IL-12 Fc fusion proteins

Abstract

The present invention relates to an IL-12Fc fusion protein and a use thereof in a medicament, a pharmaceutical composition comprising the IL-12Fc fusion protein, and a method of treating and / or preventing cancer using the IL-12Fc fusion protein as a medicament.

Description

TECHNICAL FIELD

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

[0002] Interleukin-12 (IL-12) is a cytokine with demonstrated anti-tumor potential that has shown promising preclinical efficacy in mouse tumor models. However, drug-related toxicities were observed in clinical trials, resulting in suboptimal IL-12 dosing regimens and lack of efficacy in patients.

[0003] To overcome drug-related toxicities, it was suggested to mask IL-12 to prevent systemic activity and toxicity, creating a therapeutic window. Masking the activity of IL-12 can be followed by local activation by, for example, fusing a domain of the IL-12 receptor to IL-12 via a protease cleavable linker and by protease-mediated IL-12 receptor removal at the tumor site of a cancer patient.

[0004] Currently, there is no approved IL-12-based therapy. Therefore, there is still a high unmet need for providing novel therapeutic IL-12-based biological molecules that can be used for the treatment of cancer. SUMMARY

[0005] 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 the IL-12p40 subunit in the first polypeptide chain; wherein the first polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which is protease cleavable, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the 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 another embodiment related to the IL-12 Fc fusion protein according to the first aspect or any of the embodiments thereof, the binding moiety is linked to the C-terminus of the IL-12 p35 subunit or the C-terminus of the IL-12 p40 subunit, or the binding moiety is linked to the C-terminus of the masking moiety, and in each case optionally via a third polypeptide linker.

[0007] In another embodiment related to the IL-12 Fc fusion protein according to the first aspect or any of the embodiments thereof, the binding moiety is located between the IL-12 p35 subunit and the IL-12 p40 subunit, or the binding moiety is located between the C-terminus of the first Fc domain and the N-terminus of the IL-12 p35 subunit or the N-terminus of the IL-12 p40 subunit, and in either case the binding moiety can optionally be flanked on one or both sides by one or more linkers, preferably peptide linkers.

[0008] In another embodiment related to the IL-12 Fc fusion protein according to the first aspect or any of the embodiments thereof, the binding moiety is a collagen binding moiety.

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

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

[0011] In another embodiment related to the IL-12 Fc fusion protein according to the first aspect or any of the embodiments thereof, the collagen binding moiety 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 aa, or 9 aa.

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

[0013] In another embodiment related to the IL-12 Fc fusion protein according to the first aspect or any of the embodiments thereof, the binding moiety is a heparin binding moiety.

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

[0015] In another embodiment related to the IL-12 Fc fusion protein according to the first aspect or any of the embodiments thereof, the collagen-binding moiety binds to collagen IV.

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

[0017] In another embodiment related to the IL-12 Fc fusion protein according to the first aspect or any of the embodiments thereof, the binding moiety is a fibronectin-binding moiety.

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

[0019] In another embodiment related to the IL-12 Fc fusion protein according to the first aspect or any of the embodiments thereof, the IL-12 p35 subunit and the IL-12 p40 subunit are human.

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

[0021] In another embodiment related to the IL-12 Fc fusion protein according to the first aspect or any of the embodiments thereof, the IL-12 p40 subunit and the IL-12 p35 subunit are connected in a single chain with the following configuration / arrangement IL-12 p40— IL-12 p35 or IL-12 p35— IL-12 p40 (written from N- to C-terminus).

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

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

[0024] In another embodiment related to the IL-12 Fc fusion protein according to the first aspect or any one of the embodiments thereof, 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 another embodiment related to the IL-12 Fc fusion protein according to the first aspect or any one of the embodiments thereof, the second peptide linker is not protease cleavable.

[0026] In another embodiment related to the IL-12 Fc fusion protein according to the first aspect or any one of the embodiments thereof, 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, a scFv, or an immunoglobulin single variable domain, preferably a VHH.

[0027] In another embodiment related to the IL-12 Fc fusion protein according to the first aspect or any one of the embodiments thereof, the first Fc domain and the second Fc domain each comprise one or more mutations promoting heterodimerization of the Fc domain.

[0028] In another embodiment related to the IL-12 Fc fusion protein according to the first aspect or any of the embodiments thereof, (a) the first Fc domain is a human IgGl Fc domain comprising the mutations T366W and the second Fc domain is a human IgGl Fc domain comprising the mutations T366S, L368A, and Y407V, or (b) the first Fc domain is a human IgGl Fc domain comprising the mutations T366S, L368A, and Y407V and the second Fc domain is a human IgGl Fc domain comprising the mutations T366W.

[0029] In another embodiment related to the IL-12 Fc fusion protein according to the first aspect or any of the embodiments thereof, the first Fc domain and the second Fc domain are human IgGl Fc domains, and one of the first Fc domain or the second Fc domain comprises the mutations H435R and Y436F.

[0030] In another embodiment related to the IL-12 Fc fusion protein according to the first aspect or any of the embodiments thereof, the first Fc domain and the second Fc domain are human IgGl Fc domains, and one of the first Fc domain or the second Fc domain or both Fc domains comprise the mutations L234A and L235A.

[0031] In another embodiment related to the IL-12 Fc fusion protein according to the first aspect or any of the embodiments thereof, 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 another embodiment related to the IL-12 Fc fusion protein according to the first aspect or any of the embodiments thereof, the protease-cleavable linker is cleavable by a matrix metalloprotease (MMP), preferably MMP-2, MMP-9, or MMP-13.

[0033] In another embodiment related to the IL-12 Fc fusion protein according to the first aspect or any of the embodiments thereof, 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 application relates to an IL-12 Fc fusion protein comprising a first polypeptide chain and a second polypeptide chain, wherein 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) 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 another embodiment related to the IL-12 Fc fusion protein according to the first aspect or any of the embodiments thereof, the masking moiety comprises an IL-12 binding immunoglobulin single variable domain comprising the three CDRs contained within any of the sequences of SEQ ID NOs: 61-109.

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

[0037] In a third aspect, the present application relates to a cleavage product capable of binding to a human IL-12 receptor, the cleavage product comprising an IL-12 cytokine upon proteolytic cleavage of the cleavable linker of any of the IL-12 Fc fusion proteins of the preceding aspect and related embodiments thereof.

[0038] In another embodiment related to the cleavage product according to the third aspect or any of the embodiments thereof, the cleavage product comprises the IL-12 cytokine and the binding moiety.

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

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

[0041] In another embodiment related to the IL-12 binding immunoglobulin single variable domain according to the fourth aspect or any of the embodiments thereof, the immunoglobulin single variable domain is a VHH.

[0042] In another embodiment related to the IL-12 binding immunoglobulin single variable domain according to the fourth aspect or any of the embodiments thereof, the immunoglobulin single variable domain comprises the amino acid sequence of any of SEQ ID NOs: 61-109.

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

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

[0045] In a seventh aspect, the present application 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 the IL-12 Fc fusion protein.

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

[0047] In a ninth aspect, the present application relates to a composition comprising the IL-12 Fc fusion protein of the preceding aspect or any of the embodiments related thereto.

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

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

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

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

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

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

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

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

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

[0058] Figures 1A-1H : Exemplary formats for assembling IL-12 Fc fusion proteins with cleavable IL-12 (p35 in kidney shape, p40 in three separate spheres), antibody fragment masking domain, cleavable linker to link IL-12 to Fc (light gray with asterisks), and knob-in-hole or wild-type Fc.

[0059] Figure 2 : Functional characterization of 47VHH-Fc discovered via llama immunization followed by subsequent phage panning. Only one of the constructs showed >90% IL12 inhibition over control molecule in Promega Bioassay.

[0060] Figures 3A-3B : Serial dilutions of single chain chimeric IL-12 (BI-066) (Figure 3A) or MMP9-cleaved chimeric IL-12 Fc fusion protein (BI-057) (Figure 3B) were added to Promega IL-12 bioassay cells. After incubation, bio Bio-Glo TMReagents and luminescence was measured. Data was analyzed in GraphPad Prism and EC 50 .

[0061] 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. Subsequently, both samples were serially diluted and added to Promega IL-12 bioassay cells. After incubation, Bio-Glo TM Reagents and luminescence was measured. Data was analyzed in GraphPad Prism and EC 50 values were calculated.

[0062] Figures 5A-5D : C57B1 / 6 mice were injected with B16.F10 melanoma cells. When tumors reached a volume of 70-100 mm 3 , treatment was initiated. Animals were treated with vehicle or chimeric IL-12 Fc fusion protein at the doses indicated in the legend. Treatment schedule is depicted by the dashed line. Tumor growth (Figures 5A and 5C) and body weight changes (Figures 5B and 5D) were monitored twice per week and presented as spaghetti plots depicting individual mice.

[0063] Figures 6A-6B : C57B1 / 6 mice were injected with B16.F10 melanoma cells. When tumors reached a volume of 70-100 mm 3 , treatment was initiated. Animals were treated with vehicle or non-masked chimeric IL-12 Fc fusion protein at the doses indicated in the legend (Figure 6A: 0.08 mg / kg; Figure 6B: 1.6 mg / kg). Treatment schedule is depicted by the dashed line. Body weight changes were monitored twice per week and presented as spaghetti plots depicting individual mice.

[0064] Figures 7A-7B : C57B1 / 6 mice were injected with MC38 colon carcinoma cells. When tumors reached a volume of 70-100 mm 3 , treatment was initiated. Animals were treated with vehicle or chimeric IL-12 Fc fusion protein at the doses indicated in the legend. Treatment schedule is depicted by the dashed line. Tumor growth (Figure 7A) and body weight changes (Figure 7B) were monitored twice per week and presented as the mean values of the groups.

[0065] Figures 8A-8B : C57B1 / 6 mice were injected with B16.F10 melanoma cells. When tumors reached a volume of 70-100 mm 3treatment. Animals were treated with vehicle or chimeric IL-12 Fc fusion protein at the doses indicated in the legend. Treatment schedule is delineated by the dashed line. Tumor growth (Figure 8A) and body weight changes (Figure 8B) were monitored twice a week and presented as spaghetti plots depicting individual mice.

[0066] Figures 9A-9C : On day 1, cynomolgus monkeys were injected with three different doses of human IL-12 Fc fusion protein. Blood was collected on day 1 (pre-dose), day 8 and day 15. Values for ALT (Figure 9A), bilirubin (Figure 9B) and creatinine (Figure 9C) for each animal are shown on the graphs. Dotted lines delineate reference values.

[0067] Figures 10A-10F : Gene expression of MMP2 (Figure 10A), MMP9 (Figure 10B), MMP13 (Figure 10C), TIMP1 (Figure 10D), TIMP2 (Figure 10E), TIMP3 (Figure 10F) in normal and cancer tissues. White fill, thin line, GTEX normal tissue corresponds to TCGA cancer tissue, white fill, thick line, TCGA adjacent normal tissue, gray fill, thick line, TCGA cancer tissue.

[0068] Figure 11 : 5 variants show that the parent molecule is efficiently cleaved into the component IL-12 and Fc masking domain upon addition of MMP9.

[0069] Figure 12 : MMP9 treatment using anti-p40 Western antibody shows that the single chain IL12 is released from the complete prodrug with the released IL-12 migrating to a ~62 kD band in reducing environment. Detection antibodies used in the analysis are: anti-IL12 p40 (R&D Systems, AF309) 1:2500; anti-goat IgG HRP (R&D Systems, HAF017) 1:1000.

[0070] 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. Subsequently, all samples were serially diluted and added to Promega IL-12 bioassay cells. After incubation, Bio-Glo TM reagent was added and luminescence was measured. Data was analyzed in GraphPad Prims and EC50 values were calculated (see Table 15).

[0071] Figures 14A-14B: 2.5 mM of BI-059 was incubated with buffer control, 6.5 nM (0.025 pg) of activated recombinant human MMP9 or 5 pg 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 using antibodies against human Fc (Figure 14A) and human IL12p40 (Figure 14B) showed a size shift after cleavage of the full-length IL-12 Fc fusion protein by MMPs, corresponding to the released masking domain / Fc fragment and free IL-12, respectively.

[0072] Figure 15 : C57B1 / 6 mice were injected with MC38 colon cancer cells. When tumors reached 70-100 mm 3 in volume, treatment was initiated. Animals were treated twice with vehicle or chimeric IL-12 Fc fusion at the doses indicated in the legend on day 1 and day 4. Animals were sacrificed on day 5 and tumors were collected. Tissues were digested followed by IFNy assessment.

[0073] Figures 16A-16F : C57B1 / 6 mice were injected with MC38 colon cancer cells. When tumors reached 70-100 mm 3 in volume, treatment was initiated. Animals were treated twice with vehicle or chimeric IL-12 Fc fusion at the doses indicated in the legend on day 1 and day 4. Animals were sacrificed on day 5 and tumors were collected. Tissues were digested followed by flow cytometry evaluation of tumor infiltrating leukocytes for marker expression as indicated.

[0074] Figures 17A-17I : Gene expression of Collagen I Al (Figure 17A), Collagen I A2 (Figure 17B), Fibronectin (Figure 17C), Collagen IV Al (Figure 17D), Collagen IV A2 (Figure 17E), Collagen IV A3 (Figure 17F), Collagen IV A4 (Figure 17G), Collagen IV A5 (Figure 17H), Collagen IV A6 (Figure 171) in normal and cancer tissues. White fill, thin line, GTEX normal tissue corresponds to TCGA cancer tissue, white fill, thick line, TCGA adjacent normal tissue, gray fill, thick line, TCGA cancer tissue.

[0075] 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 wash step, biotinylated anti-human Fc Ab was used to detect bound protein. SA-HRP was added to the wells followed by substrate and OD was measured in a Tecan plate reader (Figure 18A). OD values are presented. A Biacore T200 equipped with a CM5 chip was used to measure the binding of human IL-12 Fc fusion protein BI-051 to collagen I (Figure 18B).Figure 18B )Interaction analysis was performed where human collagen type 1 (Merck, CC050) was amine coupled (3000 RU) and reference cells were amine coupled without any ligand. BI-051 and mAb lgG1 (negative control) were diluted to a final concentration of 5 mM in Biacore running buffer (phosphate buffered saline, pH 7.4, containing 0.05% Tween-20). Biacore measurements were performed using reference subtraction. Interactions only occurred on the active surface of immobilized human collagen type 1.

[0076] Figure 19 : Independent chains (knob chain / lobe chain and hole chain / cave chain) containing 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, biotinylated anti-human Fc Ab was used to detect bound protein. SA-HRP was added to the wells followed by substrate and OD was measured in a Tecan plate reader. OD values are presented.

[0077] Figures 20A-20D: Plates coated with fibroblast-produced collagen I were prepared as described in the Materials and Methods section. After the decellularization process, 5 pg of fluorescently labeled human IL-12 Fc fusion protein (BI-051) was added to the wells (Figure 20B). Wells incubated with PBS (Figure 20A) served as a control. After a washing step, the wells were stained with anti-collagen I antibody (Figures 20A and 20C) and visualized in the Opera Phenix system. Figure 20D

[0078] Figures 21A-21B: C57B1 / 6 mice were injected with MC38 colon cancer cells. When tumors reached 70-100 mm 3 in volume, treatment was initiated. 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 dashed line. Tumor growth was monitored twice per week and is presented as spaghetti plots depicting individual mice (Figure 21A). The percentage of surviving mice is presented in (Figure 21B). Figure 21B

[0079] Figure 22 : C57B1 / 6 mice were injected with B16.F10 melanoma cells. When tumors reached 70-100 mm 3 ​​Treatment was initiated when tumors reached 70-100 mm3in volume. 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 1.5 mg / kg. Treatment schedule is delineated by the dashed line. Tumor growth was monitored twice weekly and presented as spaghetti plots delineating individual mice.

[0080] Figures 23A-23B MC38 colon carcinoma cells were injected into C57B1 / 6 mice. When tumors reached 70-100 mm3in volume, treatment was initiated. 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. Treatment schedule is delineated by the dashed line. Tumor growth (Figure 23A) and body weight changes (Figure 23B) were monitored twice weekly and presented as spaghetti plots delineating individual mice. 3 MC38 colon carcinoma cells were injected into C57B1 / 6 mice. When tumors reached 70-100 mm3in volume, treatment was initiated. 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. Treatment schedule is delineated by the dashed line. Tumor growth (Figure 23A) and body weight changes (Figure 23B) were monitored twice weekly and presented as spaghetti plots delineating individual mice.

[0081] Figures 24A-24B MC38 colon carcinoma cells were injected into C57B1 / 6 mice. When tumors reached 70-100 mm3in volume, treatment was initiated. 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. Treatment schedule is delineated by the dashed line. Tumor growth (Figure 23A) and body weight changes (Figure 23B) were monitored twice weekly and presented as spaghetti plots delineating individual mice. 3 MC38 colon carcinoma cells were injected into C57B1 / 6 mice. When tumors reached 70-100 mm3in volume, treatment was initiated. 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. Treatment schedule is delineated by the dashed line. Tumor growth (Figure 23A) and body weight changes (Figure 23B) were monitored twice weekly and presented as spaghetti plots delineating individual mice.

[0082] 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 wash step, biotinylated anti-human Fc Ab was used to detect bound protein. SA-HRP was added to the wells followed by substrate and OD was measured in a Tecan plate reader. OD values are presented.

[0083] Figures 26A-26B: Precision-cut liver slices were prepared from fibrotic rat livers. IL-12 Fc fusion proteins (BI-057 or BI-065; at the amounts indicated in the legend) were added to the slices and incubated for 24 hours (Figure 26A) or 2 hours followed by a 22 hour recovery period (Figure 26B). After the 2 hour incubation, the fusion proteins were washed thoroughly and incubated in culture media for an additional 22 hours, after which the slices were harvested. After 24 hours, the slices were collected, lysed and homogenized. The amount of IL-12 Fc fusion protein recovered from the slices was determined using the MSD U-PLEX BioMarker Assay using IL-12 Fc fusion protein as a standard.

[0084] Figures 27A-27D : C57B1 / 6 albino mice were injected subcutaneously with KPCY pancreatic cancer cells. Animals were injected intravenously with chimeric IL-12 Fc fusion protein containing collagen I TME linker (BI-202) or Dylight 650 labeled chimeric IL-12 Fc fusion protein (BI-201) at a dose of 30 pg. To assess the kinetics of protein retention, mice were imaged with IVIS at the indicated time points under auto-exposure drop-in illumination fluorescence settings (Figure 27A). Image analysis was performed using Living Image to determine total radiant efficiency. Statistical analysis at the final experimental day was performed using the Mann-Whitney test (Figure 27B). Half-life (Figure 27C) and clearance (Figure 27D) were calculated using the fluorescence data.

[0085] Figures 28A-28B : Balb / c mice were injected subcutaneously with EMT6 breast cancer cells. Animals were injected intravenously with chimeric IL-12 Fc fusion protein containing collagen I TME linker (BI-051) or Dylight 650 labeled chimeric IL-12 Fc fusion protein (BI-200) at a dose of 50 pg. To assess the kinetics of protein retention, mice were imaged with IVIS at the indicated time points under auto-exposure drop-in illumination fluorescence settings (Figure 28A). Image analysis was performed using Living Image to determine total radiant efficiency. Statistical analysis at 30 hours was performed using the Mann-Whitney test (Figure 27B).

[0086] Figures 29A-29B : C57B1 / 6 mice were injected subcutaneously with PDA30364 pancreatic cancer cells. Animals were injected intratumorally with chimeric IL-12 Fc fusion protein (BI-065) or chimeric IL-12 Fc fusion protein containing collagen I TME linker (BI-057) at a dose of 150 pmol. Blood was collected 48 hours post-injection and levels of IFNy (Figure 29A) and CXCL10 (Figure 29B) were determined by LegendPlex. Statistical analysis was performed using the Mann-Whitney test.

[0087] Figures 30A-30B C57B1 / 6 mice were injected in situ with EMT6 mammary carcinoma cells. When tumors reached 50-120 mm3in volume, treatment was initiated. Animals were treated with vehicle or chimeric IL-12 Fc fusion protein (BI-065) or chimeric IL-12 Fc fusion protein with collagen ITME linker (BI-057) at the doses indicated in the legend. Blood was collected 24 hours (Figure 30A) or 72 hours (Figure 30B) after injection and levels of IFNy were determined by LegendPlex. Statistical analysis was performed using the Mann-Whitney test. 3 C57B1 / 6 mice were injected in situ with EMT6 mammary carcinoma cells. When tumors reached 50-120 mm3in volume, treatment was initiated. Animals were treated with vehicle or chimeric IL-12 Fc fusion protein (BI-065) or chimeric IL-12 Fc fusion protein with collagen ITME linker (BI-057) at the doses indicated in the legend. Blood was collected 24 hours (Figure 30A) or 72 hours (Figure 30B) after injection and levels of IFNy were determined by LegendPlex. Statistical analysis was performed using the Mann-Whitney test. DETAILED DESCRIPTION

[0089] The present inventors set out to design conditionally active IL-12 fusion proteins that would allow systemic administration of IL-12, known to be toxic, to patients for the treatment of tumors. In doing so, a number of challenges needed to be overcome, including selecting an appropriate molecular design, finding an appropriate way to block the activity of IL-12 to allow systemic administration, aligning chemical, manufacturing and control (CMC) properties with molecular function and ensuring that IL-12 reaches the tumor and subsequently restores its activity within or near the tumor.

[0090] The present invention is based on the concept of providing 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 the IL-12p40 subunit in the first polypeptide chain; wherein the first polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which is protease-cleavable, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second peptide linker, and wherein the first polypeptide chain or the 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.

[0091] The Fc-based fusion protein approach is selected to increase the half-life of the fusion protein following systemic administration. The Fc fusion protein comprises two polypeptide chains. The first polypeptide chain comprises a first Fc domain and subunits IL-12p35 and IL-12p40 of IL-12 that collectively form an active IL-12 cytokine and is connected 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 connected to the C-terminus of the second Fc domain. The two polypeptide chains are together dimerized via their respective Fc domains to form a dimeric Fc fusion protein, i.e., the polypeptide chains are connected via the binding of the two Fc domains that together form the Fc portion of the fusion protein. In this dimeric Fc fusion protein, the masking moiety on the second chain binds to the IL-12 cytokine on the first chain and thereby blocks, inhibits, or attenuates 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.

[0092] Following 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 proposed that tumor-specific activation is then achieved via a dual mechanism involving the protease-cleavable linker and the binding moiety. The protease-cleavable linker is preferably cleaved by a protease that has tumor-specificity or is 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 to allow optimal bioactivity within the TME without dose-limiting systemic toxicity.

