Masked IL-12 cytokines and their cleavage products

Abstract

The present invention provides a masked IL-12 cytokine, a cleavage product of the masked IL-12 cytokine, a composition thereof, and a method for using the same. [Solution] A masked IL-12 cytokine is provided, comprising an IL-12 cytokine or a functional fragment thereof, a masking moiety, and a proteolytically cleavable linker. The masking moiety masks the IL-12 cytokine or its functional fragment, thereby reducing or preventing its binding to its homologous receptor. However, upon proteolytic cleavage of the cleavable linker at the target site, the IL-12 cytokine or its functional fragment is activated, making it possible or more possible to bind to its homologous receptor.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of priority to U.S. Provisional Applications No. 63 / 003,842 filed on April 1, 2020, No. 63 / 118,579 filed on November 25, 2020, and No. 63 / 127,893 filed on December 18, 2020, each of which is incorporated herein by reference in its entirety.

[0002] Submission of sequence listings in ASCII text files The contents of the following submission in ASCII text file are incorporated herein by reference in their entirety: Computer-Readable Format (CRF) of Sequence Listing (filename: 737762002840SEQLIST.TXT, date recorded: March 26, 2021, size: 1,000KB).

[0003] The present invention relates to a masked IL-12 cytokine, and to methods related to its use and production. The present invention also relates to cleavage products of the masked IL-12 cytokine, and to methods related to its use. [Background technology]

[0004] Cancer is the second leading cause of death in the United States, accounting for more deaths than the next five major causes (chronic respiratory disease, stroke, accidents, Alzheimer's disease, and diabetes). Significant progress has been made, particularly in targeted therapies, although much research remains in this field. Immunotherapy and its subfield, immuno-oncology, are generating viable and exciting treatment options for malignant tumors. One notable characteristic of cancer is immune evasion, and it is now recognized that significant efforts have been made to identify targets for reactivating the immune system to recognize and treat cancer, and to develop therapies against these targets.

[0005] Cytokines can be classified in various ways, including based on their three-dimensional structure. Some cytokines are classified as heterodimers. Examples of heterodimer cytokines include IL-12 and IL-23. The IL-12 cytokine is a heterodimer containing p35 and p40 subunits.

[0006] Cytokine therapy is an effective strategy for stimulating the immune system to induce anti-tumor cell damage. In particular, aldethleukin, a recombinant form of interleukin-2 (IL-2), is approved by the FDA for the treatment of metastatic renal cell carcinoma and melanoma. Unfortunately, cytokines administered to patients generally have very short half-lives, thereby requiring frequent administration. For example, the product label for aldethleukin, marketed under the brand name Proleukin, states that in patients receiving a 5-minute intravenous (IV) infusion, the drug has been shown to have a half-life of 85 minutes. In addition, administration of high doses of cytokines can lead to adverse health outcomes such as vascular leakage through systemic immune activation. These findings illustrate the need to develop IL-2 cytokine therapies that effectively target tumors without the side effects associated with systemic immune activation.

[0007] Provided herein to address this need are masked IL-12 cytokines, cleavage products of said masked IL-12 cytokines, compositions thereof, and methods of use thereof. [Overview of the project]

[0008] The disclosed invention relates to an IL-12 cytokine or a functional fragment thereof that is engineered to be masked by a masking moiety at one or more receptor binding sites of the IL-12 cytokine or functional fragment thereof. The IL-12 cytokine is engineered to be protease-activatable at a target site, such as within the tumor microenvironment, by including a proteolytically cleavable linker. In the masked cytokine construct, the masking moiety reduces or prevents the binding of the IL-12 cytokine or functional fragment thereof to its homologous receptor. Upon proteolytic cleavage of the cleavable linker at the target site, the IL-12 cytokine or functional fragment thereof is activated and becomes capable or more capable of binding to its homologous receptor.

[0009] Provided herein is a masked IL-12 cytokine containing a heterodimer, A first polypeptide chain including the following, JPEG2026048631000002.jpg includes a second polypeptide chain containing 7170 or less, In JPEG2026048631000003.jpg7170, HL1 is the first half-life extension domain, L1 is the first linker, MM is the masking portion, HL2 is the second half-life extension domain, L2 is the second linker, and C is the IL-12 cytokine or its functional fragment. The first half-life extension domain is associated with the second half-life extension domain. The first linker or one of the second linkers is a masked IL-12 cytokine containing a peptide that can be proteolytically cleaved.

[0010] In some embodiments, the IL-12 polypeptide or its functional fragment comprises an IL-12p40 polypeptide or its functional fragment covalently bound to the IL-12p35 polypeptide or its functional fragment.

[0011] In some embodiments, the IL-12p40-IL-12p35 linker is 5 to 20 amino acids in length.

[0012] In some embodiments, the IL-12p40-IL-12p35 linker is rich in amino acid residues G and S.

[0013] In some embodiments, the IL-12p40-IL-12p35 linker comprises SEQ ID NO: 3.

[0014] In some embodiments, the IL-12p40 polypeptide comprises SEQ ID NO: 1, or an amino acid sequence having at least one amino acid modification as compared to the amino acid sequence of SEQ ID NO: 1.

[0015] In some embodiments, the IL-12p40 polypeptide comprises SEQ ID NO: 1.

[0016] In some embodiments, the IL-12p40 polypeptide comprises at least one amino acid modification in the GAG-binding domain (KSKREKKDRV) as compared to the amino acid sequence of SEQ ID NO: 1.

[0017] In some embodiments, the IL-12p40 polypeptide comprises SEQ ID NO: 57.

[0018] In some embodiments, the IL-12p40 polypeptide comprises SEQ ID NO: 58.

[0019] In some embodiments, the IL-12p40 polypeptide comprises an amino acid sequence having one or more cytokine substitution mutations as compared to the amino acid sequence of SEQ ID NO: 1.

[0020] In some embodiments, the IL-12p40 polypeptide comprises SEQ ID NO: 59.

[0021] In some embodiments, the IL-12p40 polypeptide comprises SEQ ID NO: 60.

[0022] In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence having at least one amino acid modification compared to the amino acid sequence of SEQ ID NO: 2, or SEQ ID NO: 2.

[0023] In some embodiments, the IL-12p35 polypeptide comprises SEQ ID NO: 2.

[0024] In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises SEQ ID NO: 4.

[0025] In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises SEQ ID NO: 61.

[0026] In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises SEQ ID NO: 62.

[0027] In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises SEQ ID NO: 63.

[0028] In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises SEQ ID NO: 64.

[0029] In some embodiments, the masking portion comprises an IL-12 cytokine receptor, or a subunit or functional fragment thereof.

[0030] In some embodiments, the masking portion comprises the extracellular domain of human IL-12Rβ1, or a fragment, portion, or variant thereof, that retains or otherwise demonstrates affinity for IL-12.

[0031] In some embodiments, the masking portion comprises residues 24-237 of human IL-12Rβ1, i.e., a sequence having SEQ ID NO: 5. )]]

[0032] In some embodiments, the masking portion includes residues 24-545 of human IL-12Rβ1, i.e., the sequence having sequence number 6.

[0033] In some embodiments, the masking portion includes the extracellular domain of human IL-12Rβ2, or a fragment, part, or variant thereof, which retains or otherwise demonstrates affinity for IL-12.

[0034] In some embodiments, the masking portion includes residues 24-212 of human IL-12Rβ2, i.e., the sequence having sequence number 7.

[0035] In some embodiments, the masking portion includes residues 24-222 of human IL-12Rβ2, i.e., the sequence having sequence number 8, or the masking portion includes residues 24-227 of human IL-12Rβ2, i.e., the sequence having sequence number 11.

[0036] In some embodiments, the masking portion includes residues 24-319 of human IL-12Rβ2, i.e., the sequence having sequence number 9.

[0037] In some embodiments, the masking portion comprises at least one amino acid modification compared to the sequence of SEQ ID NO: 9, and optionally, the modification is a cysteine ​​substitution mutation.

[0038] In some embodiments, the masking portion includes SEQ ID NO: 65.

[0039] In some embodiments, the masking portion includes residues 24-622 of human IL-12Rβ2, i.e., the sequence of SEQ ID NO: 10.

[0040] In some embodiments, the cleavable peptide is 6 to 10 amino acids in length.

[0041] In some embodiments, the cleavable peptide comprises the amino acid sequence of SEQ ID NO: 15.

[0042] In some embodiments, the cleavable peptide comprises the amino acid sequence of SEQ ID NO: 41.

[0043] In some embodiments, the cleavable peptide includes the amino acid sequence of SEQ ID NO: 42.

[0044] In some embodiments, the cleavable peptide includes the amino acid sequence of SEQ ID NO: 43.

[0045] In some embodiments, the cleavable peptide comprises the amino acid sequence of SEQ ID NO: 44.

[0046] In some embodiments, the cleavable peptide comprises the amino acid sequence of SEQ ID NO: 45.

[0047] In some embodiments, the first polypeptide chain includes the following: JPEG2026048631000004.jpg8170 The second polypeptide chain includes the following: JPEG2026048631000005.jpg7170

[0048] In some embodiments, the non-cleavable linker is 3 to 18 amino acids in length.

[0049] In some embodiments, the non-cleavable linker is 3 to 15 amino acids in length.

[0050] In some embodiments, the non-cleavable linker is rich in amino acid residues G and S.

[0051] In some embodiments, the non-cuttable linker is [(G)] n It includes S, and n=4 or 5.

[0052] In some embodiments, the non-cleavable linker includes the amino acid sequence shown in SEQ ID NO: 12.

[0053] In some embodiments, the non-cleavable linker includes the amino acid sequence shown in SEQ ID NO: 13.

[0054] In some embodiments, the non-cleavable linker includes the amino acid sequence shown in SEQ ID NO: 14.

[0055] In some embodiments, the non-cleavable linker includes the amino acid sequence shown in SEQ ID NO: 54 (GGSGGSGGSGGSGGSSGP).

[0056] In some embodiments, the non-cleavable linker includes the amino acid sequence shown in SEQ ID NO: 55 (PGGSGP).

[0057] In some embodiments, the non-cleavable linker includes the amino acid sequence shown in SEQ ID NO: 56 (GGSPG).

[0058] In some embodiments, the cleavable linker comprises a proteolytically cleavable peptide (CP) having spacer domains (SDs) on both sides. SD1-CP-SD2 SD1 and SD2 have a first polypeptide chain that includes the following: JPEG2026048631000006.jpg7170 is different in that the second polypeptide chain contains the following: JPEG2026048631000007.jpg7170

[0059] In some embodiments, the first spacer domain (SD1) is 3 to 10 amino acids in length.

[0060] In some embodiments, SD1 includes sequence number 16.

[0061] In some embodiments, SD1 includes sequence number 17.

[0062] In some embodiments, the second spacer domain (SD2) is 3 to 6 amino acids in length.

[0063] In some embodiments, SD2 includes sequence number 18.

[0064] In some embodiments, the proteolytically cleavable linker comprises SD1-CP-SD2, where SD1 is a first spacer domain, CP is a cleavable peptide, and SD2 is a second spacer domain, where CP has the amino acid sequence shown in SEQ ID NO: 44, and SD2 has the amino acid sequence shown in SEQ ID NO: 18.

[0065] In some embodiments, the proteolytically cleavable linker comprises SD1-CP-SD2, where SD1 is a first spacer domain, CP is a cleavable peptide, and SD2 is a second spacer domain, where CP has the amino acid sequence shown in SEQ ID NO: 45, and SD2 has the amino acid sequence shown in SEQ ID NO: 18.

[0066] In some embodiments, the detachable linker includes sequence number 19.

[0067] In some embodiments, the detachable linker includes SEQ ID NO: 20.

[0068] In some embodiments, the detachable linker includes sequence number 46 (GGSGGSMPYDLYHPSGP).

[0069] In some embodiments, the detachable linker includes sequence number 47 (GGSGGSGGSMPYDLYHPSGP).

[0070] In some embodiments, the detachable linker includes sequence number 48 (GGSGGSDSGGFMLTSGP).

[0071] In some embodiments, the detachable linker includes sequence number 49 (GGSGGSGGSDSGGFMLTSGP).

[0072] In some embodiments, the detachable linker includes sequence number 50 (GGSGGSRAAAVKSPSGP).

[0073] In some embodiments, the detachable linker includes sequence number 51 (GGSGGSGGSRAAAVKSPSGP).

[0074] In some embodiments, the detachable linker includes sequence number 52 (GGSGGSISSGLLSGRSSGP).

[0075] In some embodiments, the detachable linker includes sequence number 53 (GGSGGSGGSISSGLLSGRSSGP).

[0076] In some embodiments, the first half-life extension domain comprises a first IgG1 Fc domain or a fragment thereof, and the second half-life extension domain comprises a second IgG1 Fc domain or a fragment thereof.

[0077] In some embodiments, each of the first and / or second Fc domains includes one or more modifications that facilitate non-covalent association of the first and second half-life extension domains.

[0078] In some embodiments, the first half-life extension domain includes SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain includes SEQ ID NO: 26 (S354C, T366W, and N297A).

[0079] In some embodiments, the first half-life extension domain includes SEQ ID NO: 27 (Y349C, T366S, L38A, Y407V, N297A, and I253A), and the second half-life extension domain includes SEQ ID NO: 28 (S354C, T366W, N297A, and I253A).

[0080] In some embodiments, the first peptide chain comprises the amino acid sequence of SEQ ID NO: 34, and the second peptide chain comprises the amino acid sequence of SEQ ID NO: 40.

[0081] Provided herein are cleavage products capable of binding to IL-12R, comprising an IL-12 cytokine or a functional fragment thereof, which can be prepared by proteolytic cleavage of a cleavable peptide in a masked IL-12 cytokine as defined in any one of the descriptions or embodiments described herein.

[0082] Provided herein are cleavage products of a masked IL-12 cytokine, the cleavage products being capable of binding to IL-12R, and the cleavage products comprising a polypeptide comprising: PCP-SD2-C PCP is part of a peptide that can be cleaved proteolytically, SD2 is the spacer domain, and C is the IL-12 cytokine or its functional fragment.

[0083] In some embodiments, PCP is a portion of a proteolytically cleavable peptide as described herein.

[0084] In some embodiments, SD2 is a spacer domain as described herein.

[0085] In some embodiments, C is an IL-12 cytokine or a functional fragment thereof as described herein.

[0086] In some embodiments, the cleavage product comprises an amino acid sequence having approximately or at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 29.

[0087] In some embodiments, the cleavage product contains the amino acid sequence of SEQ ID NO: 29.

[0088] Provided herein are nucleic acids encoding any one of the masked IL-12 cytokines described herein.

[0089] Provided herein are nucleic acids encoding one of any one of the masked IL-12 cytokine chains described herein.

[0090] Provided herein are vectors containing nucleic acids as described herein.

[0091] Provided herein is a vector comprising nucleic acids encoding the masked IL-12 cytokine described herein.

[0092] Provided herein is a vector comprising a nucleic acid encoding one of the masked IL-12 cytokine chains described herein.

[0093] Provided herein are host cells containing the nucleic acids described herein.

[0094] In one embodiment, the host cell is an HEK cell. In another embodiment, the host cell is a CHO cell.

[0095] Provided herein is a composition comprising any one of the masked IL-12 cytokines described herein.

[0096] Provided herein are pharmaceutical compositions comprising one of the masked IL-12 cytokines described herein and a pharmaceutically acceptable carrier.

[0097] In some embodiments, the pharmaceutical composition is a single-unit dosage form.

[0098] In some embodiments, the pharmaceutical composition is formulated for intravenous administration and is in a single dosage form.

[0099] In some embodiments, the pharmaceutical composition is formulated for injection and is in a single-unit dosage form.

[0100] In some embodiments, the pharmaceutical composition is a liquid and is in a single dosage form.

[0101] Provided herein is a kit comprising a masked IL-12 cytokine as described herein, or a composition as described herein, or a pharmaceutical composition as described herein.

[0102] Provided herein is a method for producing the masked IL-12 cytokine described herein, comprising culturing the host cells described herein under conditions that produce the masked IL-12 cytokine.

[0103] Provided herein are nucleic acids encoding the cleavage products described herein.

[0104] Provided herein are compositions comprising the cleavage products described herein.

[0105] Provided herein are pharmaceutical compositions comprising the cleavage products described herein and a pharmaceutically acceptable carrier.

[0106] Provided herein are masked IL-12 cytokines described herein for use in drugs.

[0107] The products provided herein are cleavage products described herein for use in pharmaceuticals.

[0108] Provided herein are methods for treating or preventing cancer in a subject, comprising administering an effective amount of a masked IL-12 cytokine described herein to the subject.

[0109] Provided herein are methods for treating or preventing cancer in a subject, comprising administering an effective amount of the composition described herein to the subject.

[0110] Provided herein are methods for treating or preventing cancer in a subject, comprising administering an effective amount of the pharmaceutical composition described herein to the subject.

[0111] Provided herein are methods for treating or preventing cancer in a subject, comprising administering an effective amount of the masked IL-12 cytokine described herein to the subject, thereby causing the masked cytokine to be proteolytically cleaved in vivo to produce the cleavage products described herein.

[0112] Provided herein are methods for treating or preventing cancer in a subject, comprising the step of producing in vivo a cleavage product capable of binding to a homologous receptor, wherein the cleavage product is the method described herein.

[0113] In some embodiments, cancer is a solid tumor.

[0114] Provided herein are masked IL-12 cytokines described herein for use in the treatment or prevention of cancer.

[0115] Provided herein is a masked IL-12 cytokine described herein for use in a method for treating or preventing cancer, wherein the method comprises administering an effective amount of the masked IL-12 cytokine to a target, thereby the masked cytokine being proteolytically cleaved in vivo to produce cleavage products described herein.

[0116] In some embodiments, cancer is a solid tumor.

[0117] The products provided herein are cleavage products described herein for use in the treatment or prevention of cancer.

[0118] Provided herein are cleavage products described herein for use in methods for treating or preventing cancer, wherein the method comprises the step of administering a masked cytokine described herein to a patient, thereby producing a cleavage product by proteolytic cleavage of the masked cytokine in vivo.

[0119] Provided herein are cleavage products described herein for use in methods of treating or preventing cancer in a subject, wherein the method comprises the step of producing a cleavage product by in vivo proteolytic cleavage from a masked cytokine described herein administered to a subject.

[0120] In some embodiments, cancer is a solid tumor.

[0121] The materials provided herein are pharmaceutical compositions described herein for use in the treatment or prevention of cancer.

[0122] In some embodiments, cancer is a solid tumor. [Brief explanation of the drawing]

