positions 66 to 94, - CDR3 comprises amino acid residues at positions 95 to 102, - FR4 comprises the amino acid residues at positions 103 to 113.

[0140] The total number of amino acid residues in a VHH domain is usually in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0141] However, as is well known in the art for VH domains and for VHH domains, it should be noted that the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e., one or more positions according to Kabat numbering may not be occupied in the actual sequence, and the actual sequence may contain more amino acid residues than allowed by Kabat numbering). This generally means that the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence.

[0142] Alternative methods for numbering the amino acid residues of VH domains, which can also be applied in an analogous manner to VHH domains, are known in the art, however in the present description, claims and figures the numbering according to Kabat and applied to VHH domains as described above will be followed unless otherwise indicated.

[0143] In some embodiments, the masking moiety comprises or consists of an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 61-109.

[0144] In some embodiments, the masking moiety is an IL-12 binding immunoglobulin single variable domain comprising the three CDRs contained within any one of the sequences of SEQ ID NOs: 61-109.

[0145] In some embodiments, the masking moiety is an IL-12 binding VHH comprising three CDRs contained within any one of the sequences of SEQ ID NOs: 61-109.

[0146] In some embodiments, the masking moiety is a VHH and comprises three CDRs contained within any one of the sequences of SEQ ID NOs: 61-109, and further comprises framework regions (FR1, FR2, FR3, FR4) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the framework regions set forth in any one of the sequences of SEQ ID NOs: 61-109.

[0147] In some embodiments, the masking moiety is a VHH and comprises three CDRs contained within any one of the sequences of SEQ ID NOs: 61-109, and further comprises framework regions (FR1, FR2, FR3, FR4) having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to 20 amino acid differences, such as substitutions, deletions, or additions in the framework regions set forth in any one of the sequences of SEQ ID NOs: 61-109.

[0148] In some embodiments, the masking moiety is a VHH, wherein the VHH further comprises an additional alanine at its C-terminus. In some embodiments, the masking moiety is a VHH having an amino acid sequence set forth in any one of SEQ ID NOs: 61-109, and further comprising an additional alanine added at its C-terminus.

[0149] It will be understood that any of the VHH sequences described above (and listed below) and the CDRs contained therein may find utility beyond their use as masking moieties in IL-12 Fc fusion proteins. Thus, the present disclosure is not to be understood as limiting the VHH sequences to their use as masking moieties, but provides the VHH sequences per se, i.e., for any and all purposes.

[0150] [Table 4] TIFF2026504906000007.tif249170 TIFF2026504906000008.tif250170 TIFF2026504906000009.tif249170 TIFF2026504906000010.tif249170 TIFF2026504906000011.tif249170 TIFF2026504906000012.tif249170 TIFF2026504906000013.tif248170 TIFF2026504906000014.tif250170 TIFF2026504906000015.tif249170 TIFF2026504906000016.tif250170 TIFF2026504906000017.tif249170 TIFF2026504906000018.tif246170 TIFF2026504906000019.tif249170 TIFF2026504906000020.tif249170 TIFF2026504906000021.tif249170 TIFF2026504906000022.tif248170 TIFF2026504906000023.tif245170 TIFF2026504906000024.tif249170 TIFF2026504906000025.tif169170

[0151] Linker Methods for linking molecules are well known in the art. The linker can be a peptide linker or a non-peptide linker. When the linker is a peptide linker, it can be composed of one or more amino acids. For peptide linkers, a small linker sequence of glycine and serine amino acids (called a GS mini-linker) is typically used. The number of amino acids in the linker can vary from 4 (GGGS) (SEQ ID NO: 19), 6 (GGSGGS) (SEQ ID NO: 20), 10 (GGGGSGGGGS) (SEQ ID NO: 21), 15 (GGGGSGGGGSGGGGS) (SEQ ID NO: 22), 20 (GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 23) or more.

[0152] In some embodiments, the linker is between 5 and 20 amino acids in length. In other embodiments, the linker is rich in the amino acid residues G and S. In another embodiment, the linker is between 5 and 20 amino acids in length and is rich in the amino acid residues G and S. In another embodiment, the linker comprises only the amino acid residues G and S. In another embodiment, the linker is between 2 and 20 amino acids in length and comprises only the amino acid residues G and S.

[0153] A peptide linker, as contemplated herein, is a (poly)peptide linker of at least one amino acid in length. Preferably, the linker is 1 to 100 amino acids in length. More preferably, the linker is 5 to 50 amino acids in length, even more preferably 10 to 40 amino acids in length, and even more preferably 15 to 30 amino acids in length. A non-limiting example of a small linker that is often used includes a sequence of glycine and serine amino acids, called the GS mini-linker. Examples of preferred linker sequences are Gly / Ser linkers of different lengths, e.g., (gly x ser y ) zLinkers such as (gly4ser)3, (gly4ser)4, (gly4ser), (gly3ser), gly3, and (gly3ser2)3 can be used. The number of amino acids in these linkers can vary, for example, they can be four (e.g., GGGS) (SEQ ID NO: 19), six (e.g., GGSGGS) (SEQ ID NO: 20), seven (e.g., GGGGSGGS), or multiples thereof, such as two, three, or more repeats of these four / six amino acids. Most preferably, such a GS minilinker has 20 amino acids and the sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 23). Further examples of such linkers include GGGGSGGGG (SEQ ID NO: 24), GSGG (SEQ ID NO: 25), or GGGGSGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 26).

[0154] Further examples of linkers include: [ka]

[0155] The linker can also be a variant described in Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448. Other linkers that can be used for the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. Immunother. 50:51-59.

[0156] In a preferred embodiment, the non-cleavable linker is selected from any of the aforementioned GS linkers. In a further preferred embodiment, the non-cleavable linker is between 2 and 20 amino acids in length and contains only the amino acid residues G and S.

[0157] joining part The binding moiety serves to promote accumulation or retention of the IL-12 Fc fusion protein and / or cleavage products within the TME, preferably the ECM, and more preferably in the vicinity of the tumor. The binding moiety can be located on either the first or second polypeptide chain of the IL-12 Fc fusion protein. Thus, in a preferred embodiment, the binding moiety is an extracellular matrix binding moiety.

[0158] In some instances, it may be desirable to place the binding moiety on the first polypeptide chain. When the binding moiety is located on the first polypeptide chain, this may additionally promote accumulation and / or retention of the cleavage product in the TME. In instances where the binding moiety is located on the first polypeptide chain, the binding moiety may be linked to the C-terminus of the IL-12p35 subunit or the C-terminus of the IL-12p40 subunit. In both cases, the binding moiety may be linked to the C-terminus directly or, optionally, via a polypeptide linker, such as any of the polypeptide linkers disclosed herein. The binding moiety may also be located between the IL-12p35 subunit and the IL-12p40 subunit. In this case, the binding moiety may optionally be flanked on one or both sides by a polypeptide linker or linkers. This can result in different configurations, such as IL-12p35(binding moiety)IL-12p40 or IL-12p40(binding moiety)IL-12p35, where the binding moiety is directly linked at its N- and C-terminus to the respective IL-12 subunit. Another configuration may include one or more linkers, such as IL-12p35(linker)(binding moiety)IL-12p40, IL-12p35(linker)(binding moiety)(linker)IL-12p40, IL-12p35(binding moiety)(linker)IL-12p40, IL-12p35(linker)(binding moiety)(linker)IL-12p40, IL-12p40(linker)(binding moiety)IL-12p35, IL-12p40(linker)(binding moiety)(linker)IL-12p35, IL-12p40(linker)(binding moiety)(linker)IL-12p35, IL-12p40(linker)(binding moiety)(linker)IL-12p35, etc. In any of these configurations, the linker may be a polypeptide linker, such as any of the polypeptide linkers disclosed herein. In these configurations, the binding moiety is expressed in a single chain together with the IL-12 subunit and remains with the IL-12 cytokine after cleavage in the TME.

[0159] In some other instances, it may be desirable to place the binding moiety on a second polypeptide chain. In this way, the properties of the binding moiety can be readily applied to the IL-12 Fc fusion protein, but after cleavage, the cleavage product is not additionally accumulated and / or retained in the TME. In some instances, the binding moiety may be linked to the C-terminus of the masking moiety. The binding moiety may be linked to the C-terminus directly or, optionally, via a polypeptide linker, such as any of the polypeptide linkers disclosed herein.

[0160] In some instances, the binding moiety may be located on the N-terminus of the first or second Fc domain. The binding moiety may be linked to the N-terminus directly or, optionally, via a polypeptide linker, such as any of the polypeptide linkers disclosed herein.

[0161] The binding moiety is selected from the list consisting of a collagen-binding moiety, a heparin-binding moiety, and a fibronectin-binding moiety. The binding moieties disclosed herein have binding specificity for collagen, heparin, or fibronectin. In the context of the present invention, the binding moiety is derived from a polypeptide or a portion thereof that binds to collagen, heparin, or fibronectin, respectively.

[0162] In a preferred embodiment, the binding moiety is a collagen binding moiety, more preferably a collagen I binding moiety. In a further preferred embodiment, the collagen I binding moiety is positioned at the C-terminus of the IL-12p35 subunit and / or IL-12p40, e.g., IL-12p35-IL-12p40(binding moiety), IL-12p35(linker)IL-12p40(binding moiety), IL-12p35(linker)IL-12p40(linker)(binding moiety), IL-12p35-IL-12p40(linker)(binding moiety), IL-12p40-IL-12p35(binding moiety), IL-12p40(linker)IL-12p35(binding moiety), IL-12p40(linker)IL-12p35(linker)(binding moiety), or IL-12p40-IL-12p35(linker)(binding moiety).

[0163] Collagen binding site Collagen is a major component of the tumor microenvironment and is involved in cancer fibrosis. Collagen biosynthesis can be regulated by cancer cells through mutated genes, transcription factors, signaling pathways, and receptors; furthermore, collagen can influence tumor cell behavior through integrins, discoidin domain receptors, tyrosine kinase receptors, and several signaling pathways. Cancer-associated fibroblasts produce high levels of extracellular matrix proteins (ECM) in the TME, leading to the overexpression of various collagens in many tumor types. The role of collagen in cancer has been extensively reviewed, including the relationship between collagen and proteases, such as MMPs, which work together to regulate the TME (Xu, S., Xu, H., Wang, W. et al. The role of collagen in cancer: from bench to bedside. J Transl Med 17, 309 (2019)).

[0164] The collagen superfamily contains 28 members (I-XXVIII) numbered using Roman numerals in vertebrates. A common structural feature of collagens is the presence of a triple helical structure, which can range from the majority of their structures (96% for collagen I) to less than 10% (collagen XII). The diversity of the collagen family is further increased by the presence of several α-chains, several molecular isoforms and supramolecular structures for the single collagen types, as well as the use of alternative promoters and alternative splicing.

[0165] Among different collagens, type I collagen is the most abundant protein in mammals. The basic structural unit of type I collagen is a long (300 nM), thin (1.5 nM diameter) protein consisting of three coiled subunits: two alpha 1 (I) chains and one alpha 2 (I) chain. Each chain contains 1,050 amino acids that are wound around each other in a characteristic right-handed triple helix. In humans, type I collagen is encoded by the COL1A1 and COL1A2 genes. The COL1A1 gene encodes the pro-alpha 1 chain of type I collagen. The COL1A2 gene encodes the pro-alpha 2 chain of type I collagen, whose triple helix contains two alpha 1 chains and one alpha 2 chain. Type I is the fibril-forming collagen found in most connective tissues and is abundant in bone, cornea, dermis, and tendon.

[0166] An exemplary amino acid sequence for the human alpha 1 chain precursor of type I collagen is shown in SEQ ID NO: 38 (NCBI Reference Sequence: NP 000079.2). An exemplary amino acid sequence for the human alpha 2 chain precursor of type I collagen is shown in SEQ ID NO: 39 (NCBI Reference Sequence: NP 000000.2).

[0167] In some embodiments, the collagen-binding moiety comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) leucine-rich repeats that bind to collagen. In some embodiments, the collagen-binding moiety comprises a proteoglycan. In some embodiments, the collagen-binding moiety comprises a proteoglycan, wherein the proteoglycan is selected from the group consisting of decorin, biglycan, testocan, bikunin, fibromodulin, lumican, chondroherin, keratin, ECM2, epiphycan, asporin, PRELP, keratocan, osteoadherin, opticin, osteoglycan, nyctalopin, horsetail, podocan, podocan-like protein 1, versican, perlecan, nidogen, neurocan, aggrecan, and brevican.

[0168] In some embodiments, the collagen binding moiety comprises a class I small leucine-rich proteoglycan (SLRP). In some embodiments, the collagen binding domain comprises a class II SLRP. In some embodiments, the collagen binding domain comprises a class III SLRP. In some embodiments, the collagen binding domain comprises a class IV SLRP. In some embodiments, the collagen binding domain comprises a class V SLRP. In some embodiments, the collagen binding domain comprises one or more leucine-rich repeats from a human proteoglycan class II member of the small leucine-rich proteoglycan (SLRP) family. In some embodiments, the SLRP is selected from lumican, decorin, biglycan, fibromodulin, keratin, epiphycan, asporin, and osteoglycin. In some embodiments, the SLRP is lumican.

[0169] It has also been hypothesized that collagen-rich tumor tissues have higher activity of MMPs, such as collagenases MMP2 and MMP9, which may further contribute to the faster cleavage of the IL-12 Fc fusion protein.

[0170] In some embodiments, the IL-12 Fc fusion protein comprises a collagen binding moiety that specifically binds to collagen. In some embodiments, the collagen binding moiety specifically binds to human type I collagen and / or human type IV collagen. In some embodiments, the collagen binding moiety specifically binds to human type I collagen. In some embodiments, the collagen binding moiety specifically binds to human type IV collagen. In some embodiments, the collagen binding moiety specifically binds to human type I collagen and human type IV collagen. In some embodiments, the collagen binding moiety specifically binds to human type I collagen or human type IV collagen.

[0171] In a further embodiment, the present disclosure provides an IL-12 Fc fusion protein, in which the collagen binding moiety binds to collagen IV and has the amino acid sequence KLWVLPK (SEQ ID NO: 40).

[0172] Binding of a collagen-binding moiety to collagen can be determined by methods known in the art. In some embodiments, the collagen-binding moiety is determined by its ability to compete with a known or reference collagen-binding protein for binding to collagen. In some embodiments, the collagen-binding moiety is derived from a naturally occurring collagen-binding protein or collagen receptor.

[0173] In some embodiments, the IL-12 Fc fusion protein specifically binds to collagen with an affinity (KD) of less than about 500 μM as determined by a collagen binding assay. In some embodiments, the IL-12 Fc fusion protein comprises a collagen binding portion that specifically binds to collagen with an affinity (KD) of less than about 100 μM as determined by a collagen binding assay. In some embodiments, the IL-12 Fc fusion protein comprises a collagen binding portion that specifically binds to collagen with an affinity (KD) of less than about 1 μM as determined by a collagen binding assay. In some embodiments, the IL-12 Fc fusion protein comprises a collagen binding portion that specifically binds to collagen with an affinity (KD) of less than about 500 nM as determined by a collagen binding assay. In some embodiments, the collagen binding portion specifically binds to collagen with an affinity (KD) of about 0.1-500 μM, 0.1-100 μM, or 0.1-1 μM as determined by a collagen binding assay. In some embodiments, the collagen-binding moiety specifically binds to collagen with an affinity (KD) of about 100-1000 nM, 100-1000 nM, 100-800 nM, 100-600 nM, or 100-500 nM as determined by a collagen-binding assay.

[0174] In some embodiments, the collagen binding assay determines the binding affinity of the collagen binding moiety for collagen. In some embodiments, the collagen binding assay determines the binding affinity of the collagen binding moiety for type I collagen. In some embodiments, the collagen binding assay determines the binding affinity for type IV collagen.

[0175] In some embodiments, the collagen binding assay is an ELISA. Methods and techniques for performing collagen-binding ELISAs are known in the art (see, e.g., Smith et al., (2000) J Biol Chem 275:4205-4209). Thus, in some embodiments, an IL-12 Fc fusion protein comprises a collagen-binding portion that specifically binds to collagen with an affinity (KD) of less than about 500 μM as determined by ELISA. In some embodiments, an IL-12 Fc fusion protein comprises a collagen-binding portion that specifically binds to collagen with an affinity (KD) of less than about 100 μM as determined by ELISA. In some embodiments, an IL-12 Fc fusion protein comprises a collagen-binding portion that specifically binds to collagen with an affinity (KD) of less than about 1 μM as determined by ELISA. In some embodiments, an IL-12 Fc fusion protein comprises a collagen-binding portion that specifically binds to collagen with an affinity (KD) of less than about 500 nM as determined by ELISA. In some embodiments, the collagen-binding moiety specifically binds to collagen with an affinity (KD) of about 0.1-500 μM, 0.1-100 μM, or 0.1-1 μM as determined by ELISA. In some embodiments, the collagen-binding moiety specifically binds to collagen with an affinity (KD) of about 100-1000 nM, 100-1000 nM, 100-800 nM, 100-600 nM, or 100-500 nM as determined by ELISA.

[0176] In some embodiments, the collagen binding assay is a surface plasmon resonance (SPR) assay. Methods and techniques for performing collagen binding SPR assays are known in the art (see, e.g., Saenko et al., (2002) Anal Biochem 302(2):252-262). Thus, in some embodiments, an IL-12 Fc fusion protein comprises a collagen-binding moiety that specifically binds to collagen with an affinity (KD) of less than about 500 μM as determined by an SPR assay. In some embodiments, an IL-12 Fc fusion protein comprises a collagen-binding moiety that specifically binds to collagen with an affinity (KD) of less than about 100 μM as determined by an SPR assay. In some embodiments, an IL-12 Fc fusion protein comprises a collagen-binding moiety that specifically binds to collagen with an affinity (KD) of less than about 1 μM as determined by an SPR assay. In some embodiments, the IL-12 Fc fusion protein comprises a collagen-binding moiety that specifically binds to collagen with an affinity (KD) of less than about 500 nM as determined by an SPR assay. In some embodiments, the collagen-binding moiety specifically binds to collagen with an affinity (KD) of about 0.1-500 μM, 0.1-100 μM, or 0.1-1 μM as determined by an SPR assay. In some embodiments, the collagen-binding moiety specifically binds to collagen with an affinity (KD) of about 100-1000 nM, 100-1000 nM, 100-800 nM, 100-600 nM, or 100-500 nM as determined by an SPR assay.