[0093] Maintaining the IL-12 Fc fusion protein within the TME can have other advantages, such as enabling the fusion protein to be exposed to the environment of the upregulated protease activity in the TME for a longer period, which can increase the cleavage efficiency and ultimately increase the level of the cleavage product to be released. Also, after cleavage, the cleavage product can be retained within the tumor, which can increase the potency of the response by preventing, reducing, or delaying the cleavage product from entering the circulation as well as reducing systemic toxicity.

[0094] In one aspect, the unmasked IL-12 provides a potent Thl polarizing stimulus to T cells at the tumor site to improve their effector function.

[0095] In another aspect, the IL-12 Fc fusion protein has improved pharmacokinetic and / or toxicology properties compared to unmasked IL-12. In another aspect, the IL-12 Fc fusion protein has improved pharmacokinetic and / or toxicology properties compared to other masked IL-12 fusion proteins.

[0096] In another aspect, the IL-12 Fc fusion protein can be produced as a stable molecule with high process performance and productivity.

[0097] In another aspect, the IL-12 Fc fusion can have tumor agonistic / agnostic properties, i.e., can be useful for treating several cancers. In a related aspect, the IL-12 Fc fusion protein can be suitable for immunomodulatory treatment of cancer or tumors.

[0098] In one aspect, the cleavage product of the IL-12 Fc fusion protein prevents, reduces, or delays entry into circulation after cleavage in the TME.

[0099] In another aspect, the cleavage product of the IL-12 Fc fusion protein shows increased retention within the tumor or TME. In a related aspect, the cleavage product of the IL-12 Fc fusion protein shows increased potency response. In a related aspect, the cleavage product of the IL-12 Fc fusion protein shows reduced systemic toxicity.

[0100] In another aspect, 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 lower than the IL-12 activity of the IL-12 Fc fusion protein after cleavage of the cleavable linker (is at most 1 / 50, 1 / 75, 1 / 100, 1 / 125, 1 / 150, 1 / 175, 1 / 200, 1 / 225, 1 / 250, 1 / 275, 1 / 300, 1 / 325, 1 / 350, 1 / 375, 1 / 400, 1 / 425, 1 / 450, 1 / 475, 1 / 500, 1 / 525, 1 / 550, 1 / 575, or 1 / 600 of the IL-12 activity of the IL-12 Fc fusion protein after cleavage of the cleavable linker). In other words, the difference in EC50 between the uncleaved IL-12 Fc fusion protein (e.g., as measured in the Promega IL-12 bioassay described in the Examples) and the cleaved 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. 50 Uncleaved IL-12 Fc fusion protein: EC50 50 Cleaved IL-12 Fc fusion protein), e.g., as measured in the Promega IL-12 bioassay described in the Examples, 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 lower than the EC50 of the cleaved IL-12 Fc fusion protein. 50 Cleaved IL-12 Fc fusion protein), e.g., as measured in the Promega IL-12 bioassay described in the Examples, 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 lower than the EC50 of the cleaved IL-12 Fc fusion protein.

[0101] Definitions

[0102] Terms not specifically defined herein have the meanings that would be given to them by a person skilled in the art in light of the disclosure and context, unless defined otherwise herein. However, as used in the present specification, the following terms are intended to have the meanings presented below and are appended with the following provisions.

[0103] As used herein, the term "comprising" and variations thereof, such as "comprises" and "comprise", are to be construed as if the term "including" or "containing" or "having" were used instead.

[0104] Unless the context requires otherwise, the term "sequence" (e.g., such as the terms "heavy chain / light chain sequence", "antibody sequence", "variable domain sequence", "constant domain sequence", or "protein sequence") as used herein is generally understood to include the relevant amino acid sequence as well as the nucleic acid sequence or nucleotide sequence encoding it, unless the context requires a more limited interpretation.

[0105] The "Fc domain" of an antibody is not directly involved in binding of the antibody to antigen, but exhibits various effector functions. "Fc part / domain of an antibody" is a term well known to the person skilled in the art and is defined on the basis of papain cleavage of antibodies. Depending on the amino acid sequence of the constant domain of the heavy chain, antibodies or immunoglobulins are assigned to the following classes: IgA, IgD, IgE, IgG, and IgM. Depending on the heavy chain constant domain, the different classes of immunoglobulins are called a, δ, ε, γ, and μ, respectively. Several of these can be further divided into subclasses (isotypes), e.g. IgGl, IgG2, IgG3, and IgG4, IgAl and IgA2. The Fc part of an antibody is directly involved in ADCC (antibody-dependent cell-mediated 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 the complement factor Clq to the Fc part of most IgG antibody subclasses. While the influence of antibodies on the complement system depends on certain conditions, binding to Clq is caused by a defined binding site within the Fc part. Such binding sites are, for example, L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to EU numbering (Edelman et al., Proc Natl Acad Sci U S A. 1969 May; 63(1): 78-85)). Of these residues, the most critical in mediating Clq and Fcy receptor binding in IgGl are L234 and L235 (Hezareh et al., J. Virology 75 (2001) 12161-12168, Shields et al. (2001) JBC, 276(9): 6591-6604). Antibodies of the subclasses IgGl and IgG3 usually show complement activation and Clq and C3 binding, while IgG2 and IgG4 do not activate the complement system and do not bind Clq and C3.

[0106] A "single-chain Fv" or "scFv" antibody fragment is a single-chain Fv variant comprising the VH and VL domains of an antibody, where the domains are present in a single polypeptide chain. Single-chain Fv is capable of recognizing and binding antigen. The scFv polypeptide can optionally further comprise a polypeptide linker between the VH and VL domains to facilitate the formation of a three-dimensional structure of the antigen binding site by the scFv (see, e.g., Pluckthun, 1994, The Pharmacology of monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315).

[0107] As used herein, "non-cleavable linker" or "protease non-cleavable linker" refers to a peptide linker that does not contain a peptide sequence or a peptide sequence mimetic that is a target of a protease. Exemplary non-cleavable linkers are described in the Linker section.

[0108] An antigen binding molecule / protein (such as an immunoglobulin, antibody, antigen binding unit, or fragment of such an antigen binding molecule / protein) that can "bind to", "bind", "specifically bind to", or "specifically bind" an epitope, antigen, or protein (or at least one portion, fragment, or epitope thereof), has "affinity for", is "is specific for", and / or "has specificity for" that epitope, antigen, or protein, or is a "binding" molecule / protein "to" or "for" that epitope, antigen, or protein, relative to such an epitope, antigen, or protein.

[0109] As used herein, the terms "bind" and "specifically bind" refer to the binding of an antibody or antigen binding portion (such as 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 in a plasmon resonance assay (Malmqvist M., "Surface plasmon resonance for detection and measurement of antibody-antigen affinity and kinetics.", Curr Opin Immunol. 1993 Apr; 5(2): 282-6.) utilizing purified wild-type antigen. Antibody affinity can also be measured using Kinetic Exclusion Assay (KinExA) technology (Darling, R.J. and Brault P-A., "Kinetic exclusion assay technology: Characterization of Molecular Interactions.", ASSAY and Drug Development Technologies. 2004 Dec 2(6): 647-657).

[0110] In general, the term "specificity" refers to the number of different types of antigens or epitopes to which a particular antigen binding molecule / protein (such as an immunoglobulin, antibody, antigen binding unit, or fragment of such an antigen binding molecule / protein) can bind. The specificity of an antigen binding molecule / protein can be determined based on its affinity and / or avidity. The dissociation equilibrium constant (Kd) of an antigen from an antigen binding protein is a measure of the affinity of the antigen binding protein for the antigen. The lower the Kd, the higher the affinity of the antigen binding protein for the antigen.D ) represents the affinity, which is a measure of the strength of binding between the epitope and the antigen binding site on the antigen binding molecule / protein: K D values are smaller, the stronger the binding between the epitope and the antigen binding molecule (alternatively, affinity can also be expressed as the affinity constant (K A ), which is 1 / K D ). As will be clear to the skilled person (e.g. based on the other disclosures herein), affinity can be determined in the manner known per se, which depends on the relevant specific antigen. The avidity is a measure of the strength of binding between the antigen binding molecule / protein (such as an immunoglobulin, an antibody, an antigen binding unit, or a fragment of such antigen binding molecule / protein) and the relevant antigen. The avidity is related to both the affinity between the epitope and the antigen binding site on the antigen binding molecule / protein and the number of relevant binding sites present on the antigen binding molecule / protein.

[0111] For use in humans, it is often desirable to reduce the immunogenicity of therapeutic molecules (such as antibodies or binding proteins comprising an antigen binding unit described herein) that are initially derived from other species, such as mice. This can be accomplished by constructing chimeric antibodies / binding proteins, or by a process known as "humanization". In this context, a "chimeric antibody"; or "chimeric antigen binding unit" is understood to be an antibody or antigen binding unit comprising a portion of the sequence (e.g. the constant domain) derived from one species (e.g. human) fused to a portion of the sequence (e.g. the variable domain) derived from a different species (e.g. mouse). In this context, a "humanized antibody", "humanized binding protein", or "humanized antigen binding unit" is an antibody, protein or antigen binding unit comprising a variable domain originally derived from a non-human species, wherein certain amino acids have been mutated to produce an overall sequence of the variable domain that more closely resembles that of a human variable domain. Methods of 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).

[0112] An "optimized antibody" or "optimized antigen binding unit or protein" is a particular type of humanized antibody or humanized antigen binding unit / protein that includes immunoglobulin amino acid sequence variants or fragments thereof that are capable of binding to a predetermined antigen and comprise 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, often referred to as the "imported" sequence, is typically obtained from the "imported" antibody domain, particularly the variable domain. Generally, an optimized antibody includes at least the CDRs (or HVLs) of a non-human antibody or derived from a non-human antibody inserted between the FRs of a human heavy or light chain variable domain. It is understood that certain mouse FR residues can be important for the function of the optimized antibody and thus certain human germline sequence heavy and light chain variable domain residues are modified to be identical to those of the corresponding mouse sequence. During this process, undesirable amino acids can also be removed or altered, for example, to avoid deamidation, undesirable charge or lipophilicity, or non-specific binding. An "optimized antibody," "optimized antibody fragment," or "optimized" can sometimes be referred to as "humanized antibody," "humanized antibody fragment," or "humanized," or as "sequence-optimized."

[0113] Moreover, technologies have been developed for generating antibodies or VH / VL domains based on sequences derived from the human genome, for example by phage display or using transgenic animals (WWW.Ablexis.com / technology-alivamab.php; WO 90 / 05144; D. Marks, H. R. Hoogenboom, T. P. Bonnert, J. McCafferty, A. D. Griffiths, and G. Winter (1991) "By-passing immunisation. 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. August 1997; 8(4): 455-8.). In the context of the present invention, such antibodies or antigen binding units or VH / VL domains are "human antibodies", "human antigen binding units" or "human VH / VL domains".

[0114] As used herein, the terms "identical" or "percent identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same with respect to the identity of their nucleotides or amino acid residues, when compared and aligned for maximum correspondence. To determine percent identity, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino 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, then 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 = number of identical positions / total number of positions (e.g., overlapping positions) x 100). In some embodiments, when appropriate, the two sequences being compared are the same in length (e.g., not including additional sequence that extends beyond the compared sequences) after introduction of gaps, as appropriate. 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 a CDR in the same position in both sequences (e.g., CDR-H1 of each sequence).

[0115] Mathematical algorithms can be used to determine percent identity or percent similarity between two sequences. A preferred, non-limiting example of a mathematical algorithm that is suitable 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 in the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to a nucleic acid encoding a protein of interest. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to a 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 which detects distant relationships between molecules (same). 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 that is suitable for comparing sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing 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 and include ADVANCE and ADAM as 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 sensitivity and speed of the search. If ktup = 2, similar regions in the two sequences being compared are observed by looking at pairs of aligned residues; if ktup = 1, single aligned amino acids are examined. For protein sequences, ktup can be set to 2 or 1, or for DNA sequences, to 1 to 6. If ktup is not specified, the default value is 2 for proteins and 6 for DNA.Alternatively, protein sequence alignment can be performed using the CLUSTAL W algorithm as described by Higgins et al., 1996, Methods Enzymol. 266:383-402.

[0116] As used herein, the term "linked" refers to coupling of different components of an IL-12 Fc fusion protein and includes (i) via a means capable of linking different components, such as a linker, or (ii) any chemical association, including covalent and non-covalent interactions, preferably covalent interactions, that links different components of an IL-12 Fc fusion protein. Covalent interactions can be, for example, a direct covalent bond between residues, such as a peptide bond or a disulfide bond. The linker can be a peptide linker or a non-peptide linker, preferably a peptide linker. If the linker is a peptide linker, it can consist of one or more amino acids.

[0117] An "immunoglobulin single variable domain (ISVD)" is an antibody fragment consisting of a single variable antibody domain. Like a complete antibody, it is capable of selectively binding to a specific antigen. The molecular weight is only 12-18 kDa, much smaller than a common antibody consisting of two heavy protein chains and two light chains (150-160 kDa), and even smaller than a Fab fragment (about 50 kDa, one light chain and half a heavy chain) and a single-chain variable fragment (about 25 kDa, two variable domains, one from the light chain and one from the heavy chain). Generally, an immunoglobulin single variable domain will have an amino acid sequence comprising four framework regions (FR1 to FR4) and three complementarity determining regions (CDR1 to CDR3), preferably according to the following formula: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. As used herein, the term immunoglobulin single variable domain includes, but is not limited to, the variable domain of a camelid heavy chain antibody (VHH), also known as Nanobodies (Nanobodies), domain antibodies (dAbs) and immunoglobulin single variable domains derived from sharks (IgNAR domains). TM

[0118] VHH domains, also known as VHH, V H ​The H domain, VHH antibody fragment, and VHH antibody were initially described as antigen-binding immunoglobulin (variable) domains of heavy chain antibodies (i.e., antibodies that do not contain light chains; 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" has been chosen to distinguish these variable domains from the heavy chain variable domains present in conventional 4-chain antibodies (referred to herein as "VHH domain"). H The "VH domain" (or "VH domain") and the light chain variable domain (referred to as "VH domain" in this paper) present in conventional 4-chain antibodies are combined with the light chain variable domain present in conventional 4-chain antibodies. L The VHH domain is distinguished from the VL domain. The VHH domain can specifically bind to the epitope without the need for an additional antigen-binding domain (as opposed to the VH or VL domain in a conventional 4-chain antibody, in which case the epitope is recognized by both the VL and VH domains). The VHH domain is a small, stable, and highly efficient antigen recognition unit formed by a single immunoglobulin domain.

[0119] In the context of this invention, the terms VHH domain, VHH, and V... H H domain, VHH antibody fragment, VHH antibody and and" The term “Nanobody” (a trademark of Ablynx NV, Ghent, and Belgium) is used interchangeably with and represents the ISVD (which has the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and specifically binds to an epitope without the presence of a second immunoglobulin variable domain), and can also be distinguished from the VH domain by so-called “marker residues” as defined, for example, in Figure 1 of WO2009 / 109635.

[0120] Earlier, for example, methods for obtaining VHH domains that bind to specific antigens or epitopes have been described in WO2006 / 040153 and WO2006 / 122786. A camel-derived VHH domain can be "humanized" by replacing one or more amino acid residues in the amino acid sequence of the initial VHH sequence with one or more of the amino acid residues appearing at corresponding positions in the VH domain of a conventional 4-chain antibody from humans. The humanized VHH domain may contain one or more fully human framework region sequences, and in one or more specific embodiments, may contain human framework region sequences derived from DP-29, DP-47, DP-51, or portions thereof, optionally combined with a JH sequence such as JH5.

[0121] IL-12

[0122] Interleukin-12 (IL12) is a heterodimeric molecule, which is covalently linked by a disulfide bridge from an alpha chain (IL-12p35 subunit) and a beta chain (IL-12p40 subunit) 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 functions of CD8+ T and NK cells. Thus, IL-12 is a major contributor to effective anti-tumor immune responses. IL-12 signals through the IL-12Rβ1 and IL-12Rβ2 receptors expressed on target cells, which allow downstream Jak2 and Tyk2 to promote phosphorylation and homodimerization of STAT4. Further studies have shown that IL-12 is not only required for activation of effector anti-tumor immune responses, but can also directly inhibit immune suppression. Thus, the use of IL-12 as a cancer immunotherapy can be beneficial in controlling tumor growth by activating anti-tumor cytotoxic immune responses. Overall, IL-12 targets and modulates T cells, NK cells, and antigen-presenting cells (APCs) that regulate the fate of anti-tumor immune responses against cancer cells.

[0123] In certain embodiments, the IL-12 cytokine comprises an IL-12p35 amino acid sequence as set forth in SEQ ID NO: 1. In certain embodiments, the IL-12 cytokine comprises an IL-12p40 amino acid sequence as set forth in SEQ ID NO: 2. In certain embodiments, the IL-12 cytokine comprises an IL-12p35 amino acid sequence as set forth in SEQ ID NO: 1 and comprises an IL-12p40 amino acid sequence as set forth in SEQ ID NO: 2.

[0124] In another embodiment, the IL-12 cytokine consists of a single chain IL-12 having the configuration (written from N- to C-terminus) IL-12p40—IL-12p35 or IL-12p35—IL-12p40, relative to maintaining IL-12 as a native heterodimer. In certain embodiments, the IL-12p40—IL-12p35 comprises an amino acid sequence as set forth in SEQ ID NO: 3. In certain embodiments, the IL-12p35—IL-12p40 comprises an amino acid sequence as set forth in SEQ ID NO: 4.

[0125] In another embodiment, the IL-12 cytokine can include subunits from different species, i.e., a chimeric IL-12 cytokine. In a related embodiment, the IL-12 p35 subunit is derived from mouse and the IL-12 p40 subunit is derived from human. In certain embodiments, the IL-12 cytokine comprises an IL-12 p35 amino acid sequence as set forth in SEQ ID NO: 5. In certain embodiments, the IL-12 cytokine comprises an IL-12 p35 amino acid sequence as set forth in SEQ ID NO: 5 and comprises an IL-12 p40 amino acid sequence as set forth in SEQ ID NO: 2.

[0126] It has been found that such chimeric molecules are extremely valuable for in vitro as well as in vivo data in mice without the need for another substitution masking moiety. The masking moiety would be specific for the human IL-12 p40 subunit and thereby block the activity of the IL-12 cytokine. The IL-12 p35 subunit derived from mouse still forms a functional IL-12 cytokine with the human IL-12 p40 subunit and is active in mouse models. For use in humans, the IL-12 p35 subunit from mouse would be substituted with the IL-12 p35 subunit from human, however, the masking moiety and all other components of the IL-12 Fc fusion protein remain the same.

[0127] In certain embodiments, the chimeric IL-12 p40— IL-12 p35 comprises an amino acid sequence as set forth in SEQ ID NO: 6. In certain embodiments, the chimeric IL-12 p35— IL-12 p40 comprises an amino acid sequence as set forth in SEQ ID NO: 7.

[0128] In related embodiments, the subunits within the single chain IL-12 cytokine can be connected to each other via a linker, e.g., IL-12 p40(linker)IL-12 p35 or IL-12 p35(linker)IL-12 p40. The linker can be a peptide linker and in particular any of the peptide linkers disclosed herein and preferably a GS linker. Thus, in related embodiments, the subunits in the single chain IL-12 cytokine comprising an amino acid sequence as set forth in any of SEQ ID NOs: 3, 4, 6, or 7 are connected to each other via a linker as disclosed herein and preferably a GS linker. In related embodiments, the GS linker has the following amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 22). In a preferred embodiment, the single chain IL-12 cytokine is provided in the configuration IL-12 p40-15GS-IL-12 p35 (SEQ ID NO: 8). In another embodiment, the single chain IL-12 cytokine is provided in the configuration IL-12 p35-15GS-IL-12 p40 (SEQ ID NO: 9).

[0129] 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).

[0130] 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.

[0131] In another embodiment, the single chain IL-12p40-IL-12p35 is connected via its IL-12p40 subunit to the C-terminus of the first Fc domain. In another embodiment, the single chain IL-12p35-IL-12p40 is connected via its IL-12p35 subunit to the first Fc domain. In both cases, the single chain IL-12p40-IL-12p35 or IL-12p35-IL-12p40 is connected via a first peptide linker to the C-terminus, which first peptide linker is protease cleavable.

[0132] In another embodiment, the IL-12p40 subunit and the IL-12p35 subunit are connected to each other via a linker rich in the amino acid residues glycine and serine. In a related embodiment, the linker has a length of 5 to 20 amino acids and comprises only the amino acids glycine and serine. In a preferred embodiment, the linker has the amino acid sequence of SEQ ID NO: 22.

[0133] 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.

[0134] The IL-12 cytokine can comprise 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 can encode an IL-12 subunit or any single chain IL-12 as disclosed herein. In one embodiment, the variant encodes an IL-12 subunit or any single chain IL-12 as shown in any one of SEQ ID NOs: 1-11, with 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 shown in any one of SEQ ID NOs: 1-11.

[0135] The functional activity of IL-12 can be measured in an assay as shown in Example 5.

[0136] Table 1:

[0137]

[0138]

[0139]

[0140]

[0141]

[0142] Fc

[0143] According to the present application, the first and second polypeptide chains of the IL-12 Fc fusion protein are connected to each other via their respective Fc domains, i.e. the two polypeptide chains are dimeric via their Fc domains.

[0144] In the context of the present application, the Fc domain is derived, for example, from the heavy chain of an IgG, e.g. IgG1, IgG2, or IgG4. For example, the Fc domain of the present application is the Fc domain of the heavy chain of IgG1 and comprises a hinge region and two constant domains (CH2 and C H3 ). An example of an Fc domain (including hinge region) is shown in SEQ ID NO: 14.

[0145] For all constant region (CL, CH1, hinge, CH2, and CH3) positions discussed in the present application, 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), which refers to the numbering of the EU antibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85), unless otherwise specified. This means that, unless otherwise specified, the amino acid numbers indicated herein correspond to the position in the heavy chain of the respective isoform (e.g., IgG1 or IgG4), according to the EU numbering system. For all variable region (VL and VH) and J segment (JH and JL) positions discussed in the present application, numbering is according to 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 will be noted. Those skilled in the art of antibodies will appreciate that these conventions consist of nonsequential numbering in specific regions of immunoglobulin sequences, enabling standardized reference to conserved positions within the immunoglobulin family. Thus, the position of any given immunoglobulin, as defined by EU numbering or Kabat numbering, will not necessarily correspond to its contiguous sequence.