[0123] [Figure 1] The structure of an exemplary embodiment of a masked cytokine is shown, comprising a masking moiety, a cytokine or a functional fragment thereof ("cytokine"), a half-life extension domain, and a first linker comprising a first cleavable peptide ("1CP"), a first N-terminal spacer domain ("1NSD"), and a first C-terminal spacer domain ("1CSD"). These exemplary embodiments also include a second linker comprising a second cleavable peptide ("2CP"), a second N-terminal spacer domain ("2NSD"), and a second C-terminal spacer domain ("2CSD"). As indicated by the arrows, the exemplary embodiments show a masking moiety linked to the first linker, and the cytokine or functional fragment thereof linked to the first and second linkers, however the masking moiety and the cytokine or functional fragment thereof can be interchanged such that the cytokine or functional fragment is linked to the first linker and the masking moiety is linked to the first and second linkers. Figure 1 shows the structure of an exemplary embodiment of a masked cytokine as a monomer. [Figure 2]The structure of an exemplary embodiment of a masked cytokine is shown, comprising a masking portion, a cytokine or a functional fragment thereof ("cytokine"), a first half-life extension domain, and a second half-life extension domain. The exemplary embodiment shown in Figure 2 also comprises a first linker comprising a first cleavable peptide ("1CP"), a first N-terminal spacer domain ("1NSD"), and a first C-terminal spacer domain ("1CSD"), and a second linker comprising a second cleavable peptide ("2CP"), a second N-terminal spacer domain ("2NSD"), and a second C-terminal spacer domain ("2CSD"). The exemplary first and second half-life extension domains include "knobs into holes" modifications that facilitate the association of the first half-life extension domain with the second half-life extension domain, as indicated by "holes" in the first half-life extension domain and "knobs" in the second half-life extension domain. The first and second half-life extension domains are also shown as at least partially associating through the formation of a disulfide bond. The “hole” is depicted as part of the first half-life extension domain (linked to the masking portion), and the “knob” is depicted as part of the second half-life extension domain (linked to the cytokine). It should be understood that the “hole” and “knob” may, alternatively, be included in the second and first half-life extension domains, respectively, such that the “hole” is part of the second half-life extension domain (linked to the cytokine) and the “knob” is part of the first half-life extension domain (linked to the masking portion). [Figure 3] Exemplary embodiments of masked cytokines before (left) and after (right) protease cleavage in the tumor microenvironment are shown. Figures 3A-3B show exemplary embodiments of masked IL-2 cytokines. Protease cleavage releases either a masking portion (e.g., IL-2Rβ as shown in Figure 3B) or IL-2 (Figure 3A). [Figure 4]This shows SDS-PAGE analysis of flow-through (FT) samples (i.e., proteins that did not bind to the Protein A column) and elution (E) samples (i.e., proteins that bound to the Protein A column and eluted from it) of IL-2 constructs (AK304, AK305, AK307, AK308, AK309, AK310, AK311, AK312, AK313, AK314, and AK315) after production and purification. [Figure 5] The results from SPR analysis testing the binding of exemplary masked IL-2 polypeptide constructs (AK168) or rhIL2 controls to CD25-Fc are shown. Figure 5A shows the interaction between AK168 and CD25-Fc, Figure 5B shows the interaction between AK168 activated with MMP and CD25-Fc, and Figure 5C shows the interaction between recombinant human IL-2 (rhIL-2) control and CD25-Fc. Figure 5D provides a table summarizing the data obtained for association constant (ka), dissociation constant (kd), equilibrium dissociation constant (KD), and Chi2 and U values ​​for each interaction. [Figure 6] The results from SPR analysis testing the binding of exemplary masked IL-2 polypeptide constructs (AK111) or rhIL-2 controls to CD122-Fc are shown. Figure 6A shows the interaction between AK111 and CD122-Fc, Figure 6B shows the interaction between protease-activated AK111 and CD122-Fc, and Figure 6C shows the interaction between recombinant human IL-2 (rhIL-2) control and CD122-Fc. Figure 6D provides a table summarizing the data obtained for association constant (ka), dissociation constant (kd), equilibrium dissociation constant (KD), and Chi2 and U values ​​for each interaction. [Figure 7A] Exemplary embodiments of masked cytokines before (left) and after (right) protease cleavage in the tumor microenvironment are shown. [Figure 7B] SDS-PAGE analysis of exemplary masked IL-2 polypeptide constructs incubated in the absence (left lane) or presence (right lane) of MMP10 protease demonstrates the release of IL-2 from the Fc region. [Figure 8A-B] As a control, STAT5 activation (%) in PBMCs treated with constructs AK032, AK035, AK041, or rhIL-2 is shown. The level of STAT5 activation (%) is shown for NK cells, CD8+ T cells, effector T cells (Teff), and regulatory T cells (Treg), determined after incubation with rhIL-2 (Figure 8A), AK032 (Figure 8B), AK035 (Figure 8C), or AK041 (Figure 8D). [Figure 8C-D] As a control, STAT5 activation (%) in PBMCs treated with constructs AK032, AK035, AK041, or rhIL-2 is shown. The level of STAT5 activation (%) is shown for NK cells, CD8+ T cells, effector T cells (Teff), and regulatory T cells (Treg), determined after incubation with rhIL-2 (Figure 8A), AK032 (Figure 8B), AK035 (Figure 8C), or AK041 (Figure 8D). [Figure 9] This shows the percentage of STAT5 activation in PBMCs treated with construct AK081 or AK032. AK081 constructs were tested with or without prior exposure to MMP10. Isotype controls and IL-2-negative controls were also tested. The level of STAT5 activation (%) is shown for NK cells (Figure 9A), CD8+ T cells (Figure 9C), and CD4+ T cells (Figure 9B). [Figure 10A-B] Results from STAT5 activation studies in PBMCs using constructs AK081 and AK111, as well as controls containing rhIL-2 and anti-RSV antibodies, are shown. An untreated control was also tested. EC50(pM) is also shown for rhIL-2, AK081, and AK111 treatments. STAT5 activation (%) is shown for CD4+FoxP3+CD25+ cells (Figure 10A), CD8+ cells (Figure 10B), and CD4+FoxP3-CD25- cells (Figure 10C). Figure 10D provides EC50(pM) and magnification change data for AK081, AK111 constructs, and rhIL-2 control. [Figure 10C-D]Results from STAT5 activation studies in PBMCs using constructs AK081 and AK111, as well as controls containing rhIL-2 and anti-RSV antibodies, are shown. An untreated control was also tested. EC50(pM) is also shown for rhIL-2, AK081, and AK111 treatments. STAT5 activation (%) is shown for CD4+FoxP3+CD25+ cells (Figure 10A), CD8+ cells (Figure 10B), and CD4+FoxP3-CD25- cells (Figure 10C). Figure 10D provides EC50(pM) and magnification change data for AK081, AK111 constructs, and rhIL-2 control. [Figure 11A-B] The results of STAT5 activation studies in PBMCs using constructs AK167 and AK168, as well as controls containing rhIL-2 and anti-RSV antibodies, are shown. An untreated control was also tested. EC50 (pM) is also shown for rhIL-2, AK167, and AK168 treatments. STAT5 activation (%) is shown for CD4+FoxP3+CD25+ cells (Figure 11A), CD8+ cells (Figure 11B), and CD4+FoxP3-CD25- cells (Figure 11C). Figure 11D provides EC50 (pM) and magnification change data for the AK167 and AK168 constructs, as well as the rhIL-2 control. [Figure 11C-D] The results of STAT5 activation studies in PBMCs using constructs AK167 and AK168, as well as controls containing rhIL-2 and anti-RSV antibodies, are shown. An untreated control was also tested. EC50 (pM) is also shown for rhIL-2, AK167, and AK168 treatments. STAT5 activation (%) is shown for CD4+FoxP3+CD25+ cells (Figure 11A), CD8+ cells (Figure 11B), and CD4+FoxP3-CD25- cells (Figure 11C). Figure 11D provides EC50 (pM) and magnification change data for the AK167 and AK168 constructs, as well as the rhIL-2 control. [Figure 12A-B]Figure 12A shows the percentage of STAT5 activation in PBMCs treated with construct AK165 or AK166, or with isotype controls or IL-2-Fc controls, which were either (+MMP10) or had not been previously exposed to MMP10 protease. The legend shown in Figure 12A also applies to Figure 12B, and the legend shown in Figure 12C also applies to Figure 12D. STAT5 activation (%) is shown for CD4+FoxP3+T regulatory cells (Figure 12A), CD4+FoxP3-T helper cells (Figure 12B), CD8+ cytotoxic T cells (Figure 12C), and CD56+ NK cells (Figure 12D). [Figure 12C-D] Figure 12A shows the percentage of STAT5 activation in PBMCs treated with construct AK165 or AK166, or with isotype controls or IL-2-Fc controls, which were either (+MMP10) or had not been previously exposed to MMP10 protease. The legend shown in Figure 12A also applies to Figure 12B, and the legend shown in Figure 12C also applies to Figure 12D. STAT5 activation (%) is shown for CD4+FoxP3+T regulatory cells (Figure 12A), CD4+FoxP3-T helper cells (Figure 12B), CD8+ cytotoxic T cells (Figure 12C), and CD56+ NK cells (Figure 12D). [Figure 13] Figure 13A shows STAT5 activation (%) in PBMCs treated with construct AK109 or AK110, or isotype control or IL-2-Fc control, which were either (+MMP10) or had not been previously exposed to MMP10 protease. The legend shown in Figure 12B also applies to Figure 13A. STAT5 activation (%) is shown for NK cells (Figure 13A), CD8 cells (Figure 13B), and CD4 cells (Figure 13C). [Figure 14A-B]The results from STAT5 activation studies in PBMCs using constructs AK211, AK235, AK253, AK306, AK310, AK314, and AK316, as well as an rhIL-2 control, are shown. STAT5 activation (%) is shown for CD3+CD4+FoxP3+ cells (Figure 14A), CD3+CD4+FoxP3- cells (Figure 14B), and CD3+CD8+ cells (Figure 14C). Figure 14D provides EC50 data for each of the tested constructs and the rhIL-2 control. [Figure 14C-D] The results from STAT5 activation studies in PBMCs using constructs AK211, AK235, AK253, AK306, AK310, AK314, and AK316, as well as an rhIL-2 control, are shown. STAT5 activation (%) is shown for CD3+CD4+FoxP3+ cells (Figure 14A), CD3+CD4+FoxP3- cells (Figure 14B), and CD3+CD8+ cells (Figure 14C). Figure 14D provides EC50 data for each of the tested constructs and the rhIL-2 control. [Figure 15A-B] The results from STAT5 activation studies in PBMCs using protease-activated constructs AK081, AK167, AK216, AK218, AK219, AK220, and AK223, as well as an rhIL-2 control, are shown. STAT5 activation (%) is shown for CD4+FoxP3+CD25+ regulatory T cells (Figure 15A), CD4+FoxP3-CD25- cells (Figure 15B), and CD8+ cells (Figure 15C). Figure 15D provides EC50 data for each of the tested constructs and the rhIL-2 control. [Figure 15C-D] The results from STAT5 activation studies in PBMCs using protease-activated constructs AK081, AK167, AK216, AK218, AK219, AK220, and AK223, as well as an rhIL-2 control, are shown. STAT5 activation (%) is shown for CD4+FoxP3+CD25+ regulatory T cells (Figure 15A), CD4+FoxP3-CD25- cells (Figure 15B), and CD8+ cells (Figure 15C). Figure 15D provides EC50 data for each of the tested constructs and the rhIL-2 control. [Figure 16] Figures 16A, 16B, and 16C show STAT5 activation (%) in PBMCs treated with construct AK081, AK189, AK190, or AK210, or with an anti-RSV control. The legend shown in Figure 16A also applies to Figures 16B and 16C. STAT5 activation (%) is shown for regulatory T cells (Figure 16A), CD4 helper T cells (Figure 16B), and CD8 cells (Figure 16C). [Figure 17] STAT5 activation (%) is shown in PBMCs treated with construct AK167, AK191, AK192, or AK193, or with an anti-RSV control. The legend shown in Figure 17A also applies to Figures 17B and 17C. STAT5 activation (%) is shown for regulatory T cells (Figure 17A), CD4 helper T cells (Figure 17B), and CD8 cells (Figure 17C). [Figure 18] The results from pharmacokinetic studies performed on tumor-bearing mice using constructs AK032, AK081, AK111, AK167, or AK168, or anti-RSV controls, are shown. Figure 18A provides a simple depiction of the structure of each of the tested constructs. Figure 18B shows plasma Fc levels (μg / mL) by detecting human IgG, Figure 18C shows plasma Fc-CD122 levels (μg / mL) by detecting human CD122, and Figure 18D shows plasma Fc-IL2 levels (μg / mL) by detecting human IL-2. Prior to the detection step, anti-human IG was used as a capture antibody. [Figure 19A-B] The results from pharmacokinetic studies performed on tumor-bearing mice using constructs AK167, AK191, AK197, AK203, AK209, or AK211, or anti-RSV controls are shown. Figure 19A provides a simple depiction of the structure of each of the tested constructs. Figure 19B shows plasma Fc levels (μg / mL) by detecting human IgG, Figure 19C shows plasma Fc-IL2 levels (μg / mL) by detecting human IL-2, and Figure 19D shows plasma Fc-CD122 levels (μg / mL) by detecting human CD122. Prior to the detection step, anti-human IG was used as a capture antibody. [Figure 19C-D] The results from pharmacokinetic studies performed on tumor-bearing mice using constructs AK167, AK191, AK197, AK203, AK209, or AK211, or anti-RSV controls are shown. Figure 19A provides a simple depiction of the structure of each of the tested constructs. Figure 19B shows plasma Fc levels (μg / mL) by detecting human IgG, Figure 19C shows plasma Fc-IL2 levels (μg / mL) by detecting human IL-2, and Figure 19D shows plasma Fc-CD122 levels (μg / mL) by detecting human CD122. Prior to the detection step, anti-human IG was used as a capture antibody. [Figure 20A-D] Results from studies testing the in vivo response rates of CD4, CD8, NK, and Treg cells in spleen, blood, and tumors using AK032, AK081, AK111, AK167, or AK168 constructs, or anti-RSV IgG controls, are shown. For spleen tissue, the percentages of CD8 cells in CD3 cells (Figure 20A), CD4 cells in CD3 cells (Figure 20B), NK cells in CD3- cells (Figure 20C), and FoxP cells in CD4 cells (Figure 20D) are shown. For blood, the percentages of CD8 cells in CD3 cells (Figure 20E), CD4 cells in CD3 cells (Figure 20F), NK cells in CD3- cells (Figure 20G), and FoxP cells in CD4 cells (Figure 20H) are shown. Regarding tumor tissue, the following are shown: % CD8 cells from CD3 cells (Figure 20I), % CD4 cells from CD3 cells (Figure 20J), % NK cells from CD3- cells (Figure 20K), and 3% FoxP cells from CD4 cells (Figure 20L). [Figure 20E-H]Results from studies testing the in vivo response rates of CD4, CD8, NK, and Treg cells in spleen, blood, and tumors using AK032, AK081, AK111, AK167, or AK168 constructs, or anti-RSV IgG controls, are shown. For spleen tissue, the percentages of CD8 cells in CD3 cells (Figure 20A), CD4 cells in CD3 cells (Figure 20B), NK cells in CD3- cells (Figure 20C), and FoxP cells in CD4 cells (Figure 20D) are shown. For blood, the percentages of CD8 cells in CD3 cells (Figure 20E), CD4 cells in CD3 cells (Figure 20F), NK cells in CD3- cells (Figure 20G), and FoxP cells in CD4 cells (Figure 20H) are shown. Regarding tumor tissue, the following are shown: % CD8 cells from CD3 cells (Figure 20I), % CD4 cells from CD3 cells (Figure 20J), % NK cells from CD3- cells (Figure 20K), and 3% FoxP cells from CD4 cells (Figure 20L). [Figure 20I-L] Results from studies testing the in vivo response rates of CD4, CD8, NK, and Treg cells in spleen, blood, and tumors using AK032, AK081, AK111, AK167, or AK168 constructs, or anti-RSV IgG controls, are shown. For spleen tissue, the percentages of CD8 cells in CD3 cells (Figure 20A), CD4 cells in CD3 cells (Figure 20B), NK cells in CD3- cells (Figure 20C), and FoxP cells in CD4 cells (Figure 20D) are shown. For blood, the percentages of CD8 cells in CD3 cells (Figure 20E), CD4 cells in CD3 cells (Figure 20F), NK cells in CD3- cells (Figure 20G), and FoxP cells in CD4 cells (Figure 20H) are shown. Regarding tumor tissue, the following are shown: % CD8 cells from CD3 cells (Figure 20I), % CD4 cells from CD3 cells (Figure 20J), % NK cells from CD3- cells (Figure 20K), and 3% FoxP cells from CD4 cells (Figure 20L). [Figure 21A-D]Results from studies testing the in vivo response rates of CD4, CD8, NK, and Treg cells in spleen, blood, and tumors using AK167, AK168, AK191, AK197, AK203, AK209, or AK211 constructs, or anti-RSV IgG controls, are shown. For spleen tissue, the percentages of CD8 cells in CD3 cells (Figure 21A), CD4 cells in CD3 cells (Figure 21B), NK cells in CD3- cells (Figure 21C), and FoxP cells in CD4 cells (Figure 21D) are shown. For blood, the percentages of CD8 cells in CD3 cells (Figure 21E), CD4 cells in CD3 cells (Figure 21F), NK cells in CD3- cells (Figure 21G), and FoxP cells in CD4 cells (Figure 21H) are shown. Regarding tumor tissue, the following are shown: % CD8 cells from CD3 cells (Figure 21I), % CD4 cells from CD3 cells (Figure 21J), % NK cells from CD3- cells (Figure 21K), and 3% FoxP cells from CD4 cells (Figure 21L). [Figure 21E-H] Results from studies testing the in vivo response rates of CD4, CD8, NK, and Treg cells in spleen, blood, and tumors using AK167, AK168, AK191, AK197, AK203, AK209, or AK211 constructs, or anti-RSV IgG controls, are shown. For spleen tissue, the percentages of CD8 cells in CD3 cells (Figure 21A), CD4 cells in CD3 cells (Figure 21B), NK cells in CD3- cells (Figure 21C), and FoxP cells in CD4 cells (Figure 21D) are shown. For blood, the percentages of CD8 cells in CD3 cells (Figure 21E), CD4 cells in CD3 cells (Figure 21F), NK cells in CD3- cells (Figure 21G), and FoxP cells in CD4 cells (Figure 21H) are shown. Regarding tumor tissue, the following are shown: % CD8 cells from CD3 cells (Figure 21I), % CD4 cells from CD3 cells (Figure 21J), % NK cells from CD3- cells (Figure 21K), and 3% FoxP cells from CD4 cells (Figure 21L). [Figure 21I-L]Results from studies testing the in vivo response rates of CD4, CD8, NK, and Treg cells in spleen, blood, and tumors using AK167, AK168, AK191, AK197, AK203, AK209, or AK211 constructs, or anti-RSV IgG controls, are shown. For spleen tissue, the percentages of CD8 cells in CD3 cells (Figure 21A), CD4 cells in CD3 cells (Figure 21B), NK cells in CD3- cells (Figure 21C), and FoxP cells in CD4 cells (Figure 21D) are shown. For blood, the percentages of CD8 cells in CD3 cells (Figure 21E), CD4 cells in CD3 cells (Figure 21F), NK cells in CD3- cells (Figure 21G), and FoxP cells in CD4 cells (Figure 21H) are shown. Regarding tumor tissue, the following are shown: % CD8 cells from CD3 cells (Figure 21I), % CD4 cells from CD3 cells (Figure 21J), % NK cells from CD3- cells (Figure 21K), and 3% FoxP cells from CD4 cells (Figure 21L). [Figure 22A-D] Results from studies testing the in vivo response rates of CD4, CD8, NK, and Treg cells in spleen, blood, and tumors using AK235, AK191, AK192, AK193, AK210, AK189, AK190, or AK211 constructs, or anti-RSV IgG controls, are shown. For spleen tissue, the percentages of CD8 cells in CD3 cells (Figure 22A), CD4 cells in CD3 cells (Figure 22B), NK cells in CD3- cells (Figure 22C), and FoxP cells in CD4 cells (Figure 22D) are shown. For blood, the percentages of CD8 cells in CD3 cells (Figure 22E), CD4 cells in CD3 cells (Figure 22F), NK cells in CD3- cells (Figure 22G), and FoxP cells in CD4 cells (Figure 22H) are shown. For spleen tissue, the percentages of CD3 cells that are CD8 cells (Figure 22I), CD3 cells that are CD4 cells (Figure 22J), CD3- cells that are NK cells (Figure 22K), and CD4 cells that are FoxP 3% (Figure 22L) are shown. [Figure 22E-H]Results from studies testing the in vivo response rates of CD4, CD8, NK, and Treg cells in spleen, blood, and tumors using AK235, AK191, AK192, AK193, AK210, AK189, AK190, or AK211 constructs, or anti-RSV IgG controls, are shown. For spleen tissue, the percentages of CD8 cells in CD3 cells (Figure 22A), CD4 cells in CD3 cells (Figure 22B), NK cells in CD3- cells (Figure 22C), and FoxP cells in CD4 cells (Figure 22D) are shown. For blood, the percentages of CD8 cells in CD3 cells (Figure 22E), CD4 cells in CD3 cells (Figure 22F), NK cells in CD3- cells (Figure 22G), and FoxP cells in CD4 cells (Figure 22H) are shown. For spleen tissue, the percentages of CD3 cells that are CD8 cells (Figure 22I), CD3 cells that are CD4 cells (Figure 22J), CD3- cells that are NK cells (Figure 22K), and CD4 cells that are FoxP 3% (Figure 22L) are shown. [Figure 22I-L] Results from studies testing the in vivo response rates of CD4, CD8, NK, and Treg cells in spleen, blood, and tumors using AK235, AK191, AK192, AK193, AK210, AK189, AK190, or AK211 constructs, or anti-RSV IgG controls, are shown. For spleen tissue, the percentages of CD8 cells in CD3 cells (Figure 22A), CD4 cells in CD3 cells (Figure 22B), NK cells in CD3- cells (Figure 22C), and FoxP cells in CD4 cells (Figure 22D) are shown. For blood, the percentages of CD8 cells in CD3 cells (Figure 22E), CD4 cells in CD3 cells (Figure 22F), NK cells in CD3- cells (Figure 22G), and FoxP cells in CD4 cells (Figure 22H) are shown. For spleen tissue, the percentages of CD3 cells that are CD8 cells (Figure 22I), CD3 cells that are CD4 cells (Figure 22J), CD3- cells that are NK cells (Figure 22K), and CD4 cells that are FoxP 3% (Figure 22L) are shown. [Figure 23A-C]Results from in vivo T cell activation in the spleen, blood, and tumors using the AK235, AK191, AK192, AK193, AK210, AK189, AK190, or AK211 constructs are shown. T cell activation was measured as the mean fluorescence intensity (MFI) of CD25 in CD8+ T cells (Figures 23A, 23D, 23G), CD4+ T cells (Figures 23B, 23E, 23H), or Foxp3+ cells (Figures 23C, 23F, 23I) in the spleen, blood, and tumors. Statistical analysis was performed using one-way ANOVA compared to the non-cleavable AK211 construct. [Figure 23D-F] Results from in vivo T cell activation in the spleen, blood, and tumors using the AK235, AK191, AK192, AK193, AK210, AK189, AK190, or AK211 constructs are shown. T cell activation was measured as the mean fluorescence intensity (MFI) of CD25 in CD8+ T cells (Figures 23A, 23D, 23G), CD4+ T cells (Figures 23B, 23E, 23H), or Foxp3+ cells (Figures 23C, 23F, 23I) in the spleen, blood, and tumors. Statistical analysis was performed using one-way ANOVA compared to the non-cleavable AK211 construct. [Figure 23G-I] Results from in vivo T cell activation in the spleen, blood, and tumors using the AK235, AK191, AK192, AK193, AK210, AK189, AK190, or AK211 constructs are shown. T cell activation was measured as the mean fluorescence intensity (MFI) of CD25 in CD8+ T cells (Figures 23A, 23D, 23G), CD4+ T cells (Figures 23B, 23E, 23H), or Foxp3+ cells (Figures 23C, 23F, 23I) in the spleen, blood, and tumors. Statistical analysis was performed using one-way ANOVA compared to the non-cleavable AK211 construct. [Figure 24A-D]The results from studies testing in vivo cleavage of exemplary masked IL-2 polypeptide constructs AK168 (cleavable peptide sequence: MPYDLYHP) and AK209 (cleavable peptide sequence: VPLSLY, SEQ ID NO: 15) are shown. Figure 24E shows the results from pharmacokinetic studies of total plasma IgG concentration (μg / mL) for the combined levels of the AK167, AK168, and AK209 constructs, as well as the levels of the uncleaved forms of each construct. [Figure 24E] The results from studies testing in vivo cleavage of exemplary masked IL-2 polypeptide constructs AK168 (cleavable peptide sequence: MPYDLYHP) and AK209 (cleavable peptide sequence: VPLSLY, SEQ ID NO: 15) are shown. Figure 24E shows the results from pharmacokinetic studies of total plasma IgG concentration (μg / mL) for the combined levels of the AK167, AK168, and AK209 constructs, as well as the levels of the uncleaved forms of each construct. [Figure 25A-D] Results from in vivo studies evaluating vascular leakage using exemplary masked IL-2 polypeptide constructs AK111 or AK168, or unmasked IL-2 polypeptide constructs AK081 or AK167, or anti-RSV controls are shown. Figure 25A shows the percentage of weight loss, and Figures 25B, 25C, and 25D show the weights of the liver, lungs, and spleen in grams, respectively. [Figure 26A-B] The results from in vivo studies evaluating vascular leakage, indicated by measuring the degree of dye leakage into liver and lung tissue after administration of AK081, AK111, AK167, or AK168 constructs, or anti-RSV controls, are shown. The degree of dye leakage into the liver (Figure 26A) and lungs (Figure 26B) was measured based on absorbance at 650 nm. [Figure 27A-B] Results from in vivo studies evaluating vascular leakage, indicated by measuring the degree of mononuclear cell perivascular infiltration into liver and lung tissue after administration of the AK081, AK111, AK167, or AK168 construct, or an anti-RSV control, are shown. The mean number of mononuclear cells in the liver (Figure 27A) and the mean number of mononuclear cells in the lungs (Figure 27B) are depicted for each. [Figure 28A] Results from syngeneic tumor model studies evaluating tumor volume and body weight over the course of treatment using the AK032, AK081, AK111, AK167, or AK168 constructs, or anti-RSV controls, are shown. Figure 28A shows data for tumor volume over the course of treatment, and Figure 28B shows data for the percentage change in body weight over the course of treatment. [Figure 28B] Results from syngeneic tumor model studies evaluating tumor volume and body weight over the course of treatment using the AK032, AK081, AK111, AK167, or AK168 constructs, or anti-RSV controls, are shown. Figure 28A shows data for tumor volume over the course of treatment, and Figure 28B shows data for the percentage change in body weight over the course of treatment. [Figure 29A-B] This study shows that AK471 with the I253A FcRn mutation induced robust CD8 T cell expansion in the TME, while remaining inactive in the surrounding areas. [Figure 30A-C] This shows that AK471 has a slightly shorter half-life compared to aglyco-hIgG1. [Figure 31A-C] The presence of AK471 in plasma indicates no evidence of cleavage or excision. [Figure 32] An exemplary IL-12 structure is shown. [Figure 33A-B] Exemplary cleavage processes for exemplary molecules AK380, AK381, and AK384 (Figure 34A), as well as AK383, AK386, AK434, AK447, AK448, AK446, AK528, and AK529 (Figure 34B) are shown. [Figure 34A-D]The masking of IL-12 toward IL-12RB1 is depicted using SPR analysis that tested the binding of exemplary masked IL-12 polypeptide constructs (AK384 and AK386) to rhIL-12RB1-Fc. Figure 34A depicts the interaction between AK384 and IL-12RB1-Fc, Figure 34B depicts the interaction between AK386 and IL-12RB1-Fc, and Figure 34C depicts the interaction between recombinant human IL-12 (rhIL-12) control and IL-12RB1-Fc. Figure 34D provides a table summarizing the data obtained for the association constant (ka), dissociation constant (kd), equilibrium dissociation constant (KD), and Chi2 and U values ​​for each interaction. Due to the shape of the curves, the exact rate could not be determined, and therefore, KD is estimated based on the on-rate. These results demonstrate that these exemplary masked IL-12 polypeptide constructs (AK384 and AK386) did not demonstrate detectable binding to IL-12RB1-Fc, while the wild-type rhIL-12 control did. [Figure 35A-D] The masking of IL-12 toward IL-12RB2 is depicted using SPR analysis that tested the binding of exemplary masked IL-12 polypeptide constructs (AK384 and AK386) to rhIL-12RB2-Fc. Figure 35A depicts the interaction between AK384 and IL-12RB2-Fc, Figure 35B depicts the interaction between AK386 and IL-12RB2-Fc, and Figure 35C depicts the interaction between recombinant human IL-12 (rhIL-12) control and IL-12RB2-Fc. Figure 35D provides a table summarizing the data obtained for the association constant (ka), dissociation constant (kd), equilibrium dissociation constant (KD), and Chi2 and U values ​​for each interaction. Due to the shape of the curves, the exact rate could not be determined, and therefore, KD is estimated based on the on-rate. These results demonstrate that the exemplary masked IL-12 polypeptide construct (AK386) demonstrated weak but detectable binding to IL-12RB1-Fc, while the wild-type rhIL-12 control and exemplary IL-12 polypeptide construct (AK384) demonstrated detectable binding. [Figure 36] The results from Example 6 are shown below. [Figure 37] The results from Example 6 are shown below. [Figure 38] The results from Example 6 are shown below. [Figure 39] The results from Example 6 are shown below. [Figure 40] The results from Example 6 are shown below. [Figure 41] The results from Example 7 are shown below. [Figure 42] The results from Example 7 are shown below. [Figure 43] The results from Example 7 are shown below. [Figure 44] The results from Example 8 are shown below. [Figure 45] The results from Example 8 are shown below. [Figure 46A] The results from Example 8 are shown below. [Figure 46B] The results from Example 8 are shown below. [Figure 47A-B] The results from Example 8 are shown below. [Figure 48A-B] The results from Example 8 are shown below. [Figure 48C-D] The results from Example 8 are shown below. [Figure 48E-F] The results from Example 8 are shown below. [Figure 48G-H] The results from Example 8 are shown below. [Figure 48I-J] The results from Example 8 are shown below. [Figure 48K] The results from Example 8 are shown below. [Figure 49A-B] The results from Example 8 are shown below. [Figure 50A-B] The results from Example 8 are shown below. [Figure 50C-D] The results from Example 8 are shown below. [Figure 50E-F] The results from Example 8 are shown below. [Figure 50G] The results from Example 8 are shown below. [Figure 51] The results from Example 8 are shown below. [Figure 52A] The results from Example 8 are shown below. [Figure 52B-C] The results from Example 8 are shown below. [Figure 52D-E] The results from Example 8 are shown below. [Figures 53A-53D-54A-54F] The results of SDS-PAGE and HEK-Blue IL-2 bioassays using exemplary IL-15 constructs AK904 and AK910 without peptide substrates, and constructs AK932, AK938, AK930, and AK936 with peptide substrates are shown. [Figure 53A-B] The results of the SDS-PAGE gel are shown. [Figure 53C-D] The results of the SDS-PAGE gel are shown. [Figure 54A-C] The results of the HEK-Blue IL-2 bioassay are shown. [Figure 54D-F] The results of the HEK-Blue IL-2 bioassay are shown. [Figure 55] The results of Example 11 are shown. The PK / PD of exemplary mouse-derived molecules AK944, AK945, AK947, and control AK948 were analyzed in vivo in two tumor models, MB49 and B16F10. [Figure 56] The results of Example 11 are shown. The PK / PD of exemplary mouse-derived molecules AK944, AK945, AK947, and control AK948 were analyzed in vivo in two tumor models, MB49 and B16F10. [Figure 57] The results of Example 11 are shown. The PK / PD of exemplary mouse-derived molecules AK944, AK945, AK947, and control AK948 were analyzed in vivo in two tumor models, MB49 and B16F10. [Figure 58] The results of Example 11 are shown. The PK / PD of exemplary mouse-derived molecules AK944, AK945, AK947, and control AK948 were analyzed in vivo in two tumor models, MB49 and B16F10. [Figure 59]The results of Example 11 are shown. The PK / PD of exemplary mouse-derived molecules AK944, AK945, AK947, and control AK948 were analyzed in vivo in two tumor models, MB49 and B16F10. [Figure 60] The results of Example 11 are shown. The PK / PD of exemplary mouse-derived molecules AK944, AK945, AK947, and control AK948 were analyzed in vivo in two tumor models, MB49 and B16F10. [Figure 61A] The results of Example 11 are shown. The PK / PD of exemplary mouse-derived molecules AK944, AK945, AK947, and control AK948 were analyzed in vivo in two tumor models, MB49 and B16F10. [Figure 61B] The results of Example 11 are shown. The PK / PD of exemplary mouse-derived molecules AK944, AK945, AK947, and control AK948 were analyzed in vivo in two tumor models, MB49 and B16F10. [Figure 62] The results of Example 11 are shown. The PK / PD of exemplary mouse-derived molecules AK944, AK945, AK947, and control AK948 were analyzed in vivo in two tumor models, MB49 and B16F10. [Figure 63A] The results of Example 11 are shown. The PK / PD of exemplary mouse-derived molecules AK944, AK945, AK947, and control AK948 were analyzed in vivo in two tumor models, MB49 and B16F10. [Figure 63B] The results of Example 11 are shown. The PK / PD of exemplary mouse-derived molecules AK944, AK945, AK947, and control AK948 were analyzed in vivo in two tumor models, MB49 and B16F10. [Figure 64A] The results of Example 11 are shown. The PK / PD of exemplary mouse-derived molecules AK944, AK945, AK947, and control AK948 were analyzed in vivo in two tumor models, MB49 and B16F10. [Figure 64B]The results of Example 11 are shown. The PK / PD of exemplary mouse-derived molecules AK944, AK945, AK947, and control AK948 were analyzed in vivo in two tumor models, MB49 and B16F10. [Figure 65A] The results of Example 11 are shown. The PK / PD of exemplary mouse-derived molecules AK944, AK945, AK947, and control AK948 were analyzed in vivo in two tumor models, MB49 and B16F10. [Figure 65B] The results of Example 11 are shown. The PK / PD of exemplary mouse-derived molecules AK944, AK945, AK947, and control AK948 were analyzed in vivo in two tumor models, MB49 and B16F10. [Figure 65C] The results of Example 11 are shown. The PK / PD of exemplary mouse-derived molecules AK944, AK945, AK947, and control AK948 were analyzed in vivo in two tumor models, MB49 and B16F10. [Figure 66] The results of Example 11 are shown. The PK / PD of exemplary mouse-derived molecules AK944, AK945, AK947, and control AK948 were analyzed in vivo in two tumor models, MB49 and B16F10. [Figure 67A] The results of Example 11 are shown. The PK / PD of exemplary mouse-derived molecules AK944, AK945, AK947, and control AK948 were analyzed in vivo in two tumor models, MB49 and B16F10. [Figure 67B] The results of Example 11 are shown. The PK / PD of exemplary mouse-derived molecules AK944, AK945, AK947, and control AK948 were analyzed in vivo in two tumor models, MB49 and B16F10. [Figure 67C] The results of Example 11 are shown. The PK / PD of exemplary mouse-derived molecules AK944, AK945, AK947, and control AK948 were analyzed in vivo in two tumor models, MB49 and B16F10. [Figure 68]The results of Example 11 are shown. The PK / PD of exemplary mouse-derived molecules AK944, AK945, AK947, and control AK948 were analyzed in vivo in two tumor models, MB49 and B16F10. [Figure 69] The results for Example 12 are shown. PK, PD, blood, and serum chemistry of exemplary molecules AK667, AK921, AK923, and control AK671 were analyzed in cynomolgus monkeys. [Figure 70] The results for Example 12 are shown. PK, PD, blood, and serum chemistry of exemplary molecules AK667, AK921, AK923, and control AK671 were analyzed in cynomolgus monkeys. [Figure 71A] The results for Example 12 are shown. PK, PD, blood, and serum chemistry of exemplary molecules AK667, AK921, AK923, and control AK671 were analyzed in cynomolgus monkeys. [Figure 71B] The results for Example 12 are shown. PK, PD, blood, and serum chemistry of exemplary molecules AK667, AK921, AK923, and control AK671 were analyzed in cynomolgus monkeys. [Modes for carrying out the invention]

[0124] By using the masking portion, systemic side effects of administered IL-12 cytokines or their functional fragments can be reduced by interfering with the ability of IL-12 cytokines or their functional fragments to bind to their homologous receptors.

[0125] The interleukin-12 receptor is a type I cytokine receptor that binds to interleukin-12. It consists of beta-1 and beta-2 subunits.

[0126] By masking IL-12 cytokines or their functional fragments using a linker containing a proteolytically cleavable peptide, the binding ability that is hindered by the use of the masking moiety can be restored by cleavage of the cleavable peptide in the tumor microenvironment. Therefore, the masked IL-12 cytokines provided herein are engineered to precisely target their pharmacological activity in the tumor microenvironment by leveraging one of the characteristics of cancer: high local concentrations of active proteases. This characteristic of the tumor microenvironment is used to transform systemically inactive molecules into locally active IL-12 cytokines or their functional fragments in the form of IL-12 cleavage products. Activation of IL-12 cytokines or their functional fragments in the tumor microenvironment significantly reduces the systemic toxicity that may be associated with drugs administered to a target in their active form. Therefore, the masked IL-2 cytokines of the present invention may be considered prodrugs.

[0127] The masked IL-12 cytokines described herein have been found to exhibit a variety of advantageous properties. The masked IL-12 cytokines described anywhere in this specification have been found to be able to preferentially activate (proliferate and expand) immune cells within the tumor microenvironment and at lower levels in the periphery during proteolytic cleavage. The masked IL-12 cytokines described anywhere in this specification have been found to promote tumor eradication (i.e., exhibit antitumor activity) and inhibit metastasis during proteolytic cleavage. The masked IL-12 cytokines described anywhere in this specification have been found to demonstrate advantageous long-term drug exposure. The masked IL-12 cytokines described herein have been found to demonstrate advantageous stability. The masked IL-12 cytokines described herein have been found to demonstrate advantageous tolerability. Furthermore, the masked IL-12 cytokines described herein have been found to demonstrate advantageous efficacy.

[0128] 1. Masked cytokines of "heterodimers" In some embodiments, what is provided herein is a masked cytokine comprising a masking portion in a first polypeptide chain and an IL-12 cytokine or a functional fragment thereof in a second polypeptide chain. Such a masked cytokine may be referred to as a “heterodimarian” masked cytokine.

[0129] In some embodiments, the masked cytokines are a) A first polypeptide chain comprising a masking portion linked to a first half-life extension domain via a first linker, b) A protein heterodimer comprising a second polypeptide chain containing an IL-12 cytokine or a functional fragment thereof linked to a second half-life extension domain via a second linker, The first half-life extension domain is associated with the second half-life extension domain. One of the first or second linkers contains a peptide that can be cleaved proteolytically.

[0130] The masking portion, half-life extension domain, IL-12 cytokine or its functional fragment, linker, and the type of association between the first half-life extension domain and the second half-life extension domain may be any one of those described herein, or any combination thereof.

[0131] In some embodiments, in the first polypeptide chain, the first half-life extension domain is linked to the amino terminus of the first linker, and the carboxyl terminus of the first linker is linked to the amino terminus of the masking moiety; in the second polypeptide chain, the second half-life extension domain is linked to the amino terminus of the second linker, and the carboxyl terminus of the second linker is linked to the amino terminus of the IL-12 cytokine or its functional fragment. This is schematically shown below: The first polypeptide chain includes the following: JPEG2026048631000008.jpg7170 The second polypeptide chain includes the following: JPEG2026048631000009.jpg7170HL1 is the first half-life extension domain, L1 is the first linker, MM is the masking portion, HL2 is the second half-life extension domain, L2 is the second linker, and C is the IL-12 cytokine or its functional fragment.

[0132] 1.1 IL-12 cytokines Provided herein are IL-12 cytokines or functional fragments thereof for use in masked cytokines or their cleavage products. Cytokines play a role in cellular signaling, particularly within cells of the immune system. IL-12 is an interleukin, a type of cytokine signaling molecule in the immune system that modulates the activity of leukocytes.

[0133] Endogenous IL-12 exists as two distinct molecules, IL-12 p40 and IL-12 p35, which dimerize within the cell during biosynthesis.

[0134] The complete sequences of IL-12 p40 and IL-12 p35 are shown below (propeptides cleaved during biosynthesis are shown in bold). JPEG2026048631000010.jpg79170JPEG2026048631000011.jpg54170

[0135] The mature forms are as follows: IL-12 p40 subunit: IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATL SAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS IL-12 p35 subunit: RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS

[0136] They are expressed as two chains that covalently dimerize during biosynthesis via a disulfide bond between the two subunits. Cysteine ​​C199 of the p40 subunit associates with cysteine ​​C96 of the p35 subunit.

[0137] A "functional fragment" of the IL-12 cytokine includes a portion of the full-length cytokine protein that retains or modifies cytokine receptor binding ability (for example, within 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the full-length cytokine protein). Cytokine receptor binding ability can be indicated, for example, by the cytokine's ability to bind to its homologous receptor or a component thereof.

[0138] In some embodiments, the IL-12 cytokine or its functional fragment is any naturally occurring interleukin-2 (IL-12) protein or a modified variant thereof that is capable of binding to the interleukin-12 receptor.

[0139] In some embodiments, the IL-12 polypeptide or its functional fragment comprises an IL-12p40 polypeptide or its functional fragment covalently bound to the IL-12p35 polypeptide or its functional fragment.

[0140] Since the IL-12p40 polypeptide or its functional fragment can attach to the first half-life extension domain, the first polypeptide chain includes the following: JPEG2026048631000012.jpg7170 The second polypeptide chain includes the following: JPEG2026048631000013.jpg7170 "IL-12p40" is the IL-12p40 polypeptide or a functional fragment thereof, and "IL-12p35" is the IL-12p35 polypeptide or a functional fragment thereof.

[0141] In some embodiments, the IL-12p40 polypeptide comprises SEQ ID NO: 1. In some embodiments, the IL-12p40 polypeptide or its functional fragment comprises an amino acid sequence having at least one amino acid modification compared to the amino acid sequence of SEQ ID NO: 1. Each of the at least one amino acid modification may be any amino acid modification such as substitution, insertion, or deletion. In some embodiments, the IL-12 cytokine or its functional fragment comprises an amino acid sequence having at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the IL-12 cytokine or its functional fragment comprises an amino acid sequence having at least five amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 1.

[0142] The IL-12p40 polypeptide contains a glycosaminoglycan (GAG) binding domain. GAGs such as heparin and heparan sulfate have been shown to bind to numerous growth factors and cytokines, including IL-12. The physiological significance of this binding is 2:1. Firstly, GAGs act as co-receptors on the cell surface, maintaining high local concentrations of cytokines. Secondly, GAGs can regulate the bioactivity of growth factors and cytokines through multiple mechanisms, including protection from dimerization and proteolysis.

[0143] The GAG-binding domain of the mature form of the IL-12 p40 subunit is shown below in bold. JPEG2026048631000014.jpg67170

[0144] Modification of the GAG-binding domain (KSKREKKDRV) is shown herein to increase the PK profile of constructs containing the IL-12 cytokine with the mutated GAG-binding domain without any decrease in cytokine activity. Therefore, in some embodiments, the IL-12p40 polypeptide includes at least one amino acid modification to the GAG-binding domain. In some embodiments, the modification to the GAG-binding domain is a deletion mutation. In some embodiments, the modification to the GAG-binding domain is a deletion mutation and at least one substitution mutation.