[0177] The term "surface plasmon resonance" refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, New Jersey). For further explanation, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51: 19-26; Jonsson, U., et al. (1991) Biotechniques 11:620-627; Johnson, B., et al. (1995) J. Mol. Recognit. 8: 125-131; and Johnson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0178] In some embodiments, an IL-12 Fc fusion protein comprises a collagen-binding moiety that specifically binds to collagen but not to one or more non-collagenous extracellular matrix (ECM) components, including, but not limited to, fibronectin, heparin, vitronectin, tenascin-C, osteopontin, and fibrinogen. In some embodiments, the collagen-binding moiety binds to collagen with a lower KD than to one or more non-collagenous ECM components. In some embodiments, the KD of the collagen-binding moiety for type I collagen is less than the KD of the collagen-binding moiety for an extracellular matrix component selected from fibronectin, heparin, vitronectin, osteopontin, tenascin-C, or fibrinogen. In some embodiments, the KD of the collagen-binding moiety for type I collagen is less than the KD of the collagen-binding moiety for any other type of collagen. In some embodiments, the collagen-binding moiety binds to collagen with a KD that is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% lower than that of one or more non-collagenous ECM components, hi some embodiments, the collagen-binding moiety binds to collagen with a KD that is about 2-fold, about 3-fold, about 4-fold, about 5-fold, or about 10-fold lower than that of one or more non-collagenous ECM components.

[0179] In some embodiments, the collagen-binding moiety binds to type I collagen with a lower KD than to type IV collagen. In some embodiments, the collagen-binding moiety competes with a reference collagen-binding moiety for binding to collagen. In some embodiments, the collagen-binding moiety competes with a reference collagen-binding moiety for binding to type I collagen.

[0180] In some embodiments, the reference collagen-binding moiety comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) leucine-rich repeats that bind to collagen. In some embodiments, the reference collagen-binding domain comprises a proteoglycan. In some embodiments, the reference collagen-binding moiety comprises a proteoglycan, wherein the proteoglycan is selected from the group consisting of decorin, biglycan, fibromodulin, lumican, chondroadherin, asporin, PRELP, osteoadherin / osteomodulin, optisin, osteoglycin / mimecan, podocan, perlecan, and nidogen. In some embodiments, the reference collagen-binding moiety is lumican. In some embodiments, the reference collagen-binding moiety comprises a class I small leucine-rich proteoglycan (SLRP). SLRPs are known to bind to collagen (Chen and Birk (2013) FEBS Journal 2120-2137). In some embodiments, the reference collagen-binding moiety comprises a class II SLRP. In some embodiments, the reference collagen binding moiety comprises a class III SLRP. In some embodiments, the reference collagen binding moiety comprises a class IV SLRP. In some embodiments, the reference collagen binding moiety comprises a class V SLRP.

[0181] In some embodiments, the reference collagen-binding moiety comprises a leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1) protein.

[0182] In some embodiments, the reference collagen binding moiety comprises a leukocyte-associated immunoglobulin-like receptor 2 (LAIR-2) protein. In some embodiments, the reference collagen binding moiety comprises glycoprotein IV.

[0183] In its broadest form, the disclosure provides an IL-12 Fc fusion protein, in which the collagen binding moiety binds to collagen. Preferably, the collagen binding moiety specifically binds to type I collagen.

[0184] In a further preferred embodiment, the present disclosure provides an IL-12 Fc fusion protein, wherein the collagen binding moiety binds to collagen I and has the sequence LxxLxLxxN (SEQ ID NO: 41), where L is leucine, N is asparagine, and x is any amino acid.

[0185] In further embodiments, the present disclosure provides IL-12 Fc fusion proteins in which the collagen binding moiety comprises or consists of any of the following sequences: LSELRLHEN (SEQ ID NO: 42), LTELHLDNN (SEQ ID NO: 43), LSELRLHNN (SEQ ID NO: 44), LSELRLHAN (SEQ ID NO: 45), LRELHLNNN (SEQ ID NO: 46), or LRELHLDNN (SEQ ID NO: 47). In a related most preferred embodiment, the collagen binding moiety comprises or consists of the sequence LRELHLDNN (SEQ ID NO: 47).

[0186] In another embodiment, the disclosure provides an IL-12 Fc fusion protein, wherein the collagen binding portion has a length of 20 amino acids (aa), 19 aa, 18 aa, 17 aa, 16 aa, 15 aa, 14 aa, 13 aa, 12 aa, 11 aa, 10 a, or 9 aa, and comprises the sequence LxxLxLxxN (SEQ ID NO: 41), where L is leucine, N is asparagine, and x is any amino acid.

[0187] Fibronectin binding moiety Fibronectin is a high molecular weight (approximately 500–600 kDa) glycoprotein of the extracellular matrix that binds to transmembrane receptor proteins called integrins. Fibronectin also binds to other extracellular matrix proteins, such as collagen, fibrin, and heparan sulfate proteoglycans (e.g., syndecans). Fibronectin exists as a protein dimer consisting of two nearly identical monomers linked by a pair of disulfide bonds. Fibronectin protein is produced from a single gene; however, alternative splicing of its pre-mRNA leads to the production of several isoforms. Two types of fibronectin exist in vertebrates: soluble plasma fibronectin (formerly called "cold-insoluble globulin," or CIg), which is the major protein component of plasma (300 μg / ml) and is produced in the liver by hepatocytes; insoluble cellular fibronectin is a major component of the extracellular matrix. It is secreted by various cells, mainly fibroblasts, as a soluble protein dimer, and then assembled into an insoluble matrix in a complex cell-mediated process. Fibronectin plays a key role in cell adhesion, growth, migration, and differentiation, and is important for processes such as wound healing and embryonic development. Altered fibronectin expression, degradation, and organization have been associated with many pathologies, including cancer, arthritis, and fibrosis. It has been suggested that fibronectin expression may be upregulated in cancer tissues.

[0188] An exemplary amino acid sequence for fibronectin is shown in SEQ ID NO: 48 (UNIPROT Reference Sequence: P02751).

[0189] Thus, in some embodiments, the IL-12 Fc fusion protein comprises a fibronectin binding moiety that specifically binds to fibronectin. In some embodiments, the fibronectin binding moiety specifically binds to human fibronectin.

[0190] The binding of a fibronectin-binding moiety to fibronectin can be determined by methods known in the art. In some embodiments, the fibronectin-binding moiety is determined by its ability to compete with a known or reference fibronectin-binding protein for binding to fibronectin. In some embodiments, the fibronectin-binding moiety is derived from a naturally occurring fibronectin-binding protein or fibronectin receptor.

[0191] In some embodiments, the IL-12 Fc fusion protein specifically binds to fibronectin with an affinity (KD) of less than about 500 μM as determined by a fibronectin binding assay. In some embodiments, the IL-12 Fc fusion protein comprises a fibronectin binding portion that specifically binds to fibronectin with an affinity (KD) of less than about 100 μM as determined by a fibronectin binding assay. In some embodiments, the IL-12 Fc fusion protein comprises a fibronectin binding portion that specifically binds to fibronectin with an affinity (KD) of less than about 1 μM as determined by a fibronectin binding assay. In some embodiments, the IL-12 Fc fusion protein comprises a fibronectin binding portion that specifically binds to fibronectin with an affinity (KD) of less than about 500 nM as determined by a fibronectin binding assay. In some embodiments, the fibronectin binding moiety specifically binds to fibronectin with an affinity (KD) of about 0.1-500 μM, 0.1-100 μM, or 0.1-1 μM as determined by a fibronectin binding assay. In some embodiments, the fibronectin binding moiety specifically binds to fibronectin with an affinity (KD) of about 100-1000 nM, 100-1000 nM, 100-800 nM, 100-600 nM, or 100-500 nM as determined by a fibronectin binding assay.

[0192] In some embodiments, the binding affinity of the fibronectin binding moiety for fibronectin is determined by a fibronectin binding assay.

[0193] In some embodiments, the fibronectin binding assay is an ELISA. Methods and techniques for performing fibronectin-binding ELISAs are known in the art (see, e.g., Gao et al., (1998) European Journal of Pharmaceutics and Biopharmaceutics, Volume 45, Issue 3, Pages 275-284). Thus, in some embodiments, an IL-12 Fc fusion protein comprises a fibronectin-binding portion that specifically binds to fibronectin with an affinity (KD) of less than about 500 μM as determined by ELISA. In some embodiments, an IL-12 Fc fusion protein comprises a fibronectin-binding portion that specifically binds to fibronectin with an affinity (KD) of less than about 100 μM as determined by ELISA. In some embodiments, an IL-12 Fc fusion protein comprises a fibronectin-binding portion that specifically binds to fibronectin with an affinity (KD) of less than about 1 μM as determined by ELISA. In some embodiments, the IL-12 Fc fusion protein comprises a fibronectin binding moiety that specifically binds to fibronectin with an affinity (KD) of less than about 500 nM as determined by ELISA. In some embodiments, the fibronectin binding moiety specifically binds to fibronectin with an affinity (KD) of about 0.1-500 μM, 0.1-100 μM, or 0.1-1 μM as determined by ELISA. In some embodiments, the fibronectin binding moiety specifically binds to fibronectin with an affinity (KD) of about 100-1000 nM, 100-1000 nM, 100-800 nM, 100-600 nM, or 100-500 nM as determined by ELISA.

[0194] In some embodiments, the fibronectin binding assay is a surface plasmon resonance (SPR) assay. Methods and techniques for performing fibronectin binding SPR assays are known in the art (see, e.g., Makogonenko et al. (2002) Biochemistry, 41, 25, 7907-7913). Thus, in some embodiments, an IL-12 Fc fusion protein comprises a fibronectin binding moiety that specifically binds to fibronectin with an affinity (KD) of less than about 500 μM as determined by an SPR assay. In some embodiments, an IL-12 Fc fusion protein comprises a fibronectin binding moiety that specifically binds to fibronectin with an affinity (KD) of less than about 100 μM as determined by an SPR assay. In some embodiments, an IL-12 Fc fusion protein comprises a fibronectin binding moiety that specifically binds to fibronectin with an affinity (KD) of less than about 1 μM as determined by an SPR assay. In some embodiments, the IL-12 Fc fusion protein comprises a fibronectin binding moiety that specifically binds to fibronectin with an affinity (KD) of less than about 500 nM as determined by SPR assay. In some embodiments, the fibronectin binding moiety specifically binds to fibronectin with an affinity (KD) of about 0.1-500 μM, 0.1-100 μM, or 0.1-1 μM as determined by SPR assay. In some embodiments, the fibronectin binding moiety specifically binds to fibronectin with an affinity (KD) of about 100-1000 nM, 100-1000 nM, 100-800 nM, 100-600 nM, or 100-500 nM as determined by SPR assay.

[0195] In some embodiments, an IL-12 Fc fusion protein comprises a fibronectin-binding moiety that specifically binds to fibronectin but not to one or more non-fibronectin extracellular matrix (ECM) components, including, but not limited to, collagen, heparin, vitronectin, tenascin-C, osteopontin, and fibrinogen. In some embodiments, the fibronectin-binding moiety binds to fibronectin with a lower KD than to one or more non-collagen ECM components. In some embodiments, the KD of the fibronectin-binding moiety for fibronectin is lower than the KD of the fibronectin-binding moiety for an extracellular matrix component selected from collagen, heparin, vitronectin, osteopontin, tenascin-C, or fibrinogen. In some embodiments, the fibronectin binding moiety binds to fibronectin with a KD that is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% lower than that of one or more non-collagenous ECM components, hi some embodiments, the fibronectin binding moiety binds to fibronectin with a KD that is about 2-fold, about 3-fold, about 4-fold, about 5-fold, or about 10-fold lower than that of one or more non-collagenous ECM components.

[0196] In some embodiments, the fibronectin binding moiety competes with a reference fibronectin binding moiety for binding to fibronectin. In some embodiments, the reference fibronectin binding moiety comprises, for example, a peptide disclosed in Sipes et al., (1993) Journal Cell Biol., 121(2):469-77.

[0197] In its broadest form, the disclosure provides an IL-12 Fc fusion protein in which the fibronectin binding moiety binds to fibronectin.

[0198] In a further preferred embodiment, the present disclosure provides an IL-12 Fc fusion protein, wherein the fibronectin binding moiety binds to fibronectin and has the sequence GGWSHW (SEQ ID NO: 49).

[0199] In another embodiment, the present disclosure provides an IL-12 Fc fusion protein, wherein the fibronectin binding portion has a length of 20 amino acids (aa), 19 aa, 18 aa, 17 aa, 16 aa, 15 aa, 14 aa, 13 aa, 12 aa, 11 aa, 10 a, or 9 aa, and comprises the sequence GGWSHW (SEQ ID NO: 49).

[0200] Heparin-binding moiety Heparin, also known as unfractionated heparin (UFH), is a pharmaceutical, naturally occurring glycosaminoglycan. Native heparin is a polymer with a molecular weight of 3-30 kDa, but the average molecular weight of most commercially available heparin preparations is in the range of 12-15 kDa. Heparin is a member of the glycosaminoglycan family of carbohydrates (which includes the closely related molecule, heparan sulfate), and consists of repeating disaccharide units that are variably sulfated.

[0201] Thus, in some embodiments, the IL-12 Fc fusion protein comprises a heparin-binding moiety that specifically binds heparin. In some embodiments, the heparin-binding moiety specifically binds human heparin.

[0202] The binding of the heparin-binding moiety to heparin can be determined by methods known in the art. In some embodiments, the heparin-binding moiety is determined by its ability to compete with a known or reference heparin-binding protein for binding to heparin. In some embodiments, the heparin-binding moiety is derived from a naturally occurring heparin-binding protein or heparin receptor.

[0203] In some embodiments, the IL-12 Fc fusion protein specifically binds to heparin with an affinity (KD) of less than about 500 μM as determined by a heparin binding assay. In some embodiments, the IL-12 Fc fusion protein comprises a heparin binding portion that specifically binds to heparin with an affinity (KD) of less than about 100 μM as determined by a heparin binding assay. In some embodiments, the IL-12 Fc fusion protein comprises a heparin binding portion that specifically binds to heparin with an affinity (KD) of less than about 1 μM as determined by a heparin binding assay. In some embodiments, the IL-12 Fc fusion protein comprises a heparin binding portion that specifically binds to heparin with an affinity (KD) of less than about 500 nM as determined by a heparin binding assay. In some embodiments, the heparin binding moiety specifically binds heparin with an affinity (KD) of about 0.1-500 μM, 0.1-100 μM, or 0.1-1 μM as determined by a heparin binding assay. In some embodiments, the heparin binding moiety specifically binds heparin with an affinity (KD) of about 100-1000 nM, 100-1000 nM, 100-800 nM, 100-600 nM, or 100-500 nM as determined by a heparin binding assay.

[0204] In some embodiments, the binding affinity of the heparin-binding moiety for heparin is determined by a heparin-binding assay.

[0205] In some embodiments, the heparin binding assay is an ELISA. Methods and techniques for performing heparin-binding ELISAs are known in the art. Thus, in some embodiments, an IL-12 Fc fusion protein comprises a heparin-binding portion that specifically binds to fibronectin with an affinity (KD) of less than about 500 μM as determined by ELISA. In some embodiments, an IL-12 Fc fusion protein comprises a heparin-binding portion that specifically binds to heparin with an affinity (KD) of less than about 100 μM as determined by ELISA. In some embodiments, an IL-12 Fc fusion protein comprises a heparin-binding portion that specifically binds to heparin with an affinity (KD) of less than about 1 μM as determined by ELISA. In some embodiments, an IL-12 Fc fusion protein comprises a heparin-binding portion that specifically binds to heparin with an affinity (KD) of less than about 500 nM as determined by ELISA. In some embodiments, the heparin-binding moiety specifically binds to heparin with an affinity (KD) of about 0.1-500 μM, 0.1-100 μM, or 0.1-1 μM as determined by ELISA. In some embodiments, the heparin-binding moiety specifically binds to heparin with an affinity (KD) of about 100-1000 nM, 100-1000 nM, 100-800 nM, 100-600 nM, or 100-500 nM as determined by ELISA.

[0206] In some embodiments, the heparin binding assay is a surface plasmon resonance (SPR) assay. Methods and techniques for performing heparin-binding SPR assays are known in the art (see, e.g., Rusnati et al., (2016) Methods Mol Biol., 1464:73-84). Thus, in some embodiments, an IL-12 Fc fusion protein comprises a heparin-binding portion that specifically binds to heparin with an affinity (KD) of less than about 500 μM as determined by an SPR assay. In some embodiments, an IL-12 Fc fusion protein comprises a heparin-binding portion that specifically binds to heparin with an affinity (KD) of less than about 100 μM as determined by an SPR assay. In some embodiments, an IL-12 Fc fusion protein comprises a heparin-binding portion that specifically binds to heparin with an affinity (KD) of less than about 1 μM as determined by an SPR assay. In some embodiments, the IL-12 Fc fusion protein comprises a heparin-binding moiety that specifically binds heparin with an affinity (KD) of less than about 500 nM as determined by SPR assay. In some embodiments, the heparin-binding moiety specifically binds heparin with an affinity (KD) of about 0.1-500 μM, 0.1-100 μM, or 0.1-1 μM as determined by SPR assay. In some embodiments, the heparin-binding moiety specifically binds heparin with an affinity (KD) of about 100-1000 nM, 100-1000 nM, 100-800 nM, 100-600 nM, or 100-500 nM as determined by SPR assay.

[0207] In some embodiments, an IL-12 Fc fusion protein comprises a heparin-binding moiety that specifically binds to heparin but not to one or more non-heparin extracellular matrix (ECM) components, including, but not limited to, collagen, fibronectin, vitronectin, tenascin-C, osteopontin, and fibrinogen. In some embodiments, the heparin-binding moiety binds to heparin with a lower KD than to one or more non-heparin ECM components. In some embodiments, the KD of the heparin-binding moiety for heparin is lower than the KD of the heparin-binding moiety for an extracellular matrix component selected from collagen, fibronectin, vitronectin, osteopontin, tenascin-C, or fibrinogen. In some embodiments, the heparin-binding moiety binds heparin with a KD that is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% lower than that of one or more non-heparin ECM components, hi some embodiments, the heparin-binding moiety binds heparin with a KD that is about 2-fold, about 3-fold, about 4-fold, about 5-fold, or about 10-fold lower than that of one or more non-heparin ECM components.

[0208] In some embodiments, the heparin-binding moiety competes with a reference heparin-binding moiety for binding to heparin. In some embodiments, the reference heparin-binding moiety comprises a peptide disclosed, for example, by Luria-Perez et al., (2019) Cytokine, 120:220-226.

[0209] In its broadest form, the disclosure provides an IL-12 Fc fusion protein in which the heparin-binding moiety binds to heparin.

[0210] In a further preferred embodiment, the present disclosure provides an IL-12 Fc fusion protein, in which the heparin binding moiety binds to heparin and has the sequence VRIQRKKEKMKET (SEQ ID NO: 50).

[0211] In another embodiment, the disclosure provides an IL-12 Fc fusion protein, wherein the heparin binding portion has a length of 20 amino acids (aa), 19 aa, 18 aa, 17 aa, 16 aa, 15 aa, 14 aa, 13 aa, 12 aa, 11 aa, 10 a, or 9 aa, and comprises the sequence VRIQRKKEKMKET (SEQ ID NO: 50).