[0146] In some embodiments, each of the first and second Fc domains in the fusion proteins of the application comprises one or more amino acid changes that reduce the formation of homodimers of the first or second polypeptide chain over heterodimers of the first and second polypeptide chains. Through these changes, a "protuberance" is created in one Fc domain by replacing one or more small amino acid side chains from the interface with a larger side chain, such as tyrosine or tryptophan. A compensating "cavity" of the same or similar size is created on the interface of the other Fc domain by replacing a large amino acid side chain with a smaller amino acid side chain, such as alanine or threonine. This provides a mechanism for increasing the yield of heterodimers over other undesired end products, such as homodimers, particularly homodimers of the Fc domain with the "protuberance" (see, e.g., Ridgway et al. Protein Eng, 1996. 9(7): p. 617-21; Atwell et al., JMB, 1997, 270, 26-35).

[0147] In some embodiments, such amino acid changes 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 preferred embodiments, 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 an Fc domain according to EU numbering corresponds to amino acid position 151 in the human IgGl Fc sequence of SEQ ID NO: 14, which changes 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 acid positions 151, 153, and 192 in SEQ ID NO: 14, which change 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 can be in the second Fc domain and the respective amino acid changes of the second Fc domain can be in the first Fc domain. In other words, the terms "first" and "second" are interchangeable in these embodiments. In some embodiments, such Fc domains are Fc domains derived from the heavy chain of IgGl.

[0148] 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 366 [T366S], an alanine (A) at position 368 [L368A], and a valine (V) at position 407 [Y407V]. In one aspect, such Fc domains are Fc domains derived from an IgGl heavy chain.

[0149] In some embodiments, the first and / or second Fc domain of the application derived from IgGl also includes "KO" mutations (L234A, L235A).

[0150] In some embodiments, the first Fc domain or the second Fc domain in the Fc fusion protein of the application further comprises one or more amino acid changes that reduce binding of the Fc domain to Protein A. In some embodiments, such amino acid changes 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 in the CH3 domain and were 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): p. 25-34). These two changes facilitate removal of homodimers of heavy chains comprising these changes during protein purification.

[0151] In some embodiments, in the fusion protein of the application, the Fc domain comprising a threonine (T) at position 407 [Y407T] 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 not the two changes at positions 435 and 436. Alternatively, in some embodiments, in the protein of the application, the Fc domain comprising a serine (S) at position 366 [T366S], an alanine (A) at position 368 [L368A] and a valine (V) at position 407 [Y407V] further comprises an arginine at position 435 [H435R] and a phenylalanine at position 436 [Y436F]. In this case, the other Fc domain comprises a tryptophan (W) at position 366 [T366W] but not the two changes at positions 435 and 436. Thus, the Fc domain comprising the amino acid changes that create a "cavity" as described above also comprises amino acid changes that reduce binding to Protein A. A homodimer comprising this Fc domain is removed by reducing binding to Protein A. The production of a homodimer comprising the other Fc domain of the "protuberance" is reduced by the presence of the "protuberance".

[0152] In preferred embodiments, the first Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14. In preferred embodiments, the first Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15. In preferred embodiments, the first Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16. In preferred embodiments, 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.

[0153] In any of the foregoing embodiments, the serine at position 5 of the amino acid sequences SEQ ID NOs: 14-18 can be replaced with a cysteine.

[0154] In some embodiments, the Fc domain of the fusion proteins of the present application can or can not further comprise the YTE mutations (M252Y / S254T / T256E, EU numbering (Dall'Acqua, Kiener et al. 2006)). These mutations have been shown to improve the pharmacokinetic properties of the Fc domain by preferentially increasing the binding affinity to the neonatal FcRn receptor at pH 6.0.

[0155] Table 2:

[0156]

[0157] Protease-cleavable linker

[0158] The protease-cleavable linker connects the Fc domain to the IL-12 cytokine, i.e., the protease-cleavable linker is disposed between the C-terminus of the Fc domain and the IL-12 cytokine. Once the protease-cleavable linker is cleaved by its corresponding protease, the IL-12 cytokine is released and no longer connected to the Fc fusion protein.

[0159] On the Fc domain side, the protease-cleavable linker can be directly connected to the C-terminus of the Fc domain (i.e., no linker), or it can be connected to the C-terminus of the Fc domain via a linker, such as any of the linkers described in the Linker section below, and preferably a peptide linker, e.g., of about 4 to 20 amino acids in length. In a preferred embodiment, the protease-cleavable linker is connected to the C-terminus of the Fc domain via a linker having the sequence GGGGSGGGG (SEQ ID NO: 24).

[0160] On the IL-12 cytokine side, the protease-cleavable linker can be directly connected to the IL-12 cytokine or it can be connected to the IL-12 cytokine via a linker, such as any of the linkers described in the Linker section below, and preferably a peptide linker, e.g., of about 4 to 20 amino acids in length. Depending on the configuration of the IL-12 cytokine, the protease-cleavable linker is connected to either the IL-12 p40 subunit or the IL-12 p35 subunit. In a preferred embodiment, the IL-12 cytokine is provided in a single chain configuration, and the protease-cleavable linker is connected to (a) the IL-12 p40 subunit of a single chain IL-12 p40— IL-12 p35, or (b) the IL-12 p35 subunit of a single chain IL-12 p35— IL-12 p40. In a related embodiment, the protease-cleavable linker is connected to the IL-12 p35 subunit or the IL-12 p40 subunit via a linker having the sequence GGGGS (SEQ ID NO: 27).

[0161] The protease-cleavable linker generally has 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, the protease-cleavable linker can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 aa in length.

[0162] Preferably, the desired protease is enriched, selectively expressed, and / or more active at the desired site of cytokine activity, e.g., TME. Thus, the IL-12 Fc fusion protein is selectively or preferably cleaved at the desired site of cytokine activity.

[0163] Proteases known to be associated with diseased cells or tissues include, but are not limited to, serine proteases, cysteine proteases, aspartate proteases, threonine proteases, glutamic acid proteases, metalloproteases, asparagine peptide lyases, serum proteases, cathepsins, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, kallikrein, hK1, hK10, hK15, fibrinolysin, collagenase, collagenase type IV, stromelysin, factor Xa, chymotrypsin-like proteases, trypsin-like proteases, elastase-like proteases, subtilisin-like proteases, actinidain, bromelain, calpains, caspases, caspase-3, Mirl-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, cysteine proteases (Plasmepsin), nepenthesin, metalloexopeptidases, metalloendopeptidases, matrix metalloproteases (MMPs), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, urokinase plasminogen activator (uPA), enteropeptidase, prostate specific antigen (PSA, hK3), interleukin-1 beta converting enzyme, thrombin, FAP (FAPa), dipeptidyl peptidases, Meprins, granzymes, and dipeptidyl peptidase IV (DPPIV / CD26).

[0164] Proteases capable of cleaving amino acid sequences (encoded by the protease-cleavable linker sequences provided herein) may be selected, for example, from the group consisting of: prostate-specific antigen (PSA), matrix metalloproteinases (MMPs), deintegrin and metalloproteinases (ADAMs), plasminogen activator, cathepsins, caspases, tumor cell surface proteases, and elastases. MMPs may be, for example, matrix metalloproteinase 2 (MMP2) or matrix metalloproteinase 9 (MMP9). Preferably, the protease-cleavable linker is cleaved via MMP2, MMP9, or MMP13.

[0165] Table 3:

[0166] Cleavable MMP Linker SEQ ID NO: GPLGVRG 232 GPLGLRG 233 GPLGLAR 234 GPAALVGA 235 GPAALIGG 236 GPLNLVGR 237 GPAGLVAD 238 GPANLVAP 239 VPLSLYSG 240 SGESPAYYTA 241

[0167] In a preferred embodiment, the protein cleavable linker sequence is GPLGVRG (SEQ ID NO:232).

[0168] The cleavage of the protease-cleavable linker can be readily determined, as shown in Examples 7 and 8.

[0169] Concealed parts

[0170] As used herein, the masking portion refers to the portion of the IL-12 p35 and / or IL-12 p40 subunits that bind to the IL-12 cytokine. In one embodiment, by binding the masking portion to the IL-12 p35 and / or IL-12 p40 subunits, the affinity of the IL-12 cytokine for its homologous receptor is reduced. In another embodiment, by binding the masking portion to the IL-12 p35 and / or IL-12 p40 subunits, the functional activity of the IL-12 cytokine is blocked, inhibited, or attenuated.

[0171] The binding of the masked portion to the IL-12p35 and / or IL-12p40 subunits of the IL-12 cytokine can be readily measured using methods well-known in the art, see, for example, Example 2. The strength or affinity of the specific binding can be determined based on the dissociation constant of the interaction (K). D ) indicates that the smaller K D This indicates greater affinity and a larger K value. D This indicates lower affinity. Binding characteristics can be determined using methods such as biolayer interferometry and surface plasmon resonance-based methods, including the Biacore and Octet methods. One such method requires measuring the rates of binding and dissociation of antigen-binding site / antigen or receptor / ligand complexes, where the rates depend on the concentration of the complex coupler, the affinity of the interaction, and geometric parameters that equally affect the rates in both directions. Therefore, the binding rate (k) can be determined. a ) and dissociation rate (k d ), and ka / k d The ratio of the dissociation constant K D .

[0172] Specific binding to the IL-12p35 and / or IL-12p40 subunit can be exhibited, for example, by a masking moiety having a K D of 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 greater.

[0173] In one embodiment, the masking moiety can be an IL-12 receptor or an IL-12p35 or IL-12p40 binding fragment thereof. In one embodiment, the masking moiety can be an IL-12p40 binding fragment of an IL-12 receptor. In one embodiment, the masking moiety can be an IL-12p35 binding fragment of an IL-12 receptor.

[0174] IL-12 receptor is a type I cytokine receptor that binds IL-12. It consists of beta 1 and beta 2 subunits. Briefly, IL-12 receptor beta 1 or IL-12R beta 1 is a subunit of the interleukin 12 receptor. IL12RB1 is the name of its human gene. IL-12R beta 1 is also known as CD212 (cluster of differentiation 212). Human IL-12R beta 1 has an amino acid sequence as set forth 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-12R beta 2 has an amino acid sequence as set forth in SEQ ID NO: 13. 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 human IL-12R beta 1 having an amino acid sequence as set forth 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 human IL-12R beta 2 having an amino acid sequence as set forth in SEQ ID NO: 13.

[0175] In some embodiments, the masking moiety comprises an extracellular domain of IL-12Rβ1 or IL-12Rβ2 or a fragment, portion, or variant thereof that retains affinity for IL-12. The extracellular domain is 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 an extracellular domain of human IL-12Rβ1 having the amino acid sequence as set forth 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 an extracellular domain of human IL-12Rβ2 having the amino acid sequence as set forth in SEQ ID NO: 13.

[0176] The masking moiety can also be a scFv or an immunoglobulin single variable domain. Preferably, the masking moiety is a VHH and more preferably a humanized VHH.

[0177] 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.

[0178] The CDRs disclosed herein and depicted in SEQ ID NOs: 61-109 are presented according to the Kabat nomenclature. The underlined sequences correspond to the respective CDR-1, CDR-2, and CDR-3 according to the Kabat nomenclature. The CDRs are again individually identified in SEQ ID NOs: 333-479 in the Kabat nomenclature.

[0179] As additional nomenclatures are known in the art, CDR sequences are also shown based on the most commonly used of these, but only for those cases where different amino acid sequences result from the application of these alternative nomenclatures. These numbering systems are based on (i) CCG (Chemical Computing Group described 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(l):55-77) and (iv) North (North B, J Mol Biol. (2011) 406:228-56).

[0180] When applied to VHH domains from camels, the amino acid residues of the VHH domain are numbered according to the general numbering given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, 91st Ed.) for VH domains, as shown in Riechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999), for example. Figure 2

[0181] According to this numbering,

[0182] FR1 comprises the amino acid residues at positions 1 to 30,

[0183] CDR1 comprises the amino acid residues at positions 31 to 35,

[0184] FR2 comprises the amino acids at positions 36 to 49,

[0185] CDR2 comprises the amino acid residues at positions 50 to 65,

[0186] FR3 comprises the amino acid residues at positions 66 to 94,

[0187] CDR3 comprises the amino acid residues at positions 95 to 102, and

[0188] FR4 comprises the amino acid residues at positions 103 to 113.

[0189] ​The total number of amino acid residues in a VHH domain will generally range from 110 to 120, typically 112-115. It should be noted, however, that smaller and longer sequences can also be suitable for the purposes described herein.

[0190] It should be noted, however, as is well known in the art for VH domains and VHH domains, the total number of amino acid residues in each of the CDRs can vary and can not correspond to the total number of amino acid residues indicated by the numbering of Kabat (i.e., one or more positions according to the numbering of Kabat can not be occupied in the actual sequence, or the actual sequence can contain more amino acid residues than allowed by the numbering of Kabat). This means that in general, the numbering according to Kabat can or can not correspond to the actual numbering of the amino acid residues in the actual sequence.

[0191] Alternative numbering methods for the amino acid residues of a VH domain are known in the art, which methods can also be applied in an analogous manner to VHH domains. In the present specification, claims and drawings, however, unless otherwise indicated, the numbering according to Kabat and as applied to VHH domains as described above will be followed.

[0192] 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.

[0193] 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.

[0194] In some embodiments, the masking moiety is an IL-12 binding VHH comprising the three CDRs contained within any one of the sequences of SEQ ID NOs: 61-109.

[0195] In some embodiments, the masking moiety is a VHH and comprises the three CDRs contained within any one of the sequences of SEQ ID NOs: 61-109 and further comprises a framework region (FR1, FR2, FR3, FR4) that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the framework region as shown in any one of the sequences of SEQ ID NOs: 61-109.

[0196] 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 a framework region (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 as shown in any one of the sequences of SEQ ID NOs: 61-109.

[0197] In some embodiments, the masking moiety is a VHH and 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 as shown in any one of SEQ ID NOs: 61-109 and further comprises an additional alanine connected to its C-terminus.

[0198] It is understood that any one of the foregoing (and hereinafter listed) VHH sequences and CDRs contained within the VHH sequences can find utility outside of their use as masking moieties in IL-12 Fc fusion proteins. Thus, the present application is not to be construed as limiting the VHH sequences to their use as masking moieties, but rather provides the sequences per se, i.e., for any and all purposes.

[0199] Table 4:

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218] Linker

[0219] Methods for linking molecules are well known in the art. The linker can be a peptide linker or a non-peptide linker. If the linker is a peptide linker, it can be composed of one or more amino acids. For peptide linkers, small linker sequences of glycine and serine amino acids are typically used (referred to as GS mini-linkers). 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.

[0220] In some embodiments, the linker is 5-20 amino acids in length. In other embodiments, the linker is rich in amino acid residues G and S. In another embodiment, the linker is 5-20 amino acids in length and is rich in amino acid residues G and S. In another embodiment, the linker includes only amino acid residues G and S. In another embodiment, the linker is 2-20 amino acids in length and includes only amino acid residues G and S.

[0221] As contemplated herein, a peptide linker is a (poly)peptide linker that is at least 1 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, more preferably 10 to 40 amino acids in length, and even more preferably the linker is 15 to 30 amino acids in length. Non-limiting examples of small linkers that are typically used include sequences of glycine and serine amino acids, referred to as GS mini-linkers. Preferred examples of linker sequences are Gly / Ser linkers of varying lengths, such as (gly x ser y ) zLinkers, including (gly4ser)3, (gly4ser)4, (gly4ser), (gly3ser), gly3, and (gly3ser2)3. The number of amino acids of these linkers can vary, for example it can be 4 (e.g. GGGS) (SEQ ID NO: 19), 6 (e.g. GGSGGS) (SEQ ID NO: 20), 7 (e.g. GGGSGGS) or multiples thereof, such as three or more repeats of these four / six amino acids. Most preferably, such GS mini-linkers have 20 amino acids and the sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 23). Other examples of such linkers include GGGGSGGGG (SEQ ID NO: 24), GSGG (SEQ ID NO: 25) or GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 26).

[0222] Other examples of linkers include the following:

[0223] 5GS linker: GGGGS (SEQ ID NO: 27)

[0224] 7GS linker: SGGSGGS (SEQ ID NO: 28)

[0225] 8GS linker: GGGSGGGS (SEQ ID NO: 29)

[0226] 9GS linker: GGGGSGGGS (SEQ ID NO: 30)

[0227] 10GS linker: GGGGSGGGGS (SEQ ID NO: 31)

[0228] 15GS linker: GGGGSGGGGSGGGGS (SEQ ID NO: 32)

[0229] 18GS linker: GGGGSGGGGSGGGGGGGS (SEQ ID NO: 33)

[0230] 20GS linker: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 34)

[0231] 25GS linker: GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 35)

[0232] 30GS linker: GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 36)

[0233] 35 GS linker: GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 37)

[0234] The linker can also be a variant described in Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448. Other linkers useful in the present application 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.

[0235] In a preferred embodiment, the non-cleavable linker is selected from any of the aforementioned GS linkers. In another preferred embodiment, the non-cleavable linker has a length of 2-20 amino acids and comprises only the amino acid residues G and S.

[0236] Binding moiety

[0237] A binding moiety can be used to facilitate accumulation and / or retention of the IL-12 Fc fusion protein and / or cleavage product in the TME, preferably the ECM, and more preferably within the vicinity of a tumor. The binding moiety can be placed on 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.

[0238] In some cases, it can be desirable to place the binding moiety on the first polypeptide chain. If the binding moiety is placed on the first polypeptide chain, this can additionally facilitate accumulation and / or retention of the cleavage product in the TME. In those cases, when the binding moiety is placed on the first polypeptide chain, the binding moiety can be linked to the C-terminus of the IL-12 p35 subunit or to the C-terminus of the IL-12 p40 subunit. In both cases, the binding moiety can be linked directly to the C-terminus or optionally via a polypeptide linker, such as any of those disclosed herein. The binding moiety can also be placed between the IL-12 p35 subunit and the IL-12 p40 subunit. In this case, the binding moiety can optionally be flanked on one or both sides by one or more polypeptide linkers. This can result in different configurations, such as IL-12 p35 (binding moiety) IL-12 p40 or IL-12 p40 (binding moiety) IL-12 p35, where the binding moiety is linked directly to each IL-12 subunit at its N-terminus and C-terminus. Another configuration can include one or more linkers, such as IL-12 p35 (linker) (binding moiety) IL-12 p40, IL-12 p35 (linker) (binding moiety) (linker) IL-12 p40, IL-12 p35 (binding moiety) (linker) IL-12 p40, IL-12 p35 (linker) (binding moiety) (linker) IL-12 p40, IL-12 p40 (linker) (binding moiety) IL-12 p35, IL-12 p40 (linker) (binding moiety) (linker) IL-12 p35, IL-12 p40 (binding moiety) (linker) IL-12 p35, IL-12 p40 (linker) (binding moiety) (linker) IL-12 p35. In any of those configurations, the linker can be a polypeptide linker, such as any of those disclosed herein. In these configurations, the binding moiety is expressed in a single chain with the IL-12 subunits and retained with the IL-12 cytokine after cleavage in the TME.

[0239] In some other cases, it can be desirable to place the binding moiety on the second polypeptide chain. In this way, the properties of the binding moiety will be readily adapted 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 cases, the binding moiety can be linked to the C-terminus of the masking moiety. The binding moiety can be linked directly to the C-terminus or optionally via a polypeptide linker, such as any of those disclosed herein.

[0240] In some cases, the binding moiety can be located at the N-terminus of the first or second Fc domain. The binding moiety can be linked directly to the N-terminus or optionally via a polypeptide linker, such as any of those disclosed herein.

[0241] 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 moiety disclosed herein has binding specificity for collagen, heparin or fibronectin. In relation to the present application, the binding moiety is derived from a polypeptide or a portion thereof that binds to collagen, heparin or fibronectin, respectively.

[0242] In a preferred embodiment, the binding moiety is a collagen binding moiety, and more preferably a collagen I binding moiety. In another preferred embodiment, the collagen I binding moiety is placed at the C-terminus of the IL-12 p35 subunit and / or IL-12 p40, such as IL-12 p35 - IL-12 p40 (binding moiety), IL-12 p35 (linker) IL-12 p40 (binding moiety), IL-12 p35 (linker) IL-12 p40 (linker) (binding moiety), IL-12 p35 - IL-12 p40 (linker) (binding moiety), IL-12 p40 - IL-12 p35 (binding moiety), IL-12 p40 (linker) IL-12 p35 (binding moiety), IL-12 p40 (linker) IL-12 p35 (linker) (binding moiety) or IL-12 p40 - IL-12 p35 (linker) (binding moiety).

[0243] Collagen binding moiety

[0244] 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 receptors, tyrosine kinase receptors and some signaling pathways. Cancer-associated fibroblasts produce high levels of additional extracellular matrix proteins (ECM) in the TME, resulting in overexpression of multiple types of collagen in many tumor types. The role of collagen in cancer has been extensively reviewed, including the relationship of collagen with proteases such as MMPs that are used 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).

[0245] The collagen superfamily comprises 28 members in vertebrates, numbered using Roman numerals (I-XXVIII). A common structural feature of collagens is the presence of a triple helix structure, which can range from most 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 alpha chains, several molecular isoforms for a single collagen type, and supramolecular structures, as well as the use of alternative promoters and alternative splicing.

[0246] Among the different collagens, type I collagen is the most abundant protein in mammals. The basic building block of type I collagen is a long (300 nm), thin (1.5 nm diameter) protein, composed of three coiled subunits: two alpha 1 (I) chains and one alpha 2 (I). Each chain contains 1050 amino acids, which are wound around each other in a characteristic right-handed triple helix. In human type I collagen, it is encoded by COL1A1 and COL1A2 genes. The COL1A1 gene encodes the pro-alpha 1 chain of type I collagen. The COL1A2 gene of type I collagen pro-alpha 2 chain, whose triple helix comprises two alpha 1 chains and one alpha 2 chain. Type I is a fibril-forming collagen found in most connective tissues and is abundant in the skeleton, cornea, dermis, and tendons.

[0247] An exemplary amino acid sequence of the human alpha 1 chain precursor of type I collagen is set forth in SEQ ID NO: 38 (NCBI Reference Sequence: NP 000079.2). An exemplary amino acid sequence of the human alpha 2 chain precursor of type I collagen is set forth in SEQ ID NO: 39 (NCBI Reference Sequence: NP 000000.2).

[0248] In some embodiments, the collagen-binding moiety comprises one or more (e.g., two, three, four, five, six, seven, eight, nine, ten or more) leucine-rich repeat sequences that bind 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, testican, bikunin, fibromodulin, lumican, chondroadherin, keratin, ECM2, epiphycan, asporin, PRELP, keratocan, osteoadherin, opticin, osteoglycin, nyctalopin, Tsukushi, podocan, podocan-like protein 1 versican, perlecan, nidogen, neurocan, aggrecan, and brevican.

