Tumor-specific cleavable linkers

Abstract

This invention provides tumor-specific cleavable linkers and their use in pharmaceuticals and prodrugs for delivering therapeutic agents to the tumor cellular environment. The invention also provides cleavage products of such pharmaceuticals and prodrugs and methods relating to their use.

Description

Technical field

[0001] The present invention relates to tumor-specific cleavable linkers and their use in drugs and prodrugs for the delivery of therapeutic agents to the tumor cell environment. The invention also relates to cleavage products of such drugs and prodrugs and methods related to their use.

Prior Technology

[0002] Cancer is the second leading cause of death in the United States, causing more deaths than the next five leading causes (chronic respiratory disease, stroke, accident, Alzheimer's disease, and diabetes) . Although great progress has been made, especially through targeted therapies, much work remains in this field. Immunotherapy and an offshoot of this field, immuno-oncology, are generating viable and exciting therapeutic options for the treatment of malignancies. In particular, one hallmark of cancer is now found to be immune evasion and a great deal of work has been done to identify targets and develop therapies against these targets to reactivate the immune system to recognize and treat cancer.

[0003] Interleukin therapy is a strategy to effectively stimulate the immune system to induce anti-tumor cytotoxicity. In particular, the FDA has approved aldesleukin, a recombinant form of interleukin-2 (IL-2), for the treatment of metastatic renal cell carcinoma and melanoma. Unfortunately, interleukins administered to patients often have very short half-lives, thus requiring frequent dosing. For example, the product label for aldesleukin, sold under the brand name Proleukin, states that the drug exhibits a half-life of 85 minutes in patients receiving a 5-minute intravenous (IV) infusion. In addition, administration of high doses of cytokines can lead to deleterious health consequences, such as vascular leakage, through systemic immune activation. These findings illustrate the need for the development of therapeutic agents, such as cytokine therapeutics, that can effectively target tumors without the side effects associated with systemic immune activation.

[0004] Prodrugs in which interleukin therapeutics are masked by masking moieties and in which the therapeutic agent is active only after cleavage of the masking part in the tumor cell environment are one approach envisioned to meet this need.

Content of invention

[0005] The present invention provides novel tumor-specific proteolytic peptide linkers comprising tumor-specific proteolytic peptides and their use in polypeptide drug constructs for delivery of therapeutic moieties to the tumor cell environment. The portion of the construct other than the therapeutic portion can be considered a carrier portion.

[0006] Tumor-specific proteolytic peptides serve as substrates for proteases present in the tumor cell environment. The proteolytically cleavable peptide linker is located within the polypeptide drug construct such that the linker is cleaved by the action of a protease in the tumor cell environment and the polypeptide drug construct separates to form cleavage products, one of which will comprise the therapeutic moiety. The invention also relates to cleavage products of these drug constructs and methods related to their use.

[0007] Provided herein is a polypeptide drug construct comprising (i) a therapeutic moiety; (ii) a carrier moiety; and (iii) a linker comprising a peptide having DLLAVVAAS (SEQ ID NO: 248) or ISSGLLSGRS (SEQ ID NO: 249) amino acid sequence of tumor-specific protein cleavage peptide.

[0008] In some embodiments, the protein cleavable peptide (CP) is flanked by spacer domains (SD1 and SD2) flanked by the following formula: SD1-CP-SD2.

[0009] In some embodiments, the spacer domain is enriched in amino acid residues G, S, and P.

[0010] In some embodiments, the proteolytic peptide linker is 10 to 25 amino acids in length.

[0011] In some embodiments, the spacer domain includes only types of amino acid residues selected from the group consisting of G, S, and P.

[0012] In some embodiments, the first spacer domain (SD1) is between 3 and 6 amino acids in length.

[0013] In some embodiments, the second spacer domain (SD2) is between 3 and 6 amino acids in length.

[0014] In some embodiments, SD2 comprises the amino acid sequence SGP (SEQ ID NO: 29).

[0015] In some embodiments, SD2 has the amino acid sequence SGP (SEQ ID NO: 29).

[0016] In some embodiments, the protein cleavable peptide linker comprises the sequence GGPSDLLAVVAASSGP (SEQ ID NO: 245).

[0017] In some embodiments, the protein cleavable peptide linker comprises the sequence GSGPSDLLAVVAASSGP (SEQ ID NO: 246).

[0018] In some embodiments, the protein cleavable peptide linker comprises the sequence GSSGGPDLLAVVAASSGP (SEQ ID NO: 247).

[0019] In some embodiments, the protein cleavable peptide linker comprises the sequence GSPDLLAVVAASSGP (SEQ ID NO: 242).

[0020] In some embodiments, the protein cleavable peptide linker comprises the sequence GSPGDLLAVVAASSGP (SEQ ID NO: 243).

[0021] In some embodiments, the protein cleavable peptide linker comprises the sequence GSGSPSDLLAVVAASSGP (SEQ ID NO: 244).

[0022] In some embodiments, the protein cleavable linker comprises the sequence GGSSGGSPISSGLLSGRSSGPGSGS (SEQ ID NO: 112).

[0023] In some embodiments, the protein cleavable linker comprises the sequence GPPSGSSPISSGLLSGRSSGGG (SEQ ID NO: 113).

[0024] In some embodiments, the protein cleavable linker comprises the sequence GGSGGSISSGLLSGRSSGP (SEQ ID NO: 114).

[0025] In some embodiments, the protein cleavable linker comprises the sequence GGSGGSGGSISSGLLSGRSSGP (SEQ ID NO: 115).

[0026] In some embodiments, the proteolytic peptide linker is directly covalently bonded to the therapeutic moiety.

[0027] In some embodiments, a protein cleavable peptide linker is located within the drug construct between the therapeutic moiety and the carrier moiety.

[0028] In some embodiments, the protein cleavable peptide linker is located within the carrier moiety.

[0029] In some embodiments, a polypeptide drug construct comprises a single polypeptide chain. This means that the therapeutic moiety, carrier moiety and protein cleavable peptide linker are present in the same polypeptide chain.

[0030] In some embodiments, a polypeptide drug construct comprises more than one polypeptide chain. In some embodiments, the protein cleavable peptide linker is present in the same polypeptide chain as the therapeutic moiety. In some embodiments, the proteolytic peptide linker is present in a different polypeptide chain than the therapeutic moiety.

[0031] In some embodiments, the Polypeptide Drug Construct is a prodrug. In some embodiments, where the Polypeptide Drug Construct is a prodrug, the remainder of the molecule (which is separated from the therapeutic moiety following cleavage of the proteolytic peptide linker) comprises a masking moiety that inhibits the therapeutic moiety in the prodrug. The biological activity of the moiety is such that the therapeutic moiety is only biologically active after cleavage of the proteolytic peptide linker in the tumor cell environment, in some embodiments, the masking moiety is present in the same polypeptide chain as the therapeutic moiety. In some embodiments, the masking moiety is present in a different polypeptide chain than the therapeutic moiety.