[0212] [Table 5] TIFF2026504906000028.tif248170 TIFF2026504906000029.tif249170 TIFF2026504906000030.tif70170

[0213] IL-12 Fc fusion protein Described and disclosed herein are IL-12 Fc fusion proteins, as well as compositions and articles of manufacture comprising the IL-12 Fc fusion proteins of the invention. The inventors have devised the IL-12 Fc fusion proteins of the invention shown below, and a selection of these fusion proteins are provided and discussed in the accompanying Examples. The cleavage products, including the IL-12 cytokine and binding moieties after proteolytic cleavage, are underlined in Table 5.

[0214] [Table 6] TIFF2026504906000032.tif250170 TIFF2026504906000033.tif250170 TIFF2026504906000034.tif250170 TIFF2026504906000035.tif249170 TIFF2026504906000036.tif225170

[0215] In the broadest sense, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein a) the first polypeptide chain comprises a first Fc domain and the IL-12p35 and IL-12p40 subunits of IL-12, and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunits in the first polypeptide chain; wherein the first and second polypeptide chains comprise the first Fc domain and the second Fc domain. wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first or second polypeptide chain further comprises a binding moiety selected from the group consisting of a collagen-binding moiety, a heparin-binding moiety, and a fibronectin-binding moiety.

[0216] In one embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain; wherein the first and second polypeptide chains comprise the first Fc domain and and wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which is protease-cleavable, wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first or second polypeptide chain further comprises a collagen-binding moiety, preferably selected from the group consisting of SEQ ID NOs: 41-47.

[0217] In one embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein a) the first polypeptide chain comprises a first Fc domain, and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain; wherein the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109, and wherein the first and second polypeptide chains comprise a first Fc domain and and wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which is protease-cleavable; and wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker; and wherein the first or second polypeptide chain further comprises a binding moiety selected from the group consisting of a collagen-binding moiety, a heparin-binding moiety, and a fibronectin-binding moiety, and the masking moiety may further comprise an additional alanine at its C-terminus.

[0218] In one embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12; and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the first and second polypeptide chains are linked via the first Fc domain and the second Fc domain, and wherein the IL-12p35 the subunit or IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, wherein the protease-cleavable linker is selected from the group consisting of any one of the amino acid sequences of SEQ ID NOs: 232-241; and wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, wherein the first or second polypeptide chain further comprises a binding moiety selected from the group consisting of a collagen-binding moiety, a heparin-binding moiety, and a fibronectin-binding moiety.

[0219] In another embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, wherein the first Fc domain comprises the amino acid sequence of SEQ ID NO: 15; and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the second Fc domain comprises the amino acid sequence of SEQ ID NO: 16. wherein the first and second polypeptide chains are linked via a first Fc domain and a second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first or second polypeptide chain further comprises a binding moiety selected from the group consisting of a collagen-binding moiety, a heparin-binding moiety, and a fibronectin-binding moiety.

[0220] In another embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, wherein the first Fc domain comprises the amino acid sequence of SEQ ID NO: 17; and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the second Fc domain comprises the amino acid sequence of SEQ ID NO: 18; wherein the first and second polypeptide chains are linked via a first Fc domain and a second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first or second polypeptide chain further comprises a binding moiety selected from the group consisting of a collagen-binding moiety, a heparin-binding moiety, and a fibronectin-binding moiety.

[0221] In one embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12; and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109, wherein the first and second polypeptide chains The polypeptide chain is linked via a first Fc domain and a second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which is protease-cleavable, and wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first or second polypeptide chain further comprises a collagen-binding moiety preferably selected from the group consisting of SEQ ID NOs: 41 to 47. The masking moiety may further comprise an additional alanine added to its C-terminus.

[0222] In one embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12; and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109, wherein the first and second polypeptide chains are coupled via the first Fc domain and the second Fc domain. wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, wherein the protease-cleavable linker is selected from the group consisting of any one of the amino acid sequences of SEQ ID NOs: 232-241, and wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, wherein the first or second polypeptide chain further comprises a collagen-binding moiety, preferably selected from the group consisting of SEQ ID NOs: 41-47. The masking moiety may further comprise an additional alanine added to its C-terminus.

[0223] In one embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, wherein the first Fc domain comprises the amino acid sequence of SEQ ID NO: 15; and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the second Fc domain comprises the amino acid sequence of SEQ ID NO: 16 and the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109, wherein the first and a second polypeptide chain linked via a first Fc domain and a second Fc domain, wherein the IL-12p35 subunit or IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, wherein the protease-cleavable linker is selected from the group consisting of any one of the amino acid sequences of SEQ ID NOs: 232-241, and wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, wherein the first or second polypeptide chain further comprises a collagen-binding moiety preferably selected from the group consisting of SEQ ID NOs: 41-47. The masking moiety may further comprise an additional alanine added to its C-terminus.

[0224] In one embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, wherein the first Fc domain comprises the amino acid sequence of SEQ ID NO: 17; and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the second Fc domain comprises the amino acid sequence of SEQ ID NO: 18 and the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109, wherein the first and a second polypeptide chain linked via a first Fc domain and a second Fc domain, wherein the IL-12p35 subunit or IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, wherein the protease-cleavable linker is selected from the group consisting of any one of the amino acid sequences of SEQ ID NOs: 232-241, and wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, wherein the first or second polypeptide chain further comprises a collagen-binding moiety preferably selected from the group consisting of SEQ ID NOs: 41-47. The masking moiety may further comprise an additional alanine added to its C-terminus.

[0225] In one embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein: a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, wherein the first Fc domain comprises the amino acid sequence of SEQ ID NO: 17; and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the second Fc domain comprises the amino acid sequence of SEQ ID NO: 18 and the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109; wherein the first and second polypeptide chains are linked via a first Fc domain and a second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, wherein the protease-cleavable linker is selected from the group consisting of any one of the amino acid sequences of SEQ ID NOs: 232 to 241, and wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, wherein the first or second polypeptide chain further comprises a collagen-binding moiety having the amino acid sequence of SEQ ID NO: 41. The masking moiety may further comprise an additional alanine added to its C-terminus.

[0226] In one embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein: a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, wherein the first Fc domain comprises the amino acid sequence of SEQ ID NO: 17; and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the second Fc domain comprises the amino acid sequence of SEQ ID NO: 18 and the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109; wherein the first and second polypeptide chains are linked via a first Fc domain and a second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, wherein the protease-cleavable linker is selected from the group consisting of any one of the amino acid sequences of SEQ ID NOs: 232 to 241, wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, wherein the first or second polypeptide chain further comprises a collagen-binding moiety having the amino acid sequence of SEQ ID NO: 42. The masking moiety may further comprise an additional alanine added to its C-terminus.

[0227] In one embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein: a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, wherein the first Fc domain comprises the amino acid sequence of SEQ ID NO: 17; and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the second Fc domain comprises the amino acid sequence of SEQ ID NO: 18 and the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109; wherein the first and second polypeptide chains are linked via a first Fc domain and a second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, wherein the protease-cleavable linker is selected from the group consisting of any one of the amino acid sequences of SEQ ID NOs: 232 to 241, wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, wherein the first or second polypeptide chain further comprises a collagen-binding moiety having the amino acid sequence of SEQ ID NO: 43. The masking moiety may further comprise an additional alanine added to its C-terminus.

[0228] In one embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein: a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, wherein the first Fc domain comprises the amino acid sequence of SEQ ID NO: 17; and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the second Fc domain comprises the amino acid sequence of SEQ ID NO: 18 and the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109; wherein the first and second polypeptide chains are linked via a first Fc domain and a second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, wherein the protease-cleavable linker is selected from the group consisting of any one of the amino acid sequences of SEQ ID NOs: 232 to 241, and wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, wherein the first or second polypeptide chain further comprises a collagen-binding moiety having the amino acid sequence of SEQ ID NO: 44. The masking moiety may further comprise an additional alanine added to its C-terminus.

[0229] In one embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein: a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, wherein the first Fc domain comprises the amino acid sequence of SEQ ID NO: 17; and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the second Fc domain comprises the amino acid sequence of SEQ ID NO: 18 and the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109; wherein the first and second polypeptide chains are linked via a first Fc domain and a second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, wherein the protease-cleavable linker is selected from the group consisting of any one of the amino acid sequences of SEQ ID NOs: 232 to 241, wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, wherein the first or second polypeptide chain further comprises a collagen-binding moiety having the amino acid sequence of SEQ ID NO: 45. The masking moiety may further comprise an additional alanine added to its C-terminus.

[0230] In one embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein: a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, wherein the first Fc domain comprises the amino acid sequence of SEQ ID NO: 17; and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the second Fc domain comprises the amino acid sequence of SEQ ID NO: 18 and the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109; wherein the first and second polypeptide chains are linked via a first Fc domain and a second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, wherein the protease-cleavable linker is selected from the group consisting of any one of the amino acid sequences of SEQ ID NOs: 232 to 241, wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, wherein the first or second polypeptide chain further comprises a collagen-binding moiety having the amino acid sequence of SEQ ID NO: 46. The masking moiety may further comprise an additional alanine added to its C-terminus.

[0231] In one embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein: a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, wherein the first Fc domain comprises the amino acid sequence of SEQ ID NO: 17; and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the second Fc domain comprises the amino acid sequence of SEQ ID NO: 18 and the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109; wherein the first and second polypeptide chains are linked via a first Fc domain and a second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, wherein the protease-cleavable linker is selected from the group consisting of any one of the amino acid sequences of SEQ ID NOs: 232 to 241, wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, wherein the first or second polypeptide chain further comprises a collagen-binding moiety having the amino acid sequence of SEQ ID NO: 47. The masking moiety may further comprise an additional alanine added to its C-terminus.

[0232] In one embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109; and a second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, wherein the protease-cleavable linker is selected from the group consisting of any one of the amino acid sequences of SEQ ID NOs: 232 to 241, and wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, wherein the first or second polypeptide chain further comprises a collagen-binding moiety having the amino acid sequence of SEQ ID NO: 41. The masking moiety may further comprise an additional alanine added to its C-terminus.

[0233] In one embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109; and a second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, wherein the protease-cleavable linker is selected from the group consisting of any one of the amino acid sequences of SEQ ID NOs: 232 to 241, and wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, wherein the first or second polypeptide chain further comprises a collagen-binding moiety having the amino acid sequence of SEQ ID NO: 42. The masking moiety may further comprise an additional alanine added to its C-terminus.

[0234] In one embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109; and a second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, wherein the protease-cleavable linker is selected from the group consisting of any one of the amino acid sequences of SEQ ID NOs: 232 to 241, and wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, wherein the first or second polypeptide chain further comprises a collagen-binding moiety having the amino acid sequence of SEQ ID NO: 43. The masking moiety may further comprise an additional alanine added to its C-terminus.

[0235] In one embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109; and a second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, wherein the protease-cleavable linker is selected from the group consisting of any one of the amino acid sequences of SEQ ID NOs: 232 to 241, and wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, wherein the first or second polypeptide chain further comprises a collagen-binding moiety having the amino acid sequence of SEQ ID NO: 44. The masking moiety may further comprise an additional alanine added to its C-terminus.

[0236] In one embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109; and a second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, wherein the protease-cleavable linker is selected from the group consisting of any one of the amino acid sequences of SEQ ID NOs: 232 to 241, and wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, wherein the first or second polypeptide chain further comprises a collagen-binding moiety having the amino acid sequence of SEQ ID NO: 45. The masking moiety may further comprise an additional alanine added to its C-terminus.

[0237] In one embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109; and a second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, wherein the protease-cleavable linker is selected from the group consisting of any one of the amino acid sequences of SEQ ID NOs: 232 to 241, and wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, wherein the first or second polypeptide chain further comprises a collagen-binding moiety having the amino acid sequence of SEQ ID NO: 46. The masking moiety may further comprise an additional alanine added to its C-terminus.

[0238] In one embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109; and a second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, wherein the protease-cleavable linker is selected from the group consisting of any one of the amino acid sequences of SEQ ID NOs: 232 to 241, and wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, wherein the first or second polypeptide chain further comprises a collagen-binding moiety having the amino acid sequence of SEQ ID NO: 47. The masking moiety may further comprise an additional alanine added to its C-terminus.

[0239] In one embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109; and wherein the first and second polypeptide chains comprise a first Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109. and a second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, wherein the protease-cleavable linker comprises or consists of the amino acid sequence of SEQ ID NO: 232, and wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, wherein the first or second polypeptide chain further comprises a collagen-binding moiety having the amino acid sequence of SEQ ID NO: 41. The masking moiety may further comprise an additional alanine added to its C-terminus.

[0240] In one embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109; and wherein the first and second polypeptide chains comprise a first Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109. and a second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, wherein the protease-cleavable linker comprises or consists of the amino acid sequence of SEQ ID NO: 232, and wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, wherein the first or second polypeptide chain further comprises a collagen-binding moiety having the amino acid sequence of SEQ ID NO: 42. The masking moiety may further comprise an additional alanine added to its C-terminus.

[0241] In one embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109; and wherein the first and second polypeptide chains comprise a first Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109. and a second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, wherein the protease-cleavable linker comprises or consists of the amino acid sequence of SEQ ID NO: 232, and wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, wherein the first or second polypeptide chain further comprises a collagen-binding moiety having the amino acid sequence of SEQ ID NO: 43. The masking moiety may further comprise an additional alanine added to its C-terminus.

[0242] In one embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109; and wherein the first and second polypeptide chains comprise a first Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109. and a second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, wherein the protease-cleavable linker comprises or consists of the amino acid sequence of SEQ ID NO: 232, and wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, wherein the first or second polypeptide chain further comprises a collagen-binding moiety having the amino acid sequence of SEQ ID NO: 44. The masking moiety may further comprise an additional alanine added to its C-terminus.

[0243] In one embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109; and wherein the first and second polypeptide chains comprise a first Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109. and a second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, wherein the protease-cleavable linker comprises or consists of the amino acid sequence of SEQ ID NO: 232, and wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, wherein the first or second polypeptide chain further comprises a collagen-binding moiety having the amino acid sequence of SEQ ID NO: 45. The masking moiety may further comprise an additional alanine added to its C-terminus.

[0244] In one embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109; and wherein the first and second polypeptide chains comprise a first Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109. and a second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, wherein the protease-cleavable linker comprises or consists of the amino acid sequence of SEQ ID NO: 232, and wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, wherein the first or second polypeptide chain further comprises a collagen-binding moiety having the amino acid sequence of SEQ ID NO: 46. The masking moiety may further comprise an additional alanine added to its C-terminus.

[0245] In one embodiment, an interleukin-12 (IL-12) Fc fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein a) the first polypeptide chain comprises a first Fc domain and an IL-12p35 subunit and an IL-12p40 subunit of IL-12, and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109; and wherein the first and second polypeptide chains comprise a first Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunit in the first polypeptide chain, wherein the masking moiety is selected from the group consisting of any one of SEQ ID NOs: 61-109. and a second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, which first peptide linker is protease-cleavable, wherein the protease-cleavable linker comprises or consists of the amino acid sequence of SEQ ID NO: 232, and wherein the masking moiety is linked to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, wherein the first or second polypeptide chain further comprises a collagen-binding moiety having the amino acid sequence of SEQ ID NO: 47. The masking moiety may further comprise an additional alanine added to its C-terminus.

[0246] In one embodiment related to any of the preceding embodiments, the IL-12 activity of the uncleaved IL-12 Fc fusion protein is at least 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, 175-fold, 200-fold, 225-fold, 250-fold, 275-fold, 300-fold, 325-fold, 350-fold, 375-fold, 400-fold, 425-fold, 450-fold, 475-fold, 500-fold, 525-fold, 550-fold, 575-fold, or 600-fold less than the IL-12 activity of the IL-12 Fc fusion protein after cleavage of the cleavable linker. In other words, the delta EC 50 (EC 50 Uncleaved IL-12 Fc fusion protein:EC50 The cleaved IL-12 Fc fusion protein) is at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 400, 425, 450, 475, 500, 525, 550, 575, or 600, for example, as in the Promega IL-12 bioassay described in the Examples.

[0247] The following table discloses additional IL-12 Fc fusion proteins or VHH masking moieties that have been conceived or generated, e.g., as intermediate molecules during optimization or as further alternatives. Each IL-12 Fc fusion protein is composed of two chains identified by appropriate identifiers, with the understanding that, for example, the BI-062 knob pairs only with the BI-062 hole, and the knob chain BI-067 pairs only with the hole chain BI-067. Thus, in another embodiment, the present invention relates to an IL-12 Fc fusion protein comprising or consisting of two polypeptide chains identified by an appropriate identifier pair disclosed in Table 6, e.g., SEQ ID NOs: 242 and 243, SEQ ID NOs: 245 and 246, SEQ ID NOs: 247 and 248, etc.

[0248] [Table 7] TIFF2026504906000038.tif249170 TIFF2026504906000039.tif250170 TIFF2026504906000040.tif250170 TIFF2026504906000041.tif250170 TIFF2026504906000042.tif249170 TIFF2026504906000043.tif249170 TIFF2026504906000044.tif250170 TIFF2026504906000045.tif250170 TIFF2026504906000046.tif249170 TIFF2026504906000047.tif250170 TIFF2026504906000048.tif249170 TIFF2026504906000049.tif250170 TIFF2026504906000050.tif249170 TIFF2026504906000051.tif248170 TIFF2026504906000052.tif249170 TIFF2026504906000053.tif250170 TIFF2026504906000054.tif249170 TIFF2026504906000055.tif249170 TIFF2026504906000056.tif249170 TIFF2026504906000057.tif122170

[0249] Treatment method In its broadest form, the present invention provides an IL-12 Fc fusion protein for use in medicine.

[0250] The IL-12 Fc fusion proteins of the present invention are useful in cancer immunotherapy and are beneficial in controlling tumor growth by activating anti-tumor cytotoxic immune responses, and therefore are useful for the treatment and / or prevention of cancer.

[0251] In a further aspect, the IL-12 Fc fusion proteins of the present invention can be used in a method for treating and / or preventing cancer and / or reducing the incidence of cancer, comprising administering a therapeutically effective amount of an IL-12 Fc fusion protein to an individual suffering from cancer, thereby ameliorating one or more symptoms of the cancer.

[0252] In a further aspect, the present invention further provides the use of an IL-12 Fc fusion protein according to the present invention for the manufacture of a medicament for the treatment and / or prevention of cancer.

[0253] In a further aspect, the IL-12 Fc fusion proteins of the present invention can be used in a method for treating and / or preventing and / or reducing the incidence of melanoma, non-small cell lung cancer (NSCLC), cutaneous squamous cell carcinoma (cSCC), or bladder cancer, comprising administering a therapeutically effective amount of IL-12 Fc fusion protein to an individual afflicted with melanoma, non-small cell lung cancer (NSCLC), cutaneous squamous cell carcinoma (cSCC), or bladder cancer, thereby ameliorating one or more symptoms of the melanoma, non-small cell lung cancer (NSCLC), cutaneous squamous cell carcinoma (cSCC), or bladder cancer.