[0249] In some embodiments, the collagen-binding moiety comprises a small leucine- rich proteoglycan (SLRP) class I. In some embodiments, the collagen-binding domain comprises a SLRP class II. In some embodiments, the collagen-binding domain comprises a SLRP class III. In some embodiments, the collagen-binding domain comprises a SLRP class IV. In some embodiments, the collagen-binding domain comprises a SLRP class V. In some embodiments, the collagen-binding domain comprises one or more leucine-rich repeat sequences 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 can be lumican.

[0250] It is also hypothesized that collagen-rich tumor tissue will show higher activity of MMPs (such as MMP2 and MMP9), which are collagenases, that can further contribute to faster cleavage of the IL-12 Fc fusion protein.

[0251] In some embodiments, the IL-12 Fc fusion protein comprises a collagen binding moiety that specifically binds collagen. In some embodiments, the collagen binding moiety specifically binds human collagen type I and / or human collagen type IV. In some embodiments, the collagen binding moiety binds human collagen type I. In some embodiments, the collagen binding moiety binds human collagen type IV. In some embodiments, the collagen binding moiety specifically binds human collagen type I and human collagen type IV. In some embodiments, the collagen binding moiety specifically binds human collagen type I or human collagen type IV.

[0252] In another embodiment, the present application provides an IL-12 Fc fusion protein, wherein the collagen binding moiety binds to collagen IV and has the amino acid sequence KLWVLPK (SEQ ID NO: 40).

[0253] Binding of a collagen binding moiety to collagen can be determined by methods known in the art. In some embodiments, a 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, a collagen binding moiety is derived from a naturally occurring collagen binding protein or collagen receptor.

[0254] In some embodiments, the IL-12 Fc fusion protein specifically binds collagen with an affinity (KD) of less than about 500 μΜ, as determined by a collagen binding assay. In some embodiments, the IL-12 Fc fusion protein comprises a collagen binding moiety that specifically binds collagen with an affinity (KD) of less than about 100 μΜ, as determined by a collagen binding assay. In some embodiments, the IL-12 Fc fusion protein comprises a collagen binding moiety that specifically binds collagen with an affinity (KD) of less than about 1 μΜ, as determined by a collagen binding assay. In some embodiments, the IL-12 Fc fusion protein comprises a collagen binding moiety that specifically binds collagen with an affinity (KD) of less than about 500 nM, as determined by a collagen binding assay. In some embodiments, the collagen binding moiety specifically binds collagen with an affinity (KD) of less than about 0.1-500 μΜ, 0.1-100 μΜ, or 0.1-1 μΜ, as determined by a collagen binding assay. In some embodiments, the collagen binding moiety specifically binds collagen with an affinity (KD) of less than about 100-1000 nM, 100-1000 nM, 100-800 nM, 100-600 nM, or 100-500 nM, as determined by a collagen binding assay.

[0255] 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 collagen type I. In some embodiments, the collagen binding assay determines the binding affinity for collagen type IV.

[0256] 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, the IL-12 Fc fusion protein comprises a collagen binding moiety that specifically binds collagen with an affinity (KD) of less than about 500 µM, as determined by ELISA. In some embodiments, the IL-12 Fc fusion protein comprises a collagen binding moiety that specifically binds collagen with an affinity (KD) of less than about 100 µM, as determined by ELISA. In some embodiments, the IL-12 Fc fusion protein comprises a collagen binding moiety that specifically binds collagen 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 collagen binding moiety that specifically binds collagen with an affinity (KD) of less than about 500 nM, as determined by ELISA. In some embodiments, the collagen binding moiety specifically binds 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 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.

[0257] 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, the IL-12 Fc fusion protein comprises a collagen binding moiety that specifically binds collagen with an affinity (KD) of less than about 500 µM, as determined by SPR assay. In some embodiments, the IL-12 Fc fusion protein comprises a collagen binding moiety that specifically binds collagen with an affinity (KD) of less than about 100 µM, as determined by SPR assay. In some embodiments, the IL-12 Fc fusion protein comprises a collagen binding moiety that specifically binds collagen with an affinity (KD) of less than about 1 µM, as determined by SPR assay. In some embodiments, the IL-12 Fc fusion protein comprises a collagen binding moiety that specifically binds collagen with an affinity (KD) of less than about 500 nM, as determined by SPR assay. In some embodiments, the collagen binding moiety specifically binds collagen with an affinity (KD) of less than about 0.1-500 µM, 0.1-100 µM, or 0.1-1 µM, as determined by SPR assay. In some embodiments, the collagen binding moiety specifically binds collagen with an affinity (KD) of less than about 100-1000 nM, 100-1000 nM, 100-800 nM, 100-600 nM, or 100-500 nM, as determined by SPR assay.

[0258] The phrase "surface plasmon resonance" includes an optical phenomenon that allows for the detection of changes in protein concentrations within a biosensor matrix by, for example, using a BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ) to detect alterations in real-time biospecific interactions. For further description, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51 :19-26; Jonsson, U., et al. (1991) Biotechniques 11 :620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0259] In some embodiments, the IL-12 Fc fusion protein comprises a collagen binding moiety that specifically binds collagen and does not specifically bind to one or more non-collagen extracellular matrix (ECM) components including, but not limited to, fibronectin, heparin, entactin, decorin, bone bridge protein, and fibrillin. In some embodiments, the collagen binding moiety binds to collagen with a KD that is lower than one or more non-collagen ECM components. In some embodiments, the collagen binding moiety of type I collagen has a KD that is lower than the collagen binding moiety of an extracellular matrix component selected from fibronectin, heparin, entactin, decorin, bone bridge protein, fibrillin, or fibrillin. In some embodiments, the collagen binding moiety of type I collagen has a KD that is lower than the collagen binding moiety of 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%, about 99% lower than one or more non-collagen ECM components. In 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, about 10-fold lower than one or more non-collagen ECM components.

[0260] In some embodiments, the collagen binding moiety binds to type I collagen with a KD that is lower than 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.

[0261] In some embodiments, the reference collagen binding moiety comprises one or more (e.g., two, three, four, five, six, seven, eight, nine, ten or more) leucine-rich repeat sequences that bind 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: core proteoglycan, biglycan, fibromodulin, lumican, recombinant human cartilage proteoglycan, asporin, PRELP, osteoadherin / osteomodulin, visinin, osteoglycin / mimecan, podocan, perlecan, nidogen. In some embodiments, the reference collagen binding moiety is lumican. In some embodiments, the reference collagen binding moiety comprises a small leucine-rich proteoglycan (SLRP) of class I. SLRPs are known to bind collagen (Chen and Birk (2013) FEBS Journal 2120-2137). In some embodiments, the reference collagen binding moiety comprises a SLRP of class II. In some embodiments, the reference collagen binding moiety comprises a SLRP of class III. In some embodiments, the reference collagen binding moiety comprises a SLRP of class IV. In some embodiments, the reference collagen binding moiety comprises a SLRP of class V.

[0262] In some embodiments, the reference collagen binding moiety comprises a leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1) protein.

[0263] 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.

[0264] In its broadest form, the present application provides IL-12 Fc fusion proteins, wherein the collagen binding moiety binds to collagen. Preferably, the collagen binding moiety specifically binds to collagen type I.

[0265] In another preferred embodiment, the present application provides IL-12 Fc fusion proteins, wherein the collagen binding moiety binds to collagen I and has the sequence LxxLxLxxN (SEQ ID NO: 41), wherein L is leucine and N is asparagine and x is any amino acid.

[0266] In another embodiment, the present application provides an IL-12 Fc fusion protein, wherein the collagen binding moiety comprises or consists of any one 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 most preferred related embodiment, the collagen binding moiety comprises or consists of the sequence LRELHLDNN (SEQ ID NO: 47).

[0267] In another embodiment, the present application provides an IL-12 Fc fusion protein, wherein the collagen binding moiety is 20 amino acids (aa) in length, 19 aa, 18 aa, 17 aa, 16 aa, 15 aa, 14 aa, 13 aa, 12 aa, 11 aa, 10 aa, or 9 aa in length and comprises the sequence LxxLxLxxN (SEQ ID NO: 41), wherein L is leucine and N is asparagine and x is any amino acid.

[0268] Fibronectin binding moiety

[0269] Fibronectin is a high molecular weight (about 500 to about 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., syndecan). Fibronectin exists as a protein dimer, which is composed of two nearly identical monomers linked by a pair of disulfide bonds. Fibronectin is produced from a single gene, but alternative splicing of the pre-mRNA results in the production of several isoforms. Two types of fibronectin exist in vertebrates: soluble plasma fibronectin (previously called "cold insoluble globulin" or CIg) is a major protein component of plasma (300 μg / ml) and is produced in the liver by insoluble cellular fibronectin from hepatocytes as a major component of the extracellular matrix. It is secreted as a soluble protein dimer by various cells, primarily fibroblasts, and then assembled into insoluble matrices in complex cell-mediated processes. Fibronectin plays a major 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 a variety of pathologies, including cancer, arthritis, and fibrosis. It has been suggested that fibronectin expression can be upregulated in cancerous tissue.

[0270] An exemplary amino acid sequence of fibronectin is set forth in SEQ ID NO: 48 (UNIPROT Reference Sequence: P02751).

[0271] Accordingly, in some embodiments, the IL-12 Fc fusion protein comprises a fibronectin binding moiety that specifically binds fibronectin. In some embodiments, the fibronectin binding moiety specifically binds human fibronectin.

[0272] The binding of a fibronectin binding moiety to a fibronectin binding moiety can be determined by methods known in the art. In some embodiments, a 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, a fibronectin binding moiety is derived from a naturally occurring fibronectin binding protein or fibronectin receptor.

[0273] In some embodiments, the IL-12 Fc fusion protein specifically binds fibronectin with an affinity (KD) of less than about 500 μΜ, as determined by a fibronectin binding assay. In some embodiments, the IL-12 Fc fusion protein comprises a fibronectin binding moiety that specifically binds fibronectin with an affinity (KD) of less than about 100 μΜ, as determined by a fibronectin binding assay. In some embodiments, the IL-12 Fc fusion protein comprises a fibronectin binding moiety that specifically binds fibronectin with an affinity (KD) of less than about 1 μΜ, as determined by a fibronectin binding assay. In some embodiments, the IL-12 Fc fusion protein comprises a fibronectin binding moiety that specifically binds 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 fibronectin with an affinity (KD) of less than about 0.1-500 μΜ, 0.1-100 μΜ, or 0.1-1 μΜ, as determined by a fibronectin binding assay. In some embodiments, the fibronectin binding moiety specifically binds fibronectin with an affinity (KD) of less than about 100-1000 nM, 100-1000 nM, 100-800 nM, 100-600 nM, or 100-500 nM, as determined by a fibronectin binding assay.

[0274] In some embodiments, the fibronectin binding assay determines the binding affinity of the fibronectin binding moiety for fibronectin.

[0275] 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, vol. 45, no. 3, pp. 275-284). Thus, in some embodiments, the IL-12 Fc fusion protein comprises a fibronectin binding moiety that specifically binds fibronectin with an affinity (KD) of less than about 500 μΜ, as determined by ELISA. In some embodiments, the IL-12 Fc fusion protein comprises a fibronectin binding moiety that specifically binds fibronectin with an affinity (KD) of less than about 100 μΜ, as determined by ELISA. In some embodiments, the IL-12 Fc fusion protein comprises a fibronectin binding moiety that specifically binds fibronectin with an affinity (KD) of less than about 1 μΜ, as determined by ELISA. In some embodiments, the IL-12 Fc fusion protein comprises a fibronectin binding moiety that specifically binds fibronectin with an affinity (KD) of less than about 500 nM, as determined by ELISA. In some embodiments, the fibronectin binding moiety specifically binds fibronectin with an affinity (KD) of less than about 0.1-500 μΜ, 0.1-100 μΜ, or 0.1-1 μΜ, as determined by ELISA. In some embodiments, the fibronectin binding moiety specifically binds fibronectin with an affinity (KD) of less than about 100-1000 nM, 100-1000 nM, 100-800 nM, 100-600 nM, or 100-500 nM, as determined by ELISA.

[0276] 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, the IL-12 Fc fusion protein comprises a fibronectin binding moiety that specifically binds fibronectin with an affinity (KD) of less than about 500 μΜ, as determined by SPR assay. In some embodiments, the IL-12 Fc fusion protein comprises a fibronectin binding moiety that specifically binds fibronectin with an affinity (KD) of less than about 100 μΜ, as determined by SPR assay. In some embodiments, the IL-12 Fc fusion protein comprises a fibronectin binding moiety that specifically binds fibronectin with an affinity (KD) of less than about 1 μΜ, as determined by SPR assay. In some embodiments, the IL-12 Fc fusion protein comprises a fibronectin binding moiety that specifically binds 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 fibronectin with an affinity (KD) of less than about 0.1-500 μΜ, 0.1-100 μΜ, or 0.1-1 μΜ, as determined by SPR assay. In some embodiments, the fibronectin binding moiety specifically binds fibronectin with an affinity (KD) of less than about 100-1000 nM, 100-1000 nM, 100-800 nM, 100-600 nM, or 100-500 nM, as determined by SPR assay.

[0277] In some embodiments, the IL-12 Fc fusion protein comprises a fibronectin binding moiety that specifically binds to fibronectin and does not specifically bind to one or more non-fibronectin extracellular matrix (ECM) components including, but not limited to, collagen, heparin, entactin, tenascin C, osteopontin, and fibrillin. In some embodiments, the fibronectin binding moiety binds to fibronectin with a KD that is lower than the KD of 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 a fibronectin binding moiety for an extracellular matrix component selected from collagen, heparin, entactin, osteopontin, tenascin C, or fibrillin. 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%, about 99% lower than one or more non-collagen ECM components. In 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, about 10-fold lower than one or more non-collagen ECM components.

[0278] In some embodiments, the fibronectin binding moiety competes for binding to fibronectin with a reference fibronectin binding moiety. In some embodiments, the reference fibronectin binding moiety comprises a peptide as disclosed, e.g., by Sipes et al., (1993) Journal Cell Biol., 121(2): 469-77.

[0279] In its broadest form, the present application provides an IL-12 Fc fusion protein, wherein the fibronectin binding moiety binds to fibronectin.

[0280] In another preferred embodiment, the present application provides an IL-12 Fc fusion protein, wherein the fibronectin binding moiety binds to fibronectin and has the sequence GGWSHW (SEQ ID NO: 49).

[0281] In another embodiment, the present application provides an IL-12 Fc fusion protein, wherein the fibronectin 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 and comprises the sequence GGWSHW (SEQ ID NO: 49).

[0282] Heparin binding moiety

[0283] Heparin, also known as unfractionated heparin (UFH), is a drug and a naturally occurring glycosaminoglycan. Native heparin is a polymer with a molecular weight range of 3 to 30 kDa, but most commercial heparin preparations have an average molecular weight in the range of 12 to 15 kDa. Heparin is a member of the family of carbohydrate glycosaminoglycans, which includes the closely related molecule heparan sulfate, and is composed of variably sulfated repeating disaccharide units.

[0284] 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.

[0285] 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 for binding to heparin with a known or reference heparin binding protein. In some embodiments, the heparin binding moiety is derived from a naturally occurring heparin binding protein or heparin receptor.

[0286] In some embodiments, the IL-12 Fc fusion protein specifically binds 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 moiety that specifically binds 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 moiety that specifically binds 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 moiety that specifically binds 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 less than 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 less than about 100-1000 nM, 100-1000 nM, 100-800 nM, 100-600 nM, or 100-500 nM, as determined by a heparin binding assay.

[0287] In some embodiments, the heparin binding assay determines the binding affinity of the heparin binding moiety for heparin.

[0288] 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, the IL-12 Fc fusion protein comprises a heparin binding moiety that specifically binds fibronectin with an affinity (KD) of less than about 500 μΜ, as determined by ELISA. 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 100 μΜ, as determined by ELISA. 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 1 μΜ, as determined by ELISA. 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 ELISA. In some embodiments, the heparin binding moiety specifically binds heparin with an affinity (KD) of less than about 0.1-500 μΜ, 0.1-100 μΜ, or 0.1-1 μΜ, as determined by ELISA. In some embodiments, the heparin binding moiety specifically binds heparin with an affinity (KD) of less than about 100-1000 nM, 100-1000 nM, 100-800 nM, 100-600 nM, or 100-500 nM, as determined by ELISA.

[0289] 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, the IL-12 Fc fusion protein comprises a heparin binding moiety that specifically binds heparin with an affinity (KD) of less than about 500 µM, as determined by 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 100 µM, as determined by 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 1 µM, as determined by 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 less than 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 less than about 100-1000 nM, 100-1000 nM, 100-800 nM, 100-600 nM, or 100-500 nM, as determined by SPR assay.

[0290] In some embodiments, the IL-12 Fc fusion protein comprises a heparin binding moiety that specifically binds heparin and does not specifically bind to one or more non-heparin extracellular matrix (ECM) components, including, but not limited to, collagen, fibronectin, entactin, decorin, osteonectin, and fibrillin. In some embodiments, the heparin binding moiety binds to heparin with a KD that is lower than the KD of 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 a heparin binding moiety for an ECM component selected from collagen, fibronectin, entactin, osteonectin, decorin, or fibrillin. In some embodiments, the heparin binding moiety binds to heparin with a KD that is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 99% lower than the KD of one or more non-heparin ECM components. In some embodiments, the heparin binding moiety binds to heparin with a KD that is about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold lower than the KD of one or more non-heparin ECM components.

[0291] In some embodiments, the heparin-binding moiety competes for binding to heparin with a reference heparin-binding moiety. In some embodiments, the reference heparin-binding moiety comprises a peptide as disclosed, e.g., in Luria-Perez et al. (2019) Cytokine, 120:220-226.

[0292] In its broadest form, the present application provides IL-12 Fc fusion proteins, wherein the heparin-binding moiety binds to heparin.

[0293] In another preferred embodiment, the present application provides IL-12 Fc fusion proteins, wherein the heparin-binding moiety binds to heparin and has the sequence VRIQRKKEKMKET (SEQ ID NO: 50).

[0294] In another embodiment, the present application provides IL-12 Fc fusion proteins, wherein the heparin-binding moiety is 20 amino acids (aa) in length, 19 aa, 18 aa, 17 aa, 16 aa, 15 aa, 14 aa, 13 aa, 12 aa, 11 aa, 10 aa, or 9 aa in length and comprises the sequence VRIQRKKEKMKET (SEQ ID NO: 50).

[0295]

[0296]

[0297]

[0298]

[0299]

[0300] IL-12 Fc fusion proteins

[0301] IL-12 Fc fusion proteins are described and disclosed herein, as are compositions and articles of manufacture comprising the IL-12 Fc fusion proteins of the present application. The present inventors contemplate the IL-12 Fc fusion proteins of the present application as shown below, and the selection of those fusion proteins and their preparation are discussed in the accompanying examples. The cleavage products comprising the IL-12 cytokine and binding moiety following proteolytic cleavage are underlined in Table 5.

[0302] Table 5:

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313] 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 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 the IL-12p40 subunit in the first polypeptide chain; wherein the first polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which is protease cleavable, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the 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.

[0314] 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 the IL-12p40 subunit in the first polypeptide chain; wherein the first polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which is protease cleavable, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the second polypeptide chain further comprises a collagen binding moiety, preferably selected from the group consisting of SEQ ID NOs: 41-47.

[0315] 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 polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which is protease-cleavable, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the 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. The masking moiety can further comprise an additional alanine connected to its C-terminus.

[0316] 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 polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which 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, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the 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.

[0317] 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 polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which is protease-cleavable, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the 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.

[0318] 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 polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which is protease-cleavable, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the 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.

[0319] 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 polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which is protease-cleavable, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the second polypeptide chain further comprises a collagen binding moiety, preferably selected from the group consisting of SEQ ID NOs: 41-47. The masking moiety can further comprise an additional alanine connected to its C-terminus.

[0320] 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 polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which 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, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the second polypeptide chain further comprises a collagen binding moiety, preferably selected from the group consisting of SEQ ID NOs: 41-47. The masking moiety can further comprise an additional alanine connected to its C-terminus.

[0321] In one embodiment, the 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 polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which 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, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the second polypeptide chain further comprises a collagen binding moiety, preferably selected from the group consisting of SEQ ID NOs: 41-47. The masking moiety can further comprise an additional alanine connected to its C-terminus.

[0322] In one embodiment, the 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 polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which 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, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the second polypeptide chain further comprises a collagen binding moiety, preferably selected from the group consisting of SEQ ID NOs: 41-47. The masking moiety can further comprise an additional alanine connected to its C-terminus.

[0323] In one embodiment, the 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 polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which 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, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the second polypeptide chain further comprises a collagen binding moiety having the amino acid sequence of SEQ ID NO: 41. The masking moiety can further comprise an additional alanine connected to its C-terminus.

[0324] In one embodiment, the 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 polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which 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, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the second polypeptide chain further comprises a collagen binding moiety having the amino acid sequence of SEQ ID NO: 42. The masking moiety can further comprise an additional alanine connected to its C-terminus.

[0325] In one embodiment, the 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 polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which 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, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the second polypeptide chain further comprises a collagen binding moiety having the amino acid sequence of SEQ ID NO: 43. The masking moiety can further comprise an additional alanine connected to its C-terminus.

[0326] In one embodiment, the 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 polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which 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, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the second polypeptide chain further comprises a collagen binding moiety having the amino acid sequence of SEQ ID NO: 44. The masking moiety can further comprise an additional alanine connected to its C-terminus.

[0327] In one embodiment, the 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 polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which 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, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the second polypeptide chain further comprises a collagen binding moiety having the amino acid sequence of SEQ ID NO: 45. The masking moiety can further comprise an additional alanine connected to its C-terminus.

[0328] In one embodiment, the 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 polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which 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, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the second polypeptide chain further comprises a collagen binding moiety having the amino acid sequence of SEQ ID NO: 46. The masking moiety can further comprise an additional alanine connected to its C-terminus.

[0329] 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 polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which 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, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the second polypeptide chain further comprises a collagen binding moiety having the amino acid sequence of SEQ ID NO: 47. The masking moiety can further comprise an additional alanine connected to its C-terminus.

[0330] 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 polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which 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, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the second polypeptide chain further comprises a collagen binding moiety having the amino acid sequence of SEQ ID NO: 41. The masking moiety can further comprise an additional alanine connected to its C-terminus.