[0032] In some embodiments, the masking moiety is present in the same polypeptide chain as the therapeutic moiety.

[0033] In some embodiments, the masking moiety is present in a first polypeptide chain and the therapeutic moiety is present in a second polypeptide chain.

[0034] In some embodiments, the drug construct comprises a half-life extending moiety.

[0035] In some embodiments, the half-life extending moiety comprises an antibody or fragment thereof.

[0036] In some embodiments, the half-life extending moiety comprises first and second half-life extending moieties.

[0037] In some embodiments, the prodrug is an interleukin prodrug, wherein the therapeutic moiety is an interleukin moiety.

[0038] In some embodiments, the masking portion comprises a domain of the extracellular domain of an interleukin receptor.

[0039] An interleukin prodrug as described herein, wherein the therapeutic moiety is an interleukin moiety, and the masking moiety comprises a domain of the extracellular domain of an interleukin receptor, referred to herein as a "masked cell interferons".

[0040] In some embodiments, provided herein is a masked interleukin comprising a masked portion in a first polypeptide chain and an interleukin portion thereof in a second polypeptide chain. Such masked interleukins may be referred to as "heterodimeric" masked interleukins.

[0041] In some embodiments, the masked cytokine comprises a protein heterodimer comprising: a) a first polypeptide chain comprising a masking moiety linked to a first half-life extending moiety via a first linker; and b) a second polypeptide chain comprising its cytokine portion linked to a second half-life-extending moiety via a second linker, wherein the first half-life-extending moiety is associated with the second half-life-extending moiety, and wherein at least the first The linker or second linker is a proteolytic peptide linker comprising a protein cleavable peptide (CP) consisting of the amino acid sequence DLLAVVAAS (SEQ ID NO: 248) or ISSGLLSGRS (SEQ ID NO: 249).

[0042] In some embodiments, in the first polypeptide chain, the first half-life extending domain is linked to the amino terminus of the first linker and the carboxy terminus of the first linker is linked to the amino terminus of the masking moiety, And in the second polypeptide chain, the second half-life extending domain is linked to the amino terminus of the second linker and the carboxy terminus of the second linker is linked to the amino terminus of the interleukin moiety thereof.

[0043] In some embodiments, the first polypeptide chain comprises: N'HL1-L1-MMC' and the second polypeptide chain comprises: N'HL2-L2-CC' wherein HL1 is the first half-life extending domain, L1 is the first linker, MM is the masking moiety, HL2 is the second half-life-extending domain, L2 is the second linker, and C is a cytokine moiety, wherein the first half-life-extending moiety is associated with the second half-life-extending moiety , and wherein at least the first linker or the second linker is a protein cleavable peptide comprising a protein cleavage peptide (CP) consisting of the amino acid sequence DLLAVVAAS (SEQ ID NO: 248) or ISSGLLSGRS (SEQ ID NO: 249) linker.

[0044] In some embodiments, the second linker is a protein cleavable linker and the first linker is a non-cleavable linker. This configuration is described herein as "Configuration A".

[0045] In some embodiments, the first polypeptide chain comprises: N'HL1-non-cleavable L1-MM C' and the second polypeptide chain comprises: N'HL2-cleavable L2-CC'

[0046] In some embodiments, the first linker is a protein cleavable linker and the second is a non-cleavable linker. This configuration is described herein as "Structure B".

[0047] In some embodiments, the first polypeptide chain comprises: N'HL1-cleavable L1-MM C' and the second polypeptide chain comprises: N'HL2-non-cleavable L2-CC'

[0048] In some embodiments, provided herein is a masked interleukin comprising a masking moiety and an interleukin portion thereof linked in a single polypeptide chain. In some embodiments, the masked cytokine comprises a polypeptide chain comprising the following formula: N' HL-L2-C-L1-MM C' wherein HL is a half-life-extending domain, L1 is a first linker, and MM is The masking part, L2 is the second linker, and C is the interleukin part, wherein at least the first linker comprises a proteolytically cleavable peptide linker comprising the amino acid sequence DLLAVVAAS (SEQ ID NO : 248) or a protein cleavage peptide (CP) consisting of ISSGLLSGRS (SEQ ID NO: 249). The protein cleavable peptide linker can be as described anywhere herein. In some embodiments, the first linker is a proteolytic peptide linker comprising a proteolytic peptide (CP) consisting of the amino acid sequence DLLAVVAAS (SEQ ID NO: 248). In some embodiments, the first linker is a proteolytic peptide linker comprising a proteolytic peptide (CP) consisting of the amino acid sequence ISSGLLSGRS (SEQ ID NO: 249). In some embodiments, the first linker is a protein cleavable peptide linker and the second linker is non-cleavable. A non-cleavable linker can be as described anywhere herein.

[0049] In some embodiments, the masked cytokine comprises a polypeptide chain comprising the formula: N' HL-L2-MM-L1-C C' wherein HL is a half-life extension domain and L1 is a first linker , MM is the masking part, L2 is the second linker, and C is its interleukin part, wherein at least the first linker comprises a proteolytically cleavable peptide linker comprising the amino acid sequence DLLAVVAAS (SEQ ID NO: 248) or a protein cleavage peptide (CP) consisting of ISSGLLSGRS (SEQ ID NO: 249). The protein cleavable peptide linker can be as described anywhere herein. In some embodiments, the first linker is a proteolytic peptide linker comprising a proteolytic peptide (CP) consisting of the amino acid sequence DLLAVVAAS (SEQ ID NO: 248). In some embodiments, the first linker is a proteolytic peptide linker comprising a proteolytic peptide (CP) consisting of the amino acid sequence ISSGLLSGRS (SEQ ID NO: 249). In some embodiments, the first linker is a protein cleavable peptide linker and the second linker is non-cleavable. A non-cleavable linker can be as described anywhere herein.

[0050] In some embodiments, the non-cleavable linker is 3 to 25 amino acids in length.

[0051] In some embodiments, wherein the non-cleavable linker is rich in amino acid residues G, S and P.

[0052] In some embodiments, the non-cleavable linker comprises the amino acid sequence of SEQ ID NO: 14.

[0053] In some embodiments, the non-cleavable linker comprises the amino acid sequence of SEQ ID NO: 23.

[0054] In some embodiments, the half-life-extending domain comprises a first half-life-extending domain and a second half-life-extending domain.

[0055] In some embodiments, the first half-life extending domain comprises a first Fc domain or a fragment thereof and the second Fc domain comprises an Fc domain or a fragment thereof.

[0056] In some embodiments, the first Fc domain comprises a CH3 domain or a fragment thereof and the second Fc domain comprises a CH3 domain or a fragment thereof.

[0057] In some embodiments, each of the first and second half-life-extending domains is an IgGl Fc domain or fragment thereof.

[0058] In some embodiments, each of the first and / or second Fc domain contains one or more modifications that facilitate non-covalent association of the first and second half-life-extending domains.