[0145] In some embodiments, the GAG-binding domain comprises the amino acid sequence KDNTERV. In some embodiments, the IL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO: 57. In some embodiments, the GAG-binding domain comprises the amino acid sequence KDNTEGRV. In some embodiments, the IL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO: 58.

[0146] In some embodiments, the GAG-binding domain consists of the amino acid sequence KDNTERV. In some embodiments, the IL-12p40 polypeptide includes the amino acid sequence SEQ ID NO: 57. In some embodiments, the GAG-binding domain consists of the amino acid sequence KDNTEGRV. In some embodiments, the IL-12p40 polypeptide includes the amino acid sequence SEQ ID NO: 58.

[0147] In some embodiments, the IL-12p40 polypeptide or its functional fragment comprises an amino acid sequence having one or more cysteine ​​substitutions compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the IL-12p40 polypeptide or its functional fragment comprises an amino acid sequence having an amino acid substitution at position C252 compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position C252 is C252S. In some embodiments, the IL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO: 59. In some embodiments, the IL-12p40 polypeptide comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 59. In some embodiments, the IL-12p40 polypeptide consists of the amino acid sequence of SEQ ID NO: 59.

[0148] In some embodiments, the IL-12p40 polypeptide comprises an amino acid sequence having one or more cysteine ​​substitutions compared to the amino acid sequence of SEQ ID NO: 1, and at least one amino acid modification to the GAG-binding domain. In some embodiments, the IL-12p40 polypeptide comprises an amino acid substitution at position C252S compared to the amino acid sequence of SEQ ID NO: 1, and the GAG-binding domain comprises the amino acid sequence KDNTERV. In some embodiments, the IL-12p40 polypeptide comprises an amino acid substitution at position C252S compared to the amino acid sequence of SEQ ID NO: 1, and the GAG-binding domain comprises the amino acid sequence KDNTEGRV. In some embodiments, the IL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO: 60. In some embodiments, the IL-12p40 polypeptide comprises an amino acid sequence having approximately or at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 60. In some embodiments, the IL-12p40 polypeptide consists of the amino acid sequence of SEQ ID NO: 60.

[0149] In some embodiments, the IL-12p35 polypeptide comprises SEQ ID NO: 2. In some embodiments, the IL-12p35 polypeptide or its functional fragment comprises an amino acid sequence having at least one amino acid modification compared to the amino acid sequence of SEQ ID NO: 2. Each of the at least one amino acid modification may be any amino acid modification such as substitution, insertion, or deletion. In some embodiments, the IL-12 cytokine or its functional fragment comprises an amino acid sequence having at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the IL-12 cytokine or its functional fragment comprises an amino acid sequence having at least five amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 2.

[0150] In some embodiments, the IL-12p40-IL-12p35 linker is 5 to 20 amino acids in length.

[0151] In some embodiments, the IL-12p40-IL-12p35 linker is rich in amino acid residues G and S.

[0152] In some embodiments, the IL-12p40-IL-12p35 linker comprises only amino acid residue types selected from the group consisting of G and S.

[0153] In some embodiments, the IL-12p40-IL-12p35 linker is [(G) n It includes S, and n=4 or 5.

[0154] In some embodiments, the IL-12p40-IL-12p35 linker includes (GGGGS) iterations.

[0155] In some embodiments, the IL-12p40-IL-12p35 linker includes sequence number 3 (GGGGSGGGGSGGGGS).

[0156] In some embodiments, the IL-12 cytokine or its functional fragment includes SEQ ID NO: 4. In some embodiments, the IL-12 cytokine or its functional fragment includes an amino acid sequence having at least one amino acid modification compared to the amino acid sequences of SEQ ID NOs: 1 and 2. Each of the at least one amino acid modification may be any amino acid modification such as a substitution, insertion, or deletion. In some embodiments, the IL-12 cytokine or its functional fragment includes an amino acid sequence having at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acid substitutions compared to the amino acid sequences of SEQ ID NOs: 1 and 2. In some embodiments, the IL-12 cytokine or its functional fragment includes an amino acid sequence having at least five amino acid substitutions compared to the amino acid sequences of SEQ ID NOs: 1 and 2.

[0157] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the IL-12 cytokine or its functional fragment comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 4.

[0158] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 61. In some embodiments, the IL-12 cytokine or its functional fragment comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 61.

[0159] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 62. In some embodiments, the IL-12 cytokine or its functional fragment comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 62.

[0160] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 63. In some embodiments, the IL-12 cytokine or its functional fragment comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 63.

[0161] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 64. In some embodiments, the IL-12 cytokine or its functional fragment comprises an amino acid sequence having approximately or at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 64.

[0162] 1.2 Masking area Provided herein are masking moieties for use with masked cytokines. It should be understood that the masking moieties are cleaved from the masked cytokine to form their cleavage products. The masking moieties mask the IL-12 cytokine or its functional fragment within the masked cytokine, thereby reducing or preventing the binding of the IL-12 cytokine or its functional fragment to its homologous receptor.

[0163] IL-12 receptor beta-1, or IL-12Rβ1, is a subunit of the IL-12 receptor complex. IL-12Rβ1 is also known as CD212. This protein binds to interleukin-12 (IL-12) at low affinity. This protein forms a disulfide-bonded oligomer, which is necessary for its IL-12 binding activity. IL-12 receptor beta-2, or IL-12Rβ2, is another subunit of the IL-12 receptor complex. Co-expression of IL-12Rβ1 and IL-12Rβ2 proteins has been shown to lead to the formation of a high-affinity IL-12 binding site.

[0164] Methods for determining the degree of binding of a protein (e.g., a cytokine) to a related protein (e.g., a cytokine receptor) are well known in the art.

[0165] In some embodiments, the masking portion includes an IL-12 cytokine receptor, or a subunit or functional fragment thereof.

[0166] The interleukin-12 receptor subunit beta-1, also known as CD212, has the following sequence: JPEG2026048631000015.jpg152170

[0167] The interleukin-12 receptor subunit beta-2 has the following sequence: JPEG2026048631000016.jpg193170

[0168] Bold text indicates propeptides, underlined italics indicate extracellular domains, italics indicate transmembrane domains, and underlined bold text indicates cytoplasmic domains.

[0169] In some embodiments, the masking portion includes an extracellular domain of human IL-12Rβ1, or a fragment, part, or variant thereof, that retains or otherwise demonstrates affinity for IL-12.

[0170] In some embodiments, the masking portion includes an amino acid sequence having the amino acid sequence of human IL-12Rβ1 with 1 to 4 amino acid substitutions. In some embodiments, the masking portion includes an amino acid sequence having the amino acid sequence of human IL-12Rβ1 with 1 or 2 amino acid substitutions.

[0171] In some embodiments, the masking moiety includes a sequence having SEQ ID NO: 5, or a fragment, part, or variant thereof, that retains or otherwise demonstrates affinity for human IL-12Rβ1 residues 24-237, i.e., IL-12. In some embodiments, the masking moiety includes IL-12Rβ1 having SEQ ID NO: 5. In some embodiments, the masking moiety includes an amino acid sequence having approximately or at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the amino acid sequences of SEQ ID NO: 5. In some embodiments, the masking moiety includes an amino acid sequence having the amino acid sequence of SEQ ID NO: 5 with 1 to 4 amino acid substitutions. In some embodiments, the masking moiety includes an amino acid sequence having the amino acid sequence of SEQ ID NO: 5 with 1 or 2 amino acid substitutions.

[0172] In some embodiments, the masking moiety includes a sequence having SEQ ID NO: 6, or a fragment, part, or variant thereof, that retains or otherwise demonstrates affinity for human IL-12Rβ1 residues 24-545, i.e., IL-12. In some embodiments, the masking moiety includes IL-12Rβ1 having SEQ ID NO: 6. In some embodiments, the masking moiety includes an amino acid sequence having approximately or at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the amino acid sequences of SEQ ID NO: 6. In some embodiments, the masking moiety includes an amino acid sequence having the amino acid sequence of SEQ ID NO: 6 with 1 to 4 amino acid substitutions. In some embodiments, the masking moiety includes an amino acid sequence having the amino acid sequence of SEQ ID NO: 6 with 1 or 2 amino acid substitutions.

[0173] In some embodiments, the masking moiety includes an extracellular domain of human IL-12Rβ2, or a fragment, part, or variant thereof, that retains or otherwise demonstrates affinity for IL-12. In some embodiments, the masking moiety includes an amino acid sequence having the amino acid sequence of human IL-12Rβ2 with 1 to 4 amino acid substitutions. In some embodiments, the masking moiety includes an amino acid sequence having the amino acid sequence of human IL-12Rβ2 with 1 or 2 amino acid substitutions.

[0174] In some embodiments, the masking portion includes residues 24-212 of human IL-12Rβ2, i.e., the sequence having sequence number 7. In some embodiments, the masking portion includes an amino acid sequence having approximately or at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the amino acid sequences of sequence number 7. In some embodiments, the masking portion includes an amino acid sequence having the amino acid sequence of sequence number 7 with 1 to 4 amino acid substitutions. In some embodiments, the masking portion includes an amino acid sequence having the amino acid sequence of sequence number 7 with 1 or 2 amino acid substitutions.

[0175] In some embodiments, the masking portion includes residues 24-222 of human IL-12Rβ2, i.e., the sequence having sequence number 8. In some embodiments, the masking portion includes an amino acid sequence having approximately or at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the amino acid sequences of sequence number 8. In some embodiments, the masking portion includes an amino acid sequence having the amino acid sequence of sequence number 8 with 1 to 4 amino acid substitutions. In some embodiments, the masking portion includes an amino acid sequence having the amino acid sequence of sequence number 8 with 1 or 2 amino acid substitutions.

[0176] In some embodiments, the masking portion includes residues 24-319 of human IL-12Rβ2, i.e., the sequence having sequence number 9. In some embodiments, the masking portion includes an amino acid sequence having approximately or at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the amino acid sequences of sequence number 9. In some embodiments, the masking portion includes an amino acid sequence having the amino acid sequence of sequence number 9 with 1 to 4 amino acid substitutions. In some embodiments, the masking portion includes an amino acid sequence having the amino acid sequence of sequence number 9 with 1 or 2 amino acid substitutions.

[0177] In some embodiments, the masking moiety includes residues 24-319 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 9 with one or more cysteine ​​substitutions. In some embodiments, the masking moiety includes residues 24-319 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 9 with an amino acid substitution at position C242. In some embodiments, the amino acid substitution at position C242 is C242S. In some embodiments, the masking moiety includes the amino acid sequence of SEQ ID NO: 65. In some embodiments, the masking moiety includes an amino acid sequence having approximately or at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 65. In some embodiments, the masking moiety consists of the amino acids of SEQ ID NO: 65. In some embodiments, the masking portion includes residues 24-622 of IL-12Rβ2, i.e., the sequence having sequence number 10. In some embodiments, the masking portion includes an amino acid sequence having approximately or at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the amino acid sequences of sequence number 10. In some embodiments, the masking portion includes an amino acid sequence having the amino acid sequence of sequence number 10 with 1 to 4 amino acid substitutions. In some embodiments, the masking portion includes an amino acid sequence having the amino acid sequence of sequence number 10 with 1 or 2 amino acid substitutions.

[0178] In some embodiments, the masking portion includes residues 24-227 of human IL-12Rβ2, i.e., the sequence having sequence number 11. In some embodiments, the masking portion includes an amino acid sequence having approximately or at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the amino acid sequences of sequence number 11. In some embodiments, the masking portion includes an amino acid sequence having the amino acid sequence of sequence number 11 with 1 to 4 amino acid substitutions. In some embodiments, the masking portion includes an amino acid sequence having the amino acid sequence of sequence number 11 with 1 or 2 amino acid substitutions.

[0179] 1.3 Linker Provided herein are linkers for use with masked cytokines or their cleavage products. The linkers provided herein refer to peptides of two additional amino acids used to link together two functional components in the masked cytokines described herein.

[0180] The masked cytokine comprises a first linker and a second linker, one of which contains a peptide that can be proteolytically cleaved.

[0181] In some embodiments, the second linker contains a proteolytically cleavable peptide (referred to herein as the “proteolytically cleavable linker”), and the first linker does not contain a proteolytically cleavable peptide (referred to herein as the “non-proteolytically cleavable linker”), so the first polypeptide chain includes: JPEG2026048631000017.jpg7170 The second polypeptide chain includes the following: JPEG2026048631000018.jpg7170

[0182] In some embodiments, the first linker contains a proteolytically cleavable peptide (referred herein to as a “proteolytically cleavable linker” or “cleavable linker”), and the second linker does not contain a proteolytically cleavable peptide (referred herein to as a “non-proteolytically cleavable linker” or “non-cleavable linker”), so the first polypeptide chain includes: JPEG2026048631000019.jpg7170 The second polypeptide chain includes the following: JPEG2026048631000020.jpg7170

[0183] Non-cuttable and cuttable linkers in several embodiments are described in more detail below.

[0184] 1.3.1 Linkers that can be cleaved non-proteinogenically In some embodiments, the non-cleavable linker is 3 to 18 amino acids in length.

[0185] In some embodiments, the non-cleavable linker is 3 to 15 amino acids in length.

[0186] In some embodiments, the non-cleavable linker is rich in amino acid residues G and S.

[0187] In some embodiments, the non-cleavable linker comprises only amino acid residue types selected from the group consisting of G and S.

[0188] In some embodiments, the non-cuttable linker is [(G)] n It includes S, and n=4 or 5.

[0189] In some embodiments, the non-cleavable linker includes the amino acid sequence shown in SEQ ID NO: 12 (GGGGS).

[0190] In some embodiments, the non-cleavable linker includes the amino acid sequence shown in SEQ ID NO: 13 (GGGGSGGGGS).

[0191] In some embodiments, the non-cleavable linker includes the amino acid sequence shown in SEQ ID NO: 14 (GGSGGGSGGGGGS).

[0192] In some embodiments, the non-cleavable linker includes the amino acid sequence shown in SEQ ID NO: 54 (GGSGGSGGSGGSGGSSGP).

[0193] In some embodiments, the non-cleavable linker includes the amino acid sequence shown in SEQ ID NO: 55 (PGGSGP).

[0194] In some embodiments, the non-cleavable linker includes the amino acid sequence shown in SEQ ID NO: 56 (GGSPG).

[0195] In some embodiments, the non-cleavable linker is 3 to 18 amino acids in length, such that the second linker is a proteolytically cleavable linker and contains a proteolytically cleavable peptide, while the first linker is a non-proteolytically cleavable linker and does not contain a proteolytically cleavable peptide. In some embodiments, the non-cleavable linker contains the amino acid sequence shown in SEQ ID NO: 12 (GGGGS). In some embodiments, the non-cleavable linker contains the amino acid sequence shown in SEQ ID NO: 13 (GGGGSGGGGS). In some embodiments, the non-cleavable linker contains the amino acid sequence shown in SEQ ID NO: 14 (GGSGGGSGGGGGS). In some embodiments, the non-cleavable linker contains the amino acid sequence shown in SEQ ID NO: 54 (GGSGGSGGSGGSGGSSGP). In some embodiments, the non-cleavable linker contains the amino acid sequence shown in SEQ ID NO: 55 (PGGSGP). In some embodiments, the non-cleavable linker contains the amino acid sequence shown in SEQ ID NO: 56 (GGSPG).

[0196] In some embodiments, it is desirable that the first and second polypeptide chains be the same or similar in length to facilitate the association of the first half-life extension domain with the second half-life extension domain and the masking portion masking the IL-12 cytokine or its functional fragment in the aggregate construct. Therefore, if the masking portion has a shorter amino acid sequence than the IL-12 cytokine or its functional fragment, the length difference can be fully or partially compensated by using a longer linker L1.

[0197] 1.3.2 Linkers capable of proteolytic cleavage In some embodiments, the cleavable linker is 10 to 25 amino acids in length.

[0198] In some embodiments, the cleavable linker comprises a proteolytically cleavable peptide (CP) with spacer domains (SDs) positioned on both sides, as shown below. SD-CP-SD

[0199] Cleavable peptides The cleavable linker contains a cleavable peptide.

[0200] A cleavable peptide is a polypeptide that contains a protease cleavage site so that the cleavable peptide can be proteolytically cleaved. A protease is an enzyme that cleaves and hydrolyzes peptide bonds between two specific amino acid residues of a target substrate protein. As used herein, “cleavage site” refers to a recognizable site for cleavage of a portion of the cleavable peptide, found in any of the linkers containing the cleavable peptides described herein. Thus, a cleavage site may be found in the sequence of the cleavable peptides described herein. In some embodiments, the cleavage site is an amino acid sequence that is recognized and cleaved by the cleavage agent.

[0201] In some embodiments, the protease cleavage site is a tumor-associated protease cleavage site. As used herein, a "tumor-associated protease cleavage site" is an amino acid sequence recognized by a protease, the expression of which is specific to tumor cells or their tumor cell environment or is upregulated.

[0202] The tumor cell environment is complex and can contain multiple different proteases. Thus, the exact site at which a given cleavable peptide is cleaved within the tumor cell environment can vary between tumor types, between patients with the same tumor type, and even between cleavage products formed in the same tumor depending on the particular tumor cell environment. Further, even after cleavage, further modification of the initial cleavage product, for example by removal of one or two terminal amino acids, can occur by further action of proteases within the tumor cell environment. Thus, the distribution of cleavage products can be expected to form within the tumor cell environment of a patient following administration of the single construct of masked cytokine described herein.

[0203] It should be understood that the cleavage sites referred to herein refer to the site between two specific amino acid residues within a cleavable peptide that is a target of a protease known to be associated with the tumor cell environment. In this sense, there can be more than one cleavage site within a cleavable peptide described herein where different proteases cleave the cleavable peptide at different cleavage sites. Also, more than one protease can potentially act on the same cleavage site within a cleavable peptide. Considerations regarding protease cleavage sites can be found in the art.

[0204] Thus, the cleavable peptides disclosed herein can be cleaved by one or more proteases.

[0205] In some embodiments, the cleavable peptide is a substrate for a protease that co-localizes within a region or tissue that expresses the IL-12 cytokine receptor.

[0206] In some embodiments, the cleavable peptides are 5mer (i.e., peptides with a length of 5 amino acids), 6mer (i.e., peptides with a length of 6 amino acids), 7-mer (i.e., peptides with a length of 7 amino acids), 8mer (i.e., peptides with a length of 8 amino acids), 9mer (i.e., peptides with a length of 9 amino acids), 10mer (i.e., peptides with a length of 10 amino acids), 11mer (i.e., peptides with a length of 11 amino acids), 12mer (i.e., peptides with a length of 12 amino acids), 13mer (i.e., peptides with a length of 13 amino acids), 14mer (i.e., peptides with a length of 14 amino acids), 15mer (i.e., peptides with a length of 15 amino acids), 16mer (i.e., peptides with a length of 16 amino acids), 17-mer (i.e., peptides with a length of 17 amino acids), or 18mer (i.e., peptides with a length of 18 amino acids).

[0207] In some embodiments, the cleavable peptide is 5 to 18 amino acids in length. In some embodiments, the cleavable peptide is 6 to 10 amino acids in length.

[0208] In some embodiments, the cleavable peptide within the cleavable linker contains amino acids selected from the group consisting of the following: [Table 1]

[0209] As a purely illustrative example, in the table above, * indicates known or observed protease cleavage sites within cleavable peptides.

[0210] In some embodiments, the cleavable peptide includes the amino acid sequence of SEQ ID NO: 15 (VPLS*LY), for example, the cleavable peptide may include the amino acids of SEQ ID NO: 210 (VPLSLYSG). In some embodiments, the cleavable peptide includes the amino acid sequence of SEQ ID NO: 41 (MPYD*LYHP). In some embodiments, the cleavable peptide includes the amino acid sequence of SEQ ID NO: 42 (DSGG*FMLT). In some embodiments, the cleavable peptide includes the amino acid sequence of SEQ ID NO: 43 (RAAA*VKSP). In some embodiments, the cleavable peptide includes the amino acid sequence of SEQ ID NO: 44 (ISSGLL*SGRS), for example, the cleavable peptide may include the amino acid sequence of SEQ ID NO: 211 (ISSGLLSGRSDQP). In some embodiments, the cleavable peptide includes the amino acid sequence of SEQ ID NO: 45 (DLLA*VVAAS).

[0211] In some embodiments, the cleavable peptide consists of the amino acid sequence of SEQ ID NO: 15 (VPLS*LY). In some embodiments, the cleavable peptide consists of the amino acid sequence of SEQ ID NO: 210 (VPLSLYSG). In some embodiments, the cleavable peptide consists of the amino acid sequence of SEQ ID NO: 41 (MPYD*LYHP). In some embodiments, the cleavable peptide consists of the amino acid sequence of SEQ ID NO: 42 (DSGG*FMLT). In some embodiments, the cleavable peptide consists of the amino acid sequence of SEQ ID NO: 43 (RAAA*VKSP). In some embodiments, the cleavable peptide consists of the amino acid sequence of SEQ ID NO: 44 (ISSGLL*SGRS). In some embodiments, the cleavable peptide consists of the amino acid sequence of SEQ ID NO: 211 (ISSGLLSGRSDQP). In some embodiments, the cleavable peptide consists of the amino acid sequence of SEQ ID NO: 45 (DLLA*VVAAS).

[0212] Cleavable peptides having the amino acid sequences shown in SEQ ID NO: 44 or 45 have been found to exhibit highly specific cleavage within the tumor cell environment compared to the non-tumor cell environment. Therefore, when these cleavable peptides are incorporated into masked IL-12 cytokines as disclosed elsewhere herein, any systemic side effects of the administered IL-12 cytokine or its functional fragments may be further reduced.

[0213] Spacer Domain The spacer domain may consist of one or more amino acids. The function of the spacer domain, if present, is to link a proteolytically cleavable peptide (CP) to other functional components in the constructs described herein.

[0214] It should be understood that the spacer domain does not alter the biological interaction of the proteolytically cleavable peptide with proteases in the tumor cell environment or the non-tumor cell environment. In other words, even in the presence of the spacer domain, the proteolytically cleavable peptides of the present invention disclosed herein retain their advantageous tumor specificity.

[0215] In some embodiments, the spacer domain adjacent to the proteolytically cleavable peptide is different.

[0216] In some embodiments, the spacer domain is rich in amino acid residues G, S, and P.

[0217] In some embodiments, the spacer domain contains only amino acid residue types selected from the group consisting of G, S, and P.

[0218] In some embodiments, the severable linker includes the following: JPEG2026048631000022.jpg7170SD1 is the first spacer domain, and SD2 is the second spacer domain.

[0219] In some embodiments, the cleavable linker comprises the following. JPEG2026048631000023.jpg7170

[0220] In some embodiments, the first polypeptide chain comprises the following, JPEG: JPEG2026048631000024.jpg7170 The second polypeptide chain comprises the following. JPEG2026048631000025.jpg7170

[0221] <P In some embodiments, the first polypeptide chain comprises the following, JPEG2026048631000026.jpg7170 The second polypeptide chain comprises the following. JPEG2026048631000027.jpg8170

[0222] In some embodiments, the N-terminus of SD1 is glycine (G).

[0223] In some embodiments, the first spacer domain (SD1) is 3 to 10 amino acids in length. In some embodiments, the first spacer domain (SD1) is 5 to 9 amino acids in length.

[0224] <P In some embodiments, SD1 comprises SEQ ID NO: 16 (GGSGGS).

[0225] In some embodiments, SD1 comprises SEQ ID NO: 17 (GGSGGSGGS).

[0226] In some embodiments, the C-terminal sequence of SD2 is JPEG2026048631000028.jpg7170.

[0227] In some embodiments, the second spacer domain (SD2) is 3 to 6 amino acids in length.

[0228] In some embodiments, SD2 includes sequence number 18 (SGP).

[0229] An example combination of SD1 and SD2 in a detachable linker is shown below. [Table 2]

[0230] In some embodiments, the proteolytically cleavable linker comprises SD1-CP-SD2, where SD1 is the first spacer domain, CP is the cleavable peptide, and SD2 is the second spacer domain, where CP has the amino acid sequence shown in SEQ ID NO: 44. In some embodiments, the spacer domain is rich in amino acid residues G, S, and P. In some embodiments, the spacer domain contains only amino acid residue types selected from the group consisting of G, S, and P.

[0231] In some embodiments, the proteolytically cleavable linker comprises SD1-CP-SD2, where SD1 is the first spacer domain, CP is the cleavable peptide, and SD2 is the second spacer domain, where CP has the amino acid sequence shown in SEQ ID NO: 45. In some embodiments, the spacer domain is rich in amino acid residues G, S, and P. In some embodiments, the spacer domain contains only amino acid residue types selected from the group consisting of G, S, and P.

[0232] In some embodiments, the proteolytically cleavable linker comprises SD1-CP-SD2, where SD1 is the first spacer domain, CP is the cleavable peptide, and SD2 is the second spacer domain, where CP has the amino acid sequence shown in SEQ ID NO: 44, and SD2 has the amino acid sequence shown in SEQ ID NO: 18. In some embodiments, SD1 is 3 to 6 amino acids in length. In some embodiments, the spacer domain is rich in amino acid residues G, S, and P. In some embodiments, the spacer domain contains only amino acid residue types selected from the group consisting of G, S, and P.

[0233] In some embodiments, the proteolytically cleavable linker comprises SD1-CP-SD2, where SD1 is the first spacer domain, CP is the cleavable peptide, and SD2 is the second spacer domain, where CP has the amino acid sequence shown in SEQ ID NO: 45, and SD2 has the amino acid sequence shown in SEQ ID NO: 18. In some embodiments, SD1 is 3 to 6 amino acids in length. In some embodiments, the spacer domain is rich in amino acid residues G, S, and P. In some embodiments, the spacer domain contains only amino acid residue types selected from the group consisting of G, S, and P.

[0234] In some embodiments, the severable linker is Includes JPEG2026048631000030.jpg8170.

[0235] In some embodiments, the severable linker is Includes JPEG2026048631000031.jpg7170.

[0236] In some embodiments, the severable linker is Includes JPEG2026048631000032.jpg8170.

[0237] In some embodiments, the severable linker is Includes JPEG2026048631000033.jpg7170.

[0238] In some embodiments, the severable linker is Includes JPEG2026048631000034.jpg7170.

[0239] In some embodiments, the severable linker is Includes JPEG2026048631000035.jpg8170.

[0240] In some embodiments, the severable linker is Includes JPEG2026048631000036.jpg8170.

[0241] In some embodiments, the severable linker is Includes JPEG2026048631000037.jpg7170.

[0242] In some embodiments, the severable linker is Includes JPEG2026048631000038.jpg7170.

[0243] In some embodiments, the severable linker is Includes JPEG2026048631000039.jpg7170.

[0244] In some embodiments, the non-cleavable linker comprises the amino acid sequence shown in SEQ ID NO: 12 (GGGGS), and the cleavable linker is Includes JPEG2026048631000040.jpg7170.

[0245] In some embodiments, the non-cleavable linker comprises the amino acid sequence shown in SEQ ID NO: 13 (GGGGSGGGGS), and the cleavable linker is Includes JPEG2026048631000041.jpg7170.

[0246] In some embodiments, the non-cleavable linker comprises the amino acid sequence shown in SEQ ID NO: 14 (GGSGGGSGGGGGS), and the cleavable linker is Includes JPEG2026048631000042.jpg8170.

[0247] In some embodiments, the non-cleavable linker comprises the amino acid sequence shown in SEQ ID NO: 12 (GGGGS), and the cleavable linker is Includes JPEG2026048631000043.jpg7170.

[0248] In some embodiments, the non-cleavable linker comprises the amino acid sequence shown in SEQ ID NO: 13 (GGGGSGGGGS), and the cleavable linker is Includes JPEG2026048631000044.jpg7170.

[0249] In some embodiments, the non-cleavable linker comprises the amino acid sequence shown in SEQ ID NO: 14 (GGSGGGSGGGGGS), and the cleavable linker is Includes JPEG2026048631000045.jpg7170.

[0250] In some embodiments, the cleavable linker includes SEQ ID NO: 46 and the non-cleavable linker includes SEQ ID NO: 55, such that the second linker is a proteolytically cleavable linker and the second linker includes a proteolytically cleavable peptide, while the first linker does not include a proteolytically cleavable peptide, such that the first linker is a non-proteolytically cleavable linker. In some embodiments, the cleavable linker includes SEQ ID NO: 47 and the non-cleavable linker includes SEQ ID NO: 55. In some embodiments, the cleavable linker includes SEQ ID NO: 48 and the non-cleavable linker includes SEQ ID NO: 55. In some embodiments, the cleavable linker includes SEQ ID NO: 49 and the non-cleavable linker includes SEQ ID NO: 56. In some embodiments, the cleavable linker includes SEQ ID NO: 50 and the non-cleavable linker includes SEQ ID NO: 55. In some embodiments, the cleavable linker includes SEQ ID NO: 50 and the non-cleavable linker includes SEQ ID NO: 14. In some embodiments, the cleavable linker includes SEQ ID NO: 51 and the non-cleavable linker includes SEQ ID NO: 56. In some embodiments, the detachable linker includes sequence number 51, and the non-detachable linker includes sequence number 14. In some embodiments, the detachable linker includes sequence number 52, and the non-detachable linker includes sequence number 55. In some embodiments, the detachable linker includes sequence number 52, and the non-detachable linker includes sequence number 14. In some embodiments, the detachable linker includes sequence number 53, and the non-detachable linker includes sequence number 56. In some embodiments, the detachable linker includes sequence number 53, and the non-detachable linker includes sequence number 14.

[0251] In some embodiments, the proteolytically cleavable linker includes a cleavable peptide consisting of the amino acid sequence of SEQ ID NO: 44 (ISSGLL*SGRS).

[0252] In some embodiments, the proteolytically cleavable linker includes a cleavable peptide consisting of the amino acid sequence of SEQ ID NO: 45 (DLLA*VVAAS).

[0253] The linker combinations disclosed herein and in the exemplary AK molecule may be used with any IL-12 cytokine or fragment thereof disclosed herein. The linker combinations disclosed herein and in the exemplary AK molecule may be used with any masking moiety disclosed herein. The linker combinations disclosed herein and in the exemplary AK molecule may be used with any half-life extension domain. In other words, the linkers disclosed in the exemplary AK molecule may be used in combination with the IL-12 cytokine or fragment thereof disclosed herein, the masking moiety disclosed herein, and / or the half-life extension domain disclosed herein.

[0254] 1.4 Half-life extension domains Provided herein are half-life extension domains for use with masked cytokines or their cleavage products. A long in vivo half-life is important for therapeutic proteins. Unfortunately, cytokines administered to a subject typically have a short half-life because they are rapidly removed from the subject by mechanisms including renal clearance and endocytotic degradation. Therefore, in the masked cytokines provided herein, the half-life extension domains are ligated to the masked cytokine for the purpose of extending the half-life of the cytokine in vivo.

[0255] The term "half-life extension domain" refers to a domain that extends the half-life of a target component in serum. The term "half-life extension domain" includes, for example, antibodies and antibody fragments.

[0256] The masked cytokines provided herein include a first half-life extension domain associated with a second half-life extension domain.

[0257] In some embodiments, the first half-life extension domain and the second half-life extension domain are associated non-covalently.

[0258] In some embodiments, the first half-life extension domain and the second half-life extension domain are covalently bonded.

[0259] In some embodiments, the first half-life extension domain is linked to the second half-life extension domain via one or more disulfide bonds.