[0254] For the prevention or treatment of disease, the appropriate dosage of IL-12 Fc fusion protein will depend on various factors, such as the type of disease being treated, the severity and course of the disease, whether the IL-12 Fc fusion protein is administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the IL-12 Fc fusion protein, and the judgment of the attending physician. The IL-12 Fc fusion protein is appropriately administered to the patient at one time or over a series of treatments.

[0255] In one embodiment, the cancer is a solid tumor. In another embodiment, the cancer is a lymphoma. In another embodiment, the cancer is a relapsed or refractory, advanced or metastatic solid tumor or lymphoma. In one embodiment, the lymphoma is a non-Hodgkin's lymphoma or a Hodgkin's lymphoma. In another embodiment, the lymphoma is a cutaneous T-cell lymphoma (CTCL) or Sézary syndrome / disease.

[0256] In one embodiment, the cancer is skin cancer, lung cancer or head and neck cancer, brain cancer, gastrointestinal cancer, endometrial cancer, vaginal cancer, HPV-positive tumors, HPV-positive cervical cancer, HPV-positive oropharyngeal cancer, HPV-positive anal cancer, HPV-positive penile cancer, HPV-positive vaginal cancer, HPV-positive vulvar cancer, anal cancer, colorectal cancer, oropharyngeal squamous cell carcinoma, squamous cell carcinoma, gastric cancer, gastroesophageal junction adenocarcinoma, esophageal cancer, cutaneous T-cell lymphoma, hepatocellular carcinoma, pancreatic adenocarcinoma, pancreatic cancer, bile duct cancer, bladder urothelial cancer, urothelial carcinoma, kidney cancer, metastatic melanoma, prostate cancer, breast cancer, ovarian cancer, head and neck squamous cell carcinoma (HNSCC), glioblastoma, non-small cell lung cancer, brain tumor, or small cell lung cancer. Preferred is the treatment of melanoma, non-small cell lung cancer (NSCLC), cutaneous squamous cell carcinoma (cSCC), urothelial carcinoma, or bladder cancer.

[0257] In another embodiment, the IL-12 Fc fusion proteins are useful for treating patients who have failed or responded inadequately to previous PD-1 or PD-L1 inhibitor treatment (e.g., immunotherapy-resistant advanced or metastatic solid tumors or lymphomas).

[0258] In another aspect, the IL-12 Fc fusion proteins are useful for the treatment of patients who have completed checkpoint inhibitor therapy with either a PD-1 inhibitor or a PD-L1 inhibitor, e.g., an antagonist antibody to PD-1 or PD-L1.

[0259] The IL-12 Fc fusion protein is administered by any suitable means, including oral, parenteral, subcutaneous, intratumoral, intravenous, intradermal, intraperitoneal, intrapulmonary, and intranasal. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. The IL-12 Fc fusion protein is also suitably administered by pulse infusion. In one embodiment, dosing is given by injection, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.

[0260] Depending on the particular IL-12 Fc fusion protein of the present invention and its particular pharmacokinetic and other properties, it may be administered daily, every two, three, four, five, or six days, weekly, monthly, etc. Dosing regimens may include chronic, weekly treatment. By "chronic" is meant a duration of at least two weeks, preferably several months or years.

[0261] Treatment schedules can involve a variety of regimens, but typically call for multiple doses administered to the patient over a period of 1, 2, 3, or 4 weeks, optionally followed by one or more additional treatments.

[0262] The term "suppression" is used herein in the same context as "amelioration" and "palliation" to mean the reduction or elimination of one or more characteristics of a disease. The IL-12 Fc fusion protein or pharmaceutical composition of the present invention will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to medical professionals. The "therapeutically effective amount" of the IL-12 Fc fusion protein to be administered will be governed by such considerations and is the minimum amount necessary to prevent, ameliorate, or treat the clinical symptoms of cancer, particularly the minimum amount that is effective in these disorders.

[0263] In another embodiment, the IL-12 Fc fusion proteins of the present invention can be administered multiple times and in several doses.

[0264] In another embodiment, the IL-12 Fc fusion protein is administered intravenously. In another embodiment, the IL-12 Fc fusion protein is administered subcutaneously.

[0265] As described above, the IL-12 Fc fusion protein of the present invention has great utility for stimulating immune responses against cancer cells. It has been observed that its potent immune stimulatory ability is restricted to the tumor microenvironment. Therefore, in a preferred embodiment, the IL-12 Fc fusion protein of the present invention can be administered systemically to patients. Systemic applicability is a crucial attribute because many cancers are highly metastatic, thereby enabling the treatment of difficult-to-access and inaccessible tumor lesions. Due to this unique immune stimulatory property, the IL-12 Fc fusion protein of the present invention is inherently useful for the treatment of metastatic tumors.

[0266] It has been observed that some patients develop resistance to checkpoint inhibitor therapy, and such patients appear to accumulate mutations in the IFN pathway. Thus, in one embodiment, the IL-12 Fc fusion proteins of the present invention are useful for treating patients who have developed resistance to checkpoint inhibitor therapy. Due to the inherent immunostimulatory properties of the IL-12 Fc fusion, such treated patients may be eligible for continued checkpoint inhibitor therapy.

[0267] In a preferred embodiment, the IL-12 Fc fusion proteins of the invention are useful for the treatment of patients with non-small cell lung cancer who have completed checkpoint inhibitor therapy with either a PD-1 inhibitor or a PD-L1 inhibitor, e.g., an antagonist antibody to PD-1 or PD-L1.

[0268] It will be understood that any of the above pharmaceutical formulations or methods of treatment can be carried out using any one of the IL-12 Fc fusion proteins or pharmaceutical compositions of the present invention.

[0269] combination The present invention also provides combination treatments / methods that offer certain advantages over treatments / methods currently in use and / or known in the prior art. These advantages may include in vivo efficacy (e.g., improved clinical response, prolonged response, increased rate of response, duration of response, rate of disease stabilization, duration of stabilization, time to disease progression, progression-free survival (PFS) and / or overall survival (OS), subsequent development of resistance, etc.), safe and well-tolerated administration, and reduced frequency and severity of adverse events.

[0270] The IL-12 Fc fusion proteins of the present invention may be used in combination with other pharmacologically active ingredients, such as state-of-the-art or standard of care compounds, such as, for example, cytostatic or cytotoxic agents, cell growth inhibitors, anti-angiogenic agents, steroids, immunomodulators / checkpoint inhibitors, etc. The IL-12 Fc fusion proteins of the present invention may also be used in combination with radiation therapy.

[0271] Cytostatic and / or cytotoxic active substances that may be administered in combination with the IL-12 Fc fusion proteins of the present invention include, without being limited to, hormones, hormone analogs and antihormones, aromatase inhibitors, LHRH agonists and antagonists, inhibitors of growth factors (growth factors such as, for example, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), human epidermal growth factor (HER, for example, HER2, HER3, HER4), and hepatocyte growth factor (HGF)), inhibitors of growth factors such as (anti)growth factors, e.g., growth factor antibodies, (anti) growth factor receptor antibodies and tyrosine kinase inhibitors, such as, for example, cetuximab, gefitinib, afatinib, nintedanib, imatinib, lapatinib, bosutinib and trastuzumab; antimetabolites (e.g., folate antagonists, such as methotrexate, raltitrexed, etc.; pyrimidine analogues, such as 5-fluorouracil (5-FU), gemcitabine, irinotecan, doxorubicin, TAS-102, capecitabine and gemcitabine, etc.; purine analogues and adenosine analogues, such as mercaptopurine, thioguanine, cladribine and pentostatin, cytarabine (arabinose), C), fludarabine, etc.); antitumor antibiotics (e.g., anthracyclines); platinum derivatives (e.g., cisplatin, oxaliplatin, carboplatin); alkylating agents (e.g., estramustine, mechlorethamine, melphalan, chlorambucil, busulfan, dacarbazine, cyclophosphamide, ifosfamide, temozolomide, nitrosoureas, such as, for example, carmustine and lomustine, thiotepa); antimitotic agents (e.g., vinca alkaloids, such as, for example, vinblastine, vindesine, vinorelbine, and vincristine; and taxanes, such as, for example, paclitaxel, docetaxel, etc.); angiogenesis inhibitors, tubulin inhibitors, including bevacizumab, ramucirumab, and aflibercept;DNA synthesis inhibitors, PARP inhibitors, topoisomerase inhibitors (e.g., epipodophyllotoxins, e.g., etoposide and etopophos, teniposide, amsacrine, topotecan, irinotecan, mitoxantrone), serine / threonine kinase inhibitors (e.g., PDK1 inhibitors, Raf inhibitors, A-Raf inhibitors, B-Raf inhibitors, C-Raf inhibitors, mTOR inhibitors, mTORC1 / 2 inhibitors, PI3K inhibitors, PI3Kα inhibitors, dual mTOR / PI3 K inhibitors, STK33 inhibitors, AKT inhibitors, PLK1 inhibitors (e.g., volasertib), inhibitors of CDKs including CDK9 inhibitors, Aurora kinase inhibitors), tyrosine kinase inhibitors (e.g., PTK2 / FAK inhibitors), protein-protein interaction inhibitors, MEK inhibitors, ERK inhibitors, FLT3 inhibitors, BRD4 inhibitors, IGF-1R inhibitors, Bcl-xL inhibitors, Bcl-2 inhibitors, Bcl-2 / Bcl-xL inhibitors, ErbB receptor inhibitors, BCR ABL inhibitors, ABL inhibitors, Src inhibitors, rapamycin analogues (e.g., everolimus, temsirolimus, ridaforolimus, sirolimus), androgen synthesis inhibitors, androgen receptor inhibitors, DNMT inhibitors, HDAC inhibitors, ANG1 / 2 inhibitors, CYP17 inhibitors, radiopharmaceuticals, immunotherapeutic agents such as immune checkpoint inhibitors (e.g., CTLA4, PD1, PD-L1, LAG3, and TIM3 binding molecules / immunoglobulins, e.g., ipilimumab, nivolumab, pembrolizumab, etc.), and various chemotherapeutic agents such as amifostine, anagrelide, clodrolimus, thiazolinone ... such as methylprednisolone, filgrastin, interferon, interferon alpha, leucovorin, rituximab, procarbazine, levamisole, mesna, mitotane, pamidronate, and porfimer; proteasome inhibitors (such as bortezomib), Smac and BH3 mimetics; agents that restore p53 functionality, including mdm2-p53 antagonists, inhibitors of the Wnt / beta-catenin signaling pathway; Flt3L and Flt3-stimulating antibodies or ligand mimetics; SIRP alpha and CD47 blocking therapeutics; and / or cyclin-dependent kinase 9 inhibitors.

[0272] Furthermore, the potential conversion of immunologically "cold" tumors into "hot" tumors, myeloid / dendritic cell activation in combination with T cell activation, further interacts favorably with therapeutic modalities such as T cell engagers. Thus, in one embodiment, the IL-12 Fc fusion proteins of the present invention can be used in combination treatments with one or more T cell engagers.

[0273] The IL-12 Fc fusion proteins of the present invention can be used in combination treatments with cancer vaccines or oncolytic viruses. Such combination treatments can be given as a non-fixed (e.g., free) combination of substances or in the form of a fixed combination, including a kit-of-parts. In one embodiment, the oncolytic virus is a vesicular stomatitis virus. In a preferred embodiment, the vesicular stomatitis virus is a vesicular stomatitis virus with the glycoprotein GP of lymphocytic choriomeningitis virus (LCMV), preferably the WE-HPI strain. Such a VSV is described, for example, in WO 2010 / 040526 and is designated VSV-GP.

[0274] In yet another embodiment, any of the disclosed IL-12 Fc fusion proteins can be encoded in a suitable viral vector, e.g., an oncolytic viral vector, preferably a vesicular stomatitis virus, or more preferably a vesicular stomatitis virus with the glycoprotein GP of lymphocytic choriomeningitis virus (LCMV), preferably the WE-HPI strain. Such a VSV is described, for example, in WO 2010 / 040526 and is designated VSV-GP. Such a viral vector can then be used to deliver the IL-12 Fc fusion protein (encoded in the viral vector's genome). The IL-12 Fc fusion protein can then be transcribed / translated in the patient, and the polypeptide chains can assemble in the human body to form the complete prodrug.

[0275] The IL-12 Fc fusion proteins of the present invention can be used in combination treatments with PD-1 pathway inhibitors. Such combination treatments can be given as a non-fixed (e.g., free) combination of agents or in the form of a fixed combination, including a kit of parts.

[0276] In this context, "combination" or "combined" within the meaning of the present invention includes, but is not limited to, products resulting from the mixing or combining of more than one active agent, including both fixed and non-fixed (e.g., free) combinations (including kits), as well as uses, such as simultaneous, parallel, sequential, sequential, alternating, or separate use of components or agents. The term "fixed combination" means that the active agents are both administered to a patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that the active agents are both administered to a patient as separate entities, either simultaneously, in parallel, or sequentially, without any specific time limitation, such that such administration provides therapeutically effective levels of the two compounds in the patient's body. The latter also applies to cocktail therapy, for example, the administration of three or more active agents.

[0277] The present invention provides IL-12 Fc fusion proteins in combination with a PD-1 pathway inhibitor for use in the treatment of cancers described herein, preferably for the treatment of solid cancers.

[0278] The present invention also provides the use of an IL-12 Fc fusion protein in combination with a PD-1 pathway inhibitor for the manufacture of a medicament for the treatment and / or prevention of cancers described herein, preferably for the treatment of solid cancers.

[0279] The present invention further provides a method for treating and / or preventing cancer, comprising administering to an individual suffering from cancer a therapeutically effective amount of an IL-12 Fc fusion protein of the present invention and a PD-1 pathway inhibitor, thereby ameliorating one or more symptoms of the cancer. The IL-12 Fc fusion protein of the present invention and the PD-1 pathway inhibitor can be administered simultaneously, sequentially, or alternately.

[0280] The IL-12 Fc fusion protein of the present invention and the PD-1 pathway inhibitor can be administered by the same route of administration or via different routes of administration. Preferably, the PD-1 pathway inhibitor is administered intravenously and the IL-12 Fc fusion protein of the present invention is administered intravenously or subcutaneously.

[0281] Particularly preferred is treatment with an IL-12 Fc fusion protein of the invention in combination with immunotherapeutic agents, including anti-PD-1 and anti-PD-L1 agents, and anti-LAG3 agents, such as pembrolizumab and nivolumab, and the antibodies disclosed in WO2017 / 198741.

[0282] Provided herein are combinations comprising (i) an IL-12 Fc fusion protein of the invention and (ii) a PD-1 pathway inhibitor, preferably an antagonist antibody directed against PD-1 or PD-L1. Further provided are uses of such combinations, comprising (i) and (ii), for the treatment of cancers described herein.

[0283] In another embodiment, a combination treatment is provided, comprising the use of i) an IL-12 Fc fusion protein of the present invention and (ii) a PD-1 pathway inhibitor. In such a combination treatment, the IL-12 Fc fusion protein of the present invention can be administered in combination, sequentially, or alternating with the PD-1 pathway inhibitor.

[0284] For example, "concomitant" administration includes administering active agents within the same general time period (e.g., on the same day, but not necessarily simultaneously). Alternating administration includes administering one agent for a period of time, e.g., over the course of several days or a week, followed by administering the other agent for a subsequent period of time, e.g., over the course of several days or a week, and then repeating this pattern for one or more cycles. Sequential or sequential administration includes administering one agent for a first period (e.g., over the course of several days or a week) using one or more doses, followed by administering the other agent for a second period (e.g., over the course of several days or a week) using one or more doses. Overlapping schedules may also be used, which include administering active agents on different days over the treatment period, but not necessarily in a regular order. Variations on these general guidelines may also be used, for example, depending on the agents used and the subject's condition.

[0285] A sequential treatment schedule comprises administration of an IL-12 Fc fusion protein of the present invention followed by administration of a PD-1 pathway inhibitor. A sequential treatment schedule also comprises administration of a PD-1 pathway inhibitor followed by administration of an IL-12 Fc fusion protein of the present invention. A sequential treatment schedule can comprise administration 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or 31 days after each other.

[0286] A PD-1 pathway inhibitor within the meaning of the present invention and all of its embodiments is a compound that inhibits the interaction between PD-1 and its receptor. The PD-1 pathway inhibitor is preferably capable of impairing PD-1 pathway signaling mediated by the PD-1 receptor. The PD-1 inhibitor can be any inhibitor directed against any member of the PD-1 pathway that can antagonize PD-1 pathway signaling. The inhibitor can be an antagonist antibody targeting any member of the PD-1 pathway, but is preferably directed against the PD-1 receptor, PD-L1, or PD-L2. The PD-1 pathway inhibitor can also be a fragment of the PD-1 receptor or a PD-1 receptor that blocks the activity of a PD-1 ligand.

[0287] PD-1 antagonists are well known in the art and are reviewed, for example, by Li et al., Int. J. Mol. Sci. 2016, 17, 1151 (incorporated herein by reference). Any PD-1 antagonist, particularly an antibody, such as those disclosed by Li et al., as well as the additional antibodies disclosed herein below, can be used in accordance with the present invention. Preferably, the PD-1 antagonist of the present invention and all its embodiments is selected from the group consisting of the following antibodies:

[0288] ezabenlimab (BI754091);

[0289] pembrolizumab (anti-PD-1 antibody);

[0290] Nivolumab (anti-PD-1 antibody);

[0291] pidilizumab (anti-PD-1 antibody);

[0292] PDR-001 (anti-PD-1 antibody);

[0293] PD1-1, PD1-2, PD1-3, PD1-4, and PD1-5 (anti-PD-1 antibodies) disclosed herein below

[0294] Atezolizumab (anti-PD-L1 antibody);

[0295] avelumab (anti-PD-L1 antibody);

[0296] Durvalumab (anti-PD-L1 antibody).

[0297] Pembrolizumab (formerly known as lambrolizumab; trade name: Keytruda; also known as MK-3475) is disclosed, for example, in Hamid, O. et al. (2013) New England Journal of Medicine 369(2):134-44 and is a humanized IgG4 monoclonal antibody that binds to PD-1; it contains a mutation at C228P designated to prevent Fc-mediated cytotoxicity. Pembrolizumab is disclosed, for example, in US 8,354,509 and WO2009 / 114335. It has been approved by the FDA for the treatment of patients with unresectable or metastatic melanoma and patients with metastatic NSCLC.

[0298] Nivolumab (CAS Registry Number: 946414-94-4; BMS-936558 or MDX1106b) is a fully human IgG4 monoclonal antibody that specifically blocks PD-1 and lacks detectable antibody-dependent cellular cytotoxicity (ADCC). Nivolumab is disclosed, for example, in US 8,008,449 and WO2006 / 121168. It has been approved by the FDA for the treatment of patients with unresectable or metastatic melanoma, metastatic NSCLC, and advanced renal cell carcinoma.

[0299] Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD-1. Pidilizumab is disclosed, for example, in WO2009 / 101611.