[0059] In some embodiments, the first half-life-extending domain comprises an IgG1 Fc domain or fragment thereof comprising mutations Y349C, T366S, L38A, and Y407V to form a "hole" in the first half-life-extending domain, and the second The second half-life-extending domain comprises an IgGl Fc domain or a fragment thereof comprising mutations S354C and T366W to form a "knob" in the second half-life-extending domain, numbered according to the Kabat EU numbering system.

[0060] In some embodiments, the first and second half-life extending domains are each an IgGl Fc domain or fragment thereof, and each comprise an amino acid substitution at position 297, numbered according to the Kabat EU numbering system.

[0061] In some embodiments, each of the first and second half-life-extending domains is an IgGl Fc domain or fragment thereof, and each comprises the amino acid substitution N297A, numbered according to the Kabat EU numbering system.

[0062] In some embodiments, the first and second half-life extending domains are each an IgGl Fc domain or fragment thereof, and each comprise an amino acid substitution at position 253, numbered according to the Kabat EU numbering system.

[0063] In some embodiments, each of the first and second half-life-extending domains is an IgGl Fc domain or fragment thereof, and each comprises the amino acid substitution I253A, numbered according to the Kabat EU numbering system.

[0064] In some embodiments, the first half-life-extending domain comprises the amino acid sequence of SEQ ID NO: 9, and its second half-life-extending domain comprises the amino acid sequence of SEQ ID NO: 12.

[0065] In some embodiments, the first half-life-extending domain comprises the amino acid sequence of SEQ ID NO: 10 and its second half-life-extending domain comprises the amino acid sequence of SEQ ID NO: 13.

[0066] In some embodiments, the half-life-extending domain (HL) comprises the Fc region (i.e., the C-terminal region of an immunoglobulin heavy chain) of an antibody comprising a dimeric Fc domain (HL1-HL2) or a fragment thereof . Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. In some embodiments, the dimeric Fc domain (HL1-HL2) of the antibody comprises a first half-life-extending domain and a second half-life-extending domain as described anywhere herein, wherein the first half-life-extending moiety comprises the first The Fc domain or fragment thereof and the second half-life extending moiety comprises a second Fc domain or fragment thereof. In some embodiments, HL2 is a component of the polypeptide chain and HL1 is dimerized with HL2.

[0067] In some embodiments, the first and second half-life extending moieties are each an IgGl Fc domain or fragment thereof. In some embodiments, the first half-life-extending moiety comprises an IgG1 Fc domain or fragment thereof comprising mutation I253A and the second half-life-extending moiety comprises an IgG1 Fc domain or fragment thereof comprising mutation I253A. In some embodiments, the first and second half-life-extending moieties are derived from the human IgG1 immunoglobulin constant γ1 sequence having SEQ ID NO: 6 (the "parental sequence") such that the first and second half-life-extending moieties Each comprises SEQ ID NO: 7 or a fragment thereof with one or more amino acid modifications. In some embodiments, the first and second half-life-extending moieties comprise SEQ ID NO: 7 with amino acid substitutions to facilitate association of the first and second half-life-extending moieties according to the "knob-in-the-hole" approach. In some embodiments, the sequence of SEQ ID NO: 7 contains the mutations Y349C, T366S, L38A, and Y407V (numbered according to the Kabat EU numbering system) to form the "hole" in the first half-life extending portion, and contains mutations S354C and T366W (numbered according to Kabat EU numbering system number) to form the "pestle" in the second half-life extension section. In some embodiments, each of the first and second half-life extending moieties further comprises the amino acid substitution N297A, numbered according to the Kabat EU numbering system. In some embodiments, each of the first and second half-life extending moieties further comprises the amino acid substitution I253A, numbered according to the Kabat EU numbering system. In some embodiments, each of the first and second half-life extending moieties further comprises both amino acid substitutions N297A and I253A, numbered according to the Kabat EU numbering system. In some embodiments, the first half-life extending moiety comprises about or at least about 85%, 86%, An amino acid sequence having 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the second half-life extending moiety comprises about or at least about 85%, 86%, An amino acid sequence having 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0068] In some embodiments, the cytokine moiety comprises a wild-type cytokine moiety or a variant cytokine moiety.

[0069] In some embodiments, the cytokine moiety is an IL-2 cytokine moiety as described anywhere herein.

[0070] In some embodiments, the IL-2 cytokine moiety comprises a wild-type IL-2 cytokine moiety or a variant thereof.

[0071] In some embodiments, the IL-2 cytokine portion comprises IL-2 cytokine or a fragment thereof.

[0072] In some embodiments, the IL-2 cytokine or functional fragment thereof is modified compared to the mature IL-2 sequence having SEQ ID NO:2.

[0073] In some embodiments, the modified IL-2 cytokine or functional fragment thereof comprises the modifications R38A, F42A, Y45A and E62A relative to the mature IL-2 sequence having SEQ ID NO: 2.

[0074] In some embodiments, the modified IL-2 cytokine or functional fragment thereof comprises the modification C125A relative to the mature IL-2 sequence having SEQ ID NO:2.

[0075] In some embodiments, relative to the mature IL-2 sequence having SEQ ID NO: 2, the modified IL-2 cytokine or functional fragment thereof comprises R38A, F42A, Y45A, E62A, and C125A.

[0076] In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 3.

[0077] In some embodiments, the masking moiety comprises IL-2R[beta] or a fragment, portion or variant thereof.

[0078] In some embodiments, IL-2Rβ or a fragment, portion or variant thereof comprises the amino acid sequence of SEQ ID NO:4.

[0079] In some embodiments, the IL-2Rβ or fragment, portion or variant thereof has a mutation at amino acid position C122 compared to IL-2β of SEQ ID NO:4.

[0080] In some embodiments, the IL-2Rβ or fragment, portion or variant thereof has a mutation at amino acid position C168 compared to IL-2β of SEQ ID NO:4.

[0081] In some embodiments, IL-2Rβ, or a fragment, portion or variant thereof, has mutations at amino acid positions C122 and C168 compared to IL-2β of SEQ ID NO:4.

[0082] In some embodiments, IL-2Rβ, or a fragment, portion or variant thereof, has mutations C122S and C168S compared to IL-2β of SEQ ID NO:4.

[0083] In some embodiments, wherein IL-2Rβ or its fragment, part or variant comprises the amino acid sequence of SEQ ID NO: 5.

[0084] In some embodiments, the cytokine moiety is an IL-12 cytokine moiety as described anywhere herein.

[0085] In some embodiments, the IL-12 cytokine moiety comprises a wild-type IL-12 cytokine moiety or a variant thereof.

[0086] In some embodiments, the IL-12 interleukin portion comprises IL-12 interleukin or a fragment thereof.

[0087] In some embodiments, the IL-12 interleukin or functional fragment thereof comprises an IL-12p40 polypeptide or functional fragment thereof covalently linked to an IL-12p35 polypeptide or functional fragment thereof.

[0088] In some embodiments, the IL-12p40-IL-12p35 linker is between 5 and 20 amino acids in length.