[0260] In some embodiments, a first half-life extension domain is linked to a second half-life extension domain via a half-life extension domain linker (HLDL).

[0261] In some embodiments, the first half-life extension domain and the second half-life extension domain are associated non-covalently, and furthermore, the first half-life extension domain is linked to the second half-life extension domain via a disulfide bond.

[0262] In some embodiments, the first half-life extension domain comprises a first antibody or a fragment thereof, and the second half-life extension domain comprises a second antibody or a fragment thereof.

[0263] Antibodies or fragments thereof capable of FcRn-mediated recirculation may reduce or otherwise delay the clearance of masked cytokines from a subject, thereby extending the half-life of the administered masked cytokines. In some embodiments, the antibody or fragment thereof is any FcRn-mediated recirculation-capable antibody or fragment thereof, such as any heavy chain polypeptide or a portion thereof (e.g., an Fc domain or a fragment thereof) capable of FcRn-mediated recirculation.

[0264] The antibody or fragment thereof may be any antibody or fragment thereof. However, in some embodiments of the masked cytokine, which includes a first half-life extension domain and a second half-life extension domain, either the first or second half-life extension domain may contain an antibody or fragment thereof that does not bind to the FcRn receptor, such as a light chain polypeptide. For example, in some embodiments of the masked cytokine, the first half-life extension domain contains an antibody or fragment thereof that includes a light chain polypeptide or a portion thereof that does not directly interact with the FcRn receptor; nevertheless, the masking cytokine has an extended half-life by including a second half-life extension domain that is capable of interacting with the FcRn receptor, for example, by including a heavy chain polypeptide. It is recognized in the art that FcRn-mediated recirculation requires the binding of the FcRn receptor to the Fc region of the antibody or fragment thereof. For example, studies have shown that residues I253, S254, H435, and Y436 (numbered according to the Kabat EU index numbering system) are important for the interaction between the human Fc region and the human FcRn complex. See, for example, Firan, M., et al., Int.Immunol. 13 (2001) 993-1002 and Shields, RL, et al., J.Biol.Chem. 276 (2001) 6591-6604). Various variants of residues 248-259, 301-317, 376-382, and 424-437 (numbered according to the Kabat EU index numbering system) have also been investigated and reported. Yeung, YA, et al. (J.Immunol. 182 (2009) 7667-7671).

[0265] In some embodiments, the antibody or fragment thereof comprises either a heavy chain polypeptide or a light chain polypeptide. In some embodiments, the antibody or fragment thereof comprises a portion of either a heavy chain polypeptide or a light chain polypeptide. In some embodiments, the antibody or fragment thereof comprises an Fc domain or a fragment thereof. In some embodiments, the antibody or fragment thereof comprises CH2 and CH3 domains or fragments thereof. In some embodiments, the antibody or fragment thereof comprises a constant domain of a heavy chain polypeptide. In some embodiments, the antibody or fragment thereof comprises a constant domain of a light chain polypeptide. In some embodiments, the antibody or fragment thereof comprises a heavy chain polypeptide or a fragment thereof (e.g., an Fc domain or a fragment thereof). In some embodiments, the antibody or fragment thereof comprises a light chain polypeptide.

[0266] In some embodiments, the first half-life extension domain includes a first Fc domain or a fragment thereof, and the second half-life extension domain includes a second Fc domain or a fragment thereof.

[0267] In some embodiments, the first and / or second Fc domains each include one or more modifications that facilitate non-covalent association of the first and second half-life extension domains. In some embodiments, the first half-life extension domain includes an IgG1 Fc domain or fragment thereof containing mutations Y349C, T366S, L38A, and Y407V that form a “hole” in the first half-life extension domain, and the second half-life extension domain includes an IgG1 Fc domain or fragment thereof containing mutations S354C and T366W that form a “knob” in the second half-life extension domain.

[0268] In some embodiments, the first and second half-life extension domains are each an IgG1, IgG2, or IgG4 Fc domain or a fragment thereof. In some embodiments, the first and second half-life extension domains are each an IgG1 Fc domain or a fragment thereof. The human IgG1 immunoglobulin heavy chain constant gamma 1 has the following sequence. JPEG2026048631000046.jpg79170

[0269] In some embodiments, the first and second half-life extension domains are derived from the sequence of human IgG1 immunoglobulin heavy chain constant gamma 1 having Sequence ID No. 21 ("parent sequence") such that the first and second half-life extension domains each include Sequence ID No. 21 or a fragment thereof with one or more amino acid modifications.

[0270] In some embodiments, the first and second half-life extension domains each optionally include one or more amino acid modifications, comprising the portion of SEQ ID NO: 21 shown in bold above, i.e., as follows: DKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 22)

[0271] In some embodiments, the first and second half-life extension domains include sequence number 22 with an amino substitution to facilitate the association of the first and second half-life extension domains according to a "knob into holes" approach. In some embodiments, sequence number 22 includes mutations Y349C, T366S, L38A, and Y407V (numbered according to the Kabat EU numbering system) that form a "hole" in the first half-life extension domain, and mutations S354C and T366W (numbered according to the Kabat EU numbering system) that form a "knob" in the second half-life extension domain. These modified sequences have sequence numbers 23 and 24 shown below. First half-life extension domain (Y349C, T366S, L38A, and Y407V) Sequence ID 23: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQ VSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Second half-life extension domain (S354C and T366W) Sequence ID 24: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTK NQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0272] In some embodiments, the first and second half-life extension domains each further comprise amino-substituted N297A numbered according to the Kabat EU numbering system. First half-life extension domain (Y349C, T366S, L38A, Y407V, and N297A) Sequence ID 25: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQ VSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Second half-life extension domain (S354C, T366W, and N297A) Sequence ID No. 26: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTK NQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0273] In some embodiments, the first and second half-life extension domains each further comprise an amino-substituted I253A numbered according to the Kabat EU numbering system.

[0274] In some embodiments, the first and second half-life extension domains each further comprise both amino-substituted N297A and I253A, numbered according to the Kabat EU numbering system. First half-life extension domain (Y349C, T366S, L38A, Y407V, N297A, and I253A) Sequence ID 27: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKE YKCKVSNKALAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCA VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG Second half-life extension domain (S354C, T366W, N297A, and I253A) Sequence ID 28: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEY KCKVSNKALAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0275] In some embodiments, the first half-life extension domain includes an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of the amino acid sequences of SEQ ID NOs. 22, 23, 25, and 27.

[0276] In some embodiments, the second half-life extension domain includes an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of the amino acid sequences of sequence numbers 22, 24, 26, and 28.

[0277] In some embodiments, the first half-life extension domain includes an amino acid sequence having one or more modifications, such as one or more amino acid substitutions, additions, or deletions, compared to any one of the amino acid sequences of SEQ ID NOs: 22, 23, 25, and 27. In some embodiments, the second half-life extension domain includes an amino acid sequence having one or more modifications, such as one or more amino acid substitutions, additions, or deletions, compared to any one of the amino acid sequences of SEQ ID NOs: 22, 24, 26, and 28. The one or more modifications may be any modifications or modifications described herein, including any modifications or modifications that promote heterodimerization of the polypeptide chain and / or inhibit homodimerization of the polypeptide chain, or that alter or enhance the effector function.

[0278] In some embodiments, the Fc domain or fragment thereof includes one or more amino acid substitutions that modify the effector function. In some embodiments, the half-life extension domain is the IgG1 Fc domain or fragment thereof and includes one or more amino acid substitutions selected from the group consisting of N297A, N297G, N297Q, L234A, L235A, C220S, C226S, C229S, P238S, E233P, L234V, L234F, L235E, P331S, S267E, L328F, D265A, and P329G, numbered according to the Kabat EU numbering system. In some embodiments, the half-life extension domain is an IgG2 Fc domain or fragment thereof, and includes amino acid substitutions numbered according to the Kabat EU numbering system: V234A and G237A, H268Q, V309L, A330S, and A331S, and / or V234A, G237A, P238S, H268A, V309L, and A330S. In some embodiments, the half-life extension domain is an IgG2 Fc domain or fragment thereof, and includes one or more amino acid substitutions selected from the group consisting of V234A, G237A, H268Q, V309L, A330S, A331S, P238S, H268A, and V309L, and numbered according to the Kabat EU numbering system. In some embodiments, the half-life extension domain is an IgG4 Fc domain or fragment thereof, and includes amino acid substitutions numbered according to the Kabat EU numbering system: L235A, G237A, and E318A, S228P, L234A, and L235A, H268Q, V309L, A330S, and P331S, and / or S228P and L235A. In some embodiments, the half-life extension domain is an IgG2 Fc domain or fragment thereof, and includes one or more amino acid substitutions selected from the group consisting of L235A, G237A, E318A, S228P, L234A, H268Q, V309L, A330S, and P331S, numbered according to the Kabat EU numbering system.

[0279] In some embodiments, the half-life extension domain comprises an Fc domain or fragment thereof containing one or more amino acid substitutions that enhance effector function. In some embodiments, the half-life extension domain is an IgG1 Fc domain or fragment thereof, numbered according to the Kabat EU numbering system: S298A, E333A, and K334A; S239D and I332E; S239D, A330L, and I332E; P247I and A339D or A339Q; D280H and K290S; D280H, K290S, and any of S298D or S298V; F243L, R292P, and Y300L; F243L, R292P, Y300L, and P396L; F243L, R292P, Y300L, V305I, and P396L; G 236A, S239D, and I332E; K326A and E333A; K326W and E333S; K290E, S298G, and T299A; K290E, S298G, T299A, and K326E; K290N, S298G, and T299A; K290N, S298G, T299A, and K326E; K334V; L235S, S239D, and K334V; K334V and Q331M, S239D, F243V, E294L, or S298T; E233L, Q311M, and K33 4V; L234I, Q311M, and K334V; K334V and S298T, A330M or A330F; K334V, Q311M, and any of A330M or A330F; K334V, S298T, and any of A330M or A330F; K334V, S239D, and any of A330M or S298T; L234Y, Y296W, and K290Y, F243V or E294L; Y296W and any of L234Y or K290Y; S239D, A330S, and I3 32E, V264I;F243L and V264I;L328M;I332E;L328M and I332E;V264I and I332E;S239E and I332E;S239Q and I332D;S239E;A330Y;I332D;L328I and I332E;L328Q and I332E;V264T;V240I;V266I;S239D;S239D and I332D;S239D and I332N;S239D and I332Q;S239E and I332D;S239E and I332N;S239E and I332Q; S239N and I332D; S239N and I332E; S239Q and I332D; A330Y and I332E; V264I, A330Y and I332E; A330L and I332E; V264I, A330L and I332E; L234E, L234Y, or L234I; L235D, L235S, L235Y, or L235I; S239T; V240M; V264Y; A330I; N325T; I332E and L328D, L328V, L328T, or L328I; V264I, I332E, and any of S239E or S239Q; S239E, V264I, A330Y, and I332E; any of A330Y, I332E, S239D, or S239N; any of A330L, I332E, S239D, or S239N; V264I, S298A, and I332E; S298A, I332 E, S239D, or S239N; S239D, V264I, and I332E; S239D, V264I, S298A, and I332E; S239D, V264I, A330L, and I332E; S239D, I332E, and A330I; P230A, P230A, E233D, and I332E; E272Y; K274T, K274E, K274R, K274L, or K274Y; F275W; N276L ;Y278T;V302I;E318R;S324D, S324I or S324V;K326I or K326T;T335D, T335R or T335Y;V240I and V266I;S239D, A330Y, I332E, and L234I;S239D, A330Y, I332E, and L235D;S239D, A330Y, I332E, and V240I;S239D, A330Y, I332E, and V264T;and / or S239D, A330Y, I332E, and K326E or K326T. In some embodiments, the half-life extension domain is an IgG1 Fc domain or a fragment thereof, and includes P230A, E233D, L234E, L234Y, L234I, L235D, L235S, L235Y, L235I, S239D, S239E, S239N, S239Q, S239T, V240I, V240M, F243L, V264I, V264T, V264Y, V266I, E272Y, K274T, K274E, K274R, K274L, K274Y, F275W, This includes one or more amino acid substitutions selected from the group consisting of N276L, Y278T, V302I, E318R, S324D, S324I, S324V, N325T, K326I, K326T, L328M, L328I, L328Q, L328D, L328V, L328T, A330Y, A330L, A330I, I332D, I332E, I332N, I332Q, T335D, T335R, and T335Y.

[0280] In some embodiments, the half-life extension domain includes one or more amino acid substitutions that enhance the binding of the half-life extension domain to FcRn. In some embodiments, one or more amino acid substitutions increase the binding affinity of an Fc-containing polypeptide (e.g., a heavy chain polypeptide or an Fc domain or fragment thereof) to FcRn at an acidic pH. In some embodiments, the half-life extension domain includes one or more amino acid substitutions selected from the group consisting of M428F, T250Q and M428F; M252Y, S254T and T256E; P257I and N434H; D376V and N434H; P257I and Q3111; N434A; N434W; M428F and N434S; V259I and V308F; M252Y, S254T and T256E; V259I, V308F and M428F; T307Q and N434A; T307Q and N434S; T307Q, E380A and N434A; V308P and N434A; N434H; and V308P.

[0281] For manufacturing purposes, signal peptides may be manipulated upstream of the half-life domain to improve protein secretion. The signal peptide is selected according to the requirements of the cell line, as is known in the art. It should be understood that the signal peptide is not expressed as part of a protein that is purified and formulated as a pharmaceutical.

[0282] 1.4.1 Heterodimization Modification The half-life extension domains described herein may include one or more modifications that promote heterodimerization of two different half-life extension domains. In some embodiments, it is desirable to promote heterodimerization of the first and second half-life extension domains so that the production of masked cytokines in their correct heterodimeric form is efficiently produced. Therefore, one or more amino acid modifications can be made to the first half-life extension domain and one or more amino acid modifications can be made to the second half-life extension domain using any strategy available in the art, including any strategy described in Klein et al. (2012), MAbs, 4(6):653-663. Exemplary strategies and modifications are described in detail below.

[0283] Knob-into-hole approach One strategy for promoting heterodimerization of two different half-life extension domains is an approach called "knobs-into-holes."

[0284] In some embodiments, the masked cytokine comprises a first half-life extension domain and a second half-life extension domain, each containing a CH3 domain. In some embodiments, the half-life extension domain containing the CH3 domain is a heavy chain polypeptide or a fragment thereof (e.g., an Fc domain or a fragment thereof). The CH3 domains of the two half-life extension domains can be modified, for example, by the “knob-into-hole” technique, which is described in detail with several examples, for example, in WO 1996 / 027011, Ridgway, JB et al, Protein Eng. (1996) 9(7):617-621, Merchant, AM, et al, Nat. Biotechnol. (1998) 16(7):677-681. See also Klein et al. (2012), MAbs, 4(6):653-663. Using the knob-into-hole method, the interaction surface of two CH3 domains is modified, increasing the heterodimerization of two half-life extension domains containing the two modified CH3 domains. This occurs by introducing a bulky residue into one of the CH3 domains of the half-life extension domains, which acts as a "knob." A "hole" capable of accommodating the knob is then formed in the other half-life extension domain to accommodate the bulky residue. One of the modified CH3 domains can be the "knob," while the other can be the "hole." The introduction of disulfide bridges further stabilizes the heterodimer (Merchant, AM, et al, Nat. Biotechnol. (1998) 16(7), Atwell, S., et al, J. Mol. Biol. (1997) 270(1):26-35) and increases the yield.

[0285] It has been reported that heterodimerization yields exceeding 97% can be achieved by creating "knobs" by introducing the S354C and T366W mutations into the heavy chain, and by creating "holes" by introducing the Y349C, T366S, L368A, and Y407V mutations into the heavy chain (residue numbering by the Kabat EU numbering system). Carter et al. (2001), J.Immunol.Methods, 248:7-15, Klein et al. (2012), MAbs, 4(6):653-663.

[0286] In some embodiments, which include a first half-life extension domain and a second half-life extension domain, the first half-life extension domain comprises a heavy chain polypeptide or a portion thereof (e.g., an Fc domain or a fragment thereof) containing amino acid mutations S354C and T366W (numbered according to the Kabat EU numbering system), and the second half-life extension domain comprises a heavy chain polypeptide or a portion thereof (e.g., an Fc domain or a fragment thereof) containing amino acid mutations Y349C, T366S, L368A, and Y407V (numbered according to the Kabat EU numbering system). In some embodiments, which include a first half-life extension domain and a second half-life extension domain, the first half-life extension domain comprises a heavy chain polypeptide or a portion thereof (e.g., an Fc domain or a fragment thereof) containing amino acid mutations Y349C, T366S, L368A, and Y407V (numbered according to the Kabat EU numbering system), and the second half-life extension domain comprises a heavy chain polypeptide or a portion thereof (e.g., an Fc domain or a fragment thereof) containing amino acid mutations S354C and T366W (numbered according to the Kabat EU numbering system).

[0287] Additional examples of substitutions that can be fabricated to form knobs and holes include those described in US20140302037A1, the contents of which are incorporated herein by reference. For example, in some embodiments, any of the following amino acid substitutions can be made in a first half-life extension domain ("first domain") and a paired second half-life extension domain ("second domain"), each containing an Fc domain: (a) Y407T in the first domain and T366Y in the second domain, (b) Y407A in the first domain and T366W in the second domain, (c) F405A in the first domain and T394W in the second domain, (d) F405W in the first domain and T394S in the second domain, (e) Y407T in the first domain and T366Y in the second domain, (f) first (g) T366Y and F405A in the first domain, and T394W and Y407T in the second domain, (h) T366W and F405W in the first domain, and T394S and Y407A in the second domain, (h) F405W and Y407A in the first domain, and T366W and T394S in the second domain, or (i) T366W in the first domain, and T366S, L368A, and Y407V in the second domain.

[0288] In some embodiments, any of the following amino acid substitutions can be made in a first half-life extension domain ("first domain") and a paired second half-life extension domain ("second domain"), each containing an Fc domain: (a) Y407T in the second domain and T366Y in the first domain, (b) Y407A in the second domain and T366W in the first domain, (c) F405A in the second domain and T394W in the second domain, (d) F405W in the second domain and T394S in the first domain, (e) Y407T in the second domain and T366Y in the first domain, (f) (g) T366Y and F405A in domain 2, and T394W and Y407T in domain 1, (h) T366W and F405W in domain 2, and T394S and Y407A in domain 1, (h) F405W and Y407A in domain 2, and T366W and T394S in domain 1, or (i) T366W in domain 2, and T366S, L368A, and Y407V in domain 1.

[0289] In embodiments comprising a first half-life extension domain and a second half-life extension domain, each containing an Fc domain, any of the heterodimerization modifications described herein can be used in the Fc domain to promote heterodimerization of any of the masked cytokines described herein.

[0290] 1.5 Exemplary Masked Cytokines The masked cytokines according to this disclosure may be a combination of an IL-12 cytokine or a functional fragment thereof as described anywhere in this specification, a masking portion as described anywhere in this specification, first and second half-life domains as described anywhere in this specification, and cleavable and non-cleavable linkers as described anywhere in this specification.

[0291] Furthermore, in some embodiments, any particular sequence disclosed herein may optionally include further amino acid substitutions, such as one, two, or three substitutions. In other embodiments, sequences having at least 90%, preferably 95%, and more preferably 99% homology to any particular sequence disclosed herein with respect to a masked cytokine domain are also included by the present invention.

[0292] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 4, the masking portion comprises human IL-12Rβ1 or its fragment, part, or variant, which retains or otherwise demonstrates affinity for IL-12, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0293] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 4, the masking portion comprises the sequence, fragment, part, or variant thereof having SEQ ID NO: 5, i.e., residues 24-237 of human IL-12Rβ1, i.e., retaining or otherwise demonstrating affinity for IL-12, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0294] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 4, the masking portion comprises the sequence, fragment, part, or variant thereof having SEQ ID NO: 6, i.e., residues 24-545 of human IL-12Rβ1, i.e., retaining or otherwise demonstrating affinity for IL-12, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0295] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 4, the masking portion comprises human IL-12Rβ2 or its fragment, part, or variant, which retains or otherwise demonstrates affinity for IL-12, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0296] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 61, the masking portion comprises human IL-12Rβ2 or its fragment, part, or variant, which retains or otherwise demonstrates affinity for IL-12, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0297] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 62, the masking portion comprises human IL-12Rβ2 or its fragment, part, or variant, which retains or otherwise demonstrates affinity for IL-12, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0298] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 63, the masking portion comprises human IL-12Rβ2 or its fragment, part, or variant, which retains or otherwise demonstrates affinity for IL-12, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0299] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 64, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0300] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 64, the masking portion comprises human IL-12Rβ2 or its fragment, part, or variant, which retains or otherwise demonstrates affinity for IL-12, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0301] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 4, the masking portion comprises residues 24-212 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 7, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0302] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 4, the masking portion comprises residues 24-222 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 8, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0303] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 4, the masking portion comprises residues 24-319 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 9, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0304] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 4, the masking portion comprises residues 24-319 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 65, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0305] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 63, the masking portion comprises residues 24-319 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 65, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0306] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 64, the masking portion comprises residues 24-319 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 65, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0307] In some embodiments, the masking portion comprises residues 24-319 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 65, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0308] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 4, the masking portion comprises residues 24-622 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 10, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0309] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 4, the masking portion comprises residues 24-227 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 11, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0310] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 4, the masking portion comprises human IL-12Rβ1 or its fragment, part, or variant, which retains or otherwise demonstrates affinity for IL-12, the first half-life extension domain comprises SEQ ID NO: 27 (Y349C, T366S, L38A, Y407V, N297A, and I253A), and the second half-life extension domain comprises SEQ ID NO: 28 (S354C, T366W, N297A, and I253A).

[0311] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 4, the masking portion comprises the sequence, fragment, part, or variant thereof having SEQ ID NO: 5, i.e., residues 24-237 of human IL-12Rβ1, i.e., retaining or otherwise demonstrating affinity for IL-12, the first half-life extension domain comprises SEQ ID NO: 27 (Y349C, T366S, L38A, Y407V, N297A, and I253A), and the second half-life extension domain comprises SEQ ID NO: 28 (S354C, T366W, N297A, and I253A).

[0312] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 4, the masking portion comprises the sequence, fragment, part, or variant thereof having SEQ ID NO: 6, i.e., residues 24-545 of human IL-12Rβ1, i.e., retaining or otherwise demonstrating affinity for IL-12, the first half-life extension domain comprises SEQ ID NO: 27 (Y349C, T366S, L38A, Y407V, N297A, and I253A), and the second half-life extension domain comprises SEQ ID NO: 28 (S354C, T366W, N297A, and I253A).

[0313] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 4, the masking portion comprises human IL-12Rβ2 or its fragment, part, or variant, which retains or otherwise demonstrates affinity for IL-12, the first half-life extension domain comprises SEQ ID NO: 27 (Y349C, T366S, L38A, Y407V, N297A, and I253A), and the second half-life extension domain comprises SEQ ID NO: 28 (S354C, T366W, N297A, and I253A).

[0314] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 4, the masking portion comprises residues 24-212 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 7, the first half-life extension domain comprises SEQ ID NO: 27 (Y349C, T366S, L38A, Y407V, N297A, and I253A), and the second half-life extension domain comprises SEQ ID NO: 28 (S354C, T366W, N297A, and I253A).

[0315] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 4, the masking portion comprises residues 24-222 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 8, the first half-life extension domain comprises SEQ ID NO: 27 (Y349C, T366S, L38A, Y407V, N297A, and I253A), and the second half-life extension domain comprises SEQ ID NO: 28 (S354C, T366W, N297A, and I253A).

[0316] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 4, the masking portion comprises residues 24-319 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 9, the first half-life extension domain comprises SEQ ID NO: 27 (Y349C, T366S, L38A, Y407V, N297A, and I253A), and the second half-life extension domain comprises SEQ ID NO: 28 (S354C, T366W, N297A, and I253A).

[0317] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 4, the masking portion comprises residues 24-622 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 10, the first half-life extension domain comprises SEQ ID NO: 27 (Y349C, T366S, L38A, Y407V, N297A, and I253A), and the second half-life extension domain comprises SEQ ID NO: 28 (S354C, T366W, N297A, and I253A).

[0318] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 4, the masking portion comprises residues 24-227 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 11, the first half-life extension domain comprises SEQ ID NO: 27 (Y349C, T366S, L38A, Y407V, N297A, and I253A), and the second half-life extension domain comprises SEQ ID NO: 28 (S354C, T366W, N297A, and I253A).

[0319] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 64, and the masking portion comprises residues 24-319 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 65.

[0320] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 64, the masking portion comprises residues 24-319 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 65, the non-cleavable linker comprises the amino acid sequence of SEQ ID NO: 14, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0321] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 64, the masking portion comprises residues 24-319 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 65, the non-cleavable linker comprises the amino acid sequence of SEQ ID NO: 55, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0322] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 64, the masking portion comprises residues 24-319 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 65, the non-cleavable linker comprises the amino acid sequence of SEQ ID NO: 56, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0323] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 64, the masking portion comprises residues 24-319 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 65, the cleavable linker comprises the amino acid sequence of SEQ ID NO: 41, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0324] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 64, the masking portion comprises residues 24-319 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 65, the cleavable linker comprises the amino acid sequence of SEQ ID NO: 43, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0325] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 64, the masking portion comprises residues 24-319 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 65, the cleavable linker comprises the amino acid sequence of SEQ ID NO: 44, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0326] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 64, the masking portion comprises residues 24-319 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 65, the cleavable linker comprises the amino acid sequence of SEQ ID NO: 51, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0327] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 64, the masking portion comprises residues 24-319 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 65, the cleavable linker comprises the amino acid sequence of SEQ ID NO: 53, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0328] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 64, the masking portion comprises residues 24-319 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 65, the cleavable linker comprises the amino acid sequence of SEQ ID NO: 46, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0329] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 64, the masking portion comprises residues 24-319 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 65, the non-cleavable linker comprises the amino acid sequence of SEQ ID NO: 56, the cleavable linker comprises the amino acid sequence of SEQ ID NO: 51, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0330] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 64, the masking portion comprises residues 24-319 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 65, the non-cleavable linker comprises the amino acid sequence of SEQ ID NO: 14, the cleavable linker comprises the amino acid sequence of SEQ ID NO: 53, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0331] In some embodiments, the IL-12 cytokine or its functional fragment comprises the amino acid sequence of SEQ ID NO: 64, the masking portion comprises residues 24-319 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 65, the non-cleavable linker comprises the amino acid sequence of SEQ ID NO: 55, the cleavable linker comprises the amino acid sequence of SEQ ID NO: 46, the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

[0332] In some embodiments of the masked cytokine, the first polypeptide chain includes: JPEG2026048631000047.jpg7170 The second polypeptide chain includes the following: JPEG2026048631000048.jpg7170 "IL-12p40" is the IL-12p40 polypeptide or a functional fragment thereof, and "IL-12p35" is the IL-12p35 polypeptide or a functional fragment thereof. The first half-life elongation domain (HL1), the first linker (L1), the masking moiety (MM), the second half-life elongation domain (HL2), the second linker (L2), and the IL-12 cytokine or fragment thereof ([IL-12p35-linker-IL-12p40]) may be as defined anywhere herein.

[0333] In some embodiments, the masked cytokine comprises a first polypeptide chain containing the amino acid sequence of SEQ ID NO: 34 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 40.

[0334] In some embodiments, the masked cytokine comprises a first polypeptide chain containing the amino acid sequence of SEQ ID NO: 81 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 40.

[0335] In some embodiments, the masked cytokine comprises a first polypeptide chain containing the amino acid sequence of SEQ ID NO: 34 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 88.

[0336] In some embodiments, the masked cytokine comprises a first polypeptide chain containing the amino acid sequence of SEQ ID NO: 81 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 88.

[0337] In some embodiments, the masked cytokine comprises a first polypeptide chain containing the amino acid sequence of SEQ ID NO: 82 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 89.

[0338] In some embodiments, the masked cytokine comprises a first polypeptide chain containing the amino acid sequence of SEQ ID NO: 83 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 90.

[0339] In some embodiments, the masked cytokine comprises a first polypeptide chain containing the amino acid sequence of SEQ ID NO: 83 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 91.

[0340] In some embodiments, the masked cytokine comprises a first polypeptide chain containing the amino acid sequence of SEQ ID NO: 82 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 92.

[0341] In some embodiments, the masked cytokine comprises a first polypeptide chain containing the amino acid sequence of SEQ ID NO: 83 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 93.

[0342] In some embodiments, the masked cytokine comprises a first polypeptide chain containing the amino acid sequence of SEQ ID NO: 82 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 94.

[0343] In some embodiments, the masked cytokine comprises a first polypeptide chain containing the amino acid sequence of SEQ ID NO: 84 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 93.

[0344] In some embodiments, the masked cytokine comprises a first polypeptide chain containing the amino acid sequence of SEQ ID NO: 84 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 94.

[0345] In some embodiments, the masked cytokine comprises a first polypeptide chain containing the amino acid sequence of SEQ ID NO: 83 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 95.

[0346] In some embodiments, the masked cytokine comprises a first polypeptide chain containing the amino acid sequence of SEQ ID NO: 82 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 96.

[0347] In some embodiments, the masked cytokine comprises a first polypeptide chain containing the amino acid sequence of SEQ ID NO: 83 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 97.

[0348] In some embodiments, the masked cytokine comprises a first polypeptide chain containing the amino acid sequence of SEQ ID NO: 82 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 98.

[0349] In some embodiments, the masked cytokine comprises a first polypeptide chain containing the amino acid sequence of SEQ ID NO: 84 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 97.

[0350] In some embodiments, the masked cytokine comprises a first polypeptide chain containing the amino acid sequence of SEQ ID NO: 84 and a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 98.

[0351] 2. Cutting products The products provided herein are the cleavage products of the “dimeric” masked IL-12 cytokine as described herein.

[0352] The masked IL-12 cytokine described herein includes a cleavable linker. Upon proteolytic cleavage of the cleavable linker at the cleavage site, a cleavage product is formed containing the IL-12 cytokine or its functional fragment. The IL-12 cytokine or its functional fragment in the cleavage product is activated because it is no longer masked by the masking moiety. Therefore, the IL-12 cytokine or its functional fragment in the cleavage product is capable of binding to a target protein.

[0353] The tumor cell environment is complex and can contain multiple different proteases. Therefore, the exact site where a given cleavable peptide within a masked IL-12 cytokine is cleaved within the tumor cell environment can vary between tumor types, between patients with the same tumor type, and even between cleavage products formed in the same tumor. Furthermore, even after cleavage, further modification of the initial cleavage product, for example by the removal of one or two terminal amino acids, can occur due to further action of proteases within the tumor cell environment. Thus, the distribution of cleavage products can be expected to be formed within the patient's tumor cell environment after administration of the masked cytokine described herein.

[0354] Provided herein are cleavage products capable of binding to IL-12R, comprising an IL-12 cytokine or a functional fragment thereof, which can be prepared by proteolytic cleavage of a cleavable peptide in a masked IL-12 cytokine as described elsewhere herein.

[0355] Furthermore, provided herein are cleavage products of a masked IL-12 cytokine, the cleavage products being capable of binding to IL-12R, and the cleavage products comprising an IL-2 cytokine or a functional fragment thereof as defined anywhere herein. Also provided herein is a distribution of cleavage products obtained or obtainable from a single structure of a masked IL-12 cytokine, each cleavage product in the distribution of cleavage products comprising (i) being capable of binding to IL-12R, and (ii) an IL-12 cytokine or a functional fragment thereof as defined anywhere herein.