[0331] 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 polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which 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, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the second polypeptide chain further comprises a collagen binding moiety having the amino acid sequence of SEQ ID NO: 42. The masking moiety can further comprise an additional alanine connected to its C-terminus.

[0332] 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 polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which 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, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the second polypeptide chain further comprises a collagen binding moiety having the amino acid sequence of SEQ ID NO: 43. The masking moiety can further comprise an additional alanine connected to its C-terminus.

[0333] 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 polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which 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, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the second polypeptide chain further comprises a collagen binding moiety having the amino acid sequence of SEQ ID NO: 44. The masking moiety can further comprise an additional alanine connected to its C-terminus.

[0334] 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 polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which 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, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the second polypeptide chain further comprises a collagen binding moiety having the amino acid sequence of SEQ ID NO: 45. The masking moiety can further comprise an additional alanine connected to its C-terminus.

[0335] 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 polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which 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, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the second polypeptide chain further comprises a collagen binding moiety having the amino acid sequence of SEQ ID NO: 46. The masking moiety can further comprise an additional alanine connected to its C-terminus.

[0336] 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 polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which 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, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the second polypeptide chain further comprises a collagen binding moiety having the amino acid sequence of SEQ ID NO: 47. The masking moiety can further comprise an additional alanine connected to its C-terminus.

[0337] 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 polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which is protease-cleavable, wherein the protease-cleavable linker comprises or consists of the amino acid sequence of SEQ ID NO: 232, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the second polypeptide chain further comprises a collagen binding moiety having the amino acid sequence of SEQ ID NO: 41. The masking moiety can further comprise an additional alanine connected to its C-terminus.

[0338] 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 polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which is protease-cleavable, wherein the protease-cleavable linker comprises or consists of the amino acid sequence of SEQ ID NO: 232, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the second polypeptide chain further comprises a collagen binding moiety having the amino acid sequence of SEQ ID NO: 42. The masking moiety can further comprise an additional alanine connected to its C-terminus.

[0339] 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 polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which is protease-cleavable, wherein the protease-cleavable linker comprises or consists of the amino acid sequence of SEQ ID NO: 232, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the second polypeptide chain further comprises a collagen binding moiety having the amino acid sequence of SEQ ID NO: 43. The masking moiety can further comprise an additional alanine connected to its C-terminus.

[0340] 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 polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which is protease-cleavable, wherein the protease-cleavable linker comprises or consists of the amino acid sequence of SEQ ID NO: 232, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the second polypeptide chain further comprises a collagen binding moiety having the amino acid sequence of SEQ ID NO: 44. The masking moiety can further comprise an additional alanine connected to its C-terminus.

[0341] 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 polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which is protease-cleavable, wherein the protease-cleavable linker comprises or consists of the amino acid sequence of SEQ ID NO: 232, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the second polypeptide chain further comprises a collagen binding moiety having the amino acid sequence of SEQ ID NO: 45. The masking moiety can further comprise an additional alanine connected to its C-terminus.

[0342] 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 polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain, wherein the IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which is protease-cleavable, wherein the protease-cleavable linker comprises or consists of the amino acid sequence of SEQ ID NO: 232, wherein the masking moiety is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the second polypeptide chain further comprises a collagen binding moiety having the amino acid sequence of SEQ ID NO: 46. The masking moiety can further comprise an additional alanine connected to its C-terminus.

[0343] In one embodiment, the 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 IL-12p35 and IL-12p40 subunits of IL-12, and b) the second polypeptide chain comprises a second Fc domain and a masking portion bound to the IL-12p35 and / or IL-12p40 subunits of the first polypeptide chain, wherein the masking portion is selected from the group consisting of any of SEQ ID NO: 61-109; wherein the first polypeptide chain and the second polypeptide chain are linked via the first Fc domain and the second Fc domain, wherein the IL-12p35 or IL-12p40 subunit is linked to the C-terminus of the first Fc domain via a first peptide linker, the first peptide linker being protease-cleavable, wherein the protease-cleavable linker comprises SEQ ID NO: 61-109. The amino acid sequence of NO:232 or is composed of that amino acid sequence, wherein the masking portion is connected to the C-terminus of the second Fc domain via a second linker, preferably a peptide linker, and wherein the first polypeptide chain or the second polypeptide chain further comprises a collagen-binding portion having the amino acid sequence of SEQ ID NO:47. The masking portion may further comprise an additional alanine residue connected to its C-terminus.

[0344] In any of the foregoing embodiments, the IL-12 activity of the uncleaved IL-12 Fc fusion protein is at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 600 times lower than the IL-12 activity of the cleaved IL-12 Fc fusion protein after cleavage of the cleavable linker (the IL-12 activity of the uncleaved IL-12 Fc fusion protein is lower than the IL-12 activity of the cleavable linker after cleavage of the cleavable linker). The IL-12 activity of the Fc fusion protein is at most 1 / 50, 1 / 75, 1 / 100, 1 / 125, 1 / 150, 1 / 175, 1 / 200, 1 / 225, 1 / 250, 1 / 275, 1 / 300, 1 / 325, 1 / 350, 1 / 375, 1 / 400, 1 / 425, 1 / 450, 1 / 475, 1 / 500, 1 / 525, 1 / 550, 1 / 575, or 1 / 600. In other words, in IL-12 bioassay (ECG) 50 Uncleaved IL-12Fc fusion protein: EC 50 The difference in EC5 between cleaved and cleaved IL-12Fc fusion proteins, measured, for example, in the PromegaIL-12 bioassay described in the examples. 50at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 400, 425, 450, 475, 500, 525, 550, 575, or 600.

[0345] In the following table, additional IL-12 Fc fusion proteins or VHH masking moieties are disclosed that have been conceived or generated, for example, as intermediate molecules during optimization or as other alternatives. It is understood that each IL-12 Fc fusion protein consists of two chains identified by an appropriate identifier, for example, BI-062 PAMBOA® knob pairs only with BI-062 POCMA® hole; or knob chain BI-067 pairs only with hole chain BI-067. Thus, in another embodiment, the present application relates to an IL-12 Fc fusion protein comprising or consisting of two polypeptide chains identified by an appropriate identifier pair as disclosed in Table 6, for example, SEQ ID NOs: 242 and 243; SEQ ID NOs: 245 and 246; SEQ ID NOs: 247 and 248; and the like.

[0346] Table 6:

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382] Therapeutic methods

[0383] In its broadest form, the present application provides IL-12 Fc fusion proteins for use in medicine.

[0384] The IL-12 Fc fusion proteins of the present application are useful in cancer immunotherapy and are beneficial in controlling tumor growth by activating anti-tumor cytotoxic immune responses. Thus, the IL-12 Fc fusion proteins of the present application are useful in the treatment and / or prevention of cancer.

[0385] In another aspect, the IL-12 Fc fusion proteins of the present application are useful in a method of treating and / or preventing cancer and / or reducing the incidence of cancer, comprising administering to an individual having cancer a therapeutically effective amount of the IL-12 Fc fusion protein, whereby one or more symptoms of the cancer are ameliorated.

[0386] In yet another aspect, the present application further provides the use of the IL-12 Fc fusion proteins according to the present application for the manufacture of a medicament for the treatment and / or prevention of cancer.

[0387] In yet another aspect, the IL-12 Fc fusion proteins of the present application are useful in a method of treating and / or preventing melanoma, non-small cell lung cancer (NSCLC), cutaneous squamous cell carcinoma (cSCC), or bladder cancer and / or reducing the incidence of melanoma, non-small cell lung cancer (NSCLC), cutaneous squamous cell carcinoma (cSCC), or bladder cancer, comprising administering to an individual having melanoma, non-small cell lung cancer (NSCLC), cutaneous squamous cell carcinoma (cSCC), or bladder cancer a therapeutically effective amount of the IL-12 Fc fusion protein, whereby one or more symptoms of melanoma, non-small cell lung cancer (NSCLC), cutaneous squamous cell carcinoma (cSCC), or bladder cancer are ameliorated.

[0388] For the prevention or treatment of disease, the appropriate dosage of the IL-12 Fc fusion protein will depend on a variety of factors, such as the type of disease to be treated, as defined above; the severity and course of the disease; whether the IL-12 Fc fusion protein is administered for preventive or therapeutic purposes; previous therapy; the patient's clinical history; and the patient's response to the IL-12 Fc fusion protein; and the discretion of the attending physician. The IL-12 Fc fusion protein is suitably administered to the patient at one time or over a series of treatments.

[0389] In one aspect, the cancer is a solid tumor. In another aspect, the cancer is a lymphoma. In another aspect, the cancer is a relapsed or refractory advanced or metastatic solid tumor or lymphoma. In one aspect, the lymphoma is a non-Hodgkin's lymphoma or a Hodgkin's lymphoma. In another aspect, the lymphoma is a cutaneous T-cell lymphoma (CTCL) or Sezary syndrome / disease.

[0390] In one aspect, the cancer is skin cancer, lung cancer or head and neck cancer, brain cancer, gastrointestinal cancer, endometrial cancer, vaginal cancer, HPV-positive tumor, 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, cholangiocarcinoma, urothelial cancer of the bladder, urothelial cancer, renal 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 cancer, or bladder cancer.

[0391] In another aspect, the IL-12 Fc fusion protein is useful for treating patients who have failed or not adequately responded to prior PD-1 or PD-L1 inhibitor therapy (e.g., anti-immunotherapy for advanced or metastatic solid tumors or lymphomas).

[0392] In another aspect, the IL-12 Fc fusion protein is useful for treating patients who have completed checkpoint inhibitor therapy with a PD-1 or PD-L1 inhibitor (e.g., an antagonistic antibody against PD-1 or PD-L1).

[0393] The IL-12 Fc fusion protein is administered by any suitable means, including oral, parenteral, subcutaneous, intratumoral, intravenous, intradermal, intraperitoneal, intrapulmonary, and intranasal. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. In addition, the IL-12 Fc fusion protein is suitably administered by pulse infusion. In one aspect, the administration can be provided by injection, most preferably intravenous or subcutaneous injection, depending in part on whether the short- or long-term administration.

[0394] Depending on the particular IL-12 Fc fusion protein of the application and its particular pharmacokinetics and other characteristics, it can be administered daily, every other day, two, three, four, five, or six days per week, weekly, monthly, and the like. The course of administration can include long-term treatment once a week. By "long-term" is meant a duration of at least two weeks and preferably months or years.

[0395] The treatment schedule can include various courses of treatment and will typically require that a plurality of doses be administered to a patient over a period of one, two, three, or four weeks, optionally followed by further treatment for one or more rounds.

[0396] The term "inhibit" is used herein in the same context as "ameliorate" and "alleviate" and means to lessen or reduce one or more characteristics of a disease. The IL-12 Fc fusion proteins or pharmaceutical compositions of the present application will be formulated, dosed, and administered in a fashion 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 part of the body to be treated, the method of administration, the scheduling of administration, the routing of administration, and other factors known to medical practitioners. The therapeutically effective amount of the IL-12 Fc fusion protein to be administered will be

[0397] In another aspect, the IL-12 Fc fusion proteins of the present application can be administered multiple times and in several doses.

[0398] In another aspect, the IL-12 Fc fusion proteins are administered intravenously. In another aspect, the IL-12 Fc fusion proteins are administered subcutaneously.

[0399] As stated above, the IL-12 Fc fusion proteins of the present application have numerous utilities for stimulating an immune response against cancer cells. The strong immune activation potential observed is limited to the tumor microenvironment. Thus, in a preferred aspect, the IL-12 Fc fusion proteins of the present application can be administered systemically to a patient. Systemic applicability is a key attribute because many cancers are highly metastatic and it allows for treatment of difficult-to-reach and unreachable tumor lesions. Due to this unique immune stimulation property, the IL-12 Fc fusion proteins according to the present application are particularly useful for treating metastatic tumors.

[0400] Some patients develop resistance to checkpoint inhibitor therapy and such patients are observed to have accumulated mutations in the IFN pathway. Thus, in one aspect, the IL-12 Fc fusion proteins of the present application are useful for treating patients that develop resistance to checkpoint inhibitor therapy. Due to the unique immune promoting property of the IL-12 Fc fusion, such treated patients can become eligible for continuation of checkpoint inhibitor therapy.

[0401] In a preferred embodiment, the IL-12 Fc fusion proteins of the present application are useful for treating patients having non-small cell lung cancer that have completed checkpoint inhibitor therapy with a PD-1 or PD-L1 inhibitor, such as an antagonistic antibody to PD-1 or PD-L1.

[0402] It should be understood that any of the above pharmaceutical formulations or methods of treatment can be practiced using any of the IL-12 Fc fusion proteins or pharmaceutical compositions of the present application.

[0403] Combination

[0404] The present application also provides combination treatments / methods that provide certain advantages over current treatments / methods and / or known in the art. These advantages can include in vivo efficacy (e.g., improved clinical response, lengthened 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), later emergence of resistance, etc.), safe and well-tolerated administration, and reduced frequency and severity of adverse events.

[0405] The IL-12 Fc fusion proteins of the present application can be used in combination with other pharmacologically active agents, such as current state-of-the-art or standard-of-care compounds, such as cytostatic or cytotoxic substances, cell proliferation inhibitors, anti-angiogenic substances, steroids, immunomodulators / check point inhibitors, and the like. The IL-12 Fc fusion proteins of the present application can also be used in combination with radiation therapy.

[0406] Cell growth inhibitory and / or cytotoxic active substances that can be administered in combination with the IL-12 Fc fusion proteins of the present application include, but are not limited to, hormones, hormone analogs and anti-hormones, aromatase inhibitors, LHRH agonists and antagonists, inhibitors of 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, e.g. HER2, HER3, HER4), and hepatocyte growth factor (HGF), inhibitors of said inhibitors being, for example, (anti) 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. antifolates such as methotrexate, raltitrexed; pyrimidine analogs such as 5-fluorouracil (5-FU), gemcitabine, irinotecan, doxorubicin, TAS-102, capecitabine, and gemcitabine; purine and adenosine analogs such as mercaptopurine, thioguanine, cladribine, and pentostatin, cytarabine (ara C), fludarabine); antitumor antibiotics (e.g. anthracyclines); platinum derivatives (e.g. cisplatin, oxaliplatin, carboplatin); alkylating agents (e.g. estramustin, meclorethamine, melphalan, chlorambucil, busulphan, dacarbazin, cyclophosphamide, ifosfamide, temozolomide, nitrosoureas such as, for example, carmustin and lomustin, thiotepa);anti-mitotic agents (e.g., vinca alkaloids such as, for example, vinblastine, vindesine, vinorelbin, and vincristine; and taxanes such as paclitaxel, docetaxel); angiogenesis inhibitors, including bevacizumab, ramucirumab, and aflibercept; tubulin inhibitors;DNA synthesis inhibitors, PARP inhibitors, topoisomerase inhibitors (e.g., epipodophyllotoxins (e.g., etoposide and etoposide phosphate), teniposide, amsacrin, 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, PI3K alpha inhibitors, dual mTOR / PI3K inhibitors, STK33 inhibitors, AKT inhibitors, PLK1 inhibitors (e.g., volasertib), CDK inhibitors (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 analogs (e.g., everolimus, temsirolimus, ridaforolimus, sirolimus), androgene synthesis inhibitors, androgen receptor inhibitors, DNMT inhibitors, HDAC inhibitors, ANG1 / 2 inhibitors, CYP17 inhibitors, radiopharmaceuticals, immunotherapeutics (e.g., immune checkpoint inhibitors (e.g., CTLA4, PD1, PD-L1, LAG3, and TIM3 binding molecules / immunoglobulins, such as ipilimumab, nivolumab, pembrolizumab) and various chemotherapeutics (e.g., amifostin, anagrelid, clodronat, 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; SIRPa and CD47 blocking therapeutics; and / or cyclin-dependent kinase 9 inhibitors.

[0407] Furthermore, the potential conversion of immune "cold" to "hot" tumors, myeloid / dendritic cell activation, and T cell activation further advantageously interact with therapeutic modalities such as T cell engagers. Thus, in one embodiment, the IL-12 Fc fusion proteins of the present application can be used in combination with one or more T cell engagers for treatment.

[0408] The IL-12 Fc fusion proteins of the present application can be used in combination therapy with a cancer vaccine or an oncolytic virus. Such combination therapy can be given in the form of a non-fixed (e.g., not bound) combination of substances or in the form of a fixed combination, including a kit of parts for separate administration. 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 the lymphocytic choriomeningitis virus (LCMV), preferably with the strain WE-HPI. Such VSV is described, for example, in WO 2010 / 040526 and designated VSV-GP.

[0409] In yet another embodiment, any of the disclosed IL-12 Fc fusion proteins can be encoded in an appropriate viral vector, for example in an oncolytic viral vector, and preferably in a vesicular stomatitis virus, or more preferably in a vesicular stomatitis virus with the glycoprotein GP of the lymphocytic choriomeningitis virus (LCMV), preferably with the strain WE-HPI. Such VSV is described, for example, in WO 2010 / 040526 and designated VSV-GP. Such viral vector can then be used for delivery of the IL-12 Fc fusion protein (encoded in the genome of the viral vector). The IL-12 Fc fusion protein will then be transcribed / translated in the patient and the polypeptide chains will assemble in the human body to form the complete prodrug.

[0410] The IL-12 Fc fusion proteins of the present application can be used in combination therapy with a PD-1 pathway inhibitor. Such combination therapy can be given in the form of a non-fixed (e.g., not bound) combination of substances or in the form of a fixed combination, including a kit of parts for separate administration.

[0411] In this context, "combination" or "combined" within the meaning of the present application includes, but is not limited to, products that result from mixing or combining more than one active agent and includes both fixed and non-fixed (e.g., not limited) combinations (including kits) and uses, such as, for example, the simultaneous, concurrent, sequential, continuous, alternating, or separate use of the components or agents. The term "fixed combination" means that the active agents are arranged in a single entity or dosage form for administration to a patient simultaneously. The term "non-fixed combination" means that the active agents are administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limitations in their administration, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient. The latter also applies to mixtures therapies, e.g. the administration of three or more active agents.

[0412] The present application provides a combination of an IL-12 Fc fusion protein and a PD-1 pathway inhibitor for use in the treatment of a cancer described herein, preferably for use in the treatment of a solid cancer.

[0413] The present application also provides the use of a combination of an IL-12 Fc fusion protein and a PD-1 pathway inhibitor for the manufacture of a medicament for the treatment and / or prevention of a cancer described herein, preferably for the treatment of a solid cancer.

[0414] The present application further provides a method for the treatment and / or prevention of a cancer, comprising administering to an individual having a cancer a therapeutically effective amount of an IL-12 Fc fusion protein of the present application and a PD-1 pathway inhibitor, thereby ameliorating one or more symptoms of the cancer. The IL-12 Fc fusion protein of the present application and the PD-1 pathway inhibitor can be administered simultaneously, sequentially or alternately.

[0415] The IL-12 Fc fusion protein of the present application 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 application is administered intravenously or subcutaneously.

[0416] Especially preferred is the use of the IL-12 Fc fusion protein of the present application in combination with immunotherapeutic agents, including anti-PD-1 and anti-PD-L1 agents and anti-LAG3 agents, such as pembrolizumab and nivolumab, and antibodies as disclosed in WO2017 / 198741.

[0417] The combination as provided herein comprises (i) an IL-12 Fc fusion protein of the present application and (ii) a PD-1 pathway inhibitor, preferably an antagonistic antibody against PD-1 or PD-L1. Further provided is the use of such combination comprising (i) and (ii) for the treatment of a cancer described herein.

[0418] In another aspect, a combination therapy comprising the use of (i) an IL-12 Fc fusion protein of the present application and (ii) a PD-1 pathway inhibitor is provided. In such combination therapy, the IL-12 Fc fusion protein of the present application can be administered simultaneously, sequentially or alternately with the PD-1 pathway inhibitor.

[0419] For example, "simultaneous" administration includes administration of the active agents within the same general time period, e.g., on the same day, but not necessarily at the same time. Alternating administration includes administration of one agent during one period, e.g., the course of several days or a week, followed by administration of the other agent during a subsequent period, e.g., the course of several days or a week, and then repeating the pattern for one or more cycles. Sequential or consecutive administration includes administration of one agent using one or more doses during a first period, e.g., the course of several days or a week, followed by administration of the other agent using one or more doses during a second and / or additional period, e.g., the course of several days or a week. Overlapping schedules can also be employed, including administration of the active agents on different days within a treatment period, not necessarily in the regular order. Variations of these general guidelines can also be employed, e.g., depending on the agents used and the condition of the individual.

[0420] Sequential treatment schedules include administration of the IL-12 Fc fusion protein of the present application followed by administration of the PD-1 pathway inhibitor. Sequential treatment schedules also include administration of the PD-1 pathway inhibitor followed by administration of the IL-12 Fc fusion protein of the present application. Sequential treatment schedules can include 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.

[0421] A PD-1 pathway inhibitor in the meaning of the present application and all embodiments thereof is a compound that inhibits the interaction of PD-1 with its receptor. A PD-1 pathway inhibitor is capable of impairing PD-1 pathway signaling, preferably mediated by the PD-1 receptor. A PD-1 inhibitor can be any inhibitor against any member of the PD-1 pathway that is capable of antagonizing PD-1 pathway signaling. The inhibitor can be an antagonistic antibody targeting any member of the PD-1 pathway, preferably against the PD-1 receptor, PD-L1 or PD-L2. Further, a PD-1 pathway inhibitor can be a fragment of the PD-1 receptor or a PD-1 receptor that blocks the activity of the PD1 ligand.

[0422] PD-1 antagonists are well known in the art, e.g. reviewed by Li et al., Int. J. Mol. Sci. 2016, 17, 1151 (incorporated herein by reference). According to the present application, any PD-1 antagonist (especially antibodies) can be used (such as those disclosed by Li et al.) as well as other antibodies disclosed herein below. Preferably, the PD-1 antagonists of the present application and all embodiments thereof are selected from the group consisting of the following antibodies:

[0423] Ezabenlimab (BI 754091 );

[0424] Pembrolizumab (anti-PD-1 antibody);

[0425] Nivolumab (anti-PD-1 antibody);

[0426] Pembrolizumab (anti-PD-1 antibody);

[0427] PDR-001 (anti-PD-1 antibody);

[0428] PD1-1, PD1-2, PD1-3, PD1-4 and PD1-5 (anti-PD-1 antibodies) as disclosed below

[0429] Avelumab (anti-PD-L1 antibody);

[0430] Avelumab (anti-PD-L1 antibody);

[0431] Durvalumab (anti-PD-L1 antibody).