[0089] In some embodiments, the IL-12p40-IL-12p35 linker is enriched in amino acid residues G and S.

[0090] In some embodiments, the IL-12p40-IL-12p35 linker comprises SEQ ID NO: 116 (GGGGSGGGGSGGGGS).

[0091] In some embodiments, the IL-12p40 polypeptide comprises SEQ ID NO: 204 (as shown in the IL-12 cytokine part table in this specification) or the amino acid sequence corresponding to SEQ ID NO: 204 than an amino acid sequence having at least one amino acid modification (as shown in the IL-12 cytokine section table in this specification).

[0092] In some embodiments, the IL-12p40 polypeptide comprises SEQ ID NO: 204 (as shown in the IL-12 cytokines section of the specification).

[0093] In some embodiments, the IL-12p40 polypeptide comprises a GAG binding domain compared to the amino acid sequence of SEQ ID NO: 204 (as shown in the IL-12 cytokines section of the specification) (KSKREKKDRV (SEQ ID NO: 117)) at least one amino acid modification.

[0094] In some embodiments, the IL-12p40 polypeptide comprises SEQ ID NO: 205 (as shown in the IL-12 cytokines section of the specification).

[0095] In some embodiments, the IL-12p40 polypeptide comprises SEQ ID NO: 206 (as shown in the IL-12 cytokines section of the specification).

[0096] In some embodiments, the IL-12p40 polypeptide comprises an amino acid sequence having one or more cysteine ​​substitution mutations compared to the amino acid sequence of SEQ ID NO: 204 (as described herein IL-12 cytokines shown in the table).

[0097] In some embodiments, the IL-12p40 polypeptide comprises SEQ ID NO: 207 (as shown in the IL-12 cytokines section of the specification).

[0098] In some embodiments, the IL-12p40 polypeptide comprises SEQ ID NO: 208 (as shown in the IL-12 cytokines section of the specification).

[0099] In some embodiments, the IL-12p35 polypeptide comprises SEQ ID NO: 209 (as shown in the IL-12 cytokine part table in this specification) or the amino acid sequence corresponding to SEQ ID NO: 209 than an amino acid sequence having at least one amino acid modification (as shown in the IL-12 cytokine section table in this specification).

[0100] In some embodiments, the IL-12p35 polypeptide comprises SEQ ID NO: 209 (as shown in the IL-12 cytokines section of the specification).

[0101] In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises SEQ ID NO: 210 (as shown in the IL-12 cytokine section table in this specification).

[0102] In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises SEQ ID NO: 211 (as shown in the IL-12 cytokine section table in this specification).

[0103] In some embodiments, the IL-12 cytokine or functional fragment thereof comprises SEQ ID NO: 212 (shown in the IL-12 cytokine section table in this specification).

[0104] In some embodiments, the IL-12 cytokine or functional fragment thereof comprises SEQ ID NO: 213 (shown in the IL-12 cytokine section table in this specification).

[0105] In some embodiments, the IL-12 cytokine or functional fragment thereof comprises SEQ ID NO: 214 (shown in the IL-12 cytokine section of the specification).

[0106] In some embodiments, the masking moiety comprises IL-12 interleukin receptor or a subunit or functional fragment thereof.

[0107] 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 exhibits affinity for IL-12.

[0108] In some embodiments, the masked portion comprises residues 24 to 237 of human IL-12Rβ1, ie, the sequence having SEQ ID NO: 215 (shown in the IL-12 masked portion table in this specification).

[0109] In some embodiments, the masked portion comprises residues 24 to 545 of human IL-12Rβ1, ie, the sequence having SEQ ID NO: 216 (as shown in the IL-12 masked portion table in this specification).

[0110] In some embodiments, the masking portion comprises the extracellular domain of human IL-12Rβ2, or a fragment, portion or variant thereof, that retains or otherwise exhibits affinity for IL-12.

[0111] In some embodiments, the masked portion comprises residues 24 to 212 of human IL-12Rβ2, ie, the sequence having SEQ ID NO: 217 (shown in the IL-12 masked portion table in this specification).

[0112] In some embodiments, the masked portion comprises residues 24 to 222 of human IL-12Rβ2, i.e., the sequence having SEQ ID NO: 218 (as shown in the IL-12 masked portion table in this specification), Or the masked portion comprises residues 24 to 227 of human IL-12Rβ2, ie the sequence having SEQ ID NO: 222 (as shown in the IL-12 masked portion table in this specification).

[0113] In some embodiments, the masked portion comprises residues 24 to 319 of human IL-12Rβ2, ie, the sequence having SEQ ID NO: 219 (as shown in the IL-12 masked portion table in this specification).

[0114] In some embodiments, the masking moiety comprises at least one amino acid modification compared to the sequence of SEQ ID NO: 219 (as shown in the IL-12 Masking Moieties table in this specification), optionally wherein the Modifications such as cysteine ​​substitution mutations.

[0115] In some embodiments, the masking moiety comprises SEQ ID NO: 220 (as shown in the IL-12 Blocking Moieties table in this specification).

[0116] In some embodiments, the masked portion comprises residues 24 to 622 of human IL-12Rβ2, ie, the sequence having SEQ ID NO: 221 (as shown in the IL-12 masked portion table in this specification).

[0117] In some embodiments, the cytokine moiety is an IL-15 cytokine moiety as described anywhere herein.

[0118] In some embodiments, the IL-15 cytokine moiety comprises a wild-type IL-15 cytokine moiety or a variant thereof.

[0119] In some embodiments, the interleukin moiety is an IL-15 interleukin moiety, and the masked interleukin further comprises a domain comprising an IL-15Rα subunit or a functional fragment thereof ("IL- 15Rα domain"). In some embodiments, the interleukin moiety is an IL-15 interleukin moiety, and the masked interleukin further comprises a domain comprising an IL-15Rα subunit or a functional fragment thereof ("IL-15Rα domain ′), and the IL-15Rα domain and the IL-15 interleukin moiety are present in different polypeptide chains in the construct and the IL-15Rα domain is non-covalently linked to the IL-15 interleukin moiety.

[0120] The "IL-15Rα domain" herein may be substituted by the sequence of the wild-type sushi domain sIL-15Rα or a variant thereof, such as having one or more (eg 1, 2, 3 or 4) amino acids Sequence composition of the wild-type sushi domain sIL-15Rα. In some embodiments, the IL-15Rα domain comprises an amino acid substitution at position R26. In some embodiments, the IL-15Rα domain comprises the amino acid substitution R26N. In some embodiments, the IL-15Rα domain comprises the amino acid substitution R26S. In some embodiments, the IL-15Rα domain comprises an amino acid substitution at position R35. In some embodiments, the IL-15Rα domain comprises the amino acid substitution R35Q. In some embodiments, the IL-15Rα domain comprises the amino acid substitution R35S. In some embodiments, the IL-15Rα domain comprises amino acid substitutions at positions R26 and R35. In some embodiments, the IL-15Rα domain comprises the amino acid substitutions R26S or R26N and R35Q or R35S. In some embodiments, the IL-15Rα domain comprises the amino acid substitutions R26N and R35Q.