[0300] PDR-001 or PDR001 is a high-affinity, ligand-blocking, humanized anti-PD-1 IgG4 antibody that blocks the binding of PD-L1 and PD-L2 to PD-1. PDR-001 is disclosed in WO2015 / 112900 and WO2017 / 019896.

[0301] Antibodies PD1-1 to PD1-5 are antibody molecules defined by the sequences shown in Table 7, where HC means (full length) heavy chain and LC means (full length) light chain.

[0302] [Table 8] TIFF2026504906000059.tif118170

[0303] Specifically, the anti-PD-1 antibody molecules described herein above have:

[0304] (PD1-1:) a heavy chain comprising the amino acid sequence of SEQ ID NO: 51 and a light chain comprising the amino acid sequence of SEQ ID NO: 52; or

[0305] (PD1-2:) a heavy chain comprising the amino acid sequence of SEQ ID NO: 53 and a light chain comprising the amino acid sequence of SEQ ID NO: 54; or

[0306] (PD1-3:) a heavy chain comprising the amino acid sequence of SEQ ID NO: 55 and a light chain comprising the amino acid sequence of SEQ ID NO: 56; or

[0307] (PD1-4:) a heavy chain comprising the amino acid sequence of SEQ ID NO: 57 and a light chain comprising the amino acid sequence of SEQ ID NO: 58; or

[0308] (PD1-5:) A heavy chain comprising the amino acid sequence of SEQ ID NO: 59 and a light chain comprising the amino acid sequence of SEQ ID NO: 60.

[0309] Atezolizumab (Tecentriq, also known as MPDL3280A) is a phage-derived human IgG1k monoclonal antibody that targets PD-L1 and is described, for example, in Deng et al. mAbs 2016;8:593-603. It has been approved by the FDA for the treatment of patients with urothelial carcinoma.

[0310] Avelumab is a fully human anti-PD-L1 IgG1 monoclonal antibody described, for example, in Boyerinas et al. Cancer Immunol. Res. 2015;3:1148-1157.

[0311] Durvalumab (MEDI4736) is a human IgG1k monoclonal antibody with high specificity for PD-L1 and is described, for example, in Stewart et al. Cancer Immunol. Res. 2015;3:1052-1062 or in Ibrahim et al. Semin. Oncol. 2015;42:474-483.

[0312] Additional PD-1 antagonists disclosed by Li et al. (supra) or known to be in clinical trials, such as AMP-224, MEDI0680 (AMP-514), REGN2810, BMS-936559, JS001-PD-1, SHR-1210, BMS-936559, TSR-042, JNJ-63723283, MEDI4736, MPDL3280A, and MSB0010718C, may be used instead of, or in addition to, the antagonists mentioned above.

[0313] As used herein, INN is also meant to encompass all biosimilar antibodies that have the same or substantially the same amino acid sequence as the original antibody, including, but not limited to, biosimilar antibodies approved under 42 U.S.C. §262 subsection (k) in the United States and equivalent regulations in other jurisdictions.

[0314] The PD-1 antagonists listed above are known in the art, along with their respective manufacture, therapeutic uses and properties.

[0315] In one embodiment, the PD-1 antagonist is ezabenlimab.

[0316] In one embodiment, the PD-1 antagonist is pembrolizumab.

[0317] In another embodiment, the PD-1 antagonist is nivolumab.

[0318] In another embodiment, the PD-1 antagonist is pidilizumab.

[0319] In another embodiment, the PD-1 antagonist is atezolizumab.

[0320] In another embodiment, the PD-1 antagonist is avelumab.

[0321] In another embodiment, the PD-1 antagonist is durvalumab.

[0322] In another embodiment, the PD-1 antagonist is PDR-001.

[0323] In another embodiment, the PD-1 antagonist is PD1-1.

[0324] In another embodiment, the PD-1 antagonist is PD1-2.

[0325] In another embodiment, the PD-1 antagonist is PD1-3.

[0326] In another embodiment, the PD-1 antagonist is PD1-4.

[0327] In another embodiment, the PD-1 antagonist is PD1-5.

[0328] Pharmaceutical Composition and Formulation The present invention relates to pharmaceutical compositions for the treatment of diseases (as specified in more detail below), wherein such compositions comprise at least one IL-12 Fc fusion protein of the present invention. The present invention further encompasses methods of treating diseases (as specified in more detail below) using at least one IL-12 Fc fusion protein of the present invention or a pharmaceutical composition as set forth below, and further encompasses the preparation of a medicament for the treatment of such diseases by using such an IL-12 Fc fusion protein or pharmaceutical composition of the present invention.

[0329] The IL-12 Fc fusion proteins of the present invention (e.g., any of those shown in the disclosed sequences) and / or compositions comprising the same can be administered to a patient in need thereof in any suitable manner, depending on the particular pharmaceutical formulation or composition used. Thus, the IL-12 Fc fusion proteins of the present invention and / or compositions comprising the same can be administered in an effective amount or dose, for example, intravenously (iv), subcutaneously (sc), intramuscularly (im), intraperitoneally (ip), transdermally, orally, sublingually (e.g., in the form of a sublingual tablet, spray, or drops placed under the tongue and absorbed through the mucous membrane into the sublingual capillary network), intranasally (e.g., in the form of a nasal spray and / or as an aerosol), topically, using a suppository, by inhalation, or any other suitable manner. The IL-12 Fc fusion proteins can be administered by infusion, bolus, or injection. In a preferred embodiment, administration is by intravenous infusion or subcutaneous injection.

[0330] The IL-12 Fc-fusion proteins of the present invention and / or compositions comprising the same are administered according to a treatment regimen appropriate to treat and / or alleviate the disease, disorder, or condition to be treated or alleviated. A clinician will generally be able to determine an appropriate treatment regimen depending on factors such as the disease, disorder, or condition to be treated or alleviated, the severity of the disease, the severity of its symptoms, the specific binding protein of the present invention used, the specific route of administration and pharmaceutical formulation or composition used, the patient's age, sex, weight, diet, general condition, and similar factors well known to clinicians. Generally, a treatment regimen will involve administration of a therapeutically effective amount or dose of an IL-12 Fc-fusion protein of the present invention or one or more compositions comprising the same.

[0331] Generally, for the treatment and / or alleviation of the diseases, disorders, and symptoms mentioned herein, the IL-12 Fc fusion proteins of the present invention are administered continuously (e.g., by infusion) or more preferably as a single dose (e.g., twice-weekly, weekly, or monthly doses; see below) in an amount between 0.005 and 20.0 mg per kilogram of body weight and dose, preferably between 0.05 and 10.0 mg / kg / dose, depending on the particular disease, disorder, or condition being treated, the potency of the particular IL-12 Fc fusion protein of the present invention used, the particular route of administration, and the particular pharmaceutical formulation or composition used, but this can vary significantly depending, inter alia, on the aforementioned parameters. Thus, in some cases, it may be sufficient to use amounts less than the minimum doses given above, while in other cases the upper limit may have to be exceeded. When administering larger amounts, it may be advisable to divide them into multiple smaller doses spread over the day.

[0332] Depending on the particular IL-12 Fc fusion protein of the present invention and its pharmacokinetic and other properties, it may be administered daily, every two, three, four, five, or six days, weekly, monthly, etc. Dosing regimens may include chronic, weekly treatment. By "chronic" is meant a duration of at least two weeks, preferably several months or years.

[0333] The efficacy of the IL-12 Fc fusion proteins of the present invention and compositions comprising the same can be tested using any suitable in vitro assay, cell-based assay, in vivo assay, and / or animal model known per se, or any combination thereof, depending on the particular disease involved. Suitable assays and animal models will be apparent to those skilled in the art and include, for example, the assays and animal models used in the Examples below.

[0334] For pharmaceutical use, the IL-12 Fc fusion proteins of the present invention can be formulated as pharmaceutical preparations containing (i) at least one IL-12 Fc fusion protein of the present invention (e.g., any one shown in the disclosed sequences) and (ii) at least one pharmacologically acceptable carrier, diluent, excipient, adjuvant, and / or stabilizer, and (iii) optionally one or more additional pharmacologically active polypeptides and / or compounds. By "pharmaceutically acceptable," it is meant that the respective material does not exhibit any biological or other undesirable effects when administered to an individual and does not interact in a deleterious manner with any of the other components (e.g., pharmaceutically active ingredients) of the pharmaceutical composition in which it is contained. Specific examples can be found in standard handbooks, such as Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Company, USA (1990). For example, the IL-12 Fc-fusion proteins of the invention can be formulated and administered in any manner known per se for conventional antibodies and antibody fragments, as well as other pharmaceutically active proteins and fusion proteins. Thus, according to a further embodiment, the present invention relates to pharmaceutical compositions or preparations comprising at least one IL-12 Fc-fusion protein of the invention and at least one pharmaceutically acceptable carrier, diluent, excipient, adjuvant and / or stabilizer, and optionally one or more additional pharmacologically active substances, in the form of a lyophilized or otherwise dried formulation or an aqueous or non-aqueous solution or suspension.

[0335] Pharmaceutical preparations for parenteral administration, such as intravenous, intramuscular, subcutaneous, or intravenous infusion, may be, for example, sterile solutions, suspensions, dispersions, emulsions, or powders containing the active ingredient, optionally suitable for infusion or injection after a further dissolution or dilution step. Suitable carriers or diluents for such preparations include, but are not limited to, sterile water and pharmaceutically acceptable aqueous buffers and solutions, such as physiological phosphate-buffered saline, Ringer's solution, dextrose solution, and Hank's solution; water, oil; glycerol; ethanol; glycols, such as propylene glycol, as well as mineral oil, animal oil, vegetable oil, such as peanut oil, soybean oil, and suitable mixtures thereof.

[0336] The solution of the IL-12 Fc fusion protein of the present invention may also contain preservatives to prevent the growth of microorganisms, such as antibacterial and antifungal agents, for example, p-hydroxybenzoic acid, parabens, chlorobutanol, phenol, sorbic acid, thiomersal, ethylenediaminetetraacetic acid (alkali metal salts), and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers, or sodium chloride. Optionally, emulsifying and / or dispersing agents may be used. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants. Other agents that delay absorption, such as aluminum monostearate and gelatin, may also be added. The solution may be filled into injection vials, ampoules, infusion bottles, etc.

[0337] In all cases, the final dosage form must be sterile, fluid and stable under the conditions of manufacture and storage.Sterile injectable solution is prepared by incorporating the active compound in the required amount in a suitable solvent with various other active ingredients as listed above, as required, followed by filtration sterilization.In the case of sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying technology and freeze-drying technology, which can produce a powder of active ingredient plus any additional desired active ingredient present in the previously sterile-filtered solution.

[0338] Typically, an aqueous solution or suspension will be preferred. Generally, a suitable formulation for a therapeutic protein, such as an IL-12 Fc fusion protein of the present invention, will comprise a buffered protein solution, such as a protein at an appropriate concentration (e.g., 0.001 to 400 mg / ml, preferably 0.005 to 200 mg / ml, more preferably 0.01 to 200 mg / ml, more preferably 1.0 to 100 mg / ml, such as 1.0 mg / ml (iv administration) or 100 mg / ml (sc administration)), and an aqueous buffer, such as:

[0339] - Phosphate buffered saline, pH 7.4,

[0340] - Other phosphate buffers, pH 6.2-8.2,

[0341] Acetate buffer, pH 3.2-7.5, preferably pH 4.8-5.5

[0342] - Histidine buffer, pH 5.5-7.0,

[0343] - succinate buffer, pH 3.2 to 6.6, and

[0344] - Citrate buffer, pH 2.1-6.2,

[0345] And optionally, the solution may contain salts (e.g., NaCl) and / or sugars (e.g., sucrose and trehalose) and / or other polyhydric alcohols (e.g., mannitol and glycerol) to provide isotonicity to the solution.

[0346] Other agents, such as surfactants, e.g., 0.02% Tween-20 or Tween-80, may also be included in such solutions. Formulations for subcutaneous application may contain significantly higher concentrations of the IL-12 Fc fusion protein of the present invention, e.g., up to or even exceeding 100 mg / ml. However, it will be apparent to those skilled in the art that the components and amounts thereof given above represent only one preferred option. Alternatives and variations thereof will be readily apparent to those skilled in the art or can be readily envisioned from the above disclosure. The formulations described above can optionally be provided as lyophilized formulations to be reconstituted in a solution, e.g., water for injection (WFI).

[0347] In accordance with a further aspect of the present invention, the IL-12 Fc fusion proteins of the present invention may be used in combination with a device useful for administering proteins, such as a syringe, injector pen, micropump, or other device.

[0348] kit The present invention also encompasses kits comprising at least an IL-12 Fc fusion protein of the present invention (e.g., any shown in the disclosed sequences) and, optionally, one or more other components selected from the group consisting of other drugs used for the treatment of diseases and disorders as described above.

[0349] In one embodiment, the kit comprises a composition comprising an effective amount of an IL-12 Fc fusion protein of the invention in a unit dosage form.

[0350] The present invention also encompasses kits comprising at least an IL-12 Fc fusion protein of the present invention and one or more other components selected from the group consisting of other drugs used for the treatment of diseases and disorders as described above.

[0351] In one embodiment, the kit comprises a composition comprising an effective amount of an IL-12 Fc fusion protein of the invention in unit dosage form. In a further embodiment, the kit comprises both a composition comprising an effective amount of an IL-12 Fc fusion protein of the invention in unit dosage form and a composition comprising an effective amount of a PD-1 antagonist, such as an anti-PD-1 antibody, most preferably PD1-1, PD1-2, PD1-3, PD1-4, and PD1-5, as described herein (e.g., Table 7) and in WO2017 / 198741, in unit dosage form.

[0352] In some embodiments, the kit comprises a sterile container containing such a composition; such a container may include a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container form known in the art. Such a container may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding pharmaceuticals. Additionally, the kit may comprise a pharmaceutical composition in a first container with an IL-12 Fc fusion protein of the present invention in lyophilized form and a second container with a pharmaceutically acceptable diluent for injection (e.g., sterile water). The pharmaceutically acceptable diluent can be used for reconstitution or dilution of the IL-12 Fc fusion protein.

[0353] If desired, the IL-12 Fc fusion proteins of the present invention are provided with instructions for administering the IL-12 Fc fusion protein to a subject with cancer. The instructions typically include information regarding the use of the composition for the treatment or prevention of cancer. In other embodiments, the instructions include at least one of the following: therapeutic agent description; dosing schedule and administration for the treatment or prevention of cancer or its symptoms; precautions; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical trials; and / or references. The instructions may be printed directly on the container (if present), as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

[0354] Methods of production and purification A further aspect of the present invention is a method of producing an IL-12 Fc fusion protein as described herein, comprising: (a) culturing a host cell of the invention under conditions that allow expression of the molecule; and (b) recovering the molecule; and optionally

[0355] In an embodiment of this aspect of the invention, the method of production further comprises step (c) further purifying and / or modifying and / or formulating the Fc-fusion protein.

[0356] To produce an IL-12 Fc fusion protein, a DNA molecule encoding a first polypeptide chain is inserted into an expression vector, operably linking the sequence to transcriptional and translational control sequences. A DNA molecule encoding a second polypeptide chain can be inserted into the same expression vector or a different expression vector. These vectors contain the usual elements required for expression of a polypeptide in a cell, such as a promoter, regulatory elements, selection elements, etc. After introducing the vector into a suitable host cell, both chains are expressed individually from the vector and secreted by the cell. Both the first and second polypeptide chains then associate via their respective Fc domains to form the complete IL-12 Fc fusion protein.

[0357] To produce an IL-12 Fc fusion protein, one skilled in the art can choose from a wide variety of expression systems known in the art, such as those reviewed by Kipriyanov and Le Gall, Curr Opin Drug DiscovDevel. 2004 Mar;7(2):233-42.

[0358] Expression vectors include plasmids, retroviruses, cosmids, EBV-derived episomes, and the like. Expression vectors and expression control sequences are selected to be compatible with the host cell. As outlined, the first and second polypeptide chain sequences can be inserted into separate vectors. In certain embodiments, both DNA sequences, the first and second polypeptide chain sequences, are inserted into the same expression vector. A convenient vector is one that encodes functionally complete human first and second polypeptide sequences, and into which suitable restriction enzyme sites have been engineered to allow for the easy insertion and expression of any first or second polypeptide sequence, as described above.

[0359] The recombinant expression vector may also encode a signal peptide that facilitates secretion of the polypeptide chain from the host cell. DNA encoding the polypeptide chains may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the mature first and / or second polypeptide chain DNA. The signal peptide may be an immunoglobulin signal peptide or a heterologous peptide from a non-immunoglobulin protein. Alternatively, the DNA sequence encoding the first or second polypeptide chain may already contain a signal peptide sequence.

[0360] In addition to the DNA sequence encoding the IL-12 Fc fusion protein chain, the recombinant expression vector carries regulatory sequences including promoters, enhancers, termination and polyadenylation signals, and other expression control elements that control the expression of the IL-12 Fc fusion protein chain in host cells. Examples of promoter sequences (exemplified for expression in mammalian cells) include CMV (e.g., CMV Simian Virus 40 (SV40) (e.g., the SV40 promoter / enhancer), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), polyoma, and strong mammalian promoters such as the native immunoglobulin and actin promoters. Examples of polyadenylation signals include BGH polyA, SV40 late or early polyA; alternatively, the 3'UTR of an immunoglobulin gene, etc., can be used.

[0361] Recombinant expression vectors may also carry sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The nucleic acid molecules encoding the IL-12 Fc fusion protein chains described herein, and vectors containing these DNA molecules, can be introduced into host cells, e.g., bacterial cells, or higher eukaryotic cells, e.g., mammalian cells, according to transfection methods well known in the art, including liposome-mediated transfection, polycation-mediated transfection, protoplast fusion, microinjection, calcium phosphate precipitation, electroporation, or transfer with viral vectors.

[0362] Preferably, both nucleic acid molecules encoding the IL-12 Fc fusion protein chains described herein are inserted onto one vector that is transfected into a host cell, preferably a mammalian cell.

[0363] Thus, a further embodiment provides a host cell comprising an expression vector comprising a nucleic acid molecule encoding an IL-12 Fc fusion protein chain as described herein.

[0364] Mammalian cell lines available as hosts for expression are well known in the art and include, among others, Chinese hamster ovary cells (CHO, CHO-DG44 cells), NSO cells, SP2 / 0 cells, HeLa cells, baby hamster kidney cells (BHK cells), monkey kidney cells (COS), human cancer cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK cells, HEK293 cells, or derivatives / progeny of any such cell line. Other mammalian cells, including but not limited to, human, mouse, rat, monkey, and rodent cell lines, or other eukaryotic cells, including but not limited to, yeast cells, insect cells, and plant cells, or prokaryotic cells, such as bacteria, may also be used. The IL-12 Fc fusion proteins of the present invention are produced by culturing host cells for a period of time sufficient to allow expression of the IL-12 Fc fusion protein in the host cells.