[0432] Pembrolizumab (previously also known as lambrolizumab; trade name Keytruda; also known as MK-3475), e.g. disclosed in Hamid, O. et al. (2013) New England Journal of Medicine 369(2): 134-44, is a humanized IgG4 monoclonal antibody that binds to PD-1; it contains a mutation at C228P designed to prevent Fc-mediated cellular toxicity. Pembrolizumab is e.g. disclosed in US 8,354,509 and WO 2009 / 114335. It is approved by the FDA for the treatment of patients with unresectable or metastatic melanoma and patients with metastatic NSCLC.

[0433] Nivolumab (CAS Registry Number: 946414-94-4; BMS-936558 or MDX1106b) is a fully human IgG4 monoclonal antibody that specifically blocks PD-1, lacking detectable antibody-dependent cellular cytotoxicity (ADCC). Nivolumab is disclosed, e.g., in US 8,008,449 and WO 2006 / 121168. It is approved by the FDA for the treatment of patients with unresectable or metastatic melanoma, metastatic NSCLC, and advanced renal cell carcinoma.

[0434] Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD-1. Pidilizumab is disclosed, e.g., in WO 2009 / 101611.

[0435] 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 WO 2015 / 112900 and WO 2017 / 019896.

[0436] The antibody PD1-1 to PD1-5 are antibody molecules defined by the sequences as shown in Table 7, wherein HC denotes the (full-length) heavy chain and LC denotes the (full-length) light chain:

[0437] Table 7:

[0438]

[0439]

[0440]

[0441] In particular, the anti-PD-1 antibody molecules described above have:

[0442] (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

[0443] (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

[0444] (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

[0445] (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

[0446] (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.

[0447] Atezolizumab (Tecentriq, also known as MPDL3280A) is a phage-derived human IgGlk monoclonal antibody targeting 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 suffering from urothelial carcinoma.

[0448] Avelumab is a fully human anti-PD-Ll IgGl monoclonal antibody and is described, for example, in Boyerinas et al. Cancer Immunol. Res. 2015; 3:1148-1157.

[0449] Durvalumab (MEDI4736) is a human IgGlk 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 Ibrahim et al. Semin. Oncol. 2015; 42:474-483.

[0450] Other PD-1 antagonists disclosed by Li et al. (see above) or known 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) can be used instead of or in addition to the antagonists mentioned above.

[0451] As used herein, INN is meant to also encompass all biosimilar antibodies having the same or substantially the same amino acid sequence as the source antibody, including but not limited to those biosimilar antibodies authorized under the equivalent provisions of U.S. 42 USC § 262 subsection (k) and other jurisdictions.

[0452] The above listed PD-1 antagonists, as well as their respective manufacture, therapeutic uses, and properties, are known in the art.

[0453] In one embodiment, the PD-1 antagonist is ezabenlimab.

[0454] In one embodiment, the PD-1 antagonist is pembrolizumab.

[0455] In another embodiment, the PD-1 antagonist is nivolumab.

[0456] In another embodiment, the PD-1 antagonist is pidilizumab.

[0457] In another embodiment, the PD-1 antagonist is atezolizumab.

[0458] In another embodiment, the PD-1 antagonist is avelumab.

[0459] In another embodiment, the PD-1 antagonist is durvalumab.

[0460] In another embodiment, the PD-1 antagonist is PDR-001.

[0461] In another embodiment, the PD-1 antagonist is PD1-1.

[0462] In another embodiment, the PD-1 antagonist is PD1-2.

[0463] In another embodiment, the PD-1 antagonist is PD1-3.

[0464] In another embodiment, the PD-1 antagonist is PD1-4.

[0465] In another embodiment, the PD-1 antagonist is PD1-5.

[0466] Pharmaceutical compositions and formulations

[0467] The present application is further directed to pharmaceutical compositions for use in 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 application. The present application further encompasses methods of using at least one IL-12 Fc fusion protein or pharmaceutical composition described below in the treatment of diseases (as specified in more detail below), and further encompasses the use of such IL-12 Fc fusion proteins or pharmaceutical compositions of the present application in the preparation of a medicament for the treatment of such diseases.

[0468] The IL-12 Fc fusion proteins of the application (e.g., any of the ones 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 to be used. Thus, the IL-12 Fc fusion proteins of the application and / or compositions comprising the same can be administered, for example, intravenously (i.v.), subcutaneously (s.c.), intramuscularly (i.m.), intraperitoneally (i.p.), transdermally, orally, sublingually (e.g., in the form of a sublingual lozenge, spray, or droplet placed under the tongue and absorbed into the capillary network thereunder by the mucosa), nasally (intra-nasally) (trans-nasally) (e.g., in the form of a nasal spray and / or in the form of an aerosol), topically, by way of a suppository, by inhalation, or in any other suitable manner in an effective amount or dose. The IL-12 Fc fusion proteins can be administered by infusion, bolus, or injection. In preferred embodiments, administration is by intravenous infusion or subcutaneous injection.

[0469] The IL-12 Fc fusion proteins of the application and / or compositions comprising the same are administered in accordance with a treatment regimen appropriate for the disease, disorder, or condition to be treated or alleviated. The clinician generally will determine appropriate dosages, taking into consideration factors relevant to the specific IL-12 Fc fusion protein of the application to be used, the particular mode of administration, and the patient's age, size, weight, diet, general condition, and the like. Generally, the treatment regimen will comprise administration of an IL-12 Fc fusion protein of the application or one or more compositions comprising the same in a therapeutically effective amount or dose.

[0470] Generally, for the treatment and / or alleviation of the diseases, disorders, and conditions referred to herein, and depending on the particular disease, disorder, or condition to be treated, the potency of the particular IL-12 Fc fusion protein of the application to be used, the particular mode of administration and the particular pharmaceutical formulation used, the IL-12 Fc fusion proteins of the application generally will be administered in an amount of 0.005-20.0 mg, preferably 0.05-10.0 mg, per kilogram body weight per dose, either continuously (e.g., by infusion) or, more preferably, as a single dose (e.g., biweekly, weekly, or monthly doses; see below), but can vary significantly, especially depending on the parameters mentioned previously. Thus, in some cases, less than the minimum dose given above can be sufficient, while in other cases the upper limit can be exceeded. When larger amounts are administered, it can be advisable to divide it into a number of smaller doses over the course of the day.

[0471] Depending on the particular IL-12 Fc fusion protein of the application and its particular pharmacokinetic and other properties, it can be administered every day, every other day, every two days, every three days, every four days, every five days, or every six days, every week, every month, and the like. A course of administration can include once-weekly chronic treatment. By "chronic" is meant a duration of at least two weeks and preferably months or years.

[0472] The IL-12 Fc fusion proteins of the application and compositions comprising the same can be tested for efficacy 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 clear to those skilled in the art and include, for example, the assays and animal models used in the Examples below.

[0473] For pharmaceutical use, the IL-12 Fc fusion proteins of the application can be formulated as pharmaceutical preparations comprising (i) at least one IL-12 Fc fusion protein of the application (e.g., any of the sequences disclosed) and (ii) at least one pharmaceutically acceptable carrier, diluent, excipient, adjuvant, and / or stabilizer, and (iii) optionally one or more other pharmacologically active polypeptides and / or compounds. By "pharmaceutically acceptable" is meant that the respective material does not show any biological or undesired effects when administered to a subject and does not interact in a deleterious manner with any of the other components (e.g., the pharmaceutically active ingredients) contained in the pharmaceutical composition. Specific examples can be found in standard handbooks, such as Remington's Pharmaceutical Sciences, 18thEdition, Mack Publishing Company, USA (1990). For example, the IL-12 Fc fusion proteins of the application can be formulated and administered in any of the ways known per se for conventional antibodies and antibody fragments and other pharmaceutically active proteins and fusion proteins. Thus, according to a further embodiment, the application relates to a pharmaceutical composition or preparation containing at least one IL-12 Fc fusion protein of the application and at least one pharmaceutically acceptable carrier, diluent, excipient, adjuvant, and / or stabilizer, and optionally one or more other pharmacologically active substances, in a lyophilized or otherwise dried formulation or in an aqueous or non-aqueous solution or suspension.

[0474] Pharmaceutical formulations intended for non-enteral administration, such as intravenous, intramuscular, subcutaneous, or intravenous infusion, may be, for example, sterile solutions, suspensions, dispersions, emulsions, or powders containing an active ingredient and optionally suitable for infusion or injection after another dissolution or dilution step. Suitable carriers or diluents for such formulations include, but are not limited to, sterile water and pharmaceutically acceptable buffered aqueous solutions and solutions such as phosphate-buffered saline, Ringer's solution, dextran solution, and Hank's solution; water-oil; glycerol; ethanol; glycols such as propylene glycol; and mineral oils, animal oils, and vegetable oils, such as peanut oil, soybean oil, and suitable mixtures thereof.

[0475] The solution of the IL-12 Fc fusion protein of the present invention may also contain preservatives, such as antibacterial and antifungal agents to prevent microbial growth, such as p-hydroxybenzoate / paraben, chlorobutanol, phenol, sorbic acid, thimerosal, ethylenediaminetetraacetic acid (an alkali metal salt), and the like. In many cases, isotonic agents are preferred, such as sugars, buffers, or sodium chloride. Optionally, emulsifiers and / or dispersants may be used. Suitable flowability can be maintained, for example, by forming liposomes, by maintaining the desired particle size in the case of a dispersion, or by using surfactants. Other agents that delay absorption may also be added, such as aluminum monostearate and gelatin. The solution can be filled into injection vials, ampoules, infusion bottles, and the like.

[0476] In all cases, the final dosage form must be sterile, fluid, and stable under both manufacturing and storage conditions. A sterile injectable solution is prepared by incorporating the required amount of the active compound, as needed, along with various other ingredients listed above, into a suitable solvent, followed by filtration and sterilization. In the case of sterile powders used to prepare sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze-drying techniques, which produce a powder containing the active ingredient present in a pre-sterile filtered solution plus any additional desired ingredients.

[0477] Generally, aqueous solutions or suspensions are preferred. Typically, formulations suitable for therapeutic proteins (such as the IL-12 Fc fusion protein of this invention) are buffered protein solutions, such as solutions comprising protein at suitable concentrations (e.g., 0.001 to 400 mg / ml, preferably 0.005 to 200 mg / ml, more preferably 0.01 to 200 mg / ml, even more preferably 1.0 to 100 mg / ml, such as 1.0 mg / ml (intravenous administration) or 100 mg / ml (subcutaneous administration)) and an aqueous buffer solution as follows:

[0478] -Phosphate-buffered saline, pH 7.4,

[0479] - other phosphate buffers, pH 6.2 to pH 8.2,

[0480] - acetate buffers, pH 3.2 to 7.5, preferably pH 4.8 to 5.5

[0481] - histidine buffers, pH 5.5 to pH 7.0,

[0482] - succinate buffers, pH 3.2 to pH 6.6, and

[0483] - citrate buffers, pH 2.1 to 6.2,

[0484] and optionally salts (e.g. NaCl) and / or sugars (such as sucrose and trehalose) and / or other polyols (such as mannitol and glycerol) for providing the tonicity of the solution.

[0485] Further, other agents, such as detergents, e.g. 0.02% Tween-20 or Tween-80 can be included in the solution. Formulations for subcutaneous administration can include significantly higher concentrations, such as up to 100 mg / ml or even more than 100 mg / ml of the IL-12 Fc fusion protein of the application. However, it will be clear to a person skilled in the art that the ingredients and their amounts as given above represent only one preferred choice. Alternatives and variations will be directly apparent to a person skilled in the art or can easily be thought of starting from the above disclosure. The above formulations can optionally be provided as lyophilized formulations to be reconstituted in a solution, e.g. water for injection (WFI).

[0486] According to another aspect of the application, the IL-12 Fc fusion protein of the application can be used in combination with a device suitable for the administration of a protein, such as a syringe, an injection pen, a micro pump or other devices.

[0487] Kit

[0488] The present application also encompasses a kit comprising at least one IL-12 Fc fusion protein of the application (e.g. any of the ones shown in the disclosed sequences) and optionally one or more other components selected from the group consisting of other pharmaceuticals for the treatment of the diseases and conditions described above.

[0489] In one embodiment, the kit comprises a composition comprising an effective amount of an IL-12 Fc fusion protein of the application in unit dosage form.

[0490] The present application also encompasses a kit comprising at least one IL-12 Fc fusion protein of the application and one or more other components selected from the group consisting of other pharmaceuticals for the treatment of the diseases and conditions described above.

[0491] In one embodiment, the kit comprises a composition comprising an effective amount of the IL-12 Fc fusion protein of the application in unit dosage form. In another embodiment, the kit comprises a composition comprising an effective amount of the IL-12 Fc fusion protein of the application 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.

[0492] In some embodiments, the kit contains a sterile container with such compositions; the containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foils, or other materials suitable for holding pharmaceutical agents. In addition, the kit can contain a first container with a pharmaceutical composition having the IL-12 Fc fusion protein of the application 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 to reconstitute or dilute the IL-12 Fc fusion protein.

[0493] If desired, the IL-12 Fc fusion protein of the application is provided with instructions for administering the IL-12 Fc fusion protein to an individual having cancer. The instructions will generally include information on the use of the composition to treat or prevent cancer. In other embodiments, the instructions include at least one of the following: instructions for the therapeutic agent, dosage schedule and administration for treating or preventing cancer or symptoms thereof, precautions, warnings, indications, contraindications, overdose information, adverse reactions, animal pharmacology, clinical studies, and / or references. The instructions can be printed directly on the container (if present), or applied as a label to the container, or as a separate sheet, pamphlet, card, or folder supplied with the container or supplied with the container.

[0494] Methods of production and purification

[0495] Another aspect of the application provides a method of producing the IL-12 Fc fusion proteins described herein, comprising:

[0496] culturing the host cell of the application under conditions permitting expression of the molecule; and

[0497] recovering the molecule; and optionally

[0498] One embodiment of this aspect of the application is where the method of production further comprises step (c) further purifying and / or modifying and / or formulating the Fc fusion protein.

[0499] To produce an IL-12 Fc fusion protein, in practice, a DNA molecule encoding the first polypeptide chain is inserted into an expression vector such that the sequence is operably linked to transcriptional and translational control sequences. The DNA molecule encoding the second polypeptide chain can be inserted into the same expression vector or a different expression vector. The vector contains all the genetic elements needed for the expression of the polypeptide in a cell, such as a promoter, regulatory components, or components for selection. After the vector has been introduced into an appropriate host cell, both chains will be expressed separately from the vector and secreted by the cell. Both the first and second polypeptide chains will then associate via their respective Fc domains to form the complete IL-12 Fc fusion protein.

[0500] To make an IL-12 Fc fusion protein, one skilled in the art can select from a wide variety of expression systems well known in the art, such as those that can be selected from a review by Kipriyanov and Le Gall, Curr Opin Drug Discov Devel. 2004 Mar; 7(2): 233-42.

[0501] Expression vectors include plasmids, retroviruses, cosmids, EBV-derived episomes, and the like. The expression vector and expression control sequences are chosen 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, the DNA sequences, first and second polypeptide chain sequences, are inserted into the same expression vector. Suitable vectors are those which encode the functional, complete human first and second polypeptide sequences, wherein appropriate restriction sites are engineered such that either the first or second polypeptide sequences can be readily inserted and expressed, as described above.

[0502] The recombinant expression vectors can also encode a signal peptide that facilitates secretion of the polypeptide chain from a host cell. The DNA encoding the polypeptide chain can be cloned into the vector such that the signal peptide is operably linked in-frame to the amino terminus of the mature first and / or second polypeptide chain DNA. The signal peptide can 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 can already contain the signal peptide sequence.

[0503] In addition to the DNA sequences encoding the IL-12 Fc fusion protein chains, the recombinant expression vector carries sequences necessary for the transcription and translation of the IL-12 Fc fusion protein chains in a host cell. Examples of such sequences are the origin of replication, a promoter, an enhancer, a terminator, and a polyadenylation signal. The recombinant expression vector can further comprise a selection marker gene, for example, a drug resistance gene, to allow the selection of host cells into which the vector has been introduced. The nucleic acid molecules encoding the IL-12 Fc fusion protein chains described herein and the vectors comprising these DNA molecules can be introduced into host cells, such as bacterial cells or higher eukaryotic cells, such as mammalian cells, according to methods well known in the art, including transfection, lipofection, polymeric-mediated transfection, protoplast fusion, microinjection, calcium phosphate precipitation, electroporation, or transfer by viral vectors.

[0504] The recombinant expression vector can also carry sequences required for the replication of the vector in a host cell, such as an origin of replication, and a selectable marker gene. The nucleic acid molecules encoding the IL-12 Fc fusion protein chains described herein and the vectors comprising these DNA molecules can be introduced into host cells, such as bacterial cells or higher eukaryotic cells, such as mammalian cells, according to methods well known in the art, including transfection, lipofection, polymeric-mediated transfection, protoplast fusion, microinjection, calcium phosphate precipitation, electroporation, or transfer by viral vectors.

[0505] Preferably, the nucleic acid molecules encoding the IL-12 Fc fusion protein chains described herein are each inserted into a vector that is transfected into a host cell, preferably a mammalian cell.

[0506] Accordingly, in another aspect, there is provided a host cell comprising an expression vector comprising a nucleic acid molecule encoding the IL-12 Fc fusion protein chains described herein.

[0507] Mammalian cell lines that can be used as hosts for expression are well known in the art and include, inter alia, Chinese hamster ovary (CHO, CHO-DG44) cells, NSO, SP2 / 0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK, HEK293, or any derivative / progeny of such cell lines. Other mammalian cells can be used, including but not limited to human, mouse, rat, monkey, and rodent cell lines; or other eukaryotic cells, including but not limited to yeast, insect, and plant cells; or prokaryotic cells, such as bacteria. The IL-12 Fc fusion proteins of the application are produced by culturing the host cells for a period of time sufficient to allow for expression of the IL-12 Fc fusion proteins in the host cells.

[0508] The IL-12 Fc fusion proteins described herein are preferably recovered from the culture medium in a secreted polypeptide form or, if expressed for example without a secretion signal, they can be recovered from the host cell lysate. The IL-12 Fc fusion proteins described herein are purified using standard protein purification methods for recombinant proteins and host cell proteins in a manner to obtain a substantially homogeneous preparation of the IL-12 Fc fusion proteins described herein. For example, a current state-of-the-art purification method suitable for obtaining the IL-12 Fc fusion proteins of the application includes removal of cells and / or particulate cell debris from the culture medium or lysate as a first step. The IL-12 Fc fusion proteins are then purified from contaminant soluble proteins, polypeptides and nucleic acids, for example by fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, Sephadex chromatography, chromatography on silica or on a cation-exchange resin. As a final step in the process of obtaining the IL-12 Fc fusion proteins described herein, the purified IL-12 Fc fusion proteins can be dried, for example lyophilized, as described below for therapeutic applications.

[0509] It will be appreciated that immunoglobulin single variable domain molecules and preferably VHHs described herein can be produced and purified by the described methods with necessary modifications in detail.

[0510] Nucleic acids

[0511] Another aspect of the application provides an isolated nucleic acid molecule encoding an IL-12 Fc fusion protein chain of the application and / or an immunoglobulin single variable domain molecule of the application, or an expression vector comprising such a nucleic acid molecule.

[0512] As will be appreciated by those skilled in the art, 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 readily prepared.

[0513] 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 using standard methods of polymerase chain reaction (PCR). Initially, suitable oligonucleotides can be synthesized using methods known in the art (e.g. Gait, 1984) which can be used to generate synthetic genes. 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).

[0514] Nucleic acid molecules of the present application include, but are not limited to, DNA molecules encoding the polypeptide sequences shown in the sequence listing. In addition, the present application 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 WO 2007 / 042309. Preferred molecules (from the mRNA perspective) 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 to one of the DNA molecules described herein. For example, given the expression of IL-12 Fc fusion proteins in eukaryotic cells, the DNA sequences shown in the sequence listing have been designed to match the codon usage in eukaryotic cells. If expression of IL-12 Fc fusion proteins in E. coli is desired, these sequences can be altered to match the E. coli codon usage. Variants of the DNA molecules of the present application can be constructed in several different ways, as described, for example, in WO 2007 / 042309.

[0515] In another embodiment, any of the disclosed IL-12 Fc fusion proteins can be encoded in an appropriate mRNA sequence. The mRNA sequence can encode both polypeptide chains of the IL-12 Fc fusion protein, or two separate mRNA molecules each encoding one of the two polypeptide chains can be constructed, optionally including at least one secretion signal linked to either or both chains. Such mRNA can be used to treat a patient for any of the treatments described herein. After delivery of the mRNA to the patient, the mRNA will be translated and the polypeptide chains will assemble in the human body to form the complete prodrug. Examples

[0516] Materials and Methods

[0517] In the following, the materials and methods that have been used in the examples are described.

[0518] MMP cleavage analysis:

[0519] Recombinant MMP9 (R&D Systems) was activated with p-aminophenylacetic acid mercury. Activated MMP9 was incubated with IL-12 Fc fusion proteins (prepared in TCNB buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35 (w / v), pH 7.5) for 24 hours at 37°C. Digested proteins were aliquoted and stored at -80°C, followed by testing in SDS-PAGE, Western blot and IL-12 functional assays.

[0520] MMP cleavage analysis (peptides only):

[0521] The recombinant MMP was activated according to the manufacturer's recommendations (R&D). The activated MMP (2.5 nM final concentration) was then added to the Dabcyl / Edans peptide (2.5 μM final concentration) in TCNB buffer. The plate was read for 2 hours at 37°C with excitation at 340 nm / emission at 490 nm at 5 min intervals using a BioTek Synergy H1 mixed multimode reader (BioTek instrument). Gain 80 was used. Specific activity was then calculated based on parameters derived from the kinetic cleavage curves.

[0522] IL-12 Functional Analysis

[0523] NK-92 IL-12 activity analysis:

[0524] NK-92 cells (ATCC CRL-2407) were seeded at a density of 200,000 cells / well in 96-well plates, with 100 μL of culture medium containing different concentrations of lysed or unlysed MMP9 IL-12 Fc fusion protein added. The total volume per well was 200 μL, corresponding to a cell density of 100,000 cells / mL. After incubation at 37°C and 5% CO2 for 24 hours, the concentration of IFN-γ in the cell culture supernatant was determined by ELISA (Invitrogen). IFN-γ standards were included in the ELISA and linear curve fitting using GraphPad Prism 7.0, the software used to determine the IFN-γ concentration in the sample from absorbance at 450 nm (A450). The [agonist] versus response (three-parameter) fitting using GraphPad Prism 7.0 was used to evaluate EC... 50 Software is used to fit the data.