[0121] In some embodiments, the IL-15 cytokine portion comprises an IL-15 cytokine or a fragment thereof.

[0122] In some embodiments, the IL-15 cytokine or fragment thereof comprises SEQ ID NO: 224 (shown in the IL-15 cytokine section table in this specification) or a functional fragment thereof.

[0123] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 224 (shown in the IL-15 cytokine section of the specification).

[0124] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having at least one amino acid modification compared to the amino acid sequence of SEQ ID NO: 224 (as described in the specification IL-15 cytokines are shown in the table).

[0125] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises one or more amino acid substitutions at positions D22, E46, E53 compared to the amino acid sequence of SEQ ID NO:224 The amino acid sequence of (as shown in the IL-15 cytokine section table in this specification).

[0126] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having one or Amino acid sequences with multiple amino acid substitutions (as shown in the IL-15 cytokine section table in this specification).

[0127] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having amino acid substitutions at positions N71 and N79 compared to the amino acid sequence of SEQ ID NO: 224 ( As shown in the IL-15 cytokine section table in this specification).

[0128] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having amino acid substitutions at positions N71 and N112 compared to the amino acid sequence of SEQ ID NO: 224 ( As shown in the IL-15 cytokine section table in this specification).

[0129] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having amino acid substitutions at positions N79 and N112 compared to the amino acid sequence of SEQ ID NO: 224 ( As shown in the IL-15 cytokine section table in this specification).

[0130] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises amino acids having amino acid substitutions at positions N71, N79 and N112 compared to the amino acid sequence of SEQ ID NO:224 Sequence (as shown in the IL-15 cytokine section table in this specification).

[0131] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 225 (shown in the IL-15 cytokine section table in this specification).

[0132] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 226 (shown in the IL-15 cytokine section of the specification).

[0133] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 227 (shown in the IL-15 cytokine section of the specification).

[0134] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 228 (as shown in the IL-15 cytokine section table in this specification).

[0135] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 229 (shown in the IL-15 cytokine section of the specification).

[0136] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 230 (shown in the IL-15 cytokine section of the specification).

[0137] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 233 (shown in the IL-15 cytokine section table in this specification).

[0138] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 234 (shown in the IL-15 cytokine section of the specification).

[0139] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 235 (as shown in the IL-15 cytokine section table in this specification).

[0140] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 236 (as shown in the IL-15 cytokine section table in this specification).

[0141] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 237 (shown in the IL-15 cytokine section table in this specification).

[0142] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 238 (shown in the IL-15 cytokine section of the specification).

[0143] In some embodiments, the IL-15 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 239 (shown in the IL-15 cytokine section of the specification).

[0144] In some embodiments, the IL-15 interleukin or functional fragment thereof comprises an additional mutation at position N71.

[0145] In some embodiments, the IL-15 interleukin or functional fragment thereof comprises an additional mutation at position S73.

[0146] In some embodiments, the IL-15 interleukin or functional fragment thereof comprises additional mutations at one or more of amino acid positions N72, N79, V80, T81 and N112.

[0147] In some embodiments, the masking moiety comprises IL-15R[beta] or a fragment or variant thereof.

[0148] In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 240 (as shown in the IL-15 Masking Moieties table in this specification).

[0149] In some embodiments, the masking moiety comprises an IL-15Rβ variant or fragment thereof having an amino acid substitution at position C122.

[0150] In some embodiments, the masking moiety comprises an IL-15R[beta] variant or fragment thereof having the amino acid substitution C122S.

[0151] In some embodiments, the masking moiety comprises an IL-15Rβ variant or fragment thereof having an amino acid substitution at position C168.

[0152] In some embodiments, the masking moiety comprises an IL-15Rβ variant or fragment thereof having the amino acid substitution C168S.

[0153] In some embodiments, the masking moiety comprises an IL-15Rβ variant or fragment thereof having amino acid substitutions at positions C122 and C168.

[0154] Provided herein is a cleavage product capable of comprising an active therapeutic moiety prepared by protein cleavage of a protein cleavable linker in a polypeptide drug construct as described anywhere herein.

[0155] Provided herein is a nucleic acid encoding any of the polypeptide drug constructs described anywhere herein as described herein.

[0156] Provided herein is a nucleic acid encoding one of the chains of any of the polypeptide drug constructs described anywhere herein as described herein.

[0157] Provided herein is a vector comprising a nucleic acid described herein.

[0158] Provided herein is a vector comprising a nucleic acid encoding a polypeptide drug construct as described herein anywhere herein.

[0159] Provided herein is a vector comprising a nucleic acid encoding one of the chains of a polypeptide drug construct as described herein described anywhere herein.

[0160] Provided herein is a host cell comprising a nucleic acid described herein.

[0161] In one embodiment, the host cell is a HEK cell. In another embodiment, the host cell is a CHO cell.

[0162] Provided herein is a composition comprising any of the polypeptide drug constructs described anywhere herein as described herein.

[0163] Provided herein is a pharmaceutical composition comprising any of the polypeptide drug constructs described anywhere herein and a pharmaceutically acceptable carrier as described herein.

[0164] Provided herein is a kit comprising any of the polypeptide drug constructs as described anywhere herein or the compositions or pharmaceutical compositions described herein.

[0165] Provided herein is a method of making any of the polypeptide drug constructs as described anywhere herein, comprising culturing a host cell described herein under conditions for making the polypeptide drug construct.

[0166] Provided herein is a nucleic acid encoding any of the cleavage products described herein.

[0167] Provided herein is a composition comprising any of the cleavage products described herein.

[0168] Provided herein is a pharmaceutical composition comprising any of the cleavage products described herein and a pharmaceutically acceptable carrier.

[0169] Provided herein is a polypeptide drug construct as described herein for use in medicine.

[0170] Provided herein is a cleavage product as described herein for use in medicine.

[0171] Provided herein is a method of treating or preventing cancer in a subject, the method comprising administering to the subject an effective amount of a polypeptide drug construct as described herein.

[0172] Provided herein is a method of treating or preventing cancer in a subject, the method comprising administering to the subject an effective amount of a composition as described herein.

[0173] Provided herein is a method of treating or preventing cancer in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition as described herein.

[0174] Provided herein is a method of treating or preventing cancer in a subject, the method comprising administering to the subject an effective amount of the polypeptide drug construct as described herein, whereby the polypeptide drug construct is passed through the protein in vivo Cleavage to produce cleavage products as described herein.

[0175] Provided herein is a method of treating or preventing cancer in a subject, the method comprising the step of producing in vivo a cleavage product capable of binding to a protein of interest thereof, wherein the cleavage product is as described herein.

[0176] Provided herein is a polypeptide drug construct as described herein for use in the treatment or prevention of cancer.