[0356] Provided herein are cleavage products of a masked IL-12 cytokine, the cleavage products being capable of binding to IL-12R, and the cleavage products are JPEG2026048631000049.jpg7170PCP is part of a peptide that can be proteolytically cleaved, SD is the spacer domain, and C is the IL-12 cytokine or its functional fragment.

[0357] Furthermore, provided herein are cleavage products of a masked IL-12 cytokine, the cleavage products being capable of binding to IL-12R, and the cleavage products are a) A first polypeptide chain containing a first half-life extension domain, b) A second polypeptide chain comprising a polypeptide containing formula 5, The protein heterodimer, including JPEG2026048631000050.jpg14170, comprises a protein heterodimer, where HL2 is a second half-life extension domain, L2 is a non-cleavable linker, C is an IL-12 cytokine or a functional fragment thereof, and the first half-life extension domain associates with the second half-life extension domain. Also provided herein is a distribution of cleavage products obtained or obtainable from a single structure of a masked IL-12 cytokine, where each cleavage product in the distribution of cleavage products is (i) capable of binding to IL-12R, and (ii) a) A first polypeptide chain containing a first half-life extension domain, b) A second polypeptide chain comprising a polypeptide containing formula 5, The protein heterodimer, including JPEG2026048631000051.jpg13170, contains a protein heterodimer where HL2 is a second half-life extension domain, L2 is a non-cleavable linker, C is an IL-12 cytokine or a functional fragment thereof, and the first half-life extension domain is associated with the second half-life extension domain.

[0358] Furthermore, provided herein are cleavage products of a masked IL-12 cytokine, the cleavage products being capable of binding to IL-12R, and the cleavage products are a) A first polypeptide chain, JPEG2026048631000052.jpg7170HL1 is the first half-life extension domain, SD is the spacer domain, and PCP is part of a peptide that can be proteolytically cleaved, forming the first polypeptide chain. b) A second polypeptide chain, JPEG2026048631000053.jpg6170 HL2 is a second half-life extension domain, L2 is a linker, and C is a second polypeptide chain which is an IL-12 cytokine or a functional fragment thereof, comprising a protein heterodimer. The first half-life extension domain is associated with the second half-life extension domain.

[0359] Within the cleavage product, the masking moiety, half-life extension domain, IL-12 cytokine or its functional fragment, linker, spacer domain, and the type of association between the first half-life extension domain and the second half-life extension domain may be any one of those described herein, or any combination thereof.

[0360] The location of the cleavable peptide determines the structure of the resulting cleavage product, which includes the IL-12 cytokine.

[0361] "A portion of the proteolytically cleavable peptide" refers to the portion of the original proteolytically cleavable peptide sequence after cleavage has occurred at the cleavage site. Further modifications of the initial cleavage product, such as the removal of one or two terminal amino acids, may also occur after cleavage due to further action of proteases in the tumor cell environment. Therefore, cleavage products in the distribution of cleavage products that may form in the patient's tumor cell environment after administration of masked cytokines may not contain any portion of the proteolytically cleavable peptide.

[0362] In some embodiments, “part” refers to one, two, three, four, five, or six amino acids of the original proteolytically cleavable peptide sequence. In some embodiments, “part” refers to two amino acids of the original proteolytically cleavable peptide sequence. In some embodiments, “part” refers to three amino acids of the original proteolytically cleavable peptide sequence. In some embodiments, “part” refers to four amino acids of the original proteolytically cleavable peptide sequence.

[0363] In some embodiments, "part" of the protein-cleavable peptide is 3 to 6 amino acids in length. In some embodiments, "part" of the protein-cleavable peptide is 3 or 4 amino acids in length.

[0364] The cutting portions for the severable linker disclosed herein are disclosed below. [Table 3]

[0365] As a purely illustrative example, in the table above, * indicates known or observed protease cleavage sites within cleavable peptides.

[0366] Therefore, what is disclosed herein is a cleavage product of any one of the masked cytokines disclosed herein.

[0367] In some embodiments, the cleavage product comprises an amino acid sequence having approximately or at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 29.

[0368] In some embodiments, the cleavage product includes an amino acid sequence having the amino acid sequence containing SEQ ID NO: 29.

[0369] In some embodiments, the cleavage product comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 66.

[0370] In some embodiments, the cleavage product includes an amino acid sequence having the amino acid sequence containing SEQ ID NO: 66.

[0371] In some embodiments, the cleavage product comprises an amino acid sequence having approximately or at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 67.

[0372] In some embodiments, the cleavage product includes an amino acid sequence having the amino acid sequence containing SEQ ID NO: 67.

[0373] In some embodiments, the cleavage product comprises an amino acid sequence having approximately or at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 68.

[0374] In some embodiments, the cleavage product includes an amino acid sequence having the amino acid sequence containing SEQ ID NO: 68.

[0375] In some embodiments, the cleavage product comprises an amino acid sequence having approximately or at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 69.

[0376] In some embodiments, the cleavage product includes an amino acid sequence having the amino acid sequence containing SEQ ID NO: 69.

[0377] In some embodiments, the cleavage product comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 70.

[0378] In some embodiments, the cleavage product includes an amino acid sequence having the amino acid sequence containing SEQ ID NO: 70.

[0379] In some embodiments, the cleavage product comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 71.

[0380] In some embodiments, the cleavage product includes an amino acid sequence having the amino acid sequence containing SEQ ID NO: 71.

[0381] In some embodiments, the cleavage product comprises an amino acid sequence having approximately or at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 72.

[0382] In some embodiments, the cleavage product includes an amino acid sequence having the amino acid sequence containing SEQ ID NO: 72.

[0383] In some embodiments, the cleavage product comprises an amino acid sequence having approximately or at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 73.

[0384] In some embodiments, the cleavage product includes an amino acid sequence having the amino acid sequence containing SEQ ID NO: 73.

[0385] In some embodiments, the cleavage product comprises an amino acid sequence having approximately or at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 74.

[0386] In some embodiments, the cleavage product includes an amino acid sequence having the amino acid sequence containing SEQ ID NO: 74.

[0387] In some embodiments, the cleavage product comprises an amino acid sequence having approximately or at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 75.

[0388] In some embodiments, the cleavage product includes an amino acid sequence having the amino acid sequence containing SEQ ID NO: 75.

[0389] In some embodiments, the cleavage product contains an amino acid sequence having approximately or at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 76. It contains an amino acid sequence having an amino acid sequence including SEQ ID NO: 76.

[0390] In some embodiments, the cleavage product includes an amino acid sequence having the amino acid sequence containing SEQ ID NO: 76.

[0391] In some embodiments, the cleavage product comprises an amino acid sequence having approximately or at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 77.

[0392] In some embodiments, the cleavage product includes an amino acid sequence having the amino acid sequence containing SEQ ID NO: 77.

[0393] In some embodiments, the cleavage product contains an amino acid sequence having approximately or at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 78.

[0394] In some embodiments, the cleavage product includes an amino acid sequence having the amino acid sequence containing SEQ ID NO: 78.

[0395] In some embodiments, the cleavage product contains an amino acid sequence having approximately or at least approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 79.

[0396] In some embodiments, the cleavage product includes an amino acid sequence having the amino acid sequence containing SEQ ID NO: 79.

[0397] In some embodiments, the cleavage product contains an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 80.

[0398] In some embodiments, the cleavage product includes an amino acid sequence having the amino acid sequence containing SEQ ID NO: 80.

[0399] 3. Binding assay The strength or affinity of immunological binding interactions, such as those specific to a cytokine or its functional fragment, or between a cytokine or its functional fragment and a binding partner (e.g., a target protein such as a cytokine receptor), can be expressed in terms of the interaction's dissociation constant (Kd), where a smaller Kd indicates greater affinity. The binding of the IL-12 cytokine to the IL-12 cytokine receptor can be expressed in terms of Kd. In some embodiments, the immunological binding interaction is between a masked cytokine (in the presence or absence of a protease) and a target protein such as a cytokine receptor. The immunological binding properties of a protein can be quantified using methods well known in the art. For example, one method involves measuring the rates of cytokine receptor (e.g., IL-12R) / cytokine (e.g., IL-12) complex formation and dissociation, where these rates depend on the concentration of the complex partner, the affinity of the interaction, and geometric parameters that equally affect the rates in both directions. Both the "on-rate constant" (Kon) and the "off-rate constant" (Koff) can be determined by calculating the concentrations and the actual rates of association and dissociation. The Koff / Kon ratio allows for the removal of all parameters unrelated to affinity and is equal to the dissociation constant Kd. See Davies et al., Annual Rev Biochem. 59:439-473, (1990).

[0400] In some embodiments, the masked cytokines described herein bind to target proteins with nearly the same or higher affinity upon protease cleavage compared to parent cytokines that contain a masking moiety but do not contain a cleavable peptide. The target protein may be any cytokine receptor.

[0401] In some embodiments, the masked cytokines provided herein, which do not contain cleavable peptides in the linker, have a dissociation constant (Kd) with the target protein of ≤1M, ≤150nM, 100nM, ≤50nM, ≤10nM, ≤1nM, ≤0.1nM, ≤0.01nM, or ≤0.001nM (e.g., ≤10⁻⁸M, e.g., 10⁻⁸M to 10⁻¹³M, e.g., 10⁻⁹M to 10⁻¹³M). In some embodiments, the masked cytokines provided herein, which include a cleavable peptide in the linker, have a dissociation constant (Kd) of ≤1M, ≤150nM, ≤100nM, ≤50nM, ≤10nM, ≤1nM, ≤0.1nM, ≤0.01nM, or ≤0.001nM (e.g., ≤10⁻⁸M, e.g., 10⁻⁸M to 10⁻¹³M, e.g., 10⁻⁹M to 10⁻¹³M) with respect to a pre-target protein cleavable by a protease. In some embodiments, the masked cytokines provided herein, which include a cleavable peptide in the linker, have a dissociation constant (Kd) with respect to the target protein at protease cleavage of ≤1M, ≤150nM, 100nM, ≤50nM, ≤10nM, ≤1nM, ≤0.1nM, ≤0.01nM, or ≤0.001nM (e.g., ≤10⁻⁸M, e.g., 10⁻⁸M to 10⁻¹³M, e.g., 10⁻⁹M to 10⁻¹³M). In some embodiments, the cytokine or functional fragment thereof of the masked cytokine provided herein has a masking portion of the masked cytokine and a dissociation constant (Kd) of ≥500M, ≥250M, ≥200M, ≥150M, ≥100M, ≥50M, ≥10M, ≥1M, ≥500nM, ≥250nM, ≥150nM, ≥100nM, ≥50nM, ≥10nM, ≥1nM, ≥0.1nM, ≥0.01nM, or ≥0.001nM.In some embodiments, the cytokine or functional fragment thereof of the masked cytokine provided herein has a dissociation constant (Kd) of approximately 200 M to approximately 50 nM, such as approximately or at least approximately 175 M, approximately or at least approximately 150 M, approximately or at least approximately 125 M, approximately or at least approximately 100 M, approximately or at least approximately 75 M, approximately or at least approximately 50 M, approximately or at least approximately 25 M, approximately or at least approximately 5 M, approximately or at least approximately 1 M, approximately or at least approximately 750 nM, approximately or at least approximately 500 nM, approximately or at least approximately 250 nM, approximately or at least approximately 150 nM, approximately or at least approximately 100 nM, approximately or at least approximately 75 nM, or approximately or at least approximately 50 nM. Assays for evaluating binding affinity are well known in the art.

[0402] In some embodiments, masked cytokines exhibiting a desired occlusion ratio are provided. As used herein, the term “occlusion ratio” refers to the ratio of (a) the maximum detection level of a parameter under a first set of conditions to (b) the minimum detection level of that parameter under a second set of conditions. In the context of masked IL-12 polypeptides, it refers to the ratio of (a) the maximum detection level of a target protein (e.g., IL-12R protein) that binds to the masked IL-12 polypeptide in the presence of at least one protease capable of cleaving the cleavable peptide of the masked IL-12 polypeptide to (b) the minimum detection level of a target protein (e.g., IL-12R protein) that binds to the masked IL-12 polypeptide in the absence of the protease. Thus, the occlusion ratio of a masked cytokine can be calculated by dividing the EC50 of the masked cytokine before cleavage by the EC50 of the masked cytokine after cleavage. The occlusion ratio of a masked cytokine can also be calculated as the ratio of the dissociation constant of the masked cytokine before cleavage by a protease to the dissociation constant of the masked cytokine after cleavage by a protease. In some embodiments, a higher occlusion ratio of a masked cytokine indicates that the target protein bound by the masked cytokine is produced to a greater extent (e.g., mostly) in the presence of a protease capable of cleaving the cleavable peptide of the masked cytokine than in the absence of the protease.

[0403] In some embodiments, masked cytokines with optimal occlusion ratios are provided herein. In some embodiments, the optimal occlusion ratio of the masked cytokine indicates that the masked cytokine has desirable properties useful for the methods or compositions intended herein. In some embodiments, the masked cytokines provided herein exhibit an optimal occlusion ratio of about 2 to about 10,000, for example, about 80 to about 100. In any further embodiment of the masked cytokines provided herein, the occlusion ratios are about 2 to about 7,500, about 2 to about 5,000, about 2 to about 2,500, about 2 to about 2,000, about 2 to about 1,000, about 2 to about 900, about 2 to about 800, about 2 to about 700, about 2 to about 600, about 2 to about 500, about 2 to about 400, about 2 to about 300, The ratios are approximately 2 to 200, 2 to 100, 2 to 50, 2 to 25, 2 to 15, 2 to 10, 5 to 10, 5 to 15, 5 to 20, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, or 100 to 1,000. In some embodiments, the masked cytokines provided herein exhibit an optimal occlusion ratio of approximately 2 to 1,000. The binding of the masked IL-12 polypeptide to the target protein before and / or after cleavage by protease can be determined using techniques well known in the art, such as by ELISA.

[0404] In some embodiments, the masking portion described herein binds to the cytokine or functional fragment described herein with an affinity lower than the affinity between the cytokine or functional fragment thereof and the target protein (e.g., cytokine receptor). In certain embodiments, the masking portion provided herein is It binds to cytokines or their functional fragments described herein with a dissociation constant (Kd) of ≥500M, ≥250M, ≥200M, ≥150M, ≥100M, ≥50M, ≥10M, ≥1M, ≥500nM, ≥250nM, ≥150nM, ≥100nM, ≥50nM, ≥10nM, ≥1nM, ≥0.1nM, ≥0.01nM, or ≥0.001nM.

[0405] 4. Masked production of IL-12 cytokines The masked cytokines described herein are prepared using techniques available in the art, the exemplary methods described herein.

[0406] 4.1 Antibody Production Some embodiments of the masked IL-12 cytokine include an antibody or a fragment thereof. The following sections provide further details on the production of antibodies, as well as their antibody fragments, variants, and derivatives, which may be used in some embodiments of the masked IL-12 cytokine provided herein. In some embodiments, the masked cytokine is in the form of a dimer produced by two copies of the masked IL-12 cytokine associated through a disulfide bond.

[0407] 1. Antibody fragment In some embodiments, the present invention encompasses antibody fragments. The antibody fragments may be any antibody fragment, including, among several fragments, an Fc domain, a portion of the heavy chain, a portion of the light chain, Fab, Fv, or scFv. The antibody fragments may be produced by conventional means such as enzymatic digestion, or by recombinant technology. In certain circumstances, there is an advantage to ligating the antibody fragment to a masked antibody fragment as described herein, rather than to the whole antibody. For an overview of certain antibody fragments, see Hudson et al. (2003) Nat. Med. 9:129-134.

[0408] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992), and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv, and ScFv antibody fragments are all expressed and secreted in E. coli and other cell types such as HEK293 and CHO cells, thus enabling the easy production of large quantities of these fragments. Alternatively, Fab-SH fragments can be recovered directly from culture medium and chemically bound to form F(ab)2 fragments (see, Carter et al., Bio / Technology 10:163-167 (1992)). According to another approach, the F(ab)2 fragment can be isolated directly from recombinant host cell cultures. Fab and F(ab)2 fragments with increased in vivo half-life, containing an FcRN / salvage receptor-binding epitope residue, are described in U.S. Patent No. 5,869,046. Other techniques for producing antibody fragments for use with masked cytokines will be apparent to those skilled in the art. In certain embodiments, the masked antibody comprises a single-stranded Fv fragment (scFv). See WO93 / 16185, U.S. Patents No. 5,571,894 and 5,587,458. scFv fusion proteins can be constructed to result in the fusion of an effector protein at either the amino or carboxyl terminus of the scFv. See Antibody Engineering, ed. Borrebaeck, above. Also, in some embodiments, bi-scFv, containing two scFvs linked via a polypeptide linker, can be used with masked cytokines.

[0409] In some embodiments, the present invention includes a linear antibody (e.g., as described in U.S. Patent No. 5,641,870) or a single-chain immunoglobulin comprising the heavy and light chain sequences of an antibody linked via a suitable linker. Such a linear antibody or immunoglobulin may be monospecific or bispecific. Such a single-chain immunoglobulin can be dimerized to maintain a structure and activity similar to that of an antibody that is originally a tetramer. In some embodiments, the antibody or fragment thereof may be an antibody having a single heavy chain variable region and no light chain sequence. Such an antibody is called a single-domain antibody (sdAb) or nanobody. These antibodies are also encompassed in the sense of functional fragments of antibodies according to the present invention. Antibody fragments can be linked to masked cytokines as described herein, in accordance with the guidance provided herein.

[0410] 2. Humanized antibodies In some embodiments, the present invention encompasses humanized antibodies or antibody fragments thereof. In some embodiments, the humanized antibody may be any antibody, comprising any antibody fragment. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "imported" residues, typically incorporated from an "imported" variable domain. Humanization can essentially be carried out by substituting the sequence of the hypervariable region into the corresponding sequence of a human antibody, according to Winter's method (Jones et al. (1986) Nature 321:522-525, Riechmann et al. (1988) Nature 332:323-327, Verhoeyen et al. (1988) Science 239:1534-1536). Therefore, such “humanized” antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), in which the substantially intact human variable domain is replaced by a corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which several hypervariable region residues and possibly several FR residues are replaced by residues from similar sites in rodent antibodies. Humanized antibodies can be ligated to masked cytokines as described herein, in accordance with the guidance provided herein.

[0411] 3. Human antibodies Human antibodies of some embodiments of the present invention can be constructed by combining an Fv clone variable domain sequence selected from a human-derived phage display library with a known human constant domain sequence. Alternatively, human monoclonal antibodies of some embodiments of the present invention can be produced by hybridoma, for example, by using mouse, rat, bovine (e.g., dairy cow), or rabbit cells to produce human monoclonal antibodies. In some embodiments, human antibodies and human monoclonal antibodies can be antibodies that bind to any antigen. In some embodiments, human monoclonal antibodies of the present invention can be produced by immunizing a non-human animal containing a human immunoglobulin locus with a target antigen and isolating the antibody from the immunized animal or cells derived from the immunized animal. Suitable examples of non-human animals include transgenic or transchromosomal animals such as HuMAb Mouse® (Medarex, Inc.), KM Mouse®, "TC Mouse", and Xenomouse®. For example, see Lonberg, et al. (1994) Nature 368:856-859, Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851, WO2002 / 43478, U.S. Patent Nos. 5,939,598, 6,075,181, 6,114,598, 6,150,584, 6,162,963, and Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727.

[0412] Human myeloma and mouse-human heterozygous myeloma cell lines for the production of human monoclonal antibodies have been described, for example, by Kozbor J. Immunol., 133:3001 (1984), Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987), and Boerner et al., J. Immunol., 147:86 (1991). Human antibodies can be ligated to masked cytokines as described herein, in accordance with the guidance provided herein.

[0413] 4. Bispecific antibodies A bispecific antibody is a monoclonal antibody that has binding specificity to at least two different antigens. In certain embodiments, the bispecific antibody is a human or humanized antibody. In some embodiments, one of the binding specificities is to a first antigen and the other is to a second antigen, which may be either two different epitopes on the same target protein or two different epitopes on two different target proteins. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing a first antigen and / or a second antigen. Bispecific antibodies can also be used to recruit cells such as T cells or natural killer cells to kill specific cells, e.g., cancer cells. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibody). Bispecific antibodies can be ligated to masked cytokines as described herein, in accordance with the guidance provided herein.

[0414] Methods for producing bispecific antibodies are known in the art. See Milstein and Cuello, Nature, 305:537 (1983), WO93 / 08829 published May 13, 1993, Traunecker et al., EMBO J., 10:3655 (1991), and Kontermann and Brinkmann, Drug Discovery Today, 20(7):838-847. For further details on the production of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986). Bispecific antibodies include crosslinked or "heterocomplex" antibodies. For example, one antibody in a heterocomplex may be bound to avidin and the other to biotin. Heterocomplex antibodies can be produced using any convenient crosslinking method. Suitable crosslinking agents are well known in the art and are disclosed in U.S. Patent No. 4,676,980, along with several crosslinking techniques.

[0415] 5. Single-domain antibodies In some embodiments, a single-domain antibody is ligated to a masked cytokine in accordance with the guidance provided herein. A single-domain antibody can be any antibody. A single-domain antibody is a single polypeptide chain comprising all or part of the heavy-chain variable domains or all or part of the light-chain variable domains of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (see Domantis, Inc., Waltham, Mass., e.g., U.S. 6,248,516B1). In some embodiments, the single-domain antibody consists of all or part of the heavy-chain variable domains of an antibody. In some embodiments, the single-domain antibody is a camelid-derived antibody obtained by immunization of a camelid with a target antigen. In some embodiments, the single-domain antibody is a shark-derived antibody obtained by immunization of a shark with a target antigen. In some embodiments, the single-domain antibody is a nanobody (see e.g., WO2004041865A2 and US20070269422A1).

[0416] 6. Antibody Variants In some embodiments, amino acid sequence modifications of antibodies or fragments thereof as described herein are intended. For example, it may be desirable to improve the FcRn binding affinity and / or pH-dependent FcRn binding affinity of the antibody. It may also be desirable to promote heterodimerization of the antibody heavy chain by introducing specific amino acid modifications. Methods for promoting heterodimerization of antibody chains, including specific modifications that may be performed to promote heterodimerization, are described in Klein et al. (2012), MAbs, 4(6):653-663. Amino acid sequence variants of antibodies can be prepared by introducing appropriate changes to the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions of residues in the amino acid sequence of the antibody, and / or substitutions thereof. Any combination of deletions, insertions, and substitutions can be performed to arrive at the final construct, provided that the final construct possesses the desired characteristics. Amino acid modifications can be introduced into the amino acid sequence of the target antibody when the sequence is constructed.

[0417] A useful method for identifying specific residues or regions of an antibody that are preferred sites for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. Here, a group of residues or target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to affect the interaction between the amino acid and the antigen. These amino acid positions that exhibit functional sensitivity to the substitution are then purified by introducing further or other variants at or for the substitution site. Thus, the site for introducing amino acid sequence mutations is predetermined, but the nature of the mutation itself does not need to be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is performed at a target codon or region, and the expressed immunoglobulin is screened for the desired activity.

[0418] Amino acid sequence insertions include amino-terminus fusions and / or carboxyl-terminus fusions ranging in length from one residue to 100 or more residues in a polypeptide, as well as intersequential insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusion of an antibody to an enzyme or polypeptide at the N-terminus or C-terminus, which extends the serum half-life of the antibody.

[0419] In some embodiments, masked cytokines are modified to eliminate, reduce, or otherwise interfere with protease cleavage near the hinge region. The “hinge region” of IgG is generally defined as containing E216 and terminating at P230 of human IgGl according to the EU index such as Kabat, but functionally, the flexible portion of the chain may be considered to contain additional residues referred to as upper and lower hinge regions, such as E216–G237 (Roux et al., 1998 J Immunol 161:4083), with the lower hinge being referred to as residues 233–239 of the Fc region, where FcyR binding is generally attributed. Modifications to any of the masked cytokines described herein can be carried out, for example, by following the method described in US 20150139984A1, incorporated herein by reference, and by incorporating any of the modifications described herein.

[0420] In some embodiments, FcRn mutations that improve pharmacokinetics include, but are not limited to, M428L, T250Q / M428L, M252Y / S254T / T256E, P257I / N434H, D376V / N434H, P257I / Q3111, N434A, N434W, M428L / N434S, V259I / V308F, M252Y / S254T / T256E, V259I / V308F / M428L, T307Q / N434A, T307Q / N434S, T307Q / E380A / N434A, V308P / N434A, N434H, and V308P. In some embodiments, such mutations enhance antibody binding to FcRn at low pH but do not alter antibody affinity at neutral pH.

[0421] In certain embodiments, an antibody or fragment thereof is modified to increase or decrease the degree to which the antibody is glycosylated. Polypeptide glycosylation is typically either N-linked or O-linked. N-linked glycosylation refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. Asparagine-X-serine and asparagine-X-threonine, tripeptide sequences in which X is any amino acid other than proline, are recognition sequences for the enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Therefore, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of a sugar, i.e., one of N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0422] The addition or deletion of glycosylation sites to masked cytokines is conveniently achieved by modifying the amino acid sequence such that one or more of the tripeptide sequences described above (for N-linked glycosylation sites) are created or removed. Modification may also be carried out by adding, deleting, or substituting one or more serine or threonine residues into the original antibody sequence (for O-linked glycosylation sites).

[0423] If an antibody or fragment thereof contains an Fc region, the carbohydrate attached thereto may be modified. For example, an antibody having a mature carbohydrate structure lacking fucose attached to the Fc region of the antibody is described in U.S. Patent Application No. US2003 / 0157108 (Presta, L.). See also U.S.2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibodies having bisected N-acetylglucosamine (GlcNAc) in the carbohydrate attached to the Fc region of the antibody are referenced in WO 2003 / 011878, Jean-Mairet et al., and U.S. Patent No. 6,602,684, Umana et al. An antibody having at least one galactose residue in the oligosaccharide attached to the Fc region of the antibody is reported in WO 1997 / 30087, Patel et al. See also WO1998 / 58964 (Raju, S.) and WO1999 / 22764 (Raju, S.) concerning antibodies with modified carbohydrates attached to the Fc region. See also US2005 / 0123546 (Umana et al.) concerning antigen-binding molecules with modified glycosylation.

[0424] In certain embodiments, the glycosylated variant comprises an Fc region, and the carbohydrate structure attached to the Fc region lacks or has reduced fucose. Such variants have improved ADCC function. Optionally, the Fc region further comprises one or more amino acid substitutions that further improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region. Examples of publications related to "defucosylated" or "fucose-deficient" antibodies include US2003 / 0157108, WO2000 / 61739, WO2001 / 29246, US2003 / 0115614, US2002 / 0164328, US2004 / 0093621, US2004 / 0132140, US2004 / 0110704, US2004 / 0110282, US2004 / 0109865, WO2003 / 085119, WO2003 / 084570, WO2005 / 035586, WO2005 / 035778, WO2005 / 053742, Okazaki et al. References include al. J. Mol. Biol. 336:1239-1249 (2004) and Yamane-Ohnuki et al. Biotech. Bioeng. 87.614 (2004). Examples of cell lines that produce defucosylated antibodies include Lee 13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986), U.S. Patent Application No. US2003 / 0157108A1, Presta, L, and WO2004 / 056312A1, Adams et al., particularly Example 11), and knockout cell lines such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004)), as well as cells overexpressing (31,4-N-acetylglucosaminyltransferase III (GnT-III) and Golgi p-mannosidase II (ManII).

[0425] In any of the embodiments herein, masked cytokines may be manipulated to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In some embodiments, masked cytokines may be produced in cell lines having alpha-1,6-fucosyltransferase (Fut8) knockout. In some embodiments, host cells are modified to have reduced endogenous alpha-1,6-fucosylation activity. Examples of methods for modifying the fucosylation pathway in mammalian host cells can be found, for example, in Yamane-Ohnuki and Satoh, MAbs, 1(3):230-236 (2009), the contents of which are incorporated herein by reference. Examples of methods and compositions for partially or completely inactivating the expression of the FUT8 gene are described, for example, in U.S. Publication No. 20160194665A1, WO2006133148A2, the contents of which are incorporated herein by reference. In some embodiments, masked cytokines are produced in Lecl3 variants of CHO cells (see, e.g., Shields et al., J. Biol. Chem., 277(30):26733-40 (2002)) or in YB2 / 0 cell lines with reduced FUT8 activity (see, e.g., Shinkawa et al., J. Biol. Chem., 278(5):3466-73 (2003)). In some embodiments, small interfering RNAs (siRNAs) can be introduced to genes related to alpha-1,6-fucosylation (see, for example, Mori et al., Biotechnol. Bioeng. 88(7):901-908 (2004), Imai-Nishiya et al., BMC Biotechnol. 7:84 (2007), and Omasa et al., J. Biosci. Bioeng., 106(2):168-173 (2008)). In some further embodiments, the masked cytokines can be produced in cell lines overexpressing |31,4-N-acetylglucosaminyltransferase III (GnT-III). In further embodiments, the cell lines also overexpress Golgi p-mannosidase II (ManII).In some embodiments of this specification, the masked cytokine may include at least one amino acid substitution in the Fc region that improves ADCC activity.

[0426] In some embodiments, masked cytokines are modified to improve their serum half-life. To increase the serum half-life of an antibody, an FcRN / salvage receptor-binding epitope may be incorporated into a linked antibody (particularly an antibody fragment), for example, as described in U.S. Patent No. 5,739,277. As used herein, the term “salvage receptor-binding epitope” refers to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is a factor in increasing the in vivo serum half-life of the IgG molecule (US2003 / 0190311, U.S. Patents 6,821,505, 6,165,745, 5,624,821, 5,648,260, 6,165,745, and 5,834,597).

[0427] Another type of variant is the amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule that is replaced by a different residue. While hypervariable regions are often the target sites for substitutional mutagenesis, modifications of the FR (Frequency-Range) are also attempted. Conservative substitutions are shown in Table 3 under the heading "Preferred Substitutions." If such substitutions result in desirable changes to biological activity, larger substitutional changes may be introduced, indicated in Table 3 as "Exemplary Substitutions," or further described below with respect to the class of amino acids, and the product may be screened. [Table 4]

[0428] Substitutional modifications in the biological properties of antibodies are achieved by selecting substitutions that have a significantly different effect on (a) the structure of the polypeptide backbone in the substitutional region, for example, as a sheet or helical structure, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the maintenance of the side chain volume. Amino acids can be grouped according to the similarity of their side chain properties (ALLehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) Non-polar: Ala(A), Val(V), Leu(L), Ile(I), Pro(P), Phe(F), Trp(W), Met(M) (2) Non-charged electrodes: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q) (3) Acidic: Asp(D), Glu(E) (4) Basic: Lys(K), Arg(R), His(H)

[0429] Alternatively, naturally occurring residues can be grouped based on the following common side-chain characteristics: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, he, (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gin, (3) Acidic: Asp, Glu, (4) Basicity: His, Lys, Arg, (5) Residues that affect the directionality of the chain: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe.

[0430] Non-conservative substitutions involve replacing one member of one of these classes with one from another. Such substituted residues can also be introduced into conserved substitution sites or the remaining (non-conservative) sites.