[0525] Promega IL-12 bioassay:

[0526] The IL-12 bioassay (catalog numbers JA2601, 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. In short, cells are thawed, resuspended, and 50 μl of the solution is pipetted into 96-well plates. 25 μl of serially diluted lysed and unlysed IL-12 Fc fusion protein is added to the cells, followed by incubation at 37°C for 6 hours. After incubation, the plate is removed from the incubator, and after reaching ambient temperature (10–15 min), 75 μl of Bio-Globe solution is added to the cells at room temperature. TM Reagent was added to each well and allowed to stand for 5-10 minutes, followed by luminescence measurement. The fold induction was calculated using the following formula: fold induction = RLU(sample - background) / RLU(no drug control - background). Data were plotted and EC was determined using GraphPad Prism software.50 .

[0527] TME linker binding

[0528] ELISA:

[0529] IL-12 Fc fusion proteins (in the amounts indicated in the legend) were diluted in PBS and added to collagen I coated 96 well plates (Corning) for 10-30 min. Prior to the addition of the proteins, the plates were blocked with 2% BSA to minimize non-specific binding. Subsequently, the plates were washed 3 times followed by incubation with biotinylated anti-human Fc antibody (Invitrogen). After additional washing steps, streptavidin-HRP and substrate were added and the plates were read in a Tecan reader.

[0530] Scar in Jar analysis:

[0531] Primary human lung fibroblasts were seeded at a density of 1000 cells / well in poly-D-lysine coated 384 CellCarrier microtiter plates from PerkinElmer in FBM with FGM-2™ Single Quots (Lonza, Basel, Switzerland). After 24 hours, the medium was replaced by the same medium without serum (starvation medium). Forty-eight hours after cell seeding, the starvation medium was replaced with starvation medium containing a mixture of Ficoll 70 and 400 (37.5 mg / mL and 25 mg / mL, respectively), 200 mM vitamin C and IPF-RC (1 : 1000 dilution). After 72 hours, the cell medium was removed and the cells were fixed with 100% ice-cold methanol for 30 minutes. Subsequently, the cells were washed with PBS and the plates were de-cellularized, IL-12 Fc fusion proteins (5 pg and 50 pg) were added to the wells for a 2-hour incubation. Next, the plates were washed and blocked with PBS containing 3% BSA for 30 minutes. After further washing steps, collagen I was stained using a monoclonal antibody (SAB4200678, Sigma-Aldrich). For primary antibody detection, the cells were washed and incubated with a goat anti-mouse IgGl Alexa Fluor 568 secondary antibody for 30 minutes at 37°C. After a final washing step, images were acquired in an Opera Phenix (Perkin Elmer) and transferred to the Columbus Image storage and analysis system (Perkin Elmer).

[0532] In vivo model:

[0533] C57BL / 6 mice were injected with MC38 or B16.F10 cells in the left flank. Treatment was initiated when tumors reached approximately 70-100 mm 3 Tumor volume and body weight were monitored 2 to 3 times per week. Statistical analysis was performed using GraphPad Prism software. Differences between groups were analyzed using t-tests with or without Welch correction, depending on the distribution of the data. Analysis of grouped data was performed using two-way analysis of variance (ANOVA) or the Kruskal-Wallis test.

[0534] Generation of stable pools

[0535] The final molecules were cloned into a mammalian expression system, encoding a knob and hole chain on one plasmid, driven by separate CMV promoters and a metabolic selection marker. Stable pools were generated by transfecting CHO-K1 GS- / - host cells with the respective expression plasmids and banked for cell culture processes.

[0536] Production and purification

[0537] The final molecules were produced from stable transfected and characterized CHO cell pools in bioreactors. Cell culture processes were performed under controlled conditions. Cell culture harvests were processed in an automated downstream process including Protein A capture, acid treatment, cation exchange and anion mixed mode chromatography polishing and different filtration steps. Product quality was subsequently determined for each construct.

[0538] Production and purification

[0539] Expression was performed in d 3L bioreactors with stable transfected CHO cell pools. Cell cultures were harvested after 14 days of cultivation. Potency was determined using Protein A HPLC. Purification was performed with an automated representative multi-step process train. Protein A capture with subsequent viral inactivation at low pH was followed by cation exchange and mixed mode chromatography. Constructs were finally concentrated using ultra- / diafiltration.

[0540] Product quality assessment

[0541] Purity of all constructs was assessed using state-of-the-art size exclusion chromatography and non-reducing capillary gel electrophoresis. Product quality was determined by hydrophilic liquid interaction chromatography coupled online to mass spectrometry (HILIC-MS).

[0542] Overall design of IL-12 fusion proteins

[0543] In the following examples, design pathways for IL-12 Fc fusion proteins are laid out. In the context of the examples, the molecules are also sometimes referred to as prodrugs or protease-activatable prodrugs.

[0544] Example 1:

[0545] Molecular components

[0546] Various arrangements of protease-activatable prodrugs desirably include the following components are evaluated:

[0547] 1) Cytokine itself: IL-12 is a heterodimer, which is composed of p40 and p35 subunits, however, these two domains can be linked together via a connecting peptide linker to form a functional single chain cytokine.

[0548] 2) Masking moiety, which can block the function of the cytokine / payload as well as a part of the prodrug, but will also release the cytokine / payload upon enzymatic activation of the prodrug.

[0549] 3) Enzyme-cleavable linker, the sequence composition of which is recognized by enzymes that are upregulated in the tumor microenvironment.

[0550] 4) Optionally, the prodrug can contain other components, such as the constant region of an antibody (Fc) to extend the half-life of the prodrug. The Fc allows for the generation of heterodimeric Fc through techniques such as knob-into-hole (knobs-into-holes). Here, the Fc can serve as a heterodimerization domain, which allows the cytokine to be generated on one arm and the masking body to be generated "in parallel" on the other arm. Conversely, wild-type Fc can also be used, where the cytokine is directly linked to the masking domain, followed by the Fc or some combination thereof, to form a "tandem" symmetrical prodrug.

[0551] 5) Optionally, the prodrug can contain a tumor-targeting domain, which can be a domain that specifically targets the prodrug to, for example, a tumor antigen or as another alternative that preferentially targets tumor-associated structures in the vicinity of the tumor. The effect of such a tumor-targeting domain can be three-fold: A) anchor the prodrug to the tumor, allowing for increased exposure of the prodrug to upregulated enzyme activity, thereby enhancing the conversion of the prodrug to the active drug, or B) anchor the active cytokine within the tumor, thereby enhancing its residence time and / or half-life within the tumor microenvironment, and C) anchor the active cytokine within the tumor to reduce potential toxicity associated with systemic exposure.

[0552] Exploration of prodrug molecule formats

[0553] Figures 1A-1H

[0554] To explore the functional inhibition of IL-12 in prodrug format, by generating molecules with different orientations and components and measuring the differential EC50 of the prodrug format to produce IL-12 activity in a functional assay50 (ΔEC 50 ) of the prodrug form compared to the active drug after cleavage of the prodrug enzyme-cleavable linker by, for example, MMP9.

[0555] Mutations to change the following parameters to generate eight (8) initial molecules:

[0556] 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.

[0557] b. Utilization of a naturally homodimerizing Fc (wild-type interface) with the cytokine and masking domain “in tandem”, or an Fc engineered to heterodimerize, such as using a knob-into-hole technology and the cytokine remaining on the Fc chain “in parallel” to the masking domain.

[0558] c. Utilization of a single chain IL-12 or version where the p35 domain is directly linked to the Fc and the p40 domain is co-transfected simultaneously, and IL12 is formed intracellularly during protein production, and the two domains are covalently linked by a naturally occurring disulfide bond.

[0559] Functional readouts explored using versions of the NK-92 cell-based assay are shown in the table below. In each case, the masking domain is a scFv tool molecule against IL-12, with an affinity for IL-12 of approximately 250 pM.

[0560] Table 8:

[0561]

[0562]

[0563] Example 2:

[0564] Masking moieties (generating VHH)

[0565] Figure 2 :

[0566] Masking moieties can be selected from a wide range of IL-12 inhibitory molecules: receptor fragments, antibodies or antibody fragments (Fabs, scFabs, scFvs, VHHs, as examples), 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 an IL-12 binding or IL-12 fusion that does not directly inhibit IL-12, but indirectly inhibits IL-12 in the context of prodrug assembly through steric interactions.

[0567] Ensuring that the cytokine does not signal systemically, creating an effective ΔEC between the prodrug in systemic circulation 50and active cytokines in the tumor microenvironment were considered key features for designing such molecules.

[0568] Antibody selection was performed in an attempt to identify masking domains. Antibodies against IL-12 were generated using in vitro and in vivo techniques: phage panning of synthetic or immunologically derived VHH-based antibody libraries were performed. Plasma cells were collected from immunized camelids and transformed into phage display libraries and further panned to directly isolate binding fragments. Selection of appropriate antibody fragments against masking domains was based on functional inhibition of IL-12, affinity for binding to IL-12, and generation of a large therapeutic window between prodrug and activated IL-12 as measured in the Promega IL-12 bioassay.

[0569] Phage panning of synthetic VHH-based antibody libraries failed to identify any functional blockers of IL-12. Despite testing over 50 VHH-based masking domains from a synthetic VHH-based antibody library, none of them showed any functional activity in the Promega IL-12 bioassay (data not shown), indicating another hurdle for selection of the optimal masking molecule.

[0570] Forty-seven initial binders were identified from the immunologically derived VHH-based antibody library and further characterized.

[0571] Table 9:

[0572]

[0573]

[0574]

[0575]

[0576] Most of the VHHs did not exhibit functional activity in the Promega IL-12 bioassay. Only one functional VHH binder (p40 binder) was identified and further optimized and humanized (BI-039). Although other identified VHH binders did not exhibit activity in functional assays, they can still be used in other applications that require binding to IL-12.

[0577] The selected blocking domain (BI-048) presents competition with ustekinumab (a known inhibitor of IL-12 and IL-23) by blocking domain 1 of the p40 domain. The selection of a blocking domain specific to the p40 domain is an important consideration in this molecular design because human IL-12 does not signal through the mouse IL-12 receptor; however, a chimeric IL-12 that utilizes human IL-12 p40 and mouse IL-12 p35 is able to signal. Thus, a blocking domain specific to the p40 domain of IL-12 allows for the generation of surrogate prodrugs without the need for species cross-reactivity of the blocking domain itself. The selected blocking domain was subjected to three rounds of humanization and was able to maintain its ability and efficacy against IL-12. Both the selected VHH and its humanized variants have an affinity of 3.5 nM and an IC50 of about 500 pM against IL-12. 50 (Table 10).

[0578] Table 10: Variations of BI-039:

[0579] Name Identifier K D (nM) IC50 [nM] 61-Gft-106_D62E BI-117 3.0 0.36 61-Gft-106-DE_Mix13 BI-048 3.7 0.44 BI-039 BI-039 3.5 0.61 61-Gft-106 BI-063 3.4 0.89 61-Gft-106-DE_Mix17 BI-118 6.3 1.5 61-Gft-106-DE_T106R BI-119 8.5 2.4 61-Gft-106-DE_D107E BI-120 4.1 2.5 61-Gft-106-DE_Mix15 BI-049 12 5.7 61-Gft-106-DE_Mix16 BI-121 9.2 6.5 61-Gft-106-DE_Mix7 BI-122 9.5 7.9 61-Gft-106-DE_Y59S BI-123 30 10 61-Gft-106-DE_Mix12 BI-124 12 10 61-Gft-106-DE_Mix26 BI-125 36 16 61-Gft-106-DE_Lia1 BI-126 13 18 61-Gft-106-DE_Mix22 BI-127 29 20 61-Gft-106-DE_Mix21 BI-128 51 56

[0580] Example 3:

[0581] MMP expression and cleavable linker

[0582] Figures 10A-10F

[0583] MMPs are often tightly regulated systemically by their natural inhibitors, tissue inhibitors of metalloproteinases (also known as TIMPS). However, within the tumor microenvironment, not only are MMP transcripts upregulated, their activity is often aided by a decrease in TIMP activity, thus allowing for higher MMPs within the tumor itself. The expression levels of various MMPs and TIMPs in various human tumors were evaluated and are presented in Figures 10A-10F MMPs, especially MMP2, MMP9, and MMP13, are significantly upregulated in tumor tissue compared to adjacent normal or healthy organs. Additionally, TIMPs, especially TIMP3, are strongly upregulated in healthy tissue compared to tumor tissue, suggesting that MMP activity is higher within tumors than in normal tissue.

[0584] In addition to expression levels, the activity of MMPs needs to be evaluated to confirm the functionality of those enzymes in the biological sample. For this purpose, tumor samples (and adjacent normal) were collected from patients who underwent surgical removal of the tumor. The samples were evaluated in terms of expression levels of various MMPs and in functional assays to confirm the proteolytic activity of the enzymes. As presented in Table 11, MMP2 and MMP9 were abundantly expressed in tumor tissue. In addition, MMP2 and MMP9 were upregulated in some adjacent normal tissue; however, this expression level was not associated with enzyme activity, possibly due to the activity of inhibitory proteins (TIMPs). In contrast to MMP2 and MMP9, MMP12 and MMP13 were almost exclusively expressed in tumor tissue.

[0585] Table 11:

[0586] Patient data is shown comparing MMP activity from tumor samples versus adjacent normal tissue from patients who underwent surgical removal of the tumor. Individual patient data is presented in pairs, i.e., tumor sample and normal sample from the same patient are presented directly below each other. MMP expression levels are shown in pg / mg lysate and specific activity is expressed in pmol (cleaved peptide) / min / ug of lysate.

[0587]

[0588]

[0589] As shown in Table 11, there is patient-to-patient variability in the expression of various MMPs. Cleavable linkers reactive against several MMPs, such as MMP2, MMP9 and MMP13, can help mitigate the relative differences in upregulation of patient-to-patient levels, and thus broad cleavable peptides are advantageous.

[0590] As presented in Table 12, linkers that can be included in the fusion proteins have broad specificity, which should mitigate patient-to-patient variability in MMP expression. For this purpose, several short peptides were tested for MMP-mediated cleavage specificity. Due to their broad specificity, peptide 5 was used in Fc fusion constructs.

[0591] Table 12:

[0592]

[0593] Example 4:

[0594] Generation of masked IL-12 Fc fusion proteins

[0595] Based on initial version exploration, screening and optimization of masking moieties, and appropriate cleavable linker selection, several optimized, masked IL-12 Fc fusion proteins were generated and further tested.

[0596] Table 13:

[0597]

[0598]

[0599]

[0600]

[0601]

[0602]

[0603]

[0604]

[0605]

[0606]

[0607] Example 5:

[0608] Masking domain - functional activity in the context of the molecule - in vitro

[0609] Figures 3A-3B and 4

[0610] The prodrug effectively inhibits IL-12 signaling in the context of the prodrug, but effectively signals upon MMP9 cleavage in in vitro assays as well as in in vivo experiments (see Example 6 for in vivo).

[0611] IL-12 routinely shows an EC of about 15 pM in the Promega IL-12 bioassay 50 and this value is independent of whether IL-12 is provided as purified recombinant IL-12 or whether IL-12 is released from the prodrug in the presence of MMP9. Figure 3A shows chimeric single chain IL-12 (human p40-GS linker-murine p35) (BI-066) in the Promega IL-12 bioassay.

[0612] The prodrug BI-057 was also tested in this assay. Cleaved prodrug BI-057 shows an EC of about 16 pM in the Promega IL-12 bioassay after MMP9 digestion 50 (Figure 3B).

[0613] In the absence of MMP9 digestion, the EC of the molecule 50Indicated over 280-fold functional displacement compared to recombinant IL-12 in the range of about 5 nM Figure 4 In summary, the data presented indicate effective masking of IL-12 in prodrug form and full functionality of the cytokine upon cleavage from the original molecule.

[0614] Example 6:

[0615] Masking domain - functional activity in the context of the molecule - in vivo

[0616] Figures 5A-5D , 6A-6B , 7A-7B, 8A-8B and 9A-9C

[0617] In the context of in vivo, a prodrug from initial lead exploratory experiments with a configuration as shown in Figure IB was tested and showed an improved safety profile compared to unmasked IL-12. This prodrug from initial lead exploratory was based on a heterodimeric Fc (knob-in-hole) with a single chain chimeric IL-12 (human p40-GS linker-murine p35) attached to the C-terminus of one Fc chain and a masking domain (scFv) against the human p40 domain attached to the other Fc chain. A cleavable linker was placed between the Fc and the p40 domain.

[0618] As shown in Figures 5A and 5C, the chimeric IL-12 Fc fusion protein inhibited tumor growth in a dose-dependent manner in a non-immunogenic and aggressive B16.F10 melanoma model. In addition, the B16.F10 model is characterized by relatively low MMP activity, thus providing an additional hurdle for the tested molecules. Effective blockade of IL-12 functionality in the prodrug version was confirmed by the lack of body weight loss even at a 2 mg / kg dose, which is 2000-fold higher (in molar equivalent) than the murine toxic dose (Figure 5D). In contrast, the unmasked chimeric IL-12 Fc fusion protein (fusion protein with all components but without the blocking moiety) caused severe toxicity in most of the animals already at a concentration of 0.08 mg / kg (Figure 6A). Higher concentrations (1.6 mg / kg, which is the molar equivalent of the 2 mg / kg dose of the masked chimeric IL-12 Fc fusion protein) resulted in severe body weight loss in treated animals, such that all treated animals died within 8 days (Figure 6B). This data further confirms effective masking of IL-12 when in prodrug form in the periphery. It also indicates that the molecule remains in prodrug form while increasing the therapeutic index of the molecule in the bloodstream.

[0619] The safety and efficacy of the chimeric IL-12 Fc fusion protein was further evaluated in the MC38 model, which confers higher MMP activity. Figure 7A shows dose-dependent tumor growth inhibition without body weight loss, which is a surrogate for toxicity (Figure 7B).

[0620] Similar trends were observed in the B16.F10 model (Figures 8A-B), which contains much lower MMP activity within the tumor, but tumor growth inhibition was actually lower, which correlates 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 effective masking properties and lack of systemic IL-12 mediated inflammation.

[0621] To further confirm this, levels of pro-inflammatory cytokines were evaluated in the serum of MC38-bearing mice treated with the chimeric IL-12 Fc fusion protein one day after the second treatment. No pro-inflammatory cytokines were detected in the serum of treated mice, indicating a lack of systemic toxicity and confirming effective masking (Table 14).

[0622] Table 14:

[0623]

[0624] To further confirm the IL-12 inhibitory properties of the masking molecule, non-human primates (NHP) were injected with the human IL-12 Fc fusion protein (BI-051) at doses up to 3 mg / kg. NHP remained healthy, with no signs of toxic clinical symptoms. No increase in ALT, bilirubin or creatinine was detected, indicating effective masking of IL-12 in the periphery in the context of the prodrug molecule Figures 9A-9C ).

[0625] Example 7:

[0626] Cleavage site cleavage by MMPs - functional activity in the context of the molecule - in vitro

[0627] Figure 11 , 12 , 13A-13E, 14A-14B

[0628] The MMP-cleavable linker incorporated in the prodrug is susceptible to cleavage by proteases that are often upregulated within the tumor microenvironment. For the purpose of functional characterization of the prodrug, recombinant MMP9 was used for cleavage and activation of IL-12 from the prodrug.

[0629] As presented in Figure 11 , activated MMP9 effectively cleaved the prodrugs (BI-050, BI-051, BI-052, BI-054, BI-055), as shown by SDS-PAGE. Release of IL-12 was further confirmed by MS analysis (not shown) and Western blotting Figure 12 ) of another prodrug molecule (BI-062).

[0630] To further investigate the functionality of IL-12 released from the prodrug, a cell-based Promega IL-12 bioassay was employed. Prodrug molecules, after protein cleavage and sham treatment, were serially diluted and added to reporter cells to measure IL-12 activity. As shown in Figures 13A to 13E, the EC50 values ​​of all tested molecules (BI-050, BI-051, BI-052, BI-054, BI-055) at cleavage are presented. 50 The values ​​were lower than those of intact molecules, indicating efficient release of biologically active IL-12. Furthermore, all molecules achieved comparable EC values ​​upon cleavage. 50 Values, while sham-treated molecules exhibit significantly different EC values. 50 The values ​​can indicate the different masking properties of constructs that are otherwise very similar (Table 15).

[0631] Using ΔEC 50 Variants were evaluated. The tested constructs were closely related to each other, differing only in the position of the collagen I tumor-preserving peptide: at the N-terminus of IL-12 (BI-050), the C-terminus of IL-12 (BI-051), or in the IL-12 endopeptide linker connecting the p40 and p35 domains (BI-052). Similarly, the lower affinity masking domain (which differs from BI-051 only in two amino acids of the masking HCDR3, while all other components are identical) showed reduced masking ability. Finally, construct BI-055, also based on BI-051, but additionally containing S354C / Y349C CH3-stabilized disulfide bonds, which are typically used to drive heterodimerization of the knob-in-Hole formation, showed similar masking ability.

[0632] The data clearly show that, with respect to these various parameters, the ΔEC for the variants 50 It has a direct impact and therefore affects the overall security landscape.

[0633] Table 15:

[0634] Construct EC of cleaved molecules 50 (pM) EC of unlysed molecules 50 (pM) ΔEC 50 ]] BI-050 5.999 229 38 BI-051 4.743 2869 605 BI-052 3.446 211 61 BI-054 3.076 952 313 BI-055 2.718 1531 563

[0635] To further evaluate the feasibility of releasing IL-12 from the prodrug in more relevant clinical settings, a lysate derived from human tumor tissue was prepared and used as a source of MMPs. The protein was evaluated in Western blotting 2 hours after culturing the chimeric molecule BI-059 with the lysate. Figures 14A-14B The study demonstrated that BI-059 was effectively lysed to a level comparable to activated MMP9 using human tumor lysates. This data suggests the feasibility of MMP-mediated lysis and cytokine release in the tumor microenvironment.