[0177] Provided herein is a polypeptide drug construct as described herein for use in a method of treating or preventing cancer, the method comprising administering an effective amount of the polypeptide drug construct to a subject, whereby the polypeptide drug construct Protein cleavage in vivo produces cleavage products as described herein.

[0178] Provided herein is a cleavage product as described herein for use in the treatment or prevention of cancer.

[0179] Provided herein is a cleavage product as described herein for use in the treatment or prevention of cancer, the method comprising the step of administering to a patient a polypeptide drug construct as described herein, whereby interleukin Protein cleavage produces cleavage products.

[0180] Provided herein is a cleavage product as described herein for use in a method of treating or preventing cancer in a subject, the method comprising living a polypeptide drug construct as described herein by self-administering to the subject. The step in which proteins are cleaved in vivo to produce cleavage products.

Implementation

[0182] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application Serial Nos. 63 / 118,585, filed November 25, 2020, and 63 / 253,090, filed October 6, 2021; each of Incorporated herein by reference in its entirety.

[0183] 1. Polypeptide drug constructs The present invention provides novel tumor-specific proteolytically cleavable peptide linkers and their use in polypeptide drug constructs for delivery of therapeutic moieties to the tumor cell environment. A proteolytically cleavable peptide linker is located within the Polypeptide Drug Construct such that when the linker is cleaved by the action of proteases in the tumor cell environment, the Polypeptide Drug Construct dissociates. The invention also relates to cleavage products of these drug constructs and methods related to their use.

[0184] The protease substrate amino acid sequences DLLAVVAAS (SEQ ID NO: 248) and ISSGLLSGRS (SEQ ID NO: 249) have been found to exhibit very specific cleavage in the tumor cell environment as compared to the non-tumor cell environment. Thus, such proteolytic peptides can advantageously be used in proteolytic peptide linkers in polypeptide drug constructs, wherein any systemic side effects of the administered protein therapeutics can be reduced.

[0185] The protein cleavable peptide linker can be directly or indirectly bonded to the therapeutic moiety in the polypeptide drug construct. Where the polypeptide drug construct comprises more than one polypeptide chain, the protein cleavable peptide linker may be present in the same polypeptide chain as the therapeutic moiety or in a different polypeptide chain.

[0186] The portion of the construct other than the therapeutic portion may be considered a carrier portion. In cases where the proteolytic peptide linker is directly covalently bonded to the Therapeutic moiety, the proteolytic peptide linker will be located within the drug construct between the Therapeutic moiety and the carrier moiety. Alternatively, the protein cleavable peptide linker may be located within the carrier moiety such that the molecule isolated after cleavage comprises the therapeutic moiety and a portion of the carrier moiety.

[0187] A polypeptide drug construct comprising a tumor-specific protein cleavable peptide linker can be a prodrug. Where a tumor-specific cleavable linker is used in a prodrug to deliver the therapeutic moiety to the tumor cell environment, the remainder of the molecule from which the therapeutic moiety is isolated after cleavage may comprise a masking moiety that inhibits the therapeutic in the prodrug The biological activity of the moiety is such that the therapeutic moiety is only biologically active after cleavage of the proteolytic peptide linker in the tumor cell environment. The masking moiety can be present in the same polypeptide chain as the therapeutic moiety. Alternatively, the masking moiety can be present in the first polypeptide chain and the therapeutic moiety can be present in the second polypeptide chain. A protein cleavable peptide linker can be present in either the first or the second polypeptide chain.

[0188] By using a masking moiety, systemic side effects of an administered protein therapeutic can be reduced by interfering with the binding ability of the therapeutic. By masking the therapeutic agent with the proteolytic peptide linker, the binding ability interfered with by use of the masking moiety can be restored by cleavage of the protein cleavable peptide linker in the tumor microenvironment. Accordingly, provided herein are prodrugs engineered to precisely target pharmacological activity to the tumor microenvironment by exploiting one of the hallmarks of cancer, namely high local concentrations of active proteases. This feature of the tumor microenvironment serves to convert systemically inert molecules into locally active molecules in the form of cleavage products. Activation of the therapeutic moiety in the tumor microenvironment significantly reduces systemic toxicity that can be associated with the drug administered in active form to a subject.

[0189] In some embodiments, the Pharmaceutical Constructs provided herein comprise a half-life extending moiety. A long in vivo half-life is important for therapeutic proteins. Unfortunately, therapeutic agents administered to a subject can have a short half-life because they are usually rapidly cleared from the subject by mechanisms including renal clearance and endocytic degradation. Thus, in the drug constructs provided herein, a half-life extending moiety can be included for the purpose of increasing the half-life of the therapeutic moiety in vivo.

[0190] Protein cleavable peptide linkers The protein cleavable peptide linkers described herein comprise a protein cleavable peptide (CP) consisting of the amino acid sequence DLLAVVAAS (SEQ ID NO: 248) or ISSGLLSGRS (SEQ ID NO: 249) .

[0191] In some embodiments, the protein cleavable peptide linker is 9 to 25 amino acids in length.

[0192] In some embodiments, the protein cleavable peptide linker is 10 to 25 amino acids in length.

[0193] In some embodiments, the protein cleavable peptide linker is 12 to 18 amino acids in length.

[0194] In some embodiments, the proteolytic peptide linker comprises a proteolytic peptide (CP) flanked by spacer domains (SD1 and SD2) as follows: SD1-CP-SD2

[0195] In some embodiments, the protein cleavable peptide (CP) consists of the amino acid sequence DLLAVVAAS (SEQ ID NO: 248).

[0196] In some embodiments, the protein cleavable peptide (CP) consists of the amino acid sequence ISSGLLSGRS (SEQ ID NO: 249).

[0197] A spacer domain may consist of one or more amino acids. The function of the spacer domain (when present) is to link the protein cleavable peptide (CP) to other functional components in the constructs described herein.

[0198] It will be appreciated that the spacer domain does not alter the biological interaction of the proteolytic peptide with the protease in a tumor cell environment or in a non-tumor cell environment. In other words, the proteolytic peptides of the invention disclosed herein retain their favorable tumor specificity even in the presence of the spacer domain.

[0199] In some embodiments, the spacer domains flanking the proteolytically cleavable peptides differ.

[0200] In some embodiments, the spacer domain is enriched in amino acid residues G, S and P.

[0201] In some embodiments, the spacer domain includes only types of amino acid residues selected from the group consisting of G, S, and P.

[0202] In some embodiments, the first spacer domain (SD1) is between 3 and 10 amino acids in length. In some embodiments, the first spacer domain (SD1) is between 4 and 9 amino acids in length. In some embodiments, the first spacer domain (SD1) is between 3 and 6 amino acids in length.

[0203] Exemplary SD1 sequences are shown below: Sequence of SD1 GGPS (SEQ ID NO: 250) GSGPS (SEQ ID NO: 251) GSSGGP (SEQ ID NO: 252) GSP (SEQ ID NO: 253) GSGSPS (SEQ ID NO: 254)

[0204] In some embodiments, the first spacer domain (SD1) has a sequence as shown in the table above.