[0365] The IL-12 Fc fusion proteins described herein are preferably recovered from the culture medium as secreted polypeptides, or they can be recovered from host cell lysates, for example, when expressed without a secretory signal. The IL-12 Fc fusion proteins described herein must be purified using standard protein purification methods used for recombinant proteins and host cell proteins in a manner that results in substantially homogeneous preparations of the IL-12 Fc fusion proteins described herein. For example, state-of-the-art purification methods useful for obtaining the IL-12 Fc fusion proteins of the present invention include, as a first step, the removal of cells and / or particulate cell debris from the culture medium or lysate. The IL-12 Fc fusion protein is then purified from contaminating soluble proteins, polypeptides, and nucleic acids by, for example, fractionation on an immunoaffinity or ion exchange column, ethanol precipitation, reverse-phase HPLC, Sephadex chromatography, chromatography on silica, or on a cation exchange resin. As a final step in the process for obtaining the IL-12 Fc fusion proteins described herein, the purified IL-12 Fc fusion proteins may be dried, e.g., lyophilized, as described below for therapeutic applications.

[0366] It will be appreciated that the immunoglobulin single variable domain molecules, preferably the VHHs described herein, can be produced and purified using the methods described, with appropriate modifications.

[0367] nucleic acid A further aspect of the invention provides an isolated nucleic acid molecule encoding an IL-12 Fc fusion protein chain of the invention and / or an immunoglobulin single variable domain molecule of the invention, or an expression vector comprising such a nucleic acid molecule.

[0368] As can be appreciated by one of skill in the art, nucleic acid molecules can be readily prepared that encode the first and / or second polypeptide chains of an IL-12 Fc fusion protein, or immunoglobulin single variable domain sequences.

[0369] Nucleic acid molecules encoding the first and / or second polypeptide chains of an IL-12 Fc fusion protein, or immunoglobulin single variable domain sequences, can be synthesized chemically and enzymatically by polymerase chain reaction (PCR) using standard methods. First, appropriate oligonucleotides can be synthesized using methods known in the art (e.g., Gait, 1984), which can be used to generate a synthetic gene. Methods for generating synthetic genes from oligonucleotides are known in the art (e.g., Stemmer et al., 1995; Ye et al., 1992; Hayden et Mandecki, 1988; Frank et al., 1987).

[0370] Nucleic acid molecules of the present invention include, but are not limited to, DNA molecules encoding the polypeptide sequences shown in the Sequence Listing. The present invention also relates to nucleic acid molecules that hybridize to DNA molecules encoding the polypeptide sequences shown in the Sequence Listing under high stringency binding and washing conditions, as defined in WO2007 / 042309. Preferred molecules (in terms of mRNA) are those that have at least 75% or 80% (preferably at least 85%, more preferably at least 90%, and most preferably at least 95%) homology or sequence identity with one of the DNA molecules described herein. By way of example, with the IL-12 Fc fusion protein being expressed in eukaryotic cells, the DNA sequences shown in the Sequence Listing are designed to be consistent with the codon usage in eukaryotic cells. If it is desired to express the IL-12 Fc fusion protein in E. coli, these sequences can be altered to be consistent with the codon usage of E. coli. Variants of the DNA molecules of the present invention can be constructed in several different ways, for example, as described in WO2007 / 042309.

[0371] In another embodiment, any of the disclosed IL-12 Fc fusion proteins can be encoded in a suitable mRNA sequence. This mRNA sequence can encode both polypeptide chains of the IL-12 Fc fusion protein, or two separate mRNA molecules can be constructed, each encoding one of the two polypeptide chains, optionally including at least one secretion signal linked to either or both chains. Such mRNA can be used to treat patients for any of the treatments described herein. After delivery of the mRNA to a patient, the mRNA is translated and the polypeptide chains can assemble in the human body to form the complete prodrug.

[0372] Example Materials and Methods The materials and methods used in the examples are described below.

[0373] MMP cleavage assay: Recombinant MMP9 (R&D Systems) is activated using p-aminophenylmercuric acetate. Activated MMP9 is incubated with IL-12 Fc fusion protein (prepared in TCNB buffer: 50 mM Tris, 10 mM CaCl, 150 mM NaCl, 0.05% Brij-35 (w / v), pH 7.5) at 37°C for 24 hours. The digested protein is aliquoted and stored at -80°C prior to testing in SDS-PAGE, Western blot, and IL-12 functional assays.

[0374] MMP cleavage assay (peptide only): Recombinant MMP is activated according to the manufacturer's recommendations (R&D). Activated MMP (2.5 nM final concentration) is then added to Dabcyl / Edans peptide (2.5 μM final concentration) in TCNB buffer. Plates are read using a BioTek Synergy H1 Hybrid Multimode Reader (BioTek Instruments) at excitation 340 nM / emission 490 nM for 2 hours at 37°C with 5-minute intervals. A gain of 80 is used. Specific activity is then calculated based on parameters derived from the kinetic cleavage curve.

[0375] IL-12 functional assay NK-92 IL-12 Activity Assay: NK-92 cells (ATCC CRL-2407) were seeded at a density of 200,000 cells per well in 96-well plates, and 100 μL of medium containing varying concentrations of MMP9-cleaved or uncleaved IL-12 Fc fusion protein was added. The total volume per well was 200 μL, corresponding to a cell density of 100,000 cells / mL. After 24 hours of incubation at 37°C and 5% CO2, the IFN-γ concentration in the cell culture supernatant was determined by ELISA (Invitrogen). An IFN-γ standard was included in the ELISA, and the IFN-γ concentration in the sample was derived from the absorbance at 450 nm (A450) using linear curve fitting with GraphPad Prism 7.0 software. Data were fitted using the agonist vs. response (three-parameter) fit in GraphPad Prism 7.0 software, and the EC 50 Estimate.

[0376] Promega IL-12 Bioassay: The IL-12 Bioassay (Cat. Nos. JA2601 and JA2605) is a bioluminescent cell-based assay designed to measure IL-12 stimulation or inhibition. The assay is performed according to the manufacturer's protocol. Briefly, cells are thawed and resuspended, and 50 μl of the solution is pipetted into a 96-well plate. 25 μl of serially diluted cleaved and uncleaved IL-12 Fc fusion protein is added to the cells, followed by a 6-hour incubation at 37°C. After incubation, the plate is removed from the incubator, and after reaching ambient temperature (10-15 minutes), 75 μl of Bio-Glo™ reagent is added to each well for 5-10 minutes at room temperature, followed by luminescence measurement. Fold induction is calculated using the following formula: Fold induction = RLU (sample - background) / RLU (no drug control - background). Data are plotted using GraphPad Prism software, and EC 50 is determined.

[0377] TME linker binding ELISA: IL-12 Fc fusion protein (in the amounts indicated in the figure legends) is diluted in PBS and added to collagen I-coated 96-well plates (Corning) for 10-30 minutes. The plates are blocked with 2% BSA before protein addition to minimize nonspecific binding. The plates are then washed three times, followed by incubation with biotinylated anti-human Fc antibody (Invitrogen). After an additional washing step, streptavidin-HRP and substrate are added, and the plates are read in a Tecan reader.

[0378] Scar in Jar Assay: Primary human lung fibroblasts were seeded in poly-D-lysine-coated 384 CellCarrier microtiter plates (PerkinElmer) at a density of 1,000 cells per well using FGM-2™ Single Quots (Lonza, Basel, Switzerland) in FBM. After 24 hours, the medium was replaced with the same medium without serum (starvation medium). 48 hours after cell seeding, the starvation medium was replaced with a mixture of Ficoll 70 and 400 (37.5 mg / mL and 25 mg / mL, respectively), 200 μM vitamin C, and IPF-RC (1:1,000 dilution). After 72 hours, the cell culture medium was removed, and the cells were fixed with 100% ice-cold methanol for 30 minutes. The cells were then washed with PBS, the plates were decellularized, and IL-12 Fc fusion protein (5 μg and 50 μg) was added to the wells for 2 hours of incubation. The plates are then washed and blocked with 3% BSA in PBS for 30 minutes. After an additional washing step, collagen I is stained using a monoclonal antibody (SAB4200678, Sigma-Aldrich). For primary antibody detection, the cells are washed and incubated with a goat anti-mouse IgG1 Alexa Fluor 568 secondary antibody for 30 minutes at 37°C. After the final washing step, images are acquired on an Opera Phenix (Perkin Elmer) and transferred to a Columbus Image Storage and Analysis system (Perkin Elmer).

[0379] In vivo model: C57BL / 6 mice are injected with MC38 or B16.F10 cells in the left flank. Treatment is performed until tumors reach approximately 70-100 mm 3Treatment begins when the tumor reaches a median age of 100%. Mice are treated twice per week with up to six injections. The IL-12 Fc fusion protein and the specific protein dose used are provided in the figure. Tumor volume and body weight are monitored two to three times per week. Statistical analysis is performed using GraphPad Prism software. Differences between groups are analyzed using a t-test with or without Welch's correction, depending on the data distribution. Analysis of grouped data is performed using two-way analysis of variance or the Kruskal-Wallis test.

[0380] Stable Pool Creation The final molecule is cloned into a mammalian expression system, with the knob and hole strands encoded on one plasmid and driven by separate CMV promoters and metabolic selection markers. CHO-K1 GS- / - host cells are transfected with each expression plasmid to generate stable pools and store them for cell culture processing.

[0381] Production and purification The final molecules are produced from stably transfected and characterized CHO cell pools in bioreactors. The cell culture process is carried out under controlled conditions. The cell culture harvest is processed in an automated downstream process that includes Protein A capture, acid treatment, cation exchange and anion mixed-mode chromatography polishing, and different filtration steps. The product quality of each construct is then assessed.

[0382] Production and purification Expression is carried out in a 3L bioreactor using a stable transfected CHO cell pool. Cell cultures are harvested after 14 days in culture. Titers are determined using Protein A HPLC. Purification is performed using an automated, representative multi-step process train. Protein A capture with subsequent viral inactivation at low pH is performed, followed by cation exchange and mixed-mode chromatography. The construct is finally concentrated by ultrafiltration / diafiltration.

[0383] Product quality evaluation The purity of all constructs is assessed using state-of-the-art size-exclusion chromatography and non-reducing capillary gel electrophoresis. Product quality is determined by hydrophilic liquid interaction chromatography coupled online to a mass spectrometer (HILIC-MS).

[0384] Overall design of IL-12 fusion proteins The following example lays out a design path for an IL-12 Fc fusion protein. In the context of the examples, the molecules are also sometimes referred to as prodrugs or protease-activatable prodrugs.

[0385] Example 1: Molecular Components Various configurations of protease-activatable prodrugs have been evaluated, but preferably they include the following components:

[0386] 1) The cytokine itself: IL-12 is a heterodimer, consisting of a p40 subunit and a p35 subunit, however, these two domains can be linked together via a connecting peptide linker to form a functional single-chain cytokine.

[0387] 2) A masking moiety that can block the function of the cytokine / payload while it is part of the prodrug, but can also release the cytokine / payload upon enzymatic activation of the prodrug.

[0388] 3) An enzymatically cleavable linker, the sequence composition of which is recognized by an enzyme that is upregulated in the tumor microenvironment.

[0389] 4) Optionally, the prodrug may contain additional components, such as the constant region (Fc) of an antibody, to extend the half-life of the prodrug. The Fc allows for the creation of heterodimeric Fc through techniques such as knob-in-hole. Here, the Fc can act as a heterodimerization domain, allowing cytokine production in one arm and mask production in the other arm "in parallel." Conversely, wild-type Fc can also be used, where the cytokine is directly linked to a masking domain, followed by the Fc, or some combination thereof, to form a symmetrical "in-tandem" prodrug.

[0390] 5) Optionally, the prodrug may include a tumor targeting domain, which may target the prodrug specifically or preferentially to tumor antigens, as an alternative to tumor-associated structures in the vicinity of the tumor, for example. The effect of such a tumor targeting domain may be three-fold: A) anchoring the prodrug within the tumor, allowing increased exposure of the prodrug to upregulated enzyme activity and enhancing the conversion of the prodrug to active drug; or B) anchoring the active cytokine within the tumor, enhancing its retention time and / or half-life within the tumor microenvironment; and C) anchoring the active cytokine within the tumor, reducing potential toxicity associated with systemic exposure.

[0391] Format scouting of prodrug molecules Figures 1A-1H To explore functional inhibition of IL-12 in prodrug form, we have taken the above considerations into account and produced molecules with different orientations and components, and measured the delta EC of IL-12 activity in functional assays. 50 (ΔEC 50 The effect of the prodrug format on its ability to generate an enzymatically cleavable linker was measured and demonstrated by comparing the active drug and the prodrug form after cleavage using, for example, MMP9.

[0392] Eight initial molecules were generated by varying the permutations of the following parameters: a. Fusion of the cytokine and / or masking domain to the N-terminus of the Fc, or optionally to the C-terminus of the Fc. b. Use of an Fc that naturally homodimerizes (wild-type interface), with the cytokine and masking domains in "tandem," or an Fc that has been engineered to heterodimerize, for example using knobs-in-hole technology, with the cytokine remaining on the Fc chain opposite the masking domain "in parallel." c. Use of either a single chain IL-12 or a version in which the p35 domain is directly linked to Fc, the p40 domain is simultaneously co-transfected, and IL-12 forms intracellularly during protein production, and the two domains are covalently linked through a naturally occurring disulfide bond.

[0393] The functional readout of format scouting using the NK-92 cell-based assay is shown in the table below. The masking domain is in each case an scFv tool molecule against IL-12, with an affinity towards IL-12 of around 250 pM.

[0394] [Table 9]

[0395] Example 2: Masking part (VHH generation) Figure 2 The masking moiety can be selected from a wide range of inhibitory molecules against IL-12: receptor fragments, antibodies or antibody fragments (e.g., Fab, scFab, scFv, VHH), or peptides. The masking moiety can be a direct inhibitor of IL-12 activity, where the mask can block IL-12 signaling through its receptor in a functional assay. The masking moiety can also be derived from IL-12 binding or IL-12 fusions, which do not directly inhibit IL-12 but instead indirectly inhibit IL-12 through steric interactions in the context of prodrug assembly.

[0396] Prevents cytokines from signaling systemically, creating an effective ΔEC between the prodrug in the systemic circulation and the active cytokine in the tumor microenvironment 50 was considered a critical feature in designing such molecules.

[0397] We undertook antibody selection to attempt to identify the masking domain. Both in vitro and in vivo techniques were used to generate antibodies against IL-12: phage panning of synthetic or immune-derived VHH-based antibody libraries was performed. Plasma cells were collected from immunized llamas, converted into phage display libraries, and further panned to directly isolate binding fragments. Selection of suitable antibody fragments for the masking domain was based on functional inhibition of IL-12, affinity of binding to IL-12, and creation of a large therapeutic window between the prodrug and activated IL-12, as measured in a Promega IL-12 Bioassay.

[0398] Phage panning of a synthetic VHH-based antibody library was unsuccessful in identifying any functional blockers of IL-12. Although over 50 VHH-based masking domains from the synthetic VHH-based antibody library were tested, none of them showed any functional activity in the Promega IL-12 Bioassay (data not shown), suggesting an additional hurdle for selecting optimal masking molecules.

[0399] From the immunization-derived VHH-based antibody library, 47 initial binders were identified and further characterized.

[0400] [Table 10] TIFF2026504906000062.tif249170 TIFF2026504906000063.tif249170 TIFF2026504906000064.tif39170

[0401] The majority of VHHs showed no functional activity in the Promega IL-12 bioassay. Only one functional VHH binder (a p40 binder) was identified and was further pursued for optimization and humanization (BI-039). Although the other identified VHH binders did not show activity in the functional assay, they still find utility in other applications requiring binding to IL-12.

[0402] The selected masking domain (BI-048) was shown to compete with ustekinumab, a known inhibitor of IL-12 and IL-23, by blocking domain 1 of the p40 domain. Selecting a masking domain specific for the p40 domain was an important consideration in the design of this molecule because human IL-12 does not signal through the mouse IL-12 receptor; however, chimeric IL-12 utilizing human IL-12 p40 and mouse IL-12 p35 can signal. Therefore, a masking domain specific for the p40 domain of IL-12 enabled the creation of an alternative prodrug without the need for species cross-reactivity of the masking domain itself. This masking domain was able to maintain its potency and efficacy against IL-12 through three rounds of humanization. Both the selected VHH and its humanized variant exhibited an affinity of 3.5 nM and an IC of approximately 500 pM. 50 (Table 10).

[0403] [Table 11]

[0404] Example 3: MMP Expression and Cleavable Linkers Figures 10A-10F MMPs are often tightly regulated systemically by their natural inhibitors, tissue inhibitors of metalloproteinases (otherwise known as TIMPS). However, within the tumor microenvironment, not only are MMP transcripts upregulated, but their activity is often supported by a decrease in TIMP activity, thus allowing for higher MMP activity within the tumor itself. Expression levels of various MMPs and TIMPs in various human tumors were assessed and are presented in Figures 10A-10F. Expression of MMPs, particularly MMP2, MMP9, and MMP13, was clearly upregulated in tumor tissue compared with adjacent normal or healthy organs. Furthermore, in many tissues, TIMPs (especially TIMP3) were strongly upregulated in healthy tissue compared with tumor tissue, suggesting higher MMP activity within tumors compared with normal tissue.

[0405] In addition to expression levels, it was necessary to evaluate the activity of MMPs to confirm the functionality of these enzymes in biological samples. For this purpose, tumor samples (and adjacent normal) were collected from patients who underwent surgical tumor resection. The samples were evaluated for the expression levels of various MMPs and in functional assays to confirm the proteolytic activity of the enzymes. As shown in Table 11, MMP2 and MMP9 were abundantly expressed in tumor tissues. MMP2 and MMP9 were also up-regulated in some adjacent normal tissues; however, this expression level did not correlate with enzyme activity, probably due to the activity of inhibitory proteins (TIMPs). In contrast to MMP2 and MMP9, MMP12 and MMP13 were almost exclusively expressed in tumor tissues.

[0406] Table 11: Patient data are shown, comparing MMP activity in tumor samples vs. adjacent normal tissue from patients who underwent tumor resection. Individual patient data are shown in pairs, i.e., tumor and normal samples from the same patient are presented directly below each other. MMP expression levels are shown in pg / mg lysate, and specific activity is expressed as pmol (of cleaved peptide) / min / μg lysate. [Table 12]

[0407] There was intra-patient variability in expression of various MMPs, as shown in Table 11. Having a cleavable linker that is reactive against several MMPs, such as MMP-2, -9, and -13, can help mitigate the relative differences in upregulation at the patient-to-patient level; therefore, broadly cleavable peptides were preferred.

[0408] As presented in Table 12, linkers that can be included in fusion proteins should have broad specificity, thereby reducing patient-to-patient variability in MMP expression. To this end, several short peptides were tested for their MMP-mediated cleavage specificity. Due to its broad specificity, peptide 5 was used in the Fc fusion construct.

[0409] [Table 13]

[0410] Example 4: Generation of masked IL-12 Fc fusion protein Based on the initial format scouting, screening and optimization of the masking moiety, and selection of a suitable cleavable linker, several optimized masked IL-12 Fc fusion proteins were generated and further tested.