[0636] Example 8:

[0637] cleavage by MMPs - functional activity in the context of a molecule - in vivo

[0638] Figure 15 and 16A-16F

[0639] IL-12 cytokine stimulates T cells and NK cells, and those cells produce IFNy in response to the stimulation. As shown in Table 14, there was no detectable amount of IFNy in the margins after treatment with the chimeric IL-12 Fc fusion protein, indicating the effective masking property of the molecule. In other experiments using the same IL-12 Fc fusion protein as tested in Example 6, it was investigated whether this treatment would produce detectable amounts of IFNy within the tumor. To this end, MC38-bearing animals were treated twice with the IL-12 Fc fusion protein (2.8 mg / kg) and tumors were collected 1 day after the second treatment. As shown in Table 15, there was a significant increase in intra-tumoral IFNy in treated animals compared to vehicle-injected mice, indicating protease-mediated release of functional IL-12. Figure 15

[0640] To further elucidate the role of active IL-12 released at the tumor site, changes within the TME were investigated after treatment with the chimeric Fc fusion protein described above. A significant change in lymphocytic and myeloid cell infiltration was observed 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), in particular CD8 T cells (Figure 16B). Those T cells exhibited an activated phenotype as indicated by CD25 expression (Figure 16C). In addition, a significant increase in Ml -like macrophages was observed in treated animals (Figure 16D), which was accompanied by a decrease in M2-like macrophages (Figure 16E). As a result, an increase in the Ml / M2 ratio was observed (Figure 16F), which is a marker of beneficial changes within the tumor microenvironment. It should be noted that Figures 16C, 16E and 16F use a logarithmic scale in order to more clearly show the results.

[0641] Example 9:

[0642] TME-retained linker - functional activity in the context of a molecule - in vitro:

[0643] Figures 17A-17I , 18A-18B, 19 and 20A-20D

[0644] ​The primary driver of the prodrug approach is to limit systemic toxicity of IL-12 and achieve a greater safety profile while still harnessing the strong anti-tumor properties of the cytokine. This is initially done by creating a prodrug with tumor-activatable IL-12 release. However, IL-12 itself has a relatively short half-life (<9 hours) and can create additional issues with cytokine escape from the tumor microenvironment. As such low levels of IL-12 are needed to trigger toxic responses, a peptide sequence is added to further trap the cytokine within the tumor microenvironment.

[0645] The TME is rich in ECM proteins such as collagen I, collagen IV, or fibronectin that can potentially act as anchors for other fusion proteins. ECM proteins are found to be upregulated in many tumor types Figures 17A-17I ). Thus, it is hypothesized that tumor-retained peptides incorporated into IL-12 can allow for greater retention of IL-12, leading to longer exposure of immune cells to the cytokine, providing further efficacy. Additionally, a linker that binds to ECM proteins can also provide the benefits of a prodrug. A circulating prodrug with a TME linker fused to IL-12 can potentially increase the retention time of the prodrug within the TME. The direct impact of this would be longer exposure of the prodrug to enzymatic cleavage, enhancing the likelihood of the prodrug conversion to active drug.

[0646] To test the ability of the peptide to bind to ECM proteins, BI-051 binding to collagen I was evaluated in the context of an Fc fusion protein. As presented in FIG. 18A, dose-dependent binding of BI-051 to collagen I-coated plates was observed. This result was further confirmed in SPR experiments as shown in FIG. 18B. Figure 18B To achieve cytokine retention at the tumor site, a tumor-retained linker was attached to the cytokine (part of one of the knob chains). To further confirm the TME-binding linker delivers binding properties, the knob and hole chains of the molecule were tested on collagen I-coated plates in a separate ELISA-based analysis. As presented in FIG. 19A, 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, which did not contain the TME linker peptide. Figure 19

[0647] To further elucidate the binding of BI-051 to collagen I under more physiological conditions, a scratch in a JAR assay was employed. In this assay, fibroblasts are stimulated to produce different matrix proteins (mainly collagen I in this assay) in a form very similar to the native extracellular matrix. To the plates prepared, fluorescently labeled IL-12 Fc fusion proteins were added and evaluated for their binding to collagen I. As presented in FIG. 20B, BI-051 could be clearly detected along the collagen I fibers, indicating binding of the molecule to this ECM protein.

[0648] ​Example 10:

[0649] Molecular in vivo data

[0650] Figures 21A-21B, 22, 23A-23B, and 24A-24B

[0651] Chimeric molecules were further tested in in vivo settings. Due to very low collagen I content in syngeneic models (data not shown), as opposed to human tumors, testing of collagen binding motifs in those models was still challenging. In contrast, fibronectin expression in syngeneic models enabled testing of the TME linker concept in tumor-bearing mice. To this end, a chimeric IL-12 Fc fusion protein containing a fibronectin binding motif (BI-059) was tested in the MC38 model. As shown in Figure 21A, BI-059 significantly inhibited tumor growth compared to vehicle and IL-12 Fc fusion without TME linker (BI-065). Inhibition of tumor growth led to a significant prolongation of survival in BI-059 treated animals, with 50% of animals still alive at the end of the experiment Figure 21B ).

[0652] To further elucidate the ability of TME binding linkers to improve anti-tumor efficacy and / or safety of IL-12 Fc fusions, BI-059 was tested in the challenging B16.F10 melanoma model. This model is characterized by lower MMP activity and significantly lower content of fibronectin (8-fold according to expression levels, data not shown). As shown in Figure 22, BI-059 animals significantly better controlled tumor growth in this aggressive model compared to animals treated with IL-12 Fc fusion without TME linker, despite low MMP activity and low fibronectin expression. Figure 22

[0653] As mentioned above, syngeneic models are characterized by poor stromal compartments and collagen I expression is usually limited to the outer membrane of those tumors. Nonetheless, 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 time compared to 2 / 10 animals in the group receiving IL-12 Fc fusion lacking the TME linker. Importantly, no signs of toxicity were observed in those animals according to in vitro data showing a significant ΔEC 50 between cleaved and uncleaved molecules (Figure 23B).

[0654] ​BI-057 was further tested in the B16.F10 model to further evaluate its efficacy (as collagen I expression levels are very low, this model is not suitable to test the benefit of the TME linker). As shown in Figure 24A, BI-057 treatment resulted in a significant delay in tumor growth compared to vehicle treated animals. Consistent with the data presented previously, there were no signs of toxicity in the treated animals as shown by the lack of body weight loss (Figure 24B).

[0655] Those data show that molecules containing the TME-retaining linker are effective and safe in these models. In an environment where ECM proteins are widely present, which is typical for human tumors, the TME linker is expected to contribute even more significantly to both efficacy and safety profiles of the presented molecules.

[0656] Example 11:

[0657] In another approach, genetically modified models characterized by increased levels of ECM proteins within the tumor are used for further analysis of IL-12 Fc fusion proteins, and in particular the effect of the TME linker on IL-12 Fc fusion protein retention, efficacy, mode of action, and safety profile. To this end, such genetically modified models are treated with IL-12 Fc fusion proteins and assessed for TME linker retention, efficacy, mode of action, and safety profile.

[0658] In yet another approach, different tumor models are selected that generally have different levels of ECM protein expression, such as collagen (low, intermediate, or high expression). The models are treated with IL-12 Fc fusion proteins and assessed for efficacy, potency, toxicity, and / or retention.

[0659] Example 12:

[0660] Product quality

[0661] Different molecules were generated (Table 16) that varied in the type and location of the VHH masking domain and the tumor-retaining linker used. The molecules were manufactured in a small scale in stable cell pools under controlled and representative conditions. The results are summarized in Table 17. Expression in the small scale bioreactor was successful, with all constructs harvested at day 14. BI-051, BI-053, BI-054, and BI-055 had similar upstream process performance. The productivity and overall process performance of the cells expressing BI-052 were lower and additional species were observed in the size exclusion chromatography of the harvested sample. Similarly, BI-050 showed non-homogenous size exclusion, indicating instability of the construct.

[0662] The expressed constructs were then purified downstream as described and the product quality was assessed (Table 18).

[0663] BI-050 and BI-052 have elevated low molecular weight species that failed to be partially identified, indicating instability of the construct during manufacturing. BI-054 shows instability of process intermediates. BI-051 and BI-055 show 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 the additional stabilizing disulfide bridge was introduced that makes the molecule more rigid and prone to breakage.

[0664] Table 16

[0665] Summary of variant molecules. Molecules differ in their TME linker selection and position in the molecule relative to IL-12 and the VHH masking domain used.

[0666]

[0667] Table 17

[0668] Process performance of molecules in upstream processing.

[0669]

[0670] Table 18

[0671] Process performance in downstream processing and product quality of final molecules.

[0672]

[0673] Example 13:

[0674] In vitro comparison of molecules containing collagen I TME linker and those that do not.

[0675] Figures 25A-25B-26A-26B

[0676] Collagen binding properties were further evaluated in ELISA assays. Chimeric IL-12 Fc fusion protein BI-065 or chimeric IL-12 Fc fusion protein BI-057 containing a collagen I TME linker were added to collagen I coated plates. For this purpose, plates were coated with rat collagen I (Figure 25A) or with human skin collagen I (Figure 25B).

[0677] Briefly, high binding microplates (greiner) were coated overnight at 4°C with rat collagen (Corning) or human collagen (Millipore / R&D) diluted in coating buffer (Invitrogen) at a concentration of 0.005 mg / mL. IL-12 Fc fusion proteins were diluted in PBS + 1% BSA and added to the collagen pre-coated plates for 2 hours. Prior to the addition of the proteins, the plates were blocked with 1% BSA to minimize non-specific binding. Subsequently, the plates were washed 3 times followed by incubation with biotinylated anti-human Fc antibody (Invitrogen). After an additional washing step, streptavidin-HRP was added to the plates. Substrate was added to the plates after a washing step and the plates were read in an ELISA reader (Tecan).

[0678] We observed dose dependent binding of BI-057 to collagen coated plates with EC50 of 26 nM or 75 nM for rat and human collagen, respectively. BI-065 did not bind to collagen coated plates and only increased binding was observed at the highest concentration.

[0679] To further evaluate the collagen binding properties of the IL-12 Fc fusion proteins, precise cut liver sections of rat fibrotic livers were employed. The syngeneic transplant / orthotopic tumor model consists of only very limited amounts of collagen (in contrast to human tumors) and therefore the fibrotic tissue constitutes a more relevant collagen architecture.

[0680] Briefly, tissue cores were cut from fibrotic rat livers using a 5 mm cylindrical electric tissue coring press (Alabama RD, MD5000) and 300 pm thick tissue sections were cut using a tissue slicer (Alabama RD, MD6000). All tissue sections were cultured in 12-well culture plates (Nunc) in a humidified incubator, 95% 02 / 5% C02, 37°C in 1.3 mL of supplemented Williams Medium E (Life Technologies) by a floating culture system (60 rpm). IL-12 Fc fusion protein was added to the sections and incubated for 2 hours or 24 hours. After 2 hours of incubation, the sections were transferred to fresh Williams Medium E and incubated for an additional 22 hours as a wash step before harvesting them. After incubation, the sections were washed 3 times with Williams Medium E to remove unbound IL-12 Fc fusion protein. Individual sections were collected for capture (flash frozen) in lysis matrix tubes. The harvested sections were lysed with MSD Tris lysis buffer (MSD) and homogenized with a Precellys Evolution Homogenizer (Bertin Technologies) at 6800 rpm for 2 x 30 s. A centrifugation step was then performed to remove cell debris. The amount of IL-12 Fc fusion protein in the supernatant of the homogenate was determined using the MSD U-PLEX BioMarker Assay and IL-12 Fc fusion protein as a standard diluted in MSD lysis buffer (serial dilutions 1 :2). For IL-12 Fc fusion protein detection, MSD gold spot streptavidin plates were coated with biotinylated anti-IL-12 Fc fusion protein mouse capture antibody and anti-IL12 human antibody with SulfoTag was used as detection antibody. MSD read buffer was added before the plates were read with a MSD microplate reader.

[0681] Chimeric IL-12 Fc fusion proteins BI-065 or BI-057 containing collagen I TME linker were added to precisely cut liver sections for 2 or 24 hours. After 2 hours, the sections were washed thoroughly and incubated in culture medium for an additional 22 hours. As shown in Figure 26A, there was a dose-dependent recovery of IL-12 Fc fusion proteins containing collagen I TME linker. The amount of IL-12 Fc fusion protein BI-065 recovered from the sections remained at background levels and was 1 / 10th of the amount of BI-057, indicating the binding of the late molecule to collagen present in the fibrotic liver. In addition, similar results were observed when the sections were incubated in the presence of BI-057 or BI-065 for 2 hours followed by extensive washing and remaining in culture for an additional 22 hours. As shown in Figure 26B, there was a dose-dependent recovery of chimeric IL-12 Fc fusion protein BI-057 containing collagen I TME linker, which was 10-fold higher than the recovery of chimeric IL-12 Fc fusion protein BI-065, indicating increased retention of BI-057 compared to BI-065 within the sections.

[0682] Example 14:

[0683] In vivo comparison of molecules containing collagen I TME linker and those that do not.

[0684] Figures 27A-27D-28A-28B

[0685] The retention of molecules containing collagen I TME linker was further evaluated in syngeneic transplant / syngeneic models. It is important to emphasize that, in contrast to human tumors, syngeneic transplant / syngeneic models contain very low amounts of collagen. Therefore, it is still difficult to observe the full potential of collagen I TME linker in those models. To evaluate tumor retention in vivo, fluorescence measurements were performed with Dylight650-labeled chimeric IL-12 Fc fusion protein BI-201 or chimeric IL-12 Fc fusion protein BI-202 containing collagen I TME linker.

[0686] Briefly, fusion proteins were labeled with DyLight650 (Thermo Fisher Scientific). Excess dye was removed using spin columns with purified resin (Thermo Fisher Scientific) and the degree of labeling was calculated for each protein. Proteins in pharmacokinetic and retention studies contained equimolar amounts of dye. To assess protein retention, mice were imaged with IVIS in a whole-body exposure drop-in light fluorescence setting. During this time period, mice were maintained on a chlorophyll low diet (Altromin) to minimize the gastrointestinal background fluorescence. Image analysis was performed using Living Image (Perkin Elmer) to determine total radiant efficiency.

[0687] Mice bearing KPCY tumors were injected i.v. with 30 pg of protein and fluorescence was measured at different time points. As presented in Figure 27A, the amount of protein was similar in both groups of animals, as measured by fluorescence, up to 24 hours. Starting at 48 hours, fluorescence content increased in animals injected with chimeric IL-12 Fc fusion protein BI-202 containing collagen I TME linker. At the end of the experiment (at 144 hours), fluorescence content was significantly higher in the group injected with BI-202 than in the group injected with BI-201 (Figure 27B). In addition, pharmacokinetic measurements (based on fluorescence data) indicated a trend to increase half-life and showed that clearance of BI-202 was significantly reduced compared to BI-201 (Figures 27C and D).

[0688] Retention of human molecules was also evaluated in animals bearing EMT6. Mice were injected i.v. with 50 pg of Dylight650 labeled human IL-12 Fc fusion protein BI-200 or human IL-12 Fc fusion protein BI-051 containing collagen I TME linker. Fluorescence was measured at different time points. As presented in Figure 28A, 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 fluorescence content between mice injected with BI-051 and BI-200, indicating increased retention of the former (Figure 28B).

[0689] Example 15:

[0690] In vivo effect of collagen I TME linker on molecule functionality / safety.

[0691] Figures 29A-29B-30A-30B

[0692] To evaluate the effect of collagen I TME linker on molecule functionality / safety, mice bearing PDA30364 pancreatic tumors were injected i.t. with 150 pmol of chimeric IL-12 Fc fusion protein (BI-065) or chimeric IL-12 Fc fusion protein containing collagen I TME linker (BI-057). To determine the safety profile of the injected molecules, blood was collected 48 hours after injection and IL-12 induced cytokines / chemokines were evaluated.

[0693] Briefly, cytokines in serum were measured using the LegendPlex Mouse Cytokine Release Syndrome Panel (13-plex) (BioLegend) (bead-based immunoassay). Individual beads in a multi-task format can be distinguished according to size and intrinsic fluorescence intensity. Beads are coated on their surface with specific antibodies and act as capture beads for that particular analyte. Pre-mixed beads are incubated with the sample or pre-mixed standards are serially diluted for 2 hours. To determine the concentration of the particular analyte after the washing step, a biotin-labeled detection antibody cocktail is added. The detection antibody binds to the particular analyte it binds on the capture bead, thus forming a capture bead-analyte-detection antibody sandwich structure. 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 was measured on a BD LSR Fortesssa cell analyzer. The concentration of the particular analyte was determined using a standard curve. Analysis was performed using FlowJo (LLC) and GraphPad Prism (GraphPad Software Inc.).

[0694] The cytokine responsible for IL-12 related adverse effects is IFNy. As shown in Figure 29A, injection of BI-065 caused a 20-fold increase in IFNy levels in the periphery compared to animals treated with BI-057. In addition, CXCL10, a chemokine induced by IFNy, was increased 10-fold in animals treated with BI-065 compared to mice injected with BI-057.

[0695] In other steps, the effect of the chimeric molecules on IFNy induction was evaluated in animals bearing orthotopic mammary EMT6 tumors. To this end, animals were injected intravenously with different doses of the chimeric IL-12 Fc fusion protein (BI-065) or the chimeric IL-12 Fc fusion protein containing the collagen I TME linker (BI-057). Blood was collected 24 hours (Figure 30A) and 72 hours (Figure 30B) after injection and the levels of IFNy were determined. As shown in Figure 30A, there was a clear trend of increased levels of IFNy in mice treated with BI-065 24 hours after injection, reaching statistical significance at the intermediate dose tested. A better safety profile of BI-065 was also observed at later time points, as defined by lower levels of IFNy (Figure 30B).

[0696] Table 19 (additional molecules used in Examples 13-15)

[0697]

[0698]

Claims

1. An interleukin-12 (IL-12) Fc fusion protein comprising a first polypeptide chain and a second polypeptide chain, wherein... a) The first polypeptide chain contains a first Fc domain and the IL-12p35 and IL-12p40 subunits of IL-12, and b) The second polypeptide chain includes a second Fc domain and a masking portion of the IL-12p35 and / or IL-12p40 subunits bound to the first polypeptide chain; The first polypeptide chain and the second polypeptide chain are connected via the first Fc domain and the second Fc domain. The IL-12p35 subunit or the IL-12p40 subunit is connected to the C-terminus of the first Fc domain via a first peptide linker, which is protease-cleavable. The masking portion is connected to the C-terminus of the second Fc domain via a second connector, preferably a peptide connector, and The first polypeptide chain or the second polypeptide chain further includes a binding portion selected from the group consisting of: a collagen-binding portion, a heparin-binding portion, and a fibronectin-binding portion.

2. The IL-12Fc fusion protein of claim 1, wherein the binding portion is attached to the C-terminus of the IL-12p35 subunit or the C-terminus of the IL-12p40 subunit, or the binding portion is attached to the C-terminus of the masking portion, and optionally via a third polypeptide linker in various cases.

3. The IL-12Fc fusion protein according to claim 1 or 2, wherein the binding portion is located between the IL-12p35 subunit and the IL-12p40 subunit, or the binding portion 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 any case, the binding portion may optionally be attached to one or more linkers, preferably peptide linkers, on one or both sides.

4. The IL-12Fc fusion protein according to any one of the preceding claims, wherein the binding portion is a collagen-binding portion.

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

6. The IL-12Fc fusion protein according to claim 5, wherein the collagen-binding portion 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.

7. The IL-12Fc fusion protein according to 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 aa, or 9 aa.

8. The IL-12Fc fusion protein according to any one of the preceding claims, wherein the collagen-binding portion comprises or is composed of any of the amino acid sequences of SEQ ID NO:40-47.

9. The IL-12Fc fusion protein according to any one of claims 1 to 3, wherein the binding portion is a heparin-binding portion.

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

11. The IL-12Fc fusion protein according to claim 4, wherein the collagen-binding portion binds to collagen IV.

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

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

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

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

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

17. The IL-12Fc fusion protein according to any one of the preceding claims, wherein the IL-12p40 subunit and the IL-12p35 subunit are linked by a single strand having a configuration of IL-12p40—IL-12p35 or IL-12p35—IL-12p40.

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

22.

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

9.

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

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

23. The IL-12Fc fusion protein according to any one of the preceding claims, wherein the first Fc domain and the second Fc domain each contain one or more mutations that promote the heterodimerization of the Fc domain.

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

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

26. The IL-12Fc fusion protein according to any one of the preceding claims, wherein the first Fc domain and the second Fc domain are human IgG1 Fc domains, and the first Fc domain or the second Fc domain or both Fc domains contain mutants L234A and L235A.

27. The IL-12Fc fusion protein according to any one of the preceding claims, 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. The IL-12Fc fusion protein according to any one of the preceding claims, wherein the protease-cleavable linker is cleaved by matrix metalloproteinases (MMPs), preferably MMP-2, MMP-9 or MMP-13.

29. The IL-12Fc fusion protein of claim 28, wherein the protease-cleavable linker comprises or is composed of any of the amino acid sequences of SEQ ID NO: 232-241.

30. An IL-12Fc fusion protein comprising a first polypeptide chain and a second polypeptide chain, wherein... a) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:208, and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:

209. b) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:210, and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:

211. c) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:212, and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:

213. d) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:214, and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:

215. e) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:216, and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:

217. f) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:218, and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:

219. g) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:220, and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:

221. h) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:222, and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:

223. i) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:224, and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:

225. j) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:226, and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:

227. k) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:228, and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:

229. l) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:230, and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:231, or m) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:242, and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:

243.

31. The IL-12Fc fusion protein according to any one of claims 1 to 29, wherein the masking portion comprises a single variable domain of IL-12 binding immunoglobulin, which comprises three CDRs contained in any one of the sequences of SEQ ID NO:61-109.

32. The IL-12Fc fusion protein according to claim 31, wherein the immunoglobulin single variable domain comprises any of the amino acid sequences of SEQ ID NO:61-109.

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

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

35. The cleavage product of claim 34, wherein, after protein hydrolysis and cleavage of the cleavable linker, it comprises or consists of the following: The amino acid sequence of any one of SEQ ID NO: 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230 or 242.

36. A single variable domain of an IL-12 binding immunoglobulin comprising the three CDRs contained in any of the sequences of SEQ ID NO:61-109.

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

38. The IL-12 binding immunoglobulin single variable domain according to claim 36, wherein the immunoglobulin single variable domain comprises the amino acid sequence of any one of SEQ ID NO:61-109.

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

40. A vector comprising the nucleic acid of claim 39, optionally wherein the vector comprises two strands of nucleic acid encoding the IL-12Fc fusion protein.

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 two strands of the IL-12Fc fusion protein.

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

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

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

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

46. ​​The IL-12Fc fusion protein according to any one of claims 1 to 32, for use in medicine.

47. The pyrolysis product according to any one of claims 33 to 35, which is used in medicine.

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

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

50. Use of the IL-12Fc fusion protein as described in any one of claims 1 to 32 for the manufacture of a pharmaceutical agent.

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

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