[0205] In some embodiments, the C-terminal sequence of SD2 is -GPC'.

[0206] In some embodiments, the sequence of the C-terminal of SD2 is SEQ ID NO: 29.

[0207] In some embodiments, the second spacer domain (SD2) is between 3 and 6 amino acids in length.

[0208] In some embodiments, SD2 comprises the amino acid sequence SGP (SEQ ID NO: 29).

[0209] In some embodiments, SD2 has the amino acid sequence SGP (SEQ ID NO: 29).

[0210] Exemplary combinations of SD1 and SD2 in cleavable linkers are shown below: linker structure SD1 sequence SD2 sequence SD1-CP-SD2 GGPS (SEQ ID NO: 250) SGP (SEQ ID NO: 29) SD1-CP-SD2 GSGPS (SEQ ID NO: 251) SGP (SEQ ID NO: 29) SD1-CP-SD2 GSSGGP (SEQ ID NO: 220) SGP (SEQ ID NO: 29) SD1-CP-SD2 GSP (SEQ ID NO: 253) SGP (SEQ ID NO: 29) SD1-CP-SD2 GSGSPS (SEQ ID NO: 254) SGP (SEQ ID NO: 29)

[0211] In some embodiments, the second spacer domain (SD2) has a sequence as shown in the table above.

[0212] In some embodiments, the protein cleavable linker comprises SD1-CP-SD2, wherein SD1 is the first spacer domain and CP is a cleavable peptide having the amino acid sequence DLLAVVAAS (SEQ ID NO: 248) , and SD2 is the second spacer domain. In some embodiments, the spacer domain is enriched in amino acid residues G, S and P. In some embodiments, the spacer domain includes only types of amino acid residues selected from the group consisting of G, S, and P. In some embodiments, SD2 has the amino acid sequence SGP (SEQ ID NO: 29).

[0213] In some embodiments, the protein cleavable linker comprises SD1-CP-SD2, wherein SD1 is the first spacer domain and CP is a cleavable peptide having the amino acid sequence ISSGLLSGRS (SEQ ID NO: 249) , and SD2 is the second spacer domain. In some embodiments, the spacer domain is enriched in amino acid residues G, S and P. In some embodiments, the spacer domain includes only types of amino acid residues selected from the group consisting of G, S, and P. In some embodiments, SD2 has the amino acid sequence SGP (SEQ ID NO: 29).

[0214] An exemplary cleavable linker using DLLAVVAAS (SEQ ID NO: 248) to cleave the peptide is shown below: Cleavable linker sequence (cleavable peptide shown in bold) GGPSDLLAVVAASSGP (SEQ ID NO: 245) GSGPSDLLAVVAASSGP (SEQ ID NO: 246) GSSGGPDLLAVVAASSGP (SEQ ID NO: 247) GSPDLLAVVAASSGP (SEQ ID NO: 242) GSPGDLLAVVAASSGP (SEQ ID NO: 243) GSGSPSSDLLAVVAASSGP (SEQ ID NO: 244) SGSDLLAVVAASSGPGSG (SEQ ID NO: 118) SGSPSGDLLAVVAASSGPGSGSP (SEQ ID NO: 119)

[0215] In some embodiments, the cleavable linker comprises the sequence GGPSDLLAVVAASSGP (SEQ ID NO: 245).

[0216] In some embodiments, the cleavable linker comprises the sequence GSGPSDLLAVVAASSGP (SEQ ID NO: 246).

[0217] In some embodiments, the cleavable linker comprises the sequence GSSGGPDLLAVVAASSGP (SEQ ID NO: 247).

[0218] In some embodiments, the cleavable linker comprises the sequence GSPDLLAVVAASSGP (SEQ ID NO: 242).

[0219] In some embodiments, the cleavable linker comprises the sequence GSPGDLLAVVAASSGP (SEQ ID NO: 243).

[0220] In some embodiments, the cleavable linker comprises the sequence GSGSPSDLLAVVAASSGP (SEQ ID NO: 244).

[0221] In some embodiments, the cleavable linker has the sequence GGPSDLLAVVAASSGP (SEQ ID NO: 245).

[0222] In some embodiments, the cleavable linker has the sequence GSGPSDLLAVVAASSGP (SEQ ID NO: 246).

[0223] In some embodiments, the cleavable linker has the sequence GSSGGPDLLAVVAASSGP (SEQ ID NO: 247).

[0224] In some embodiments, the cleavable linker has the sequence GSPDLLAVVAASSGP (SEQ ID NO: 242).

[0225] In some embodiments, the cleavable linker has the sequence GSPGDLLAVVAASSGP (SEQ ID NO: 243).

[0226] In some embodiments, the cleavable linker has the sequence GSGSPSDLLAVVAASSGP (SEQ ID NO: 244).

[0227] In some embodiments, the cleavable linker has the sequence SGSDLLAVVAASSGPGSG (SEQ ID NO: 118).

[0228] In some embodiments, the cleavable linker has the sequence SGSPSGDLLAVVAASSGPGSGSP (SEQ ID NO: 119).

[0229] Exemplary cleavable linkers using ISSGLLSGRS (SEQ ID NO: 249) to cleave peptides are shown below: Cleavable linker sequence (cleavable peptide shown in bold) GGSSGGSPISSGLLSGRSSGPGSGS (SEQ ID NO: 112) GPPSGSSPISSGLLSGRSSGGG (SEQ ID NO: 113) GGSGGSISSGLLSGRSSGP (SEQ ID NO: 114) GGSGGSGGSISSGLLSGRSSGP (SEQ ID NO: 115)

[0230] In some embodiments, the cleavable linker has the sequence GGSSGGSPISSGLLSGRSSGPGSGS (SEQ ID NO: 112).

[0231] In some embodiments, the cleavable linker has the sequence GPPSGSSPISSGLLSGRSSGGG (SEQ ID NO: 113).

[0232] In some embodiments, the cleavable linker has the sequence GGSGGSISSGLLSGRSSGP (SEQ ID NO: 114).

[0233] In some embodiments, the cleavable linker has the sequence GGSGGSGGSISSGLLSGRSSGP (SEQ ID NO: 115).

[0234] The linker combinations disclosed in the exemplary AK molecules can be used with any of the cytokine moieties disclosed herein. The linker combinations disclosed in the exemplary AK molecules can be used with any of the masking moieties disclosed herein. The linker combinations disclosed in the exemplary AK molecules can be used with any half-life extending moiety. In other words, the linkers disclosed in the exemplary AK molecules can be used in combination with any of the cytokine moieties disclosed herein, the masking moieties disclosed herein, and / or the half-life extending moieties disclosed herein.

[0235] Half-Life Extension Section A long in vivo half-life is important for therapeutic proteins.

[0236] The term "half-life extending moiety" encompasses eg PEG, albumin, antibodies and antibody fragments.

[0237] The half-life extending moiety may comprise an antibody or fragment thereof.