[0431] Another type of substitution variant involves the substitution of a naturally occurring amino acid residue with a naturally occurring amino acid residue. Naturally occurring amino acid residues can be incorporated, for example, through tRNA recoding, or through any of the methods described in WO2016154675A1, which is incorporated herein by reference.

[0432] One type of substitution variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or human antibody). Generally, variants resulting from selection for further development have modified (e.g., improved) biological properties compared to the parent antibody from which they are produced. A convenient method for generating such substitution variants involves affinity maturation using phage display, yeast display, or mammalian display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated, generating all possible amino acid substitutions at each site. The antibodies thus produced are displayed from filamentous phage particles as fusions to at least a portion of the phage coat protein (e.g., the gene III product of M13) packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity). To identify candidate hypervariable region sites for modification, scanning mutagenesis (e.g., alanine scanning) can be performed to identify hypervariable region residues that significantly contribute to antigen binding. Alternatively, or in addition, it may be beneficial to analyze the crystalline structure of the antigen-antibody complex to identify contact sites between the antibody and the antigen. Such contact residues and adjacent residues are candidates for substitution by techniques known in the art, including those detailed herein. Once such variants are generated, a panel of variants may be screened using techniques known in the art, including those described herein, and antibodies exhibiting superior properties in one or more relevant assays may be selected for further development.

[0433] Nucleic acid molecules encoding amino acid sequence variants of masked cytokines are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants), or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of previously prepared variant or non-variant versions of antibodies.

[0434] It may be desirable to introduce one or more amino acid modifications into the Fc region of the antibody of the present invention to generate an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) that includes amino acid modifications (e.g., substitutions) at one or more amino acid positions including the amino acid position of hinge cysteine.

[0435] In some embodiments, the masked cytokine provided herein comprises an antibody or fragment thereof having an IgG1, IgG2, IgG3, or IgG4 isotype with enhanced effector function. In some embodiments, the masked cytokine provided herein comprises an antibody or fragment thereof having an IgG1 isotype with enhanced effector function. In some embodiments, the masked cytokine provided herein has an IgG1 isotype with enhanced effector function. In some embodiments, the masked cytokine is defucosylated. In some embodiments, the masked cytokine has an increased level of mannose moiety. In some embodiments, the masked cytokine has an increased level of bisected glycan moiety. In some embodiments, IgG1 comprises amino acid mutations.

[0436] In some embodiments, the masked cytokines provided herein include an antibody having an IgG1 isotype (e.g., human IgG1 isotype). In some embodiments, IgG1 includes one or more amino acid substitutions that enhance effector function. In one embodiment, IgG1 includes the amino acid substitutions S298A, E333A, and K334A, where the amino acid residues are numbered according to an EU index such as Kabat. In one embodiment, IgG1 includes the amino acid substitutions S239D and I332E, where the amino acid residues are numbered according to an EU index such as Kabat. In one embodiment, IgG1 includes the amino acid substitutions S239D, A330L, and I332E, where the amino acid residues are numbered according to an EU index such as Kabat. In one embodiment, IgG1 includes the amino acid substitutions P247I and A339D or A339Q, where the amino acid residues are numbered according to an EU index such as Kabat. In one embodiment, IgG1 includes K290S or S298V with or without the amino acid substitutions D280H and S298D, and the amino acid residues are numbered according to an EU index such as Kabat. In one embodiment, IgG1 includes amino acid substitutions F243L, R292P, and Y300L, and the amino acid residues are numbered according to an EU index such as Kabat. In one embodiment, IgG1 includes amino acid substitutions F243L, R292P, Y300L, and P396L, and the amino acid residues are numbered according to an EU index such as Kabat. In one embodiment, IgG1 includes amino acid substitutions F243L, R292P, Y300L, V305I, and P396L, and the amino acid residues are numbered according to an EU index such as Kabat. In one embodiment, IgG1 includes amino acid substitutions G236A, S239D, and I332E, and the amino acid residues are numbered according to an EU index such as Kabat. In one embodiment, IgG1 includes amino acid substitutions K326A and E333A, where the amino acid residues are numbered according to an EU index such as Kabat.In one embodiment, IgG1 includes amino acid substitutions K326W and E333S, and the amino acid residues are numbered according to an EU index such as Kabat. In one embodiment, IgG1 includes amino acid substitutions K290E, S298G, and T299A, with or without K326E, and the amino acid residues are numbered according to an EU index such as Kabat. In one embodiment, IgG1 includes amino acid substitutions K290N, S298G, and T299A, with or without K326E, and the amino acid residues are numbered according to an EU index such as Kabat. In one embodiment, IgG1 includes amino acid substitution K334V, and the amino acid residues are numbered according to an EU index such as Kabat. In one embodiment, IgG1 includes amino acid substitutions L235S, S239D, and K334V, and the amino acid residues are numbered according to an EU index such as Kabat. In one embodiment, IgG1 comprises amino acid substitutions K334V and Q331M, S239D, F243V, E294L, or S298T, and the amino acid residues are numbered according to an EU index such as Kabat. In one embodiment, IgG1 comprises amino acid substitutions E233L, Q311M, and K334V, and the amino acid residues are numbered according to an EU index such as Kabat. In one embodiment, IgG1 comprises amino acid substitutions L234I, Q311M, and K334V, and the amino acid residues are numbered according to an EU index such as Kabat. In one embodiment, IgG1 comprises amino acid substitutions K334V and S298T, A330M, or A330F, and the amino acid residues are numbered according to an EU index such as Kabat. In one embodiment, IgG1 comprises amino acid substitutions K334V, Q311M, and either A330M or A330F, and the amino acid residues are numbered according to an EU index such as Kabat. In one embodiment, IgG1 comprises the amino acid substitutions K334V, S298T, and either A330M or A330F, where the amino acid residues are numbered according to an EU index such as Kabat.In one embodiment, IgG1 comprises the amino acid substitutions K334V, S239D, and either A330M or S298T, where the amino acid residues are numbered according to an EU index such as Kabat. In one embodiment, IgG1 comprises the amino acid substitutions L234Y, Y296W, and either K290Y, F243V, or E294L, where the amino acid residues are numbered according to an EU index such as Kabat. In one embodiment, IgG1 comprises the amino acid substitutions Y296W and either L234Y or K290Y, where the amino acid residues are numbered according to an EU index such as Kabat. In one embodiment, IgG1 comprises the amino acid substitutions S239D, A330S, and I332E, where the amino acid residues are numbered according to an EU index such as Kabat.

[0437] In some embodiments, IgG1 includes one or more amino acid substitutions that reduce or inhibit effector function. In one embodiment, IgG1 includes the amino acid substitutions N297A, N297G, or N297Q, where the amino acid residues are numbered according to an EU index such as Kabat. In one embodiment, IgG1 includes the amino acid substitutions L234A or L235A, where the amino acid residues are numbered according to an EU index such as Kabat. In one embodiment, IgG1 includes the amino acid substitutions C220S, C226S, C229S, and P238S, where the amino acid residues are numbered according to an EU index such as Kabat. In one embodiment, IgG1 includes the amino acid substitutions C226S, C229S, E233P, L234V, and L235A, where the amino acid residues are numbered according to an EU index such as Kabat. In one embodiment, IgG1 comprises amino acid substitutions L234F, L235E, and P331S, where the amino acid residues are numbered according to an EU index such as Kabat. In another embodiment, IgG1 comprises amino acid substitutions S267E and L328F, where the amino acid residues are numbered according to an EU index such as Kabat.

[0438] According to this description and the teachings in the art, in some embodiments, an antibody or fragment thereof of a masked cytokine may contain one or more modifications, for example, within the Fc region, compared to the wild-type corresponding antibody. For example, certain modifications may be made to the Fc region that would result in modified (i.e., improved or reduced) C1q binding and / or complement-dependent cell-mediated cytotoxicity (CDC), as described in WO99 / 51642. See also Duncan & Winter Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO94 / 29351 for other examples of Fc region variants. WO00 / 42072 (Presta) and WO2004 / 056312 (Lowman) describe antibody variants with improved or reduced binding to FcR. The contents of these patent publications are incorporated herein by reference. See also Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001). Antibodies having an increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is a factor in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) are described in US2005 / 0014934A1 (Hinton et al.). These antibodies contain an Fc region having one or more substitutions that improves the binding of the Fc region to FcRn. Polypeptide variants having a modified Fc region amino acid sequence and exhibiting increased or decreased C1q binding ability are described in U.S. Patent No. 6,194,551B1, WO99 / 51642. The contents of these patent publications are specifically incorporated herein by reference. See also Idusogie et al. J Immunol. 164:4178-4184 (2000).

[0439] 4.2 Masked IL-12 cytokine-drug conjugates The present invention also provides masked IL-12 cytokine-drug conjugates (MCDCs) comprising masked IL-12 cytokines provided herein, which may be any IL-12 masked cytokines disclosed herein, conjugated with one or more agents. In some embodiments, one or more agents include chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioisotopes. In some embodiments, one or more agents are immunostimulants.

[0440] In some embodiments, one or more drugs conjugated to a masked IL-12 cytokine may include meitansinoids (see U.S. Patents No. 5,208,020, 5,416,064, and European Patent EP0425235B1), auristatins such as monomethyl auristatin drug portions DE and DF (MMAE and MMAF) (see U.S. Patents No. 5,635,483, 5,780,588, and 7,498,298), drastatin, calitiamycin or its derivatives (see U.S. Patents No. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296, Hinman et al., Cancer See Res.53:3336-3342 (1993) and Lode et ak, Cancer Res.58:2925-2928 (1998), anthracyclines such as daunomycin or doxorubicin (see Kratz et ak, Current Med.Chem.13:477-523 (2006), Jeffrey et ak, Bioorganic & Med.Chem.Letters 16:358-362 (2006), Torgov et ak, Bioconj.Chem.16:717-721 (2005), Nagy et ak, Proc.Natl.Acad.Sci.USA 97:829-834 (2000), Dubowchik et ak, Bioorg.& Med.Chem.Letters This includes, but is not limited to, taxanes such as methotrexate, vindesine, docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel, as well as CC1065 (see 12:1529-1532 (2002), King et al., J. Med. Chem. 45:4336-4343 (2002), and U.S. Patent No. 6,630,579), methotrexate, vindesine, docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel, trichothecenes, and CC1065.

[0441] In another embodiment, one or more drugs conjugated to a masked IL-12 cytokine include, but are not limited to, inhibitors of tubulin polymerization (e.g., meitansinoids and auristatins), DNA damaging agents (e.g., pyrrolobenzodiazepine (PBD) dimers, calitiamycin, duocalmycin, and indo-linobenzodiazepine dimers), and DNA synthesis inhibitors (e.g., exatecan derivatives Dxd).

[0442] In another embodiment, the masked IL-12 cytokine-drug conjugate includes, but is not limited to, the masked IL-12 cytokines described herein, conjugated to an enzymatically active toxin or a fragment thereof, including, diphtheria A chain, an unbound active fragment of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modexin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, geronin, mitogenin, restrictocin, phenomycin, enomycin, and trichothecene.

[0443] In another embodiment, the masked IL-12 cytokine-drug conjugate comprises the masked IL-12 cytokine described herein, conjugated to a radioactive atom, to form a radiocomplex. Various radioisotopes are available for the production of the radiocomplex. Examples include the radioisotopes At211, 1131, 1125, Y90, Rel86, Rel88, Sml53, B1212, P32, Pb212, and Lu. When the radiocomplex is used for detection, it may include, for example, a radioactive atom for scintigraphy studies such as tc99m or 1123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as MRI) such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0444] In some embodiments, the masked IL-12 cytokine-drug conjugate comprises the masked IL-12 cytokine described herein, conjugated to one or more immunostimulants. In some embodiments, the immunostimulant is an interferon gene (STING) agonist or a Toll-like receptor (TER) agonist stimulant.

[0445] The STING agonist can be any agonist of STING. In some embodiments, the STING agonist is a cyclic dinucleotide (CDN). The CDN can be any CDN or a derivative or variant thereof. In some embodiments, the STING agonist is a CDN selected from the group consisting of cGAMP, c-di-AMP, c-di-GMP, cAIMP, and c-di-IMP. In some embodiments, the STING agonist is a derivative or variant of a CDN selected from the group consisting of cGAMP, c-di-AMP, c-di-GMP, cAIMP, and c-di-IMP. In some embodiments, the STING agonist is 4-(2-chloro-6-fluorobenzyl)-N-(furan-2-ylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiadin-6-carboxamide, or a derivative or variant thereof. For example, see Sali et al. (2015) PloS Pathog., 11(12):e!005324.

[0446] A TLR agonist can be an agonist of any TLR, such as TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or TLR10. In some embodiments, the TLR agonist is an agonist of a TLR expressed on the cell surface, such as TLR1, TLR2, TLR4, or TLR5. In some embodiments, the TLR agonist is an agonist of an intracellularly expressed TLR, such as TLR3, TLR7, TLR8, TLR9, or TLR10.

[0447] Masked IL-12 cytokine and cytotoxic drug conjugates can be prepared using a variety of bifunctional protein binders, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), difunctional derivatives of imide esters (e.g., dimethyladipimidine HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, lysine immunotoxins can be prepared as described in Vitetta et ah, Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanate benzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the complexation of radionucleotides to antibodies. See W094 / 11026. Linkers may be "cleavable linkers" that facilitate the release of cytotoxic drugs in cells. For example, acid-unstable linkers, peptidase-sensitive linkers, photo-unstable linkers, dimethyl linkers, or disulfide-containing linkers (Chari et ah, Cancer Res. 52:127-131 (1992), U.S. Patent No. 5,208,020) may be used.

[0448] In this specification, MCDC refers to BMPS, EMCS, GMBS, HBVS, LC-SMCC, The express intent is to prepare such complexes using crosslinking reagents, including, but not limited to, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as SVSB (e.g., commercially available succinimidyl-(4-vinylsulfonebenzoic acid) from Pierce Biotechnology, Inc., Rockford, IL, USA).

[0449] 4.3 Vectors, host cells, and recombination methods For the recombinant production of IL-12 masked cytokines of the present invention, one or more nucleic acids encoding them are used for further cloning (DNA amplification). Alternatively, for expression, it is isolated and inserted into a replicable vector. The DNA encoding the masked IL-12 cytokine, including its components, is readily isolated and sequenced using conventional procedures. Many vectors are available. The choice of vector depends in part on the host cell used. Generally, the host cell is either prokaryotic or eukaryotic (generally mammalian) in origin. It should be understood that, when applicable, the constant regions of any isotype of an antibody or fragment thereof, including IgG, IgM, IgA, IgD, and IgE constant regions, may be used for this purpose, and such constant regions may be obtained from any human or animal species. In some embodiments, one vector is used to encode the masked IL-12 cytokine. In some embodiments, more than one vector is used to encode the masked IL-12 cytokine.

[0450] 1. Generation of masked IL-12 cytokines using prokaryotic host cells a. Vector construction The polynucleotide sequences encoding the polypeptide components of the masked cytokines of the present invention can be obtained using standard recombinant techniques. Desired polynucleotide sequences of antibodies or antibody fragments can be isolated and sequenced from antibody-producing cells, such as hybridoma cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesizers or PGR techniques, or obtained from other sources. Once obtained, the sequences encoding the components of the masked cytokines are inserted into recombinant vectors capable of replicating and expressing heterologous polynucleotides in a prokaryotic host. Many vectors available and known in the art can be used for the purposes of the present invention. The selection of a suitable vector will primarily depend on the size of the nucleic acid to be inserted into the vector and the specific host cell to be transformed with the vector. Each vector contains a variety of components depending on its function (amplification or expression of heterologous polynucleotides, or both) and its compatibility with the specific host cell in which it resides. Vector components generally include, but are not limited to, origins of replication, selection marker genes, promoters, ribosome-binding sites (RBS), signal sequences, heterologous nucleic acid insertions, and transcriptional terminator sequences.

[0451] Generally, plasmid vectors containing replicons and regulatory sequences derived from a species compatible with the host cell are used in relation to these hosts. The vectors typically carry replication sites and marking sequences that can provide phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from the E. coli species. pBR322 contains genetically coded ampicillin (Amp) and tetracycline (Tet) resistance, thus providing a convenient means for identifying transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophages may also contain, or be modified to contain, promoters that can be used by microorganisms for the expression of endogenous proteins. An example of a pBR322 derivative used for the expression of a specific antibody is described by Carter et al., U.S. Patent No. 5,648,237.

[0452] In addition, phage vectors containing replicons and regulatory sequences compatible with host microorganisms can be used as transformation vectors in relation to these hosts. For example, bacteriophages such as 7GEM.TM.-11 can be used to create recombinant vectors that can be used to transform susceptible host cells such as E. coli LE392.

[0453] The expression vector of the present invention may comprise two or more promoter-cistron pairs, each encoding a polypeptide component. The promoter is a non-translating regulatory sequence located upstream (5') of the cistron that regulates its expression. Prokaryotic promoters are typically classified into two classes: inducible and homeostatic. Inducible promoters are those that initiate an increase in the level of cistron transcription under their control in response to changes in culture conditions, such as the presence or absence of nutrients or changes in temperature.

[0454] Numerous promoters recognized by various potential host cells are well known. A selected promoter can be operably ligated to cistron DNA encoding any of the masked cytokine strands by removing the promoter from the source DNA via restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the present invention. Both native promoter sequences and many heterologous promoters can be used to direct the amplification and / or expression of target genes.

[0455] In some embodiments, heterologous promoters are used because they generally allow for higher transcription levels and higher yields of the expressed target gene compared to natural target polypeptide promoters.

[0456] Suitable promoters for use with prokaryotic hosts include the PhoA promoter, the 3-galactamase and lactose promoter system, the tryptophan (trp) promoter system, and hybrid promoters such as the tac or trc promoter. However, other promoters that function in bacteria (such as other known bacterial or phage promoters) are equally suitable. Their nucleotide sequences are publicly available, thereby enabling those skilled in the art to ligate them operationally to cistrons encoding target light and heavy chains for masked cytokines, for example, including light and heavy chains, using linkers or adapters to supply any necessary restriction sites (Siebenlist et al. (1980) Cell 20:269).

[0457] In one aspect of the present invention, each cistron in a recombinant vector contains a secretory signal sequence component that orients the translocation of the expressed polypeptide across the membrane. Generally, the signal sequence may be a component of the vector or a part of the target polypeptide DNA inserted into the vector. The signal sequence selected for the purposes of the present invention should be recognized and processed by the host cell (i.e., cleaved by a signal peptidase). For prokaryotic host cells that do not recognize and process the native signal sequence for heterologous polypeptides, the signal sequence is replaced, for example, by an alkaline phosphatase, penicillinase, Ipp, or a prokaryotic signal sequence selected from the group consisting of a heat-stable enterotoxin II (STII) reader, LamB, PhoE, PelB, OmpA, and MBP. In one embodiment of the present invention, the signal sequences used in both cistrons of the expression system are STII signal sequences or variants thereof.

[0458] In another embodiment, the production of polypeptide components according to the present invention may occur within the cytoplasm of the host cell and therefore does not require the presence of secretory signaling sequences within each cistron. In this regard, in embodiments including immunoglobulin light and heavy chains, for example, the light and heavy chains are expressed with or without masking moieties and linker sequences, and are, for example, folded and assembled to form functional immunoglobulins within the cytoplasm. Certain host strains (e.g., E. coli trxB strain) provide favorable cytoplasmic conditions for disulfide bond formation, thereby enabling proper folding and assembly of the expressed protein subunits. See Proba and Pluckthun Gene, 159:203 (1995).

[0459] The masked cytokines of the present invention can also be produced by using an expression system in which the quantitative ratio of the expressed polypeptide components can be adjusted to maximize the secretion and yield of the appropriately assembled antibodies of the present invention. Such adjustment is achieved at least partially by simultaneously adjusting the translational intensity of the polypeptide components.

[0460] Suitable prokaryotic host cells for expressing the masked cytokines of the present invention include, for example, archaea and bacteria such as Gram-negative or Gram-positive organisms. Examples of useful bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, Gram-negative cells are used. In one embodiment, E. coli cells are used as the host of the present invention. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol.2 (Washington, DC: American Society for Microbiology, 1987), pp.1190-1219; ATCC Deposit No.27,325) and its derivatives, including strain 33D3 having genotype W3110 AfhuA(AtonA)ptr3 lac Iq lacL8 AompTA(nmpc-fepE)degP41 kanR (U.S. Patent No. 5,639,635). Other strains and their derivatives such as E. coli 294 (ATCC 31,446), E. coli B, E. coli 1776 (ATCC 31,537), and E. coli RV308 (ATCC 31,608) are also suitable. These examples are illustrative and not limiting. Methods for constructing derivatives of any of the aforementioned bacteria having defined genotypes are known in the art and are described, for example, in Bass et al., Proteins, 8:309-314 (1990). It is generally necessary to select appropriate bacteria, taking into account the replication potential of the bacterial replicons within the cell.For example, when supplying replicons using well-known plasmids such as pBR322, pBR325, pACYC177, or pKN410, E. coli, Serratia, or Salmonella species can be suitably used as hosts. Typically, the host cells should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors may, if desired, be incorporated into the cell culture.

[0461] b. Masked cytokine production Host cells are transformed with the expression vectors described above and cultured in conventionally modified nutrient media to induce promoters, select transformants, or amplify genes encoding desired sequences.

[0462] Transformation refers to the introduction of DNA into a prokaryotic host so that the DNA can replicate either as an extrachromosomal element or through chromosomal integrators. Depending on the host cell used, transformation is carried out using standard techniques appropriate for such cells. Calcium treatment using calcium chloride is commonly used for bacterial cells that contain a substantial cell wall barrier. Another method for transformation involves the use of polyethylene glycol / DMSO. Yet another technique used is electroporation.

[0463] The prokaryotic cells used to produce the masked cytokines of the present invention are grown in culture media known in the art and are suitable for culturing selected host cells. Examples of suitable media include Luria broth (LB) with necessary nutritional supplements. In some embodiments, the medium also contains a selectant selected based on the construction of the expression vector, selectively allowing the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the medium for the growth of cells expressing an ampicillin resistance gene.

[0464] Any necessary auxiliary agents other than carbon, nitrogen, and inorganic phosphate sources may also be included in appropriate concentrations, either alone or in mixtures with other auxiliary agents or composite nitrogen sources in the culture medium. Optionally, the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolates, dithioerythritol, and dithiothreitol.

[0465] Prokaryotic host cells are cultured at a suitable temperature. In certain embodiments, for E. coli growth, the growth temperature is in the range of about 20°C to about 39°C, about 25°C to about 37°C, or about 30°C. The pH of the culture medium can be any pH in the range of about 5 to about 9, mainly depending on the host organism. In certain embodiments, for E. coli, the pH is about 6.8 to about 7.4, or about 7.0.

[0466] When an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for promoter activation. In one aspect of the present invention, the PhoA promoter is used to control polypeptide transcription. Thus, transformed host cells are cultured in phosphate-restricted medium for induction. In certain embodiments, the phosphate-restricted medium is CRAP medium (see, for example, Simmons et al., J. Immunol. Methods (2002), 263:133-147). As is known in the art, various other inducers may be used depending on the vector construct used.

[0467] In one embodiment, the expressed masked cytokines of the present invention are secreted into the periplasm of host cells and recovered therefrom. Protein recovery typically involves disrupting the microorganisms by means such as osmotic shock, sonication, or lysis. Once the cells are disrupted, cell fragments or whole cells can be removed by centrifugation or filtration. The proteins can be further purified, for example, by affinity resin chromatography. Alternatively, the proteins can be transported to a culture medium and isolated therein. The cells can be removed from the culture, and the culture supernatant is filtered and concentrated for further purification of the produced proteins. The expressed polypeptides can be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assays.

[0468] In one aspect of the present invention, masked cytokine production is carried out on a large scale by a fermentation process. Various large-scale fed-batch fermentation procedures are available for the production of recombinant proteins. Large-scale fermentation has a capacity of at least 1,000 liters, and in certain embodiments, a capacity of about 1,000 to 100,000 liters. These fermenters use agitator impellers to distribute oxygen and nutrients, especially glucose. Small-scale fermentation generally refers to fermentation in a fermenter with a volume of about 100 liters or less, and which can range from about 1 liter to about 100 liters.

[0469] In the fermentation process, the induction of protein expression is typically initiated after the cells have been grown under favorable conditions to a desired density, e.g., OD550 of approximately 180–220, at which point the cells are in an early quiescent phase. Various inducers are known in the art and can be used according to the vector construct used, as described above. Cells may be grown for a shorter period before induction. Cells are usually induced for about 12–50 hours, but longer or shorter induction times may be used.

[0470] Various fermentation conditions can be modified to improve the production yield and quality of the polypeptides of the present invention. For example, to improve the proper assembly and folding of secreted antibody polypeptides, additional vectors overexpressing chaperone proteins such as Dsb proteins (DsbA, DsbB, DsbC, DsbD, and / or DsbG) or FkpA (peptidyl prolyl cis,trans-isomerase with caperone activity) can be used to co-transform host prokaryotic cells. Chaperone proteins have been demonstrated to promote the proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. (1999) J.Biol.Chem.274:19601-19605, Georgiou et ak, US Patent No. 6,083,715, Georgiou et ak, US Patent No. 6,027,888, Bothmann and Pluckthun (2000) J.Biol.Chem.275:17100-17105, Ramm and Pluckthun (2000) J.Biol.Chem.275:17106-17113, Arie et ak (2001) Mol.Microbiol.39:199-210.

[0471] To minimize the proteolysis of expressed heterologous proteins (particularly proteins that are proteolytically sensitive), certain host strains lacking proteolytic enzymes can be used in the present invention. For example, host cell lines can be modified to affect genetic variations in genes encoding known bacterial proteases such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI, and combinations thereof. Several E. coli protease-deficient strains are available and are described, for example, in Joly et ak (1998), Georgeo et ak, U.S. Patent No. 5,264,365, Georgeo et ak, U.S. Patent No. 5,508,192, and Kara et ak, Microbial Drug Resistance, 2:63-72 (1996).

[0472] In one embodiment, an E. coli strain transformed with a plasmid lacking a proteolytic enzyme and overexpressing one or more chaperone proteins is used as a host cell in the expression system of the present invention.

[0473] c. Purification of masked cytokines In some embodiments, the masked cytokines produced herein are further purified to obtain a substantially homogeneous preparation for further assays and use. Standard protein purification methods known in the art can be used. The following procedure is an example of a preferred purification procedure: fractionation on an immunoaffinity column or ion exchange column, ethanol precipitation, reversed-phase HPLC, chromatography on silica or a cation exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using Sephadex G-75, for example.

[0474] In some embodiments, protein A immobilized on a solid phase is used for immunoaffinity purification of masked cytokines according to the present invention. Protein A is a 41kD cell wall protein derived from Staphylococcus aureas that binds with high affinity to the Fc region of antibodies. Lindmark et al (1983) J.Immunol.Meth.62:1-13. The solid phase on which protein A is immobilized may be a column containing a glass or silica surface, or a controlled-pore glass column or a silicate column. In some applications, the column may be coated with a reagent such as glycerol to prevent nonspecific adhesion of contaminants.

[0475] As the first step of purification, the preparation derived from the cell culture described above is applied to a protein A-immobilized solid phase to enable the specific binding of the antibody of interest to protein A. The solid phase is then washed to remove contaminants that have been nonspecifically bound to the solid phase. Finally, the masked cytokine of interest is recovered from the solid phase by elution.

[0476] Other purification methods that provide high affinity binding to masked cytokine components can be used according to standard protein purification methods known in the art.

[0477] 2. Generation of masked cytokines using eukaryotic host cells Vectors for use in eukaryotic host cells generally contain one or more of the following non-limiting components: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0478] a. Signal sequence components Vectors for use in eukaryotic host cells may also contain a signal sequence or another polypeptide having a specific cleavage site at the N-terminus of a mature protein or polypeptide of interest. The selected heterologous signal sequence may be one that is recognized and processed by the host cell (i.e., cleaved by a signal peptidase). For mammalian cell expression, mammalian signal sequences and viral secretion leaders, such as the herpes simplex gD signal, are available.

[0479] Such precursor DNA regions are ligated to DNA encoding cytokines that are masked within the reading frame.

[0480] b. Origin of replication Generally, origin of replication components are not required in mammalian expression vectors. For example, the SV40 origin may typically only be used to include an early promoter.

[0481] c. Selected gene components Expression vectors and cloning vectors may contain selectable genes, also known as selectable markers. Typical selectable genes encode (a) proteins that confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline; (b) proteins that, where relevant, complement nutritional requirement deficiencies; or (c) proteins that supply essential nutrients not available from the complex medium.

[0482] One example of a selection scheme involves using drugs to halt the proliferation of host cells. These cells, successfully transformed with heterologous genes, produce proteins that confer drug resistance and thus survive the selection regimen. Examples of such dominant selection use drugs such as neomycin, mycophenolate, and hygromycin.

[0483] Another example of a suitable selectable marker for mammalian cells is one that allows for the identification of cells capable of taking up masked cytokines encoding nucleic acids, such as DHFR, thymidine kinase, metallothionein-I and -II, primate metallothionein genes, adenosine deaminase, and ornithine decarboxylase.

[0484] For example, in some embodiments, cells transformed with the DHFR selection gene are identified by first culturing all transformants in a culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR. In some embodiments, suitable host cells when wild-type DHFR is used are Chinese hamster ovary (CHO) cell lines lacking DHFR activity (e.g., ATCC CRL-9096).

[0485] Alternatively, host cells transformed or co-transformed with DNA sequences encoding masked cytokines, wild-type DHFR protein, and other selectable markers such as aminoglycoside 3'-phosphotransferase (APH) (in particular, wild-type hosts containing endogenous DHFR) can be selected by cell growth in a medium containing aminoglycoside antibiotics, e.g., kanamycin, neomycin, and selective agents for selectable markers such as G418. See U.S. Patent No. 4,965,199. Host cells may include NS0s, including cell lines lacking glutamine synthase (GS). Methods for the use of GS as a selectable marker for mammalian cells are described in U.S. Patent Nos. 5,122,464 and 5,891,693.

[0486] d. Promoter components Expression vectors and cloning vectors typically contain a promoter that is operably ligated to a nucleic acid encoding a masked cytokine of interest, which may be any masked cytokine described herein and is recognized by the host organism. Promoter sequences are known for eukaryotes. For example, almost all eukaryotic genes have an AT-rich region located approximately 25–30 bases upstream from the transcription initiation site. Another sequence found 70–80 bases upstream from the transcription initiation of many genes is the CNCAAT region, where N can be any nucleotide. At the 3' end of most eukaryotic genes is the AATAAA sequence, which may be a signal for the addition of a polyA tail to the 3' end of the coding sequence. In certain embodiments, any or all of these sequences may be suitably inserted into a eukaryotic expression vector.

[0487] Transcription from vectors in mammalian host cells is controlled by heterologous mammalian promoters, such as actin promoters or immunoglobulin promoters, or heat shock promoters, from the genomes of viruses such as polyomaviruses, fowlpox virus, adenoviruses (such as adenovirus 2), bovine papillomavirus, aerosarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and Simian virus 40 (SV40), provided that such promoters are compatible with the host cell system.