[0411] [Table 14] TIFF2026504906000069.tif249170 TIFF2026504906000070.tif252170 TIFF2026504906000071.tif249170 TIFF2026504906000072.tif250170 TIFF2026504906000073.tif249170 TIFF2026504906000074.tif34170

[0412] Example 5: Masking domains - Functional activity in a molecular context - in vitro Figures 3A-3B and 4 The prodrug efficiently inhibited IL-12 signaling while in the prodrug context, but effectively signaled upon MMP9 cleavage, both in in vitro assays as well as in in vivo experiments (see Example 6 for in vivo).

[0413] IL-12 routinely has an EC of approximately 15 pM in the Promega IL-12 bioassay. 50 This value was independent of whether IL-12 was provided as purified recombinant IL-12 or whether IL-12 was released from a prodrug in the presence of MMP9. Figure 3A shows chimeric single-chain IL-12 (human p40-GS linker-mouse p35) (BI-066) in the Promega IL-12 bioassay.

[0414] The prodrug BI-057 was also tested in this assay. After MMP9 digestion, the cleaved prodrug BI-057 had an EC of approximately 16 pM in the Promega IL-12 bioassay. 50 was shown (Figure 3B).

[0415] In the absence of MMP9 digestion, the molecular EC 50 The prodrug activity was in the range of approximately 5 nM, indicating a more than 280-fold functional shift compared to recombinant IL-12 (Figure 4). In summary, the presented data suggest efficient masking of IL-12 in the prodrug form and full functionality of the cytokine while cleaved from the original molecule.

[0416] Example 6: Masking domains - Functional activity in a molecular context: - In vivo Figures 5A~5D, 6A~6B, 7A~7B, 8A~8B, and 9A~9C In an in vivo setting, a prodrug from an early format scouting experiment with the configuration shown in Figure 1B was tested and showed an improved safety profile compared to unmasked IL-12. The prodrug from this early format scouting experiment was based on a heterodimeric Fc (knobs-in-holes) with a single-chain chimeric IL-12 (human p40-GS linker-mouse p35) attached to the C-terminus of one Fc chain and a masking domain for the human p40 domain (scFv) attached to the other Fc chain. A cleavable linker was positioned between the Fc and p40 domains.

[0417] As shown in Figures 5A and 5C, the chimeric IL-12 Fc fusion protein inhibited tumor growth in a dose-dependent manner in the non-immunogenic and aggressive B16.F10 melanoma model. The B16.F10 model is also characterized by relatively low MMP activity, thus presenting an additional hurdle for the tested molecules. Efficient blockade of IL-12 functionality in the prodrug format was confirmed by the lack of body weight loss even at a dose of 2 mg / kg, 2000x higher than the toxic dose in mice (in molar equivalents) (Figure 5D). In contrast, the unmasked chimeric IL-12 Fc fusion protein (a fusion protein containing all components but lacking the blocking moiety) caused severe toxicity in the majority of animals already at a concentration of 0.08 mg / kg (Figure 6A). Higher concentrations (1.6 mg / kg, which is the molar equivalent of a 2 mg / kg dose of masked chimeric IL-12 Fc fusion protein) led to severe weight loss in treated animals, which resulted in death in all treated animals by day 8 (Figure 6B). This data further confirmed the efficient masking of IL-12 while in prodrug form in the periphery. It also suggests that the molecule remains in prodrug form while in the bloodstream, increasing the therapeutic index of the molecule.

[0418] The safety and efficacy of the chimeric IL-12 Fc fusion protein was further evaluated in the MC38 model, which confers high MMP activity. Figure 7A shows dose-dependent tumor growth inhibition without weight loss, a surrogate for toxicity (Figure 7B).

[0419] A similar trend was observed in the B16.F10 model (Figure 8A-B), which contained much lower MMP activity within the tumor, but the tumor growth inhibitory effect was indeed lower, which correlated with the lower MMP profile of this tumor. Importantly, no signs of toxicity were observed at the concentration used (2.8 mg / kg), further confirming the efficient masking properties and lack of systemic IL-12-mediated inflammation.

[0420] To further confirm this, the levels of inflammatory cytokines in the serum of MC38-bearing mice treated with chimeric IL-12 Fc fusion protein were assessed one day after the second treatment. Inflammatory cytokines were undetectable in the serum of treated mice, suggesting a lack of systemic toxicity and confirming an efficient masking effect (Table 14).

[0421] [Table 15]

[0422] To further confirm the IL-12 inhibitory properties of the masking molecule, nonhuman primates (NHPs) were administered human IL-12 Fc fusion protein (BI-051) at doses up to 3 mg / kg. NHPs remained healthy without clinical signs of toxicity. No increases in ALT, bilirubin, or creatinine were detected, suggesting efficient masking of IL-12 in the periphery in the context of the prodrug molecule (Figures 9A-9C).

[0423] Example 7: Cleavage site cleaved by MMPs - Functional activity in molecular context - in vitro: Figures 11, 12, 13A-13E, 14A-14B MMP-cleavable linkers incorporated into the prodrugs are typically susceptible to cleavage by proteases activated within the tumor microenvironment. For the purpose of functional characterization of the prodrugs, recombinant MMP9 was used to cleave and activate IL-12 from the prodrugs.

[0424] As shown in Figure 11, activated MMP9 efficiently cleaved the prodrugs (BI-050, BI-051, BI-052, BI-054, and BI-055) as demonstrated by SDS-PAGE. The release of IL-12 was further confirmed by Western blot (Figure 12) and MS analysis (not shown) of another prodrug molecule (BI-062).

[0425] To further investigate the functionality of the prodrug-releasing IL-12, a cell-based Promega IL-12 Bioassay was used. Proteolytically cleaved and mock-treated prodrug molecules were serially diluted and added to reporter cells to measure IL-12 activity. As shown in Figures 13A-13E, all molecules tested (BI-050, BI-051, BI-052, BI-054, and BI-055) showed a lower EC upon cleavage compared to the intact molecules. 50 All molecules had comparable EC values ​​upon cleavage, suggesting efficient release of biologically active IL-12. 50 values ​​were reached, but significantly different EC values ​​of mock-treated molecules. 50 The masking properties of these otherwise very similar constructs are also significant, with significant differences in masking properties (Table 15).

[0426] ΔEC 50 Variants were evaluated using a ELISA. The constructs tested were closely related to each other but differed in the placement of the collagen I tumor-retention peptide, either at the N-terminus of IL-12 (BI-050), the C-terminus of IL-12 (BI-051), or in the IL-12 internal linker connecting the p40 and p35 domains (BI-052). Similarly, a lower-affinity masking domain differed from BI-051 by only two amino acids in the HCDR3 of the mask, and showed reduced masking capacity with all other components identical. Finally, construct BI-055 was also based on BI-051 but additionally contained the S354C / Y349C CH3-stabilizing disulfide, often used to drive knob-in-hole heterodimerization, and showed similar masking capacity.

[0427] The data show the ΔEC 50 The results clearly demonstrated that the study had a direct impact on the safety profile and therefore had implications for the safety profile.

[0428] [Table 16]

[0429] To further evaluate the feasibility of IL-12 release from the prodrug in a more clinically relevant setting, lysates from human tumor tissues were prepared and used as a source of MMP. After 2 hours of incubation of the chimeric molecule BI-059 with the lysate, the protein was evaluated by Western blot. As shown in Figures 14A-14B, BI-059 was efficiently cleaved to a level comparable to that of activated MMP9 using human tumor lysates. This data demonstrated the feasibility of MMP-mediated cleavage and cytokine release in the tumor microenvironment.

[0430] Example 8: Cleavage site cleaved by MMPs - Functional activity in molecular context - in vivo: Figures 15 and 16A-16F The IL-12 cytokine stimulates T cells and NK cells, which produce IFNγ in response to this stimulation. As shown in Table 14, there were no detectable levels of IFNγ in the periphery upon treatment with the chimeric IL-12 Fc fusion protein, suggesting the efficient masking properties of this molecule. In further experiments using the same IL-12 Fc fusion protein tested in Example 6, we investigated whether this treatment could result in detectable levels of IFNγ within tumors. To this end, MC38-bearing animals were treated twice with the IL-12 Fc fusion protein (2.8 mg / kg), and tumors were harvested one day after the second treatment. As shown in Figure 15, there was a significant increase in intratumoral IFNγ in treated animals compared with vehicle-injected mice, suggesting protease-mediated release of functional IL-12.

[0431] To further elucidate the effects of active IL-12 released at the tumor site, changes within the TME were examined upon treatment with the chimeric Fc fusion protein, as described above. Significant changes were observed in lymphoid and myeloid cell infiltration upon treatment. As shown in Figures 16A-16F, treatment with the chimeric prodrug molecule resulted in a significant influx of CD3 T cells (Figure 16A), and particularly CD8 T cells (Figure 16B). These T cells exhibited an activated phenotype, as indicated by CD25 expression (Figure 16C). A significant increase in M1-like macrophages was also observed in treated animals (Figure 16D), which was accompanied by a decrease in M2-like macrophages (Figure 16E). Consequently, an increased M1 / M2 ratio was observed (Figure 16F), which is a sign of beneficial changes within the tumor microenvironment. Note that Figures 16C, 16E, and 16F use a logarithmic scale to more clearly illustrate the results.

[0432] Example 9: TME-retaining linkers - functional activity in the in vitro molecular context: 17A-17I, 18A-18B, 19, and 20A-20D The primary impetus for the prodrug approach was to limit IL-12's systemic toxicity, enabling a greater safety profile, while leveraging the cytokine's potent antitumor properties. This was initially achieved through the creation of prodrugs with tumor-activatable IL-12 release. However, IL-12 itself has a relatively short half-life (<9 hours), and additional concerns may arise regarding the cytokine escaping the tumor microenvironment. Due to the low levels of IL-12 required to induce a toxic response, the addition of peptide sequences to further trap the cytokine within the tumor microenvironment was explored.

[0433] The TME is rich in ECM proteins, such as collagen I, collagen IV, or fibronectin, which could potentially serve as anchors for other fusion proteins. ECM proteins have been found to be upregulated in many tumor types (Figures 17A-17I). Therefore, it was hypothesized that incorporating a tumor retention peptide onto IL-12 could allow for higher retention of IL-12, leading to longer exposure of immune cells to the cytokine and providing additional efficacy. In addition, linkers that bind to ECM proteins may also provide benefits for prodrugs. Circulating prodrugs with a TME linker fused to IL-12 could potentially increase the retention time of the prodrug within the TME. The immediate impact of this would be longer exposure of the prodrug to enzymatic degradation, increasing the likelihood that the prodrug will be converted to an active drug.

[0434] To test the peptide's ability to bind to ECM proteins while in the context of an Fc fusion protein, the binding of BI-051 to collagen I was assessed. As shown in Figure 18A, dose-dependent binding of BI-051 to collagen I-coated plates was observed. This was further confirmed in SPR experiments, as shown in Figure 18B. To achieve cytokine retention at tumor sites, a tumor-retention linker was added to the cytokine (part of the knob chain). To further confirm that the TME-binding linker conveys binding properties, the knob and hole chains of the molecule were tested in separate ELISA-based assays on collagen I-coated plates. As shown in Figure 19, the knob chain containing the TME linker peptide bound to collagen I-coated plates in a dose-dependent manner and to a significantly higher extent than the hole chain lacking the TME linker peptide.

[0435] To further elucidate the binding of BI-051 to collagen I under more physiological conditions, we used a scar in a jar assay. In this assay, fibroblasts were stimulated to produce different matrix proteins (mainly collagen I in this assay) in a morphology closely resembling the native extracellular matrix. To the plates thus prepared, a fluorescently labeled IL-12 Fc fusion protein was added to assess its binding to collagen I. As shown in Figure 20B, BI-051 was clearly detectable along collagen I fibers, suggesting binding of the molecule to this ECM protein.

[0436] Example 10: Molecular in vivo data 21A-21B, 22, 23A-23B, and 24A-24B The chimeric molecule was further tested in an in vivo setting. Due to the very low content of collagen I in syngeneic models (data not shown), in contrast to human tumors, testing of collagen-binding motifs in these models remains elusive. In contrast, fibronectin expression in syngeneic models allowed testing of the TME linker concept in tumor-bearing mice. To this end, a chimeric IL-12 Fc fusion protein (BI-059) containing a fibronectin-binding motif was tested in the MC38 model. As shown in Figure 21A, BI-059 significantly inhibited tumor growth compared with both vehicle and the IL-12 Fc fusion without a TME linker (BI-065). Tumor growth inhibition led to a significant prolongation of survival in BI-059-treated animals, with 50% of the animals still alive at the end of the experiment (Figure 21B).

[0437] To further elucidate the ability of the TME-binding linker to improve the antitumor efficacy and / or safety of IL-12 Fc fusions, BI-059 was tested in the difficult-to-treat B16.F10 melanoma model. This model is characterized by low MMP activity and significantly lower levels of fibronectin (8x according to expression levels, data not shown). As shown in Figure 22, despite the low MMP activity and low fibronectin expression, BI-059 animals controlled tumor growth significantly better in this aggressive model compared to animals treated with IL-12 Fc fusion proteins without a TME linker.

[0438] As mentioned above, syngeneic models are characterized by poor stromal compartments, and collagen I expression is usually restricted to the outer membrane of these tumors. Nevertheless, a significant delay in tumor formation was observed in BI-057-treated animals bearing MC38 tumors (Figure 23A). 4 / 10 animals in this group controlled tumors for a longer period compared with 2 / 10 animals in the group receiving IL-12 Fc fusion protein lacking the TME linker. Importantly, there was a significant ΔEC difference between the cleaved and uncleaved molecules. 50 In accordance with the in vitro data showing no signs of toxicity were observed in these animals (Figure 23B).

[0439] BI-057 was further tested in the B16.F10 model to further evaluate its efficacy (due to extremely low levels of collagen I expression, this model was not suitable for testing the benefits of TME linkers). As shown in Figure 24A, BI-057 treatment resulted in a significant delay in tumor growth compared to vehicle-treated animals. Consistent with previously presented data, there were no signs of toxicity in treated animals, as indicated by a lack of weight loss (Figure 24B).

[0440] These data demonstrated that molecules containing TME-retaining linkers were effective and safe in these models. In an environment where ECM proteins are widely present, as is typical for human tumors, it is expected that TME linkers may contribute even more significantly to both the efficacy and safety of the proposed molecules.

[0441] Example 11: In a further approach, genetically engineered models characterized by increased levels of ECM proteins in tumors are used to further analyze the effect of IL-12 Fc fusion proteins, particularly the TME linker, on the retention, efficacy, mechanism of action, and safety profile of IL-12 Fc fusion proteins. To this end, such genetically engineered models are treated with IL-12 Fc fusion proteins, and the retention, efficacy, mechanism of action, and safety profile of the TME linker are evaluated.

[0442] In yet another approach, different tumor models are chosen, typically with different expression levels (low, medium, high expression) of ECM proteins, such as collagen, and the models are treated with IL-12 Fc fusion proteins to assess efficacy, potency, toxicity, and / or retention.

[0443] Example 12: Product Quality Different molecules (Table 16) were generated and differed in the type and position of the tumor-retaining linker and the VHH masking domain used. The molecules were produced in small-scale stable cell pools under controlled, representative conditions. The results are summarized in Table 17. Expression in small-scale bioreactors was successful, and harvesting was performed on day 14 for all constructs. BI-051, BI-053, BI-054, and BI-055 had similar upstream process performance. The productivity and overall process performance of BI-052-expressing cells was lower, and additional species were observed in size-exclusion chromatography of harvested samples. Similarly, BI-050 showed heterogeneous size exclusion, indicating instability of the construct.

[0444] The expressed constructs were then purified downstream as described and product quality assessed (Table 18).

[0445] BI-050 and BI-052 had elevated low molecular weight species, which were partially unidentifiable, indicating instability of the constructs during manufacturing. BI-054 showed instability in process intermediates. BI-051 and BI-055 showed overall good product quality and reasonable manufacturability. However, during product quality analysis (HILIC-MS), BI-055 was observed to fragment due to molecular instability as a result of the introduction of additional stabilizing disulfide bonds, making the molecule more rigid and prone to fragmentation.

[0446] Table 16 Overview of variant molecules. The molecules differ in their choice of TME linker, their position in the molecule relative to IL-12, and the VHH masking domain used. [Table 17]

[0447] Table 17 Molecular process performance in upstream processing [Table 18]

[0448] Table 18 Downstream process performance and final molecule product quality. [Table 19]

[0449] Example 13: In vitro comparison of molecules containing the collagen I TME linker and those lacking it. Figures 25A-25B-26A-26B Collagen-binding properties were further evaluated in an ELISA assay. Chimeric IL-12 Fc fusion protein BI-065 or chimeric IL-12 Fc fusion protein BI-057 containing a collagen I TME linker was added to collagen I-coated plates. Plates were coated with either rat collagen I (Figure 25A) or human dermal collagen I (Figure 25B).

[0450] Briefly, high-binding microplates (greiner) are coated overnight at 4°C with rat collagen (Corning) or human collagen (Millipore / R&D) diluted in coating buffer (Invitrogen) with a concentration of 0.005 mg / mL. IL-12 Fc fusion protein is diluted in PBS + 1% BSA and added to the collagen-precoated plate for 2 hours. The plate is blocked with 1% BSA before the addition of the protein to minimize nonspecific binding. The plate is then washed three times, followed by incubation with biotinylated anti-human Fc antibody (Invitrogen). After an additional washing step, streptavidin-HRP is added to the plate. Substrate is added to the plate after the washing step, and the plate is read in an ELISA reader (Tecan).

[0451] We observed dose-dependent binding of BI-057 to collagen-coated plates, with EC values ​​of 26 nM and 75 nM for rat and human collagen, respectively. 50 BI-065 did not bind to collagen-coated plates, and increased binding was observed only at the highest concentration.

[0452] To further evaluate the collagen-binding properties of the IL-12 Fc fusion protein, precision-cut liver sections from fibrotic rat livers were used. Syngeneic tumor models, in contrast to human tumors, consist of only very limited amounts of collagen, and therefore, fibrotic tissues comprise a more relevant collagen structure.