[0488] Early and late promoters of the SV40 virus are conveniently obtained as SV40 restriction fragments that also contain the SV40 virus origin of replication. The immediate early promoter of human cytomegalovirus is conveniently obtained as the HindIII E restriction fragment. A system for expressing DNA in a mammalian host using bovine papillomavirus as a vector is disclosed in U.S. Patent No. 4,419,446. Modifications of this system are described in U.S. Patent No. 4,601,978. See also Reyes et al., Nature 297:598-601 (1982), which describes the expression of human [3-interferon cDNA in mouse cells under the control of a herpes simplex virus-derived thymidine kinase promoter. Alternatively, the Rous sarcoma virus long-terminal repeat can be used as a promoter.

[0489] e. Enhancer component Transcription of the DNA encoding the masked cytokines of the present invention by higher eukaryotes is often increased by inserting enhancer sequences into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). However, typically, enhancers derived from eukaryotic viruses will be used. Examples include the SV40 enhancer (bp100-270) on the late side of the origin of replication, the human cytomegalovirus early promoter enhancer, the mouse cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the origin of replication, and the adenovirus enhancer. See also Yaniv, Nature 297:17-18 (1982) (which describes enhancer elements for activation of eukaryotic promoters). Enhancers can be spliced ​​into the vector at position 5' or 3' relative to the masked cytokine encoding sequence, but are generally located 5' from the promoter.

[0490] f. Transcription termination component Expression vectors used in eukaryotic host cells may also contain sequences necessary for transcription termination and mRNA stabilization. Such sequences are commonly available from the 5' and optionally 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments into the untranslated portion of mRNA encoding a masked cytokine. One useful transcription termination component is the bovine growth hormone polyadenylated region. See WO94 / 11026 and the expression vectors disclosed therein.

[0491] g. Selection and transformation of host cells Suitable host cells for cloning or expressing DNA in the vectors herein include higher eukaryotic cells described herein, including vertebrate host cells. The proliferation of vertebrate cells in culture (tissue culture) is a routine procedure. Examples of useful mammalian host cell lines include the CV1 monkey kidney cell line transformed with SV40 (COS-7, ATCC CRL 1651), human embryonic kidney cell line (293 or 293 cells subcloned for growth in suspension culture, Graham et ah, J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells / -DHFR (CHO, Urlaub et ah, Proc. Natl. Acad. Sci. USA 77:4216 (1980)), mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), and human cervical cancer cells (HELA, ATCC CCL 2) Examples include canine kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BEL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), mouse mammary tumor cells (MMT 060562, ATCC CCL 51), TRI cells (Mather et ah, Annals NYAcad. Sci. 383:44-68 (1982)), MRC5 cells, FS4 cells, and human liver cancer cell line (Hep G2).

[0492] Host cells are transformed with the expression or cloning vectors described above for the production of masked cytokines and cultured in conventionally modified nutrient media to induce promoters, select transformants, or amplify genes encoding desired sequences.

[0493] h. Culture of host cells The host cells used to produce the masked cytokines of the present invention can be cultured in a variety of media. Commercial media such as Ham's F10 (Sigma), Minimum Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium ((DMEM), Sigma) are suitable for culturing host cells. In addition, any of the media described in Ham et ah, Meth. Enz. 58:44 (1979), Barnes et ah, Anal. Biochem. 102:255 (1980), U.S. Patent Nos. 4,767,704, 4,657,866, A,921,162, 4,560,655, or 5,122,469, WO 90 / 03430, WO 87 / 00195, or U.S. Patent Reissue No. 30,985 can be used as a culture medium for host cells. Any of these media may be supplemented, if necessary, with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphates), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN® drugs), trace elements (usually defined as inorganic compounds present at final concentrations within the micromolar range), and glucose or equivalent energy sources. Any other adjuvants may also be included in appropriate concentrations that would be known to those skilled in the art. Culture conditions, such as temperature, pH, and equivalents, are those previously used with host cells selected for expression and would be obvious to those skilled in the art.

[0494] i. Purification of masked cytokines When recombinant techniques are used, masked cytokines can be produced intracellularly or secreted directly into the culture medium. If masked cytokines are produced intracellularly as the first step, particulate debris, which may be either host cells or lysed fragments, can be removed, for example, by centrifugation or ultrafiltration. When masked cytokines are secreted into the culture medium, the supernatant from such an expression system can first be concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF may be included in any of the aforementioned steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of exogenous contaminants.

[0495] Masked cytokine compositions prepared from cells can be purified using, for example, hydroxyl apatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present, if any, in the masked cytokine. Protein A can be used to purify antibodies based on human IgG1, IgG2, or IgG4 heavy chains (Lindmark et ak, J.Immunol.Methods 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human y3 (Guss et ak, EMBO J.5:15671575 (1986)). The matrix to which the affinity ligand is attached may be agarose, but other matrices are also available. Mechanically stable matrices such as controlled-pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. If the masked cytokine contains a CH3 domain, Bakerbond ABX® resin (JTBaker, Phillipsburg, NJ) is useful for purification.

[0496] Other techniques for protein purification, such as fractionation on ion exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE®, chromatography on anion or cation exchange resins (such as polyaspartate columns), chromatographic focusing, SDS-PAGE, and ammonium sulfate precipitation, are also available depending on the masked cytokines to be recovered.

[0497] Following any preliminary purification steps, the mixture containing the masked cytokines and contaminants of interest may undergo further purification, for example, by low-pH hydrophobic interaction chromatography using elution buffer at a pH of approximately 2.5–4.5, performed at a low salt concentration (e.g., approximately 0–0.25 M salt).

[0498] Generally, various methodologies for preparing masked cytokines for use in research, testing, and clinical applications are consistent with the methodologies described above and / or are established in the art to be considered appropriate by those skilled in the art for masked cytokines for specific purposes.

[0499] 5. Composition In some embodiments, also provided herein, are compositions comprising any of the IL-12 masked cytokines described herein. In some embodiments, the composition comprises any of the exemplary embodiments of the masked IL-12 cytokines described herein. In some embodiments, the composition comprises a dimer of any of the masked IL-12 cytokines described herein. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a masked IL-12 cytokine and further comprises one or more of the components described in detail below. For example, in some embodiments, the composition comprises one or more pharmaceutically acceptable carriers, excipients, stabilizers, buffers, preservatives, isotonic agents, nonionic surfactants or detergents, or other therapeutic agents or active compounds, or combinations thereof. Various embodiments of the composition may also be referred to herein as formulations.

[0500] Therapeutic formulations are prepared for storage by mixing an active ingredient of desired purity with an optional pharmaceutically acceptable carrier, excipient, or stabilizer (Remington: The Science and Practice of Pharmacy, 20th Ed., Lippincott Williams & Wiklins, Pub., Gennaro Ed., Philadelphia, Pa. 2000). Acceptable carriers, excipients, or stabilizers are nontoxic to the recipient at the dosage and concentration used and include buffers, antioxidants such as ascorbic acid, methionine, vitamin E, sodium metabisulfite, preservatives, isotonic agents, stabilizers, metal complexes (e.g., Zn-protein complexes), chelating agents such as EDTA, and / or nonionic surfactants.

[0501] Buffers can be used to adjust the pH within a range that optimizes therapeutic efficacy, especially when stability is pH-dependent. Buffers may be present in concentrations ranging from approximately 50 mM to approximately 250 mM. Suitable buffers for use with the present invention include both organic and inorganic acids, as well as their salts. For example, citrates, phosphates, succinates, tartrates, fumarates, glucons, oxalates, lactates, and acetates. In addition, buffers may consist of histidine salts such as Tris and trimethylamine salts.

[0502] Preservatives can be added to prevent the growth of microorganisms and are typically present in concentrations ranging from approximately 0.2% to 1.0% (w / v). Examples of suitable preservatives commonly used with therapeutic agents include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride, thimerosal, phenol, butyl or benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, m-cresol, o-cresol, p-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate, 2-phenylethanol, ethanol, chlorobutanol, thiomerosal, bronopol, benzoic acid, imidourea, chlorohexidine, sodium dehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, and chlorphenesin (3p-chlorphenoxypropane-1,2-diol).

[0503] Isotonic agents, sometimes known as "stabilizers," may be present in a composition to adjust or maintain the isotonicity of a liquid. They are often referred to as "stabilizers" because, when used with large charged biomolecules such as proteins and antibodies, they interact with the charge groups of the amino acid side chains, thereby reducing the potential for intermolecular and intramolecular interactions.

[0504] The isotonic agent may be present in any amount, from about 0.1% to about 25% by weight or from about 1% to about 5% by weight, taking into account the relative amounts of the other components. In some embodiments, the isotonic agent may be a trivalent or higher sugar alcohol such as polyhydric sugar alcohols, glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0505] Additional excipients include agents that may act as one or more of the following: (1) volume extenders, (2) solubility enhancers, (3) stabilizers, and (4) agents that prevent denaturation or adhesion to the container wall. Such excipients include polyhydric sugar alcohols (listed above), amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, and threonine, organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myo-inititose, myo-inititol, galactose, galactitol, glycerol, cyclitol (e.g., inositol), and polyethylene glycol. This includes sulfur-containing reducing agents such as glucose, urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose), disaccharides (e.g., lactose, maltose, sucrose), trisaccharides such as raffinose, and polysaccharides such as dextrin or dextran.

[0506] Nonionic surfactants or detergents (also known as “wetting agents”) may be present to help solubilize the therapeutic agent and protect the therapeutic protein from agitation-induced aggregation, which also allows the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. Nonionic surfactants are present in concentrations ranging from about 0.05 mg / ml to about 1.0 mg / ml or from about 0.07 mg / ml to about 0.2 mg / ml. In some embodiments, nonionic surfactants are present in concentrations ranging from about 0.001% to about 0.1% w / v, or from about 0.01% to about 0.1% w / v, or from about 0.01% to about 0.025% w / v.

[0507] Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), polyoxomers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl stearate 40, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glycerol monostearate, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. Anionic detergents that can be used include sodium lauryl sulfate, sodium dioctyl sulfosuccinate, and sodium dioctyl sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0508] For a formulation to be used for in vivo administration, the formulation must be sterile. The formulation can be sterilized by filtration through a sterile filtration membrane. The therapeutic compositions described herein are generally contained in containers with a sterile access port, such as intravenous solution bags or vials with stoppers that can be punctured with a subcutaneous needle.

[0509] The route of administration shall be in a preferred manner, such as subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional, or intra-articular route, local administration, by inhalation, or by injection or infusion using a sustained-release or continuous-release means, in accordance with known and accepted methods, such as single or multiple boluses or by infusion over a prolonged period.

[0510] Any of the masked IL-12 cytokines described herein may be used alone or in combination with other therapeutic agents, such as in the manner described herein. The term “in combination with” encompasses two or more therapeutic agents (e.g., masked IL-12 cytokines) contained in the same or separate formulations. In some embodiments, “in combination with” means “concurrent” administration, in which case the administration of the masked IL-12 cytokine of the present invention occurs simultaneously with the administration of one or more additional therapeutic agents (e.g., at the same time or within one hour between the administration of the masked IL-12 cytokine and the administration of one or more additional therapeutic agents). In some embodiments, “in combination with” means sequential administration, in which case the administration of the masked IL-12 cytokine of the present invention occurs before and / or after the administration of one or more additional therapeutic agents (e.g., more than one hour between the administration of the masked IL-12 cytokine and the administration of one or more additional therapeutic agents). The agents contemplated herein include, but are not limited to, cytotoxic agents, cytokines, agents targeting immune checkpoint molecules, agents targeting immunostimulatory molecules, growth inhibitors, immunostimulants, or anticancer agents.

[0511] The formulations described herein may also contain one or more active compounds as necessary for the specific indication being treated, preferably for indications having complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may contain cytotoxic agents, cytokines, agents targeting immune checkpoint molecules or stimulating molecules, proliferation inhibitors, immunostimulants, anti-inflammatory agents, or anticancer agents. Such molecules are preferably present in combination in amounts effective for the intended purpose.

[0512] The formulations may be presented in any preferred state, such as liquid formulations, solid (lyophilized) formulations, or frozen formulations. Approaches for preparing each of these types of formulations for therapeutic use are well known in the art.

[0513] 6. Treatment Methods Provided herein are methods for treating or preventing a disease in a subject, comprising administering an effective amount of any masked IL-12 cytokine or composition thereof described herein to the subject. In some embodiments, a method is provided for treating a disease in a subject, comprising administering any composition thereof described herein to the subject. In some embodiments, the subject (e.g., a human patient) has been diagnosed with cancer or is at risk of developing such a disorder. In some embodiments, a method is provided for treating or preventing a disease in a subject, comprising administering an effective amount of any masked IL-12 cytokine or composition thereof described herein to the subject, wherein the masked IL-12 cytokine is activated upon enzymatic cleavage. In some embodiments, the masked IL-12 cytokine is activated in the tumor microenvironment. The masked IL-12 cytokine is therapeutically active after cleavage. Thus, in some embodiments, the active agent is the cleavage product.

[0514] For the prevention or treatment of a disease, the appropriate dosage of the active agent depends on the type of disease being treated as defined above, the severity and course of the disease, whether the agent is administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the agent, and the discretion of the attending physician. The agent should be administered as appropriate to the patient, either as a single dose or over a series of treatments.

[0515] In some embodiments of the methods described herein, the interval between administrations of the masked IL-12 cytokine described herein is about one week or longer. In some embodiments of the methods described herein, the interval between administrations of the masked IL-12 cytokine described herein is about two days or longer, about three days or longer, about four days or longer, about five days or longer, or about six days or longer. In some embodiments of the methods described herein, the interval between administrations of the masked IL-12 cytokine described herein is about one week or longer, about two weeks or longer, about three weeks or longer, or about four weeks or longer. In some embodiments of the methods described herein, the interval between administrations of the masked IL-12 cytokine described herein is about one month or longer, about two months or longer, or about three months or longer. As used herein, the interval between administrations refers to the period between one administration of the masked IL-12 cytokine and the next administration of the masked IL-12 cytokine. As used herein, an interval of about one month includes four weeks. In some embodiments, the treatment comprises multiple doses of masked IL-12 cytokines, and the intervals between doses may vary. For example, in some embodiments, the interval between the first and second doses is about one month, and the interval between subsequent doses is about two weeks. In some embodiments, the interval between the first and second doses is about two, three, four, five, or six days, and the interval between subsequent doses is about one week.

[0516] In some embodiments, the masked IL-12 cytokine is administered multiple times over a period of time. In some embodiments, the dose administered to the subject multiple times may be the same for each administration, or in some embodiments, the masked cytokine may be administered to the subject in two or more different doses. For example, in some embodiments, the masked IL-12 cytokine is initially administered once or more times in one dose, and then later administered once or more times in a second dose, starting at a subsequent point in time.

[0517] In some embodiments, the masked IL-12 polypeptide described herein is administered in uniform doses. In some embodiments, the masked IL-12 polypeptide described herein is administered to a subject in doses ranging from about 25 mg to about 500 mg per single dose. In some embodiments, the masked IL-12 polypeptide is administered in doses ranging from about 25 mg to about 50 mg, about 50 mg to about 75 mg, about 75 mg to about 100 mg, about 100 mg to about 125 mg, about 125 mg to about 150 mg, about 150 mg to about 175 mg, about 175 mg to about 200 mg, about 200 mg to about 225 mg, and about 225 mg to about 250 mg per single dose. The drug is administered to the target population in doses of approximately 250mg to 275mg, 275mg to 300mg, 300mg to 325mg, 325mg to 350mg, 350mg to 375mg, 375mg to 400mg, 400mg to 425mg, 425mg to 450mg, 450mg to 475mg, or 475mg to 500mg.

[0518] In some embodiments, the masked IL-12 polypeptide described herein is administered to a subject in a dose based on the subject's body weight or body surface area (BSA). Depending on the type and severity of the disease, masked IL-12 polypeptide in doses of approximately 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) may be an initial candidate dose for administration to a patient, whether by one or more separate doses or by sequential infusion. A typical daily dose may range from approximately 1 μg / kg to approximately 100 mg / kg or more, depending on the factors described above. For repeated administrations over several days or more, treatment will generally be maintained until the desired suppression of disease symptoms occurs, depending on the condition. One exemplary dose of masked IL-12 polypeptide may be in the range of approximately 0.05 mg / kg to approximately 10 mg / kg. Thus, one or more doses of approximately 0.5 mg / kg, approximately 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to a patient. In some embodiments, the masked IL-12 polypeptide described herein is administered to subjects in doses of approximately 0.1 mg / kg to approximately 10 mg / kg or approximately 1.0 mg / kg to approximately 10 mg / kg. In some embodiments, the masked IL-12 polypeptide described herein is administered to the subject in one of the following doses: approximately 0.1 mg / kg, approximately 0.5 mg / kg, approximately 1.0 mg / kg, approximately 1.5 mg / kg, approximately 2.0 mg / kg, approximately 2.5 mg / kg, approximately 3.0 mg / kg, approximately 3.5 mg / kg, approximately 4.0 mg / kg, approximately 4.5 mg / kg, approximately 5.0 mg / kg, approximately 5.5 mg / kg, approximately 6.0 mg / kg, approximately 6.5 mg / kg, approximately 7.0 mg / kg, approximately 7.5 mg / kg, approximately 8.0 mg / kg, approximately 8.5 mg / kg, approximately 9.0 mg / kg, approximately 9.5 mg / kg, or approximately 10.0 mg / kg.In some embodiments, the masked IL-12 polypeptide described herein is present in amounts of approximately or at least approximately 0.1 mg / kg, approximately or at least approximately 0.5 mg / kg, approximately or at least approximately 1.0 mg / kg, approximately or at least approximately 1.5 mg / kg, approximately or at least approximately 2.0 mg / kg, approximately or at least approximately 2.5 mg / kg, approximately or at least approximately 3.0 mg / kg, approximately or at least approximately 3.5 mg / kg, approximately or at least approximately 4.0 mg / kg, approximately or at least approximately 4.5 mg / kg, approximately or at least approximately 5.0 mg / kg, approximately or at least approximately 5.5 mg / kg, approximately or at least approximately 6.0 mg / kg, approximately or at least approximately 6.5 mg / kg, approximately, or at least approximately 7.0 The drug is administered to the subject in doses of mg / kg, approximately or at least approximately 7.5 mg / kg, approximately or at least approximately 8.0 mg / kg, approximately or at least approximately 8.5 mg / kg, approximately or at least approximately 9.0 mg / kg, approximately or at least approximately 9.5 mg / kg, approximately or at least approximately 10.0 mg / kg, approximately or at least approximately 15.0 mg / kg, approximately or at least approximately 20 mg / kg, approximately or at least approximately 30 mg / kg, approximately or at least approximately 40 mg / kg, approximately or at least approximately 50 mg / kg, approximately or at least approximately 60 mg / kg, approximately or at least approximately 70 mg / kg, approximately or at least approximately 80 mg / kg, approximately or at least approximately 90 mg / kg, or approximately or at least approximately 100 mg / kg. Any of the administration frequencies listed above may be used.

[0519] The therapeutic methods envisioned herein are the treatment of disorders or diseases, such as cancer, using any of the masked IL-12 cytokines or compositions described herein. Disorders or diseases that can be treated with the formulations of the present invention include leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma, lung cancer, ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer (e.g., Merkel cell carcinoma), or testicular cancer.

[0520] In some embodiments, what is provided herein is a method for treating or preventing cancer by administering any masked IL-12 cytokine or composition described herein. In some embodiments, what is provided herein is a method for treating or preventing cancer by administering any IL-12 masked cytokine or composition described herein in combination with an anticancer agent. The anticancer agent may be any agent capable of reducing cancer growth, interfering with cancer cell replication, directly or indirectly killing cancer cells, reducing metastasis, reducing tumor blood supply, or reducing cell viability. In some embodiments, the anticancer agent is a PD-1 inhibitor, EGFR inhibitor, HER2 inhibitor, VEGFR inhibitor, CTLA-4 inhibitor, BTLA inhibitor, B7H4 inhibitor, B7H3 inhibitor, CSFIR inhibitor, HVEM inhibitor, CD27 inhibitor, KIR inhibitor, NKG2A inhibitor, NKG2D agonist, TWEAK inhibitor, ALK inhibitor, CD52-targeted antibody, CCR4-targeted antibody, PD-L1 inhibitor, KIT inhibitor, PDGFR inhibitor, BAFF inhibitor, HD AC inhibitor, VEGF ligand inhibitor, CD19 targeting molecule, FOFR1 targeting molecule, DFF3 targeting molecule, DKK1 targeting molecule, MUC1 targeting molecule, MUG16 targeting molecule, PSMA targeting molecule, MSFN targeting molecule, NY-ES0-1 targeting molecule child, B7H3 targeting molecule, B7H4 targeting molecule, BCMA targeting molecule, CD29 targeting molecule, CD151 targeting molecule, CD123 targeting molecule, CD33 targeting molecule, CD37 targeting molecule, CDH19 targeting molecule, CEA targeting molecule, claudin 18.2-targeting molecule, CFEC12A-targeting molecule, EGFRVIII-targeting molecule, EPCAM-targeting molecule, EPHA2-targeting molecule, FCRH5-targeting molecule, FLT3-targeting molecule, GD2-targeting molecule, Glypican-3-targeting molecule, gpA33-targeting molecule, GPRC5D-targeting molecule, IL-123R-targeting molecule, IL-1RAP-targeting molecule, MCSP-targeting molecule, RON-targeting molecule, ROR1-targeting molecule, STEAP2-targeting molecule, TfR-targeting molecule, CD166-targeting molecule, TPBG-targeting molecule, TROP2-targeting molecule, proteasome inhibitor, ABE inhibitor, CD30 inhibitor, FLT3 inhibitor, MET inhibitor, RET inhibitor, IL-1(3) inhibitor, MEK inhibitor, ROS1 inhibitor, BRAE inhibitor, CD38 inhibitor, RANKE The following group of inhibitors is selected: B4GALNT1 inhibitors, SLAMF7 inhibitors, IDH2 inhibitors, mTOR inhibitors, CD20-targeted antibodies, BTK inhibitors, PI3K inhibitors, FLT3 inhibitors, PARP inhibitors, CDK4 inhibitors, CDK6 inhibitors, EGFR inhibitors, RAF inhibitors, JAK1 inhibitors, JAK2 inhibitors, JAK3 inhibitors, IL-6 inhibitors, IL-17 inhibitors, blunting inhibitors, IL-6R inhibitors, BCL2 inhibitors, PTCH inhibitors, PIGF inhibitors, TGFB inhibitors, CD28 agonists, CD3 agonists, CD40 agonists, GITR agonists, OX40 agonists, VISTA agonists, CD137 agonists, LAG3 inhibitors, TIM3 inhibitors, TIGIT inhibitors, and IL-12R inhibitors.

[0521] In some embodiments, what is provided herein is a method for treating or preventing cancer by administering any masked IL-12 cytokine described herein in combination with an anti-inflammatory agent. The anti-inflammatory agent may be any agent capable of preventing, counteracting, inhibiting, or otherwise reducing inflammation.

[0522] In some embodiments, the anti-inflammatory agent is a cyclooxygenase (COX) inhibitor. A COX inhibitor can be any agent that inhibits the activity of COX-1 and / or COX-2. In some embodiments, the COX inhibitor selectively inhibits COX-1 (i.e., the COX inhibitor inhibits the activity of COX-1 more than the activity of COX-2). In some embodiments, the COX inhibitor selectively inhibits COX-2 (i.e., the COX inhibitor inhibits the activity of COX-2 more than the activity of COX-1). In some embodiments, the COX inhibitor inhibits both COX-1 and COX-2.

[0523] In some embodiments, the COX inhibitor is a selective COX-1 inhibitor and is selected from the group consisting of SC-560, FR122047, P6, mofezolac, TFAP, flurbiprofen, and ketoprofen. In some embodiments, the COX inhibitor is a selective COX-2 inhibitor and is selected from the group consisting of celecoxib, rofecoxib, meloxicam, piroxicam, delacoxib, parecoxib, valdecoxib, etoricoxib, chromene derivatives, chromene derivatives, N-(2-cyclohexyloxynitrophenyl)methanesulfonamide, parecoxib, lumiracoxib, RS The group consists of 57067, T-614, BMS-347070, JTE-522, S-2474, SVT-2016, CT-3, ABT-963, SC-58125, nimeslid, floslid, NS-398, L-745337, RWJ-63556, L-784512, darbuferon, CS-502, LAS-34475, LAS-34555, S-33516, diclofenac, mefenamic acid, and SD-8381. In some embodiments, the COX inhibitor is selected from the group consisting of ibuprofen, naproxen, ketorolac, indomethacin, aspirin, naproxen, tolmetine, piroxicam, and meclofenamate.In some embodiments, the COX inhibitor is SC-560, FR122047, P6, mofezolac, TFAP, lurbiprofen, ketoprofen, celecoxib, rofecoxib, meloxicam, piroxicam, delacoxib, parecoxib, valdecoxib, etoricoxib, chromene derivatives, chromene derivatives, N-(2-cyclohexyloxynitrophenyl)methanesulfonamide, parecoxib, lumiracoxib, RS The following are selected from the group consisting of 57067, T-614, BMS-347070, JTE-522, S-2474, SVT-2016, CT-3, ABT-963, SC-58125, nimeslid, floslid, NS-398, L-745337, RWJ-63556, L-784512, darbuferon, CS-502, LAS-34475, LAS-34555, S-33516, diclofenac, mefenamic acid, SD-8381, ibuprofen, naproxen, ketorolac, indomethacin, aspirin, naproxen, tolmetine, piroxicam, and meclofenamate.

[0524] In some embodiments, the anti-inflammatory agent is an NF-κB inhibitor. An NF-κB inhibitor can be any agent that inhibits the activity of the NF-κB pathway. In some embodiments, the NF-κB inhibitor is selected from the group consisting of IKK complex inhibitors, IκB degradation inhibitors, NF-κB nuclear translocation inhibitors, p65 acetylation inhibitors, NF-κB DNA binding inhibitors, NF-κB transactivation inhibitors, and p53 induction inhibitors.

[0525] In some embodiments, the IKK complex inhibitor is selected from the group consisting of TPCA-1, NF-κB activator VI (BOT-64), BMS-345541, Anlexanox, SC-514 (GK-01140), IMD-0354, and IKK-16. In some embodiments, the IκB degradation inhibitor is selected from the group consisting of BAY-11-7082, MG-115, MG-132, lactacystin, epoxomycin, parthenolide, carfilzomib, and MLN-4924 (pevonesistat). In some embodiments, the NF-κB nuclear translocation inhibitor is selected from the group consisting of JSH-23 and rolipram. In some embodiments, the p65 acetylation inhibitor is selected from the group consisting of gallic acid and anacardic acid. In some embodiments, the NF-κBDNA binding inhibitor is selected from the group consisting of GYY-4137, p-XSC, CV-3988, and prostaglandin E2 (PGE2). In some embodiments, the NF-κB transactivation inhibitor is selected from the group consisting of LY-294002, woltmannin, and mesalamine. In some embodiments, the p53 induction inhibitor is selected from the group consisting of quinacrine and flavopyridol. In some embodiments, the NF-κB inhibitor is selected from the group consisting of TPCA-1, NF-κB activator VI (BOT-64), BMS-345541, Amlexanox, SC-514 (GK-01140), IMD-0354, IKK-16, BAY-11-7082, MG-115, MG-132, lactacystin, epoxycin, parthenolide, carfilzomib, MLN-4924 (pevonesistat), JSH-23 rolipram, gallic acid, anacardic acid, GYY-4137, p-XSC, CV-3988, prostaglandin E2 (PGE2), LY-294002, woltmannin, mesalamine, quinacrine, and flavopyridol.

[0526] In some embodiments, what is provided herein is a method for treating or preventing cancer by administering any masked IL-12 cytokine or composition described herein in combination with an anti-cancer therapeutic protein. The anti-cancer therapeutic protein may be any therapeutic protein capable of reducing cancer growth, interfering with cancer cell replication, directly or indirectly killing cancer cells, reducing metastasis, reducing tumor blood supply, or reducing cell viability. Exemplary anti-cancer therapeutic proteins may arise in the form of antibodies or fragments thereof, antibody derivatives, bispecific antibodies, chimeric antigen receptor (CAR) T cells, fusion proteins, or bispecific T cell engagers (BiTEs). In some embodiments, what is provided herein is a method for treating or preventing cancer by administering any masked IL-2 cytokine or composition described herein in combination with CAR-NK (natural killer) cells.

[0527] 7. Manufactured goods or kits In another embodiment, a product or kit is provided comprising any masked IL-12 cytokine as described herein. The product or kit may further include instructions for the use of the cytokine in the methods of the present invention. Thus, in certain embodiments, the product or kit may include instructions for the use of the masked cytokine in a method for treating or preventing a disorder (e.g., cancer) in an individual, comprising administering an effective amount of the masked cytokine to the individual. For example, in certain embodiments, the product or kit includes instructions for the use of the masked IL-12 polypeptide in a method for treating or preventing a disorder (e.g., cancer) in an individual, comprising administering an effective amount of the masked IL-12 polypeptide to the individual. In certain embodiments, the individual is a human. In some embodiments, the individual has a disease selected from the group consisting of leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma, lung cancer, ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer, or testicular cancer.

[0528] The manufactured article or kit may further include a container. Suitable containers include, for example, bottles, vials (e.g., dual-chamber vials), syringes (such as single or dual-chamber syringes), test tubes, and intravenous (IV) bags. The container may be formed from a variety of materials, such as glass or plastic. The container holds the formulation. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the formulation is a frozen formulation. In some embodiments, the formulation is a liquid formulation.

[0529] The manufactured article or kit may further include a label or accompanying leaflet, either on or associated with the container, which may indicate instructions for the recombination and / or use of the formulation. The label or accompanying leaflet may further indicate that the formulation is useful or intended for subcutaneous, intravenous, or other modes of administration to treat or prevent a disorder in an individual (e.g., cancer). The container holding the formulation may be a single-use vial or a multi-use vial, which allows for repeated administration of the recombined formulation. The manufactured article or kit may further include a second container containing a suitable diluent. The manufactured article or kit may further include other materials desirable from a commercial, therapeutic, and user perspective, including other buffers, diluents, filters, needles, syringes, and accompanying leaflets with instructions for use.

[0530] In specific embodiments, the present invention provides a kit for a single-dose administration unit. Such a kit includes a container for an aqueous formulation of a therapeutic cytokine, This includes both single and multi-chamber prefilled syringes. Exemplary prefilled syringes are available from Vetter GmbH (Ravensburg, Germany).

[0531] The manufactured articles or kits described herein optionally further include a container containing a second drug, the masked cytokine being the first drug, and the articles or kits further include a label or instructions on the package insert for treating a subject with an effective dose of the second drug.

[0532] In another embodiment, what is provided herein is a manufactured article or kit comprising the formulation described herein for administration by an auto-injector device. An auto-injector may be described as an injection device that, upon activation, delivers its contents without any additional necessary action from the patient or administerer. They are particularly suitable for self-medication of therapeutic formulations when the delivery rate must be constant and the delivery time is longer than a few moments.

[0533] 8. Definition Unless otherwise defined, all technical terms, notations, and other technical and scientific terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art in which the claimed subject matter relates. Where applicable, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from those commonly understood in the art.

[0534] It should be understood that the present invention is not limited to any particular composition or biological system that may vary. Furthermore, it should be understood that the terms used herein are solely for the purpose of describing specific embodiments and are not intended to be limiting. Where used herein and in the appended claims, the singular forms "a," "an," and "the" refer to multiple subjects unless the content otherwise clearly indicates. Thus, for example, the reference to "an IL-12 polypeptide (IL-12 polypeptide)" optionally includes two or more such polypeptides and their equivalents.

[0535] As used herein, the term “about” refers to the normal range of error for each value that is readily known to those skilled in the art. References to “about” values ​​or parameters herein include (and describe) embodiments that essentially concern that value or parameter.

[0536] It should be understood that the aspects and embodiments of the present invention described herein include "including," "consisting of," and "essentially consisting of."

[0537] As used herein, the term "and / or" refers to any one of the items to which the term relates, any combination of items, or all of the items. For example, the phrase "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A and B or C; B and A or C; C and A or B; A (alone); B (alone); and C (alone).

[0538] The term "antibody" includes polyclonal antibodies, monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions having polyepitope specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), and antibody fragments (e.g., Fab, F(ab')2, and Fv). The term "immunoglobulin (Ig)" is used herein as synonymous with "antibody."

[0539] The term "antibody" refers to a small antibody fragment containing two antigen-binding sites, each including a heavy chain variable (VH) domain connected to a light chain variable (VL) domain within the same polypeptide chain (VH-VL).

[0540] A basic quadruple-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five basic heterotetrameric units, along with an additional polypeptide called a J chain, containing 10 antigen-binding sites, while IgA antibodies contain 2 to 5 basic quadruple-chain units that can polymerize to form multivalent aggregates in combination with the J chain. For IgG, a quadruple-chain unit generally has a value of approximately 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds, depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide crosslinks. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for the a and y chains respectively, and four CH domains for the p and s isotypes. Each light chain (L) has a variable domain (VL) at its N-terminus, followed by a constant domain at the other end. The VL aligns with the light chain (VH), and the CL aligns with the first constant domain (CHI) of the heavy chain. Certain amino acid residues are thought to form an interface between the light chain variable domain and the heavy chain variable domain. The pairing of VH and VL together forms a single antigen-binding site. For the structures and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0541] Light chains (L chains) from any vertebrate species can be assigned to one of two distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domain (CH) of their heavy chain, immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each having a heavy chain designated as a, 8, e, y, and p, respectively. The y and a classes are further divided into subclasses based on relatively minor differences in CH sequence and function. For example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. IgG1 antibodies can exist in multiple polymorphic variants called allotypes (discussed in Jefferis and Lefranc 2009.mAbs Vol 1 Issue 4 1-7), any of which is suitable for use in the present invention. Common allotype variants in human populations are specified by the letters a, f, n, and z.

[0542] An “isolated” antibody is an antibody identified, separated, and / or recovered from components of its production environment (e.g., natural or recombinant). In some embodiments, the isolated polypeptide is independent of all other components from its production environment. Contaminating components of its production environment, such as those arising from recombinant transfected cells, are typically materials that interfere with the study, diagnostic, or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the polypeptide is purified to (1) more than 95% by weight of the antibody as determined, for example, by the Lowry method, and in some embodiments to more than 99% by weight, (1) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a rotating cup sequencer, or (3) to homogeneity by SDS-PAGE under non-reducible or reducing conditions using Coomassie blue or silver staining. Because at least one component of the antibody’s natural environment is absent, the isolated antibody contains the antibody in situ within recombinant cells. However, typically, isolated polypeptides or antibodies are prepared by at least one purification step.

[0543] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. In some embodiments, the monoclonal antibody has a C-terminal cleavage in the heavy chain and / or light chain. For example, 1, 2, 3, 4, or 5 amino acid residues are cleaved at the C-terminus of the heavy chain and / or light chain. In some embodiments, the C-terminal cleavage removes C-terminal lysine from the heavy chain. In some embodiments, the monoclonal antibody has an N-terminal cleavage in the heavy chain and / or light chain. For example, 1, 2, 3, 4, or 5 amino acid residues are cleaved at the N-terminus of the heavy chain and / or light chain. In some embodiments, a truncated form of the monoclonal antibody may be produced by recombinant technology. In some embodiments, the monoclonal antibody is highly specific and oriented to a single antigen site. In some embodiments, monoclonal antibodies are highly specific and oriented to multiple antigen sites (e.g., bispecific or multispecific antibodies). The modifier “monoclonal” characterizes the antibody as being obtained from a substantially homogeneous group of antibodies, but does not imply that antibody production by any particular method is required. For example, monoclonal antibodies used in accordance with the present invention may be produced by a variety of techniques, including, for example, hybridoma methods, recombinant DNA methods, phage display techniques, and techniques for producing human or human-like antibodies in animals having some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences.

[0544] The terms “full-length antibody,” “intact antibody,” or “whole antibody” are used synonymously to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically, a whole antibody includes those having heavy and light chains containing an Fc region. The constant domain may be a natural sequence constant domain (e.g., a human natural sequence constant domain) or an amino acid sequence variant thereof. In some cases, an intact antibody may have one or more effector functions.

[0545] An "antibody fragment" includes the antigen-binding region and / or variable region of an intact antibody, and / or a portion of an intact antibody, such as the constant region of an intact antibody. Examples of antibody fragments include the Fc region of an antibody, a portion of the Fc region, or a portion of an antibody containing the Fc region. Examples of antigen-binding antibody fragments include domain antibodies (dAbs), Fab, Fab', F(ab')2, and Fv fragments, antibodies, linear antibodies (see U.S. Patent No. 5,641,870, Example 2, Zapata et ah, Protein Eng. 8(10):1057-1062

[1995] ), single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Single heavy-chain antibodies or single light-chain antibodies can be manipulated, or, in the case of heavy-chain antibodies, can be isolated from camelids, sharks, libraries, or mice that have been manipulated to produce single heavy-chain molecules.

[0546] Papain digestion of the antibody produced two identical antigen-binding fragments called "Fab" fragments and a residual "Fc" fragment, a name reflecting its readily crystallizable ability. The Fab fragments consist of the entire light chain, along with a variable region domain (VH) on the heavy chain and a first constant domain (CHI) on one heavy chain. Each Fab fragment is monovalent with respect to antigen binding, i.e., possesses a single antigen-binding site. Pepsin treatment of the antibody produces a single large F(ab')2 fragment, roughly corresponding to two disulfide-linked Fab fragments with different antigen-binding activities, yet still capable of crosslinking antigens. The Fab' fragments differ from the Fab fragments by having several additional residues at the carboxyl terminus of the CHI domain, which contains one or more cysteines derived from the antibody hinge region. Fab'-SH is the herein designation for Fab', where the cysteine ​​residues in the constant domain have free thiol groups. The F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments, each containing a hinge cysteine ​​between them. Other chemical bonds in the antibody fragment are also known. The Fc fragment contains the carboxyl terminal portions of both H chains, which are held together by a disulfide. The effector function of the antibody is determined by the sequence and glycans within the Fc region, and the region recognized by the Fc receptor (FcR) is found on certain cell types.

[0547] The "amino acid sequence identity percentage (%)" relative to a reference polypeptide sequence is defined as the proportion of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence after the sequences have been aligned and gaps introduced, if necessary, to achieve the maximum percentage of sequence identity, and does not take into account any conservative substitutions as part of sequence identity. Alignment for determining the amino acid sequence identity percentage can be achieved using various methods within the scope of the art, such as publicly available computer software like BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm necessary to achieve maximum alignment over the full length of the sequences being compared. For example, the amino acid sequence identity % of a given amino acid sequence A with respect to a given amino acid sequence B, or with respect to a given amino acid sequence B (which can be substituted for a given amino acid sequence A that has or contains a certain amino acid sequence identity % with respect to a given amino acid sequence B, or with respect to a given amino acid sequence B) is calculated as follows. 100 × fraction X / Y

[0548] In the formula, X is the number of amino acid residues scored as identical by their sequences in the arrangement of A and B in the program, and Y is the total number of amino acid residues in B. It should be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the amino acid sequence identity percentage between A and B is not equal to the amino acid sequence identity percentage between B and A.

[0549] Antibody "effector function" refers to the biological activity resulting from the antibody's Fc region (either the Fc region of the natural sequence or the Fc region of an amino acid sequence variant), and varies depending on the antibody isotype. Examples of antibody effector functions include Clq binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0550] As used herein, “binding affinity” refers to the strength of the non-covalent interaction between a single binding site of a molecule (e.g., a cytokine) and its binding partner (e.g., a cytokine receptor). In some embodiments, the affinity of a binding protein (e.g., a cytokine) can generally be expressed by the equilibrium dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein.

[0551] The “isolated” nucleic acid molecules encoding cytokine polypeptides described herein are nucleic acid molecules identified and isolated from at least one contaminating nucleic acid molecule that is typically associated with the environment in which they are produced. In some embodiments, the isolated nucleic acid is unrelated to all components associated with the production environment. The isolated nucleic acid molecules encoding polypeptides and cytokine polypeptides herein are in forms other than those found in nature or in the environment. Thus, isolated nucleic acid molecules are distinguished from nucleic acids encoding polypeptides and cytokine polypeptides herein that are naturally present in cells.

[0552] The term "pharmaceutical preparation" refers to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and does not contain any additional ingredients that would be unacceptably toxic to the subject to which the preparation is administered.

[0553] Such preparations are sterilized.

[0554] As used herein, “carrier” includes pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to cells or mammals to which they are exposed at the doses and concentrations used. Physiologically acceptable carriers are often aqueous pH buffer solutions. Examples of physiologically acceptable carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than approximately 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

[0555] As used herein, the term “treatment” refers to a clinical intervention designed to alter the natural course of an individual or cell being treated during the course of clinicopathology. Desired effects of treatment include a decrease in the rate of disease progression, improvement or mitigation of the disease state, and remission or improved prognosis. An individual is successfully “treated” if, for example, one or more symptoms associated with a disorder (e.g., a neoplastic disease) are reduced or eliminated. An individual is successfully “treated” if, for example, the treatment results in an improvement in the quality of life of the individual with the disease, a reduction in the dosage of other drugs required to treat the disease, a decrease in the frequency of disease relapses, a decrease in the severity of the disease, a delay in the onset or progression of the disease, and / or an extension of the individual’s survival.

[0556] As used herein, “in combination with” or “in combination with” refers to the administration of one therapeutic modality in addition to another therapeutic modality. Thus, “in combination with” or “in combination with” refers to the administration of one therapeutic modality before, during, or after the administration of other therapeutic modalities to an individual.

[0557] As used herein, the term “prevention” includes providing prevention with respect to the onset or recurrence of a disease in an individual. The individual may be predisposed to, susceptible to, or at risk of developing a disorder, but has not been diagnosed with the disorder. In some embodiments, the masked cytokines described herein are used to delay the onset of the disorder.

[0558] As used herein, individuals “at risk” of developing a disorder may or may not have a detectable disease or symptoms of the disease, and may or may not exhibit a detectable disease or symptoms of the disease prior to the treatment methods described herein. “At risk” means that an individual has one or more risk factors, which are measurable parameters correlated with the development of the disease, as is known in the art. Individuals with one or more of these risk factors are more likely to develop a disorder than individuals without one or more of these risk factors.

[0559] "Effective dose" refers to the amount and duration of administration that is effective in order to achieve the desired or directed effect, including at least a therapeutic or prophylactic outcome.

[0560] An effective dose may be provided in one or more doses. A “therapeutic effective dose” is the minimum concentration required to influence at least a measurable improvement in a particular disorder. The therapeutic effective dose as used herein may vary depending on factors such as the patient’s disease state, age, sex, and weight, as well as the antibody’s ability to induce the desired response in the individual. A therapeutic effective dose may also be an amount in which the therapeutically beneficial effect outweighs any toxic or adverse effects of the cytokine. A “prophylactic effective dose” refers to an effective dose and duration required to achieve the desired prophylactic outcome. Typically, but not always, a prophylactic effective dose may be less than a therapeutic effective dose, as prophylactic doses are used in subjects before or at an early stage of the disease.

[0561] "Chronic" administration refers to the continuous administration of a drug, in contrast to the acute mode, with the primary focus being on the long-term initial therapeutic effect (activity). "Intermittent" administration is a treatment that is not carried out continuously without interruption, but rather is inherently periodic.

[0562] As used herein, “individual” or “subject” is a mammal. “Mammals” for therapeutic purposes include humans, livestock and farm animals, as well as zoo, sport, or pet animals such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, and cats. In some embodiments, the individual or subject is a human.

[0563] 9. Examples The present invention will be better understood by referring to the following examples. However, they should not be construed as limiting the scope of the invention. The examples and embodiments described herein are for illustrative purposes only, and it should be understood that various modifications or changes thereto are proposed to those skilled in the art and should be included in the spirit and purpose of this application and the appended claims.

[0564] While some examples describe the manipulation, production, and / or testing of “masked” versions of IL-2 polypeptide constructs, some examples also use other constructs containing one or more of the components described herein, such as the “unmasked” parent version of the IL-2 polypeptide construct, or a control for comparison. Therefore, a description of testing performed on a masked IL-2 polypeptide construct does not necessarily mean that the unmasked version of the construct was not also tested.

[0565] Example 1: Manipulation of a masked IL-2 polypeptide A masked IL-2 polypeptide is generated according to the teachings herein. In the subsequent examples, some experiments involve the use of a monomeric form of the masked IL-2 polypeptide construct, while others involve the use of a dimeric form of the masked IL-2 construct, such as a dimer (homodimer) formed through a disulfide bond linking two copies of the same masked polypeptide construct, or a heterodimer formed by two different polypeptides (see, for example, Table 5).

[0566] A masked IL-2 polypeptide construct is generated, comprising an IL-2 polypeptide or its functional fragment, a masking moiety, and a half-life extension domain such as an antibody or its fragment (e.g., Fc region, heavy chain, and / or light chain). Several IL-2 polypeptide constructs are also generated, comprising an IL-2 polypeptide or its functional fragment linked to a half-life extension domain, without even including a masking moiety. Some of the constructs also include a linker comprising a cleavable peptide that links the masking moiety ...

Claims

1. A masked IL-12 cytokine containing a protein heterodimer, A first polypeptide chain comprising the following, A second polypeptide chain comprising the following, HL1 is the first half-life extension domain, L1 is the first linker, MM is the masking portion, HL2 is the second half-life extension domain, L2 is the second linker, and C is the IL-12 cytokine or its functional fragment. The first half-life extension domain is associated with the second half-life extension domain, A masked IL-12 cytokine in which one of the first or second linkers contains a peptide that can be proteolytically cleaved.

2. The masked cytokine according to claim 1, wherein the IL-12 polypeptide or its functional fragment comprises an IL-12p40 polypeptide or its functional fragment covalently bound to the IL-12p35 polypeptide or its functional fragment.

3. The masked cytokine according to claim 2, wherein the IL-12p40-IL-12p35 linker is 5 to 20 amino acids in length.

4. The masked cytokine according to claim 2 or 3, wherein the IL-12p40-IL-12p35 linker is rich in amino acid residues G and S.

5. The masked cytokine according to any one of claims 2 to 4, wherein the IL-12p40-IL-12p35 linker includes SEQ ID NO: 3 (GGGGGSGGGGGGGGGS).

6. The masked cytokine according to any one of claims 2 to 5, wherein the IL-12p40 polypeptide comprises SEQ ID NO: 1, or an amino acid sequence having at least one amino acid modification compared to the amino acid sequence of SEQ ID NO:

1.

7. The masked cytokine according to claim 6, wherein the IL-12p40 polypeptide chain comprises SEQ ID NO:

1.

8. The masked cytokine according to claim 6, wherein the IL-12p40 polypeptide comprises at least one amino acid modification in the GAG-binding domain (KSKREKKDRV) compared to the amino acid sequence of SEQ ID NO:

1.

9. The masked cytokine according to claim 8, wherein the IL-12p40 polypeptide comprises SEQ ID NO:

57.

10. The masked cytokine according to claim 8, wherein the IL-12p40 polypeptide comprises SEQ ID NO:

58.

11. The masked cytokine according to any one of claims 6 to 10, wherein the IL-12p40 polypeptide comprises an amino acid sequence having one or more cytokine substitution mutations compared to the amino acid sequence of SEQ ID NO:

1.

12. The masked cytokine according to claim 11, wherein the IL-12p40 polypeptide comprises SEQ ID NO:

59.

13. The masked cytokine according to claim 11, wherein the IL-12p40 polypeptide comprises SEQ ID NO:

60.

14. The masked cytokine according to any one of claims 2 to 13, wherein the IL-12p35 polypeptide comprises SEQ ID NO: 2, or an amino acid sequence having at least one amino acid modification compared to the amino acid sequence of SEQ ID NO:

2.

15. The masked cytokine according to claim 14, wherein the IL-12p35 polypeptide comprises SEQ ID NO:

2.

16. The masked cytokine according to any one of claims 1 to 7, wherein the IL-12 cytokine or a functional fragment thereof comprises SEQ ID NO:

4.

17. The masked cytokine according to any one of claims 1 to 10, wherein the IL-12 cytokine or a functional fragment thereof comprises SEQ ID NO:

61.

18. The masked cytokine according to any one of claims 1 to 10, wherein the IL-12 cytokine or a functional fragment thereof comprises SEQ ID NO:

62.

19. The masked cytokine according to any one of claims 1 to 12, wherein the IL-12 cytokine or a functional fragment thereof comprises SEQ ID NO:

63.

20. The masked cytokine according to any one of claims 1 to 13, wherein the IL-12 cytokine or a functional fragment thereof comprises SEQ ID NO:

64.

21. The masked cytokine according to any one of the prior claims, wherein the masking portion comprises an IL-12 cytokine receptor or a subunit or variant thereof.

22. The masked cytokine according to any one of the prior claims, wherein the masking portion comprises an extracellular domain of human IL-12Rβ1 or a fragment, part, or variant thereof that maintains or otherwise demonstrates affinity for IL-12.

23. The masked cytokine according to claim 22, wherein the masking portion includes residues 24 to 237 of human IL-12Rβ1, i.e., the sequence having SEQ ID NO:

5.

24. The masked cytokine according to claim 22, wherein the masking portion includes residues 24 to 545 of human IL-12Rβ1, i.e., the sequence having SEQ ID NO:

6.

25. The masked cytokine according to any one of claims 1 to 21, wherein the masking portion comprises an extracellular domain of human IL-12Rβ2 or a fragment, part, or variant thereof that maintains or otherwise demonstrates affinity for IL-12.

26. The masked cytokine according to claim 25, wherein the masking portion includes residues 24 to 212 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO:

7.

27. The masked cytokine according to claim 25, wherein the masking portion includes residues 24 to 222 of human IL-12Rβ2, i.e., a sequence having SEQ ID NO: 8, or the masking portion includes residues 24 to 227 of human IL-12Rβ2, i.e., a sequence having SEQ ID NO:

11.

28. The masked cytokine according to claim 25, wherein the masking portion includes residues 24 to 319 of human IL-12Rβ2, i.e., the sequence having sequence number 9.

29. The masked cytokine according to claim 28, wherein the masking portion comprises at least one amino acid modification compared to the sequence of SEQ ID NO: 9, and optionally the modification is a cysteine ​​substitution mutation.

30. The masked cytokine according to claim 29, wherein the masking portion includes sequence number 65.

31. The masked cytokine according to claim 25, wherein the masking portion includes residues 24 to 622 of human IL-12Rβ2, i.e., the sequence having sequence number 10.

32. The masked cytokine according to any one of the prior claims, wherein the cleavable peptide has a length of 6 to 10 amino acids.

33. The masked cytokine according to any one of the prior claims, wherein the cleavable peptide comprises the amino acid sequence of SEQ ID NO:

15.

34. The masked cytokine according to any one of the prior claims, wherein the cleavable peptide comprises the amino acid sequence of SEQ ID NO:

41.

35. The masked cytokine according to any one of the prior claims, wherein the cleavable peptide comprises the amino acid sequence of SEQ ID NO:

42.

36. The masked cytokine according to any one of the prior claims, wherein the cleavable peptide comprises the amino acid sequence of SEQ ID NO:

43.

37. The masked cytokine according to any one of the prior claims, wherein the cleavable peptide comprises the amino acid sequence of SEQ ID NO:

44.

38. The masked cytokine according to any one of the prior claims, wherein the cleavable peptide comprises the amino acid sequence of SEQ ID NO:

45.

39. The first polypeptide chain comprises the following: The masked cytokine according to any one of the prior claims, wherein the second polypeptide chain comprises the following:

40. The masked cytokine according to claim 39, wherein the non-cleavable linker is 3 to 18 amino acids in length.

41. The masked cytokine according to claim 39, wherein the non-cleavable linker is 3 to 15 amino acids in length.

42. The masked cytokine according to any one of claims 30 to 41, wherein the non-cleavable linker is rich in amino acid residues G and S.

43. The non-cuttable linker is [(G)] ｎ A masked cytokine according to any one of claims 39 to 42, comprising [S] and having n = 4 or 5.

44. The masked cytokine according to any one of claims 39 to 43, wherein the non-cleavable linker comprises the amino acid sequence shown in SEQ ID NO: 12 (GGGGS).

45. The masked cytokine according to any one of claims 39 to 43, wherein the non-cleavable linker comprises the amino acid sequence shown in SEQ ID NO: 13 (GGGGGSGGGGGS).

46. The masked cytokine according to any one of claims 39 to 43, wherein the non-cleavable linker comprises the amino acid sequence shown as 14 (GGGGGGGGGGGG).

47. The masked cytokine according to any one of claims 39 to 42, wherein the non-cleavable linker comprises the amino acid sequence shown in 54 (GGGGGGGGGGGGGGGP).

48. The masked cytokine according to any one of claims 39 to 42, wherein the non-cleavable linker comprises the amino acid sequence shown at 55 (PGGSGP).

49. The masked cytokine according to any one of claims 39 to 42, wherein the non-cleavable linker comprises the amino acid sequence shown at 56 (GGSPG).

50. The cleavable linker comprises a proteolytically cleavable peptide (CP) having spacer domains (SDs) on both sides, SD1-CP-SD2 SD1 and SD2 are the first polypeptide chains comprising the following: A masked cytokine according to any one of claims 39 to 49, wherein the second polypeptide chain comprises the following:

51. The masked cytokine according to claim 50, wherein the first spacer domain (SD1) is 3 to 10 amino acids in length.

52. The masked cytokine according to claim 50 or 51, wherein SD1 comprises SEQ ID NO: 16 (GGSGGS).

53. The masked cytokine according to claim 50 or 51, wherein SD1 comprises SEQ ID NO: 17 (GGGGGGGS).

54. The masked cytokine according to any one of claims 50 to 53, wherein the second spacer domain (SD2) is 3 to 6 amino acids in length.

55. A masked cytokine according to any one of claims 50 to 54, wherein SD2 comprises SEQ ID NO: 18 (SGP).

56. The masked IL-2 cytokine according to claim 50, wherein the proteolytically cleavable linker comprises SD1-CP-SD2, where SD1 is a first spacer domain, CP is a cleavable peptide, SD2 is a second spacer domain, CP has the amino acid sequence shown in SEQ ID NO: 44, and SD2 has the amino acid sequence shown in SEQ ID NO:

18.

57. The masked IL-2 cytokine according to claim 50, wherein the proteolytically cleavable linker comprises SD1-CP-SD2, where SD1 is a first spacer domain, CP is a cleavable peptide, SD2 is a second spacer domain, CP has the amino acid sequence shown in SEQ ID NO: 45, and SD2 has the amino acid sequence shown in SEQ ID NO:

18.

58. The aforementioned severable linker A masked cytokine according to any one of claims 50 to 54, including the masked cytokine described in any one of claims 50 to 54.

59. The aforementioned severable linker A masked cytokine according to any one of claims 50 to 54, including the masked cytokine described in any one of claims 50 to 54.

60. The masked cytokine according to claim 50, wherein the cleavable linker includes sequence number 46 (GGGGSMPYDLYHPSGP).

61. The masked cytokine according to claim 50, wherein the cleavable linker includes sequence number 47 (GGGGSMPYDLYHPSGP).

62. The masked cytokine according to claim 50, wherein the cleavable linker includes SEQ ID NO: 48 (GGGGGSDSGGGFMLTSGP).

63. The masked cytokine according to claim 50, wherein the cleavable linker includes SEQ ID NO: 49 (GGGGGGGSDSGGGFMLTSGP).

64. The masked cytokine according to claim 50, wherein the cleavable linker includes SEQ ID NO: 50 (GGGGGSRAAAAAVKSPSGP).

65. The masked cytokine according to claim 50, wherein the cleavable linker includes SEQ ID NO: 51 (GGGGGGGSRAAAAAVKSPSGP).

66. The masked cytokine according to claim 50, wherein the cleavable linker includes SEQ ID NO: 52 (GGGGSISGLLSGRSSGP).

67. The masked cytokine according to claim 50, wherein the cleavable linker includes SEQ ID NO: 53 (GGGGGGGGSISGLLSGRSSGP).

68. The masked cytokine according to any one of the prior claims, wherein the first half-life extension domain comprises a first IgG1 Fc domain or a fragment thereof, and the second half-life extension domain comprises a second IgG1 Fc domain or a fragment thereof.

69. A masked cytokine according to any one of the prior claims, wherein each of the first and / or second Fc domains comprises one or more modifications that promote non-covalent association of the first and second half-life extension domains.

70. The masked cytokine according to any one of the prior claims, wherein the first half-life extension domain comprises SEQ ID NO: 25 (Y349C, T366S, L38A, Y407V, and N297A), and the second half-life extension domain comprises SEQ ID NO: 26 (S354C, T366W, and N297A).

71. A masked cytokine according to any one of the prior claims, wherein the first half-life extension domain comprises SEQ ID NO: 27 (Y349C, T366S, L38A, Y407V, N297A, and I253A), and the second half-life extension domain comprises SEQ ID NO: 28 (S354C, T366W, N297A, and I253A).

72. The masked cytokine according to claim 1, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 34, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:

40.

73. A cleavage product capable of binding to IL-12R, wherein the cleavage product comprises an IL-12 cytokine or a functional fragment thereof, which can be prepared by proteolytic cleavage of a cleavable peptide in a masked IL-12 cytokine as described in any one of claims 1 to 42.

74. A masked IL-12 cytokine cleavage product wherein the cleavage product is capable of binding to IL-12R, and the cleavage product comprises a polypeptide comprising the following: PCP-SD2-C A cleavage product in which PCP is part of a peptide that can be proteolytically cleaved, SD2 is a spacer domain, and C is an IL-12 cytokine or a functional fragment thereof.

75. The cleavage product according to claim 74, wherein PCP is part of a proteolytically cleavable peptide according to any one of claims 32 to 38.

76. The cutting product according to claim 74 or 75, wherein SD2 is a spacer domain as defined in any one of claims 54 to 55.

77. The cleavage product according to any one of claims 74 to 76, wherein C is an IL-12 cytokine or a fragment thereof as defined in any one of claims 2 to 20.

78. The cleavage product according to any one of claims 74 to 77, wherein the cleavage product comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO:

29.

79. The cleavage product according to claim 78, wherein the cleavage product comprises the amino acid sequence of SEQ ID NO:

29.

80. A nucleic acid encoding a masked IL-12 cytokine as described in any one of claims 1 to 72, or encoding one of the peptide chains of a masked IL-12 cytokine as described in any one of claims 1 to 72.

81. A vector comprising the nucleic acid described in claim 80.

82. A host cell comprising a nucleic acid encoding a masked IL-12 cytokine according to any one of claims 1 to 72.

83. A composition comprising a masked IL-12 cytokine according to any one of claims 1 to 72.

84. A pharmaceutical composition comprising a masked IL-12 cytokine according to any one of claims 1 to 72 and a pharmaceutically acceptable carrier.

85. The pharmaceutical composition according to claim 84, wherein the pharmaceutical composition is a single unit dosage form.

86. The pharmaceutical composition according to claim 84, wherein the pharmaceutical composition is formulated for intravenous administration and is in a single dosage form.

87. The pharmaceutical composition according to claim 84, wherein the pharmaceutical composition is formulated for injection and is in a single unit dosage form.

88. The pharmaceutical composition according to claim 84, wherein the pharmaceutical composition is a liquid and is in a single unit dosage form.

89. A kit comprising a masked IL-12 cytokine according to any one of claims 1 to 41, or the composition according to claim 83, or the pharmaceutical composition according to any one of claims 84 to 88.

90. A method for producing a masked IL-12 cytokine as defined in any one of claims 1 to 72, comprising culturing the host cell described in claim 82 under conditions for producing the masked IL-12 cytokine.

91. A nucleic acid encoding the cleavage product according to any one of claims 73 to 79.

92. A composition comprising the cutting product described in any one of claims 73 to 79.

93. A pharmaceutical composition comprising a cleavage product according to any one of claims 73 to 79 and a pharmaceutically acceptable carrier.

94. A masked IL-12 cytokine according to any one of claims 1 to 72 for use in a drug.

95. A cleavage product according to any one of claims 73 to 79 for use in pharmaceuticals.

96. A method for treating or preventing cancer in a subject, comprising administering to the subject an effective amount of a masked IL-12 cytokine as described in any one of claims 1 to 72.

97. A method for treating or preventing cancer in a subject, comprising administering an effective amount of the composition described in claim 83 or 92 to the subject.

98. A method for treating or preventing cancer in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition according to any one of claims 84 to 88 or 93.

99. A method for treating or preventing cancer in a subject, comprising administering to the subject an effective amount of a masked IL-12 cytokine described in any one of claims 1 to 72, wherein the masked cytokine is proteolytically cleaved in vivo to produce a cleavage product described in any one of claims 73 to 79.

100. A method for treating or preventing cancer in a subject, comprising the step of producing in vivo a cleavage product capable of binding to a homologous receptor, wherein the cleavage product is as defined in any one of claims 73 to 79.

101. The method according to any one of claims 96 to 100, wherein the cancer is a solid tumor.

102. A masked IL-12 cytokine according to any one of claims 1 to 72 for use in the treatment or prevention of cancer.

103. A masked IL-12 cytokine according to any one of claims 1 to 72 for use in a method for treating or preventing cancer, wherein the method comprises administering an effective amount of the masked IL-12 cytokine to the subject, thereby proteolytically cleaving the masked cytokine in vivo to produce a cleavage product according to any one of claims 73 to 79.

104. The masked IL-12 cytokine for use according to claim 103, wherein the cancer is a solid tumor.

105. A cutting product according to any one of claims 73 to 79 for use in the treatment or prevention of cancer.

106. A cleavage product according to any one of claims 73 to 79 for use in a method for treating or preventing cancer, wherein the method comprises the step of administering a masked cytokine according to any one of claims 1 to 72 to a patient, thereby producing the cleavage product by proteolytic cleavage of the masked cytokine in vivo.

107. A cleavage product according to any one of claims 73 to 79 for use in a method of treating or preventing cancer in a subject, wherein the method comprises the step of producing the cleavage product by in vivo proteolytic cleavage from a masked cytokine according to any one of claims 1 to 72 administered to the subject.

108. The cleavage product for use according to any one of claims 105 to 107, wherein the cancer is a solid tumor.

109. A pharmaceutical composition according to any one of claims 84 to 88 and 93 for use in the treatment or prevention of cancer.

110. The pharmaceutical composition for use according to claim 109, wherein the cancer is a solid tumor.

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