[0453] Briefly, tissue cores were cut from fibrotic rat livers using a 5 mm cylindrical electric tissue coring press (Alabama R&D, MD5000), and 300 μM thick tissue slices were cut using a tissue slicer (Alabama R&D, MD6000). All tissue slices were cultured in 1.3 mL supplemented William's Medium E (Life Technologies) in a humidified incubator at 95% O2 / 5% CO2 at 37°C using a suspension culture system (Nunc) in a suspension culture system (60 rpm). IL-12 Fc fusion protein was added to the slices and incubated for 2 or 24 hours. After 2 hours of incubation, the slices were transferred to fresh William's Medium E and incubated for an additional 22 hours as a washing step before harvesting. After incubation, the slices were washed three times with Williams' Medium E to remove unbound IL-12 Fc fusion protein. Single slices are collected in Lysis Matrix tubes (flash-frozen) for harvesting. The harvested sections are lysed using MSD Tris Lysis Buffer (MSD) and homogenized for 2 x 30 seconds at 6800 rpm using a PrecellysEvolution Homogenizer (Bertin Technologies). A subsequent centrifugation step is performed to remove cellular debris. The amount of IL-12 Fc fusion protein in the homogenate supernatant is determined using the MSD U-PLEX Biomarker Assay and, as a standard, IL-12 Fc fusion protein diluted in MSD Lysis buffer (serial dilution 1:2). For IL-12 Fc fusion protein detection, MSD Gold Small Spot Streptavidin plates are coated with biotinylated anti-IL12 mouse capture antibody, and anti-IL12 human antibody with a sulfotag is used as the detection antibody. MSD Read Buffer is added before the plate is read using an MSD microplate reader.

[0454] The chimeric IL-12 Fc fusion protein BI-065 or the chimeric IL-12 Fc fusion protein BI-057 containing a collagen I TME linker was added to precision-cut liver slices for 2 or 24 hours. After 2 hours, the slices were thoroughly washed and cultured in culture medium for an additional 22 hours. As shown in Figure 26A, there was a dose-dependent recovery of the IL-12 Fc fusion protein containing a collagen I TME linker. The amount of IL-12 Fc fusion protein BI-065 recovered from the slices remained at background levels and was more than 10x lower than the amount of BI-057, suggesting binding of the latter molecule to collagen present in fibrotic liver. Similar results were also observed when the slices were cultured in the presence of BI-057 or BI-065 for 2 hours, then thoroughly washed and left in culture for an additional 22 hours. As shown in Figure 26B, there was a dose-dependent recovery of the chimeric IL-12 Fc fusion protein BI-057 containing a collagen I TME linker, which was 10-fold higher than the recovery of the chimeric IL-12 Fc fusion protein BI-065, suggesting increased retention of BI-057 in slices compared to BI-065.

[0455] Example 14: In vivo comparison of molecules containing the collagen I TME linker and those lacking it. Figures 27A-27D-28A-28B The retention of molecules containing the collagen I TME linker was further evaluated in vivo in syngeneic models. It is important to emphasize that syngeneic models, in contrast to human tumors, contain very low levels of collagen. As a result, it remains difficult to observe the full potential of the collagen I TME linker in these models. To assess tumor retention in vivo, we used fluorescence measurements of Dylight650-labeled chimeric IL-12 Fc fusion protein BI-201 or chimeric IL-12 Fc fusion protein BI-202 containing the collagen I TME linker.

[0456] Briefly, fusion proteins are labeled using DyLight650 (Thermo Fisher Scientific). Excess dye is removed using a spin column with Purification Resin (Thermo Fisher Scientific), and the degree of labeling for each protein is calculated. Proteins compared in pharmacokinetic and retention studies contain equimolar amounts of dye. To assess protein retention, mice are imaged using an IVIS under an autoexposure epifluorescence setting. During this time, mice are maintained on a low-chlorophyll diet (Altromin) to minimize gastrointestinal background fluorescence. Image analysis to determine total radiant efficiency is performed using Living Image (Perkin Elmer).

[0457] Mice bearing KPCY tumors were intravenously injected with 30 μg of protein, and fluorescence was measured at different time points. As shown in Figure 27A, up to 24 hours, the amount of protein measured by fluorescence was similar in both groups of animals. From 48 hours onward, there was an increased level of fluorescence in animals injected with BI-202, a chimeric IL-12 Fc fusion protein containing a collagen I TME linker. Fluorescence levels at the end of the experiment (144 hours) in the group injected with BI-202 were significantly higher than in the group injected with BI-201 (Figure 27B). Pharmacokinetic measurements (based on fluorescence data) also indicated a significantly reduced clearance of BI-202 compared to BI-201, suggesting a trend toward an increased half-life (Figures 27C and D).

[0458] Retention of human molecules was also evaluated in EMT6 gene-carrying animals. Mice were intravenously injected with 50 μg of Dylight650-labeled human IL-12 Fc fusion protein BI-200 or human IL-12 Fc fusion protein BI-051 containing a collagen I TME linker. Fluorescence was measured at different time points. As shown in Figure 28A, already 24 hours after injection, there was increased fluorescence in mice injected with BI-051, with a peak at 30 hours. At this time point, there was a significant difference in the level of fluorescence between mice injected with BI-051 and BI-200, suggesting increased retention of the former (Figure 28B).

[0459] Example 15: In vivo effect of collagen I TME linkers on molecule functionality / safety. Figures 29A-29B and 30A-30B To evaluate the effect of the collagen I TME linker on molecule function / safety, mice bearing PDA30364 pancreatic tumors were injected intratumorally with 150 pmol of chimeric IL-12 Fc fusion protein (BI-065) or chimeric IL-12 Fc fusion protein containing the collagen I TME linker (BI-057). Blood was collected 48 hours post-injection, and IL-12-induced cytokines / chemokines were assessed to determine the safety profile of the injected molecules.

[0460] Briefly, cytokines in serum are measured using the LegendPlex Mouse Cytokine Release Syndrome Panel (13-plex) (BioLegend), a bead-based immunoassay. Each bead in the multiplex can be differentiated by size and internal fluorescence intensity. Beads are coated with specific antibodies on their surface and serve as capture beads for specific analytes. The premixed beads are incubated with samples or serially diluted premixed standards for two hours. To determine the concentration of a specific analyte after a washing step, a biotinylated detection antibody cocktail is added. The detection antibody binds to the specific analyte bound on the capture beads, thus forming a capture bead-analyte-detection antibody sandwich. Streptavidin-phycoerythrin (SA-PE) is then added, which binds to the biotinylated detection antibody. The fluorescence signal intensity is proportional to the amount of bound analyte. LegendPlex is measured on a BD LSR Fortescue Cell Analyzer. The concentration of the specific analyte is determined using a standard curve. Analyses are performed using FlowJo (LLC) and GraphPad Prism (GraphPad Software Inc.).

[0461] The cytokine implicated in IL-12-related adverse effects is IFNγ. As shown in Figure 29A, injection of BI-065 resulted in a 20-fold increase in peripheral IFNγ levels compared to animals treated with BI-057. CXCL10, an IFNγ-inducible chemokine, was also increased approximately 10-fold in BI-065-treated animals compared to mice injected with BI-057.

[0462] In a further step, the effect of the chimeric molecule on IFNγ induction was evaluated in animals bearing orthotopic mammary EMT6 tumors. To this end, animals were intravenously injected with different doses of either a chimeric IL-12 Fc fusion protein (BI-065) or a chimeric IL-12 Fc fusion protein containing a collagen I TME linker (BI-057). Blood was collected 24 hours (Figure 30A) and 72 hours (Figure 30B) after injection, and IFNγ levels were determined. As shown in Figure 30A, already 24 hours after injection, there was a clear trend toward increased IFNγ levels in mice treated with BI-065, which reached statistical significance at the moderate dose tested. A better safety profile of BI-065, defined by reduced IFNγ levels, was also observed at later time points (Figure 30B).

[0463] [Table 20] TIFF2026504906000081.tif221170

Claims

1. 1. An interleukin-12 (IL-12) Fc fusion protein comprising a first polypeptide chain and a second polypeptide chain, a) the first polypeptide chain comprises a first Fc domain, and the IL-12p35 and IL-12p40 subunits of IL-12; and b) the second polypeptide chain comprises a second Fc domain and a masking moiety that binds to the IL-12p35 and / or IL-12p40 subunits in the first polypeptide chain; wherein the first and second polypeptide chains are linked via the first Fc domain and the second Fc domain; wherein the IL-12p35 subunit or the IL-12p40 subunit is linked to the C-terminus of the first Fc domain via the first peptide linker, the first peptide linker being protease-cleavable; wherein the masking moiety is linked to the C-terminus of said second Fc domain via a second linker, preferably a peptide linker; and An interleukin-12 (IL-12) Fc fusion protein wherein the first or second polypeptide chain further comprises a binding moiety selected from the group consisting of a collagen-binding moiety, a heparin-binding moiety, and a fibronectin-binding moiety.

2. 2. The IL-12 Fc fusion protein of claim 1, wherein the binding moiety is linked to the C-terminus of the IL-12p35 subunit or the C-terminus of the IL-12p40 subunit, or the binding moiety is linked to the C-terminus of the masking moiety, in each case optionally via a third polypeptide linker.

3. 3. The IL-12 Fc fusion protein of claim 1, wherein the binding moiety is located between the IL-12p35 subunit and the IL-12p40 subunit, or the binding moiety is located between the C-terminus of the first Fc domain and the N-terminus of the IL-12p35 subunit or the N-terminus of the IL-12p40 subunit, and in either case the binding moiety may optionally be flanked on one or both sides by a linker or multiple linkers, preferably peptide linkers.

4. The IL-12 Fc fusion protein of any one of claims 1 to 3, wherein the binding moiety is a collagen binding moiety.

5. 5. The IL-12 Fc fusion protein of claim 4, wherein the collagen binding moiety binds to collagen I.

6. 6. The IL-12 Fc fusion protein of claim 5, wherein the collagen binding moiety binds to collagen I and has the sequence LxxLxLxxN (SEQ ID NO: 41), where L is leucine and N is asparagine and x is any amino acid.

7. 7. The IL-12 Fc fusion protein of claim 6, wherein the collagen binding portion has a length of 20 amino acids (aa), 19 aa, 18 aa, 17 aa, 16 aa, 15 aa, 14 aa, 13 aa, 12 aa, 11 aa, 10 a, or 9 aa.

8. 8. The IL-12 Fc fusion protein of claim 1, wherein the collagen-binding portion comprises or consists of any one of the amino acid sequences of SEQ ID NOs: 40-47.

9. 4. The IL-12 Fc fusion protein of claim 1, wherein the binding moiety is a heparin-binding moiety.

10. 10. The IL-12 Fc fusion protein of claim 9, wherein the heparin binding portion has the sequence VRIQRKKEKMKET (SEQ ID NO: 50).

11. 5. The IL-12 Fc fusion protein of claim 4, wherein the collagen binding moiety binds to collagen IV.

12. 12. The IL-12 Fc fusion protein of claim 11, wherein the collagen binding portion has the sequence KLWVLPK (SEQ ID NO: 40).

13. The IL-12 Fc fusion protein of any one of claims 1 to 3, wherein the binding moiety is a fibronectin binding moiety.

14. 14. The IL-12 Fc fusion protein of claim 13, wherein the fibronectin binding moiety has the sequence GGWSHW (SEQ ID NO: 49).

15. 15. The IL-12 Fc fusion protein of any one of claims 1 to 14, wherein the IL-12 p35 subunit and the IL-12 p40 subunit are human.

16. 16. The IL-12 Fc fusion protein of any one of claims 1 to 15, wherein the IL-12p35 subunit comprises a polypeptide having at least 95% identity to SEQ ID NO: 1, and the IL-12p40 subunit comprises a polypeptide having at least 95% identity to SEQ ID NO: 2, preferably wherein the IL-12p35 subunit comprises the polypeptide of SEQ ID NO: 1, and the IL-12p40 subunit comprises the polypeptide of SEQ ID NO:

2.

17. 17. The IL-12 Fc fusion protein of any one of claims 1 to 16, wherein the IL-12p40 subunit and the IL-12p35 subunit are linked in a single chain having the configuration IL-12p40-IL-12p35 or IL-12p35-IL-12p40.

18. 18. The IL-12 Fc fusion protein of claim 17, wherein the single-chain IL-12p40-IL-12p35 is linked to the C-terminus of the first Fc domain via its IL-12p40 subunit, or the single-chain IL-12p35-IL-12p40 is linked to the first Fc domain via its IL-12p35 subunit, and in both cases via the first peptide linker, which is protease-cleavable.

19. 18. The IL-12 Fc fusion protein of claim 17, wherein the IL-12p40 subunit and the IL-12p35 subunit are linked to each other via a linker that is rich in glycine and serine amino acid residues, preferably having a length of 5 to 20 amino acids, and containing only glycine and serine amino acids, more preferably a glycine and serine linker having the amino acid sequence of SEQ ID NO:

22.

20. 18. The IL-12 Fc fusion protein of claim 17, wherein the single chain IL-12p40-IL-12p35 comprises a polypeptide having at least 95% identity to SEQ ID NO:8, or the single chain IL-12p35-IL-12p40 comprises a polypeptide having at least 95% identity to SEQ ID NO:

9.

21. 21. The IL-12 Fc fusion protein of any one of claims 1 to 20, wherein the second peptide linker is not protease cleavable.

22. 22. The IL-12 Fc fusion protein of any one of claims 1 to 21, wherein the masking moiety binds to the mIL-12p40 subunit and is selected from the group consisting of an IL-12 receptor or an IL-12p40-binding fragment thereof, an scFv, or an immunoglobulin single variable domain, preferably a VHH.

23. 23. The IL-12 Fc fusion protein of any one of claims 1 to 22, wherein the first and second Fc domains each comprise one or more mutations that promote heterodimerization of the Fc domains.

24. 24. The IL-12 Fc fusion protein of claim 23, wherein (a) the first Fc domain is a human IgG1 Fc domain comprising the mutation T366W and the second Fc domain is a human IgG1 Fc domain comprising the mutations T366S, L368A, and Y407V, or (b) the first Fc domain is a human IgG1 Fc domain comprising the mutations T366S, L368A, and Y407V and the second Fc domain is a human IgG1 Fc domain comprising the mutation T366W.

25. 25. The IL-12 Fc fusion protein of any one of claims 1 to 24, wherein the first and second Fc domains are human IgG1 Fc domains, and one of the first or second Fc domains comprises the mutations H435R and Y436F.

26. 26. The IL-12 Fc fusion protein of any one of claims 1 to 25, wherein the first and second Fc domains are human IgG1 Fc domains, and either the first Fc domain, or the second Fc domain, or both Fc domains comprise the mutations L234A and L235A.

27. 27. The IL-12 Fc fusion protein of any one of claims 1 to 26, wherein the first Fc domain comprises the amino acid sequence of SEQ ID NO: 15 and the second Fc domain comprises the amino acid sequence of SEQ ID NO: 16, or the first Fc domain comprises the amino acid sequence of SEQ ID NO: 17 and the second Fc domain comprises the amino acid sequence of SEQ ID NO: 18, or the first Fc domain comprises the amino acid sequence of SEQ ID NO: 16 and the second Fc domain comprises the amino acid sequence of SEQ ID NO: 15, or the first Fc domain comprises the amino acid sequence of SEQ ID NO: 18 and the second Fc domain comprises the amino acid sequence of SEQ ID NO:

17.

28. 28. The IL-12 Fc fusion protein of any one of claims 1 to 27, wherein the protease-cleavable linker is cleavable by a matrix metalloprotease (MMP), preferably MMP-2, MMP-9, or MMP-13.

29. 29. The IL-12 Fc fusion protein of claim 28, wherein the protease-cleavable linker comprises or consists of any one of the amino acid sequences of SEQ ID NOs: 232-241.

30. 1. An IL-12 Fc fusion protein comprising a first polypeptide chain and a second polypeptide chain, a) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 208, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 209; b) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 210, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 211; c) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 212, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 213; d) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 214, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 215; e) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 216, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 217; f) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 218, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 219; g) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 220, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 221; h) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 222, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 223; i) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 224 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 225; j) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 226, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 227; k) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 228, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 229; l) the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 230, and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO: 231; or m) An IL-12 Fc fusion protein, wherein the first polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:242 and the second polypeptide chain comprises or consists of the amino acid sequence of SEQ ID NO:

243.

31. 30. The IL-12 Fc fusion protein of any one of claims 1 to 29, wherein the masking moiety comprises an IL-12 binding immunoglobulin single variable domain comprising three CDRs comprised within any one of the sequences of SEQ ID NOs: 61 to 109.

32. 32. The IL-12 Fc fusion protein of claim 31, wherein the immunoglobulin single variable domain comprises any one of the amino acid sequences of SEQ ID NOs: 61-109.

33. 33. A cleavage product capable of binding to the human IL-12 receptor, comprising the IL-12 cytokine after proteolytic cleavage of the cleavable linker as defined in any one of the IL-12 Fc fusion proteins of claims 1 to 32.

34. 34. The cleavage product of claim 33, comprising the IL-12 cytokine and the binding moiety.

35. 35. The cleavage product of claim 34, comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, or 242 after proteolytic cleavage of the cleavable linker.

36. An IL-12 binding immunoglobulin single variable domain comprising the three CDRs contained within any one of the sequences SEQ ID NOs: 61 to 109.

37. 37. The IL-12-binding immunoglobulin single variable domain of claim 36, wherein the immunoglobulin single variable domain is a VHH.

38. 37. The IL-12-binding immunoglobulin single variable domain of claim 36, wherein the immunoglobulin single variable domain comprises the amino acid sequence of any one of SEQ ID NOs: 61 to 109.

39. 39. A nucleic acid encoding at least one polypeptide of an IL-12 Fc fusion protein according to any one of claims 1 to 32, or encoding one of the polypeptide chains of an IL-12 Fc fusion protein according to any one of claims 1 to 32, or encoding an IL-12-binding immunoglobulin single variable domain according to any one of claims 36 to 38.

40. 40. The vector comprising the nucleic acid of claim 39, optionally wherein the vector comprises nucleic acid encoding both chains of said IL-12 Fc fusion protein.

41. 41. A host cell comprising the nucleic acid of claim 39 or the vector of claim 40, optionally wherein the cell comprises one or more nucleic acids encoding both chains of said IL-12 Fc fusion protein.

42. 42. A method of producing an IL-12 Fc fusion protein, comprising culturing the host cell of claim 41 under conditions to produce said IL-12 Fc fusion protein, and optionally purifying said IL-12 Fc fusion protein.

43. A composition comprising the IL-12 Fc fusion protein of any one of claims 1 to 32.

44. A pharmaceutical composition comprising the IL-12 Fc fusion protein of any one of claims 1 to 32 and a pharmaceutically acceptable carrier.

45. A kit comprising the IL-12 Fc fusion protein of any one of claims 1 to 32, or the composition of claim 43, or the pharmaceutical composition of claim 44.

46. IL-12 Fc fusion protein as defined in any one of claims 1 to 32 for use in medicine.

47. A cleavage product as defined in any one of claims 33 to 35 for use in medicine.

48. 33. A method of treating or reducing the incidence of cancer in a subject, comprising administering to the subject an effective amount of an IL-12 Fc fusion protein of any one of claims 1 to 32.

49. 33. The IL-12 Fc fusion protein of any one of claims 1 to 32 for use in the treatment or prevention of cancer.

50. Use of an IL-12Fc fusion protein according to any one of claims 1 to 32 for the manufacture of a medicament.

51. 33. Use of an IL-12 Fc fusion protein according to any one of claims 1 to 32 for the manufacture of a medicament for reducing the incidence of or treating cancer.

Images