[0238] Antibodies or fragments thereof capable of FcRn-mediated recycling can reduce or otherwise delay clearance of a drug construct from a subject, thereby extending the half-life of the administered drug construct. In some embodiments, the antibody or fragment thereof is any antibody or fragment thereof capable of FcRn-mediated recycling, such as any heavy chain polypeptide or portion thereof (e.g., Fc domain or fragment thereof) capable of FcRn-mediated recycling .

[0239] The antibody or fragment thereof may be any antibody or fragment thereof. However, in some embodiments of a drug construct comprising a first half-life-extending moiety and a second half-life-extending moiety, the first half-life-extending moiety or the second half-life-extending moiety may comprise an antibody or fragment thereof that does not bind to the FcRn receptor, such as light chain polypeptides. For example, in some embodiments of the drug construct, the first half-life extending moiety comprises an antibody or fragment thereof comprising a light chain polypeptide or portion thereof that does not directly interact with the FcRn receptor, but the drug construct The body is attributed to the inclusion of a second half-life extending moiety capable of interacting with the FcRn receptor, such as by including a heavy chain polypeptide and still have an extended half-life. It is recognized in the art that FcRn-mediated recycling requires binding of the FcRn receptor to the Fc region of an 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 eg Firan, M. et al., Int. Immunol. 13 (2001) 993-1002; Shields, R.L. et al., J. Biol. Chem. 276 (2001) 6591-6604). Various mutants of residues 248-259, 301-317, 376-382 and 424-437 (numbered according to the Kabat EU index numbering system) have also been examined and reported. Yeung, Y.A. et al. (J. Immunol. 182 (2009) 7667-7671.

[0240] In some embodiments, the antibody or fragment thereof comprises a heavy chain polypeptide or a light chain polypeptide. In some embodiments, the antibody or fragment thereof comprises a heavy chain polypeptide or a portion of a light chain polypeptide. In some embodiments, the antibody or fragment thereof comprises an Fc domain or 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 the 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, an antibody or fragment thereof comprises a heavy chain polypeptide or fragment thereof (eg, an Fc domain or fragment thereof). In some embodiments, the antibody or fragment thereof comprises a light chain polypeptide.

[0241] In some embodiments, the first half-life-extending moiety comprises a first Fc domain or fragment thereof and the second half-life-extending moiety comprises a second Fc domain or fragment thereof.

[0242] In some embodiments, each of the first and / or second Fc domain contains one or more modifications that facilitate non-covalent association of the first and second half-life-extending moieties. In some embodiments, the first half-life-extending moiety comprises an IgG1 Fc domain or fragment thereof comprising mutations Y349C, T366S, L38A, and Y407V to form a "hole" in the first half-life-extending moiety, and the second half-life-extending moiety comprises IgGl Fc domain or fragment thereof comprising mutations S354C and T366W to form the "knob" in the second half-life extending moiety.

[0243] In some embodiments, each of the first and second half-life extending moieties is an IgGl, IgG2 or IgG4 Fc domain or fragment thereof. In some embodiments, each of the first and second half-life extending moieties is an IgGl Fc domain or fragment thereof. Human IgG1 immunoglobulin heavy constant γ1 has the sequence:

[0244] In some embodiments, the first and second half-life extending moieties are derived from a human IgG1 immunoglobulin heavy constant γ1 sequence having SEQ ID NO: 6 ("parental sequence") such that the first and second The half-life extending moieties each comprise SEQ ID NO: 6 or a fragment thereof having one or more amino acid modifications.

[0245] In some embodiments, the first and second half-life extending moieties each comprise the portion of SEQ ID NO: 6 shown above in bold, optionally with one or more amino acid modifications, i.e.:

[0246] In some embodiments, the first and second half-life-extending moieties comprise SEQ ID NO: 7 with amino acid substitutions to facilitate association of the first and second half-life-extending moieties according to the "knob-in-hole" approach. In some embodiments, the sequence of SEQ ID NO: 7 contains the mutations Y349C, T366S, L38A and Y407V (numbering is according to the Kabat EU numbering system) to form the "hole" in the first half-life extension part, and contains the mutations S354C and T366W ( Numbering is according to the Kabat EU numbering system) to form the "pestle" in the second half-life extension section. These modified sequences have SEQ ID NOs 8 and 11 shown below: First half-life extending moiety (Y349C, T366S, L38A and Y407V) SEQ ID NO 8: Second half-life extending moiety (S354C and T366W) SEQ ID NO 11:

[0247] In some embodiments, each of the first and second half-life extending moieties further comprises an amino acid substitution N297A, numbered according to the Kabat EU numbering system: First half-life extending moieties (Y349C, T366S, L38A, Y407V, and N297A) SEQ ID NO 9: Second half-life extending moiety (S354C, T366W and N297A) SEQ ID NO 12:

[0248] In some embodiments, each of the first and second half-life extending moieties further comprises an amino acid substitution I253A, numbered according to the Kabat EU numbering system.

[0249] In some embodiments, each of the first and second half-life extending moieties further comprises both amino acid substitutions N297A and I253A, numbered according to the Kabat EU numbering system: First half-life extending moieties (Y349C, T366S, L38A, Y407V, N297A and I253A) SEQ ID NO 10: Second half-life extending moiety (S354C, T366W, N297A and I253A) SEQ ID NO 13:

[0250] In some embodiments, the first half-life extending moiety comprises about or at least about 85% or at least about 85%, An amino acid sequence having 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0251] In some embodiments, the second half-life extending moiety comprises about or at least about 85% or at least about 85%, An amino acid sequence having 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0252] In some embodiments, the first half-life extending moiety comprises one or more modifications compared to the amino acid sequence of any one of SEQ ID NOs: 7, 8, 9 and 10, such as one or more Amino acid sequence with amino acid substitutions, additions or deletions. In some embodiments, the second half-life extending moiety comprises one or more modifications compared to the amino acid sequence of any one of SEQ ID NOs: 7, 11, 12 and 13, such as one or more amine groups Acid-substituted, added or deleted amino acid sequences. The one or more modifications can be any of the modifications or changes described herein, including, in some embodiments, those disclosed herein that promote heterodimerization of polypeptide chains and / or suppress homodimerization of polypeptide chains, alter effector function, or enhance Any modification or alteration of effector function.

[0253] In some embodiments, the Fc domain or fragment thereof comprises one or more amino acid substitutions that alter effector function. In some embodiments, the half-life extending moiety is an IgG1 Fc domain or fragment thereof and comprises 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 extending moiety is an IgG2 Fc domain or fragment thereof and comprises amino acid substitutions: V234A and G237A; H268Q, V309L, A330S and A331S; and / or V234A, G237A, P238S, H268A, V309L and A330S , numbered according to the Kabat EU numbering system. In some embodiments, the half-life extending moiety is an IgG2 Fc domain or fragment thereof and comprises one or more amino acid substitutions selected from the group consisting of: V234A, G237A, H268Q, V309L, A330S, A331S, P238S, H268A and V309L, numbered according to the Kabat EU numbering system. In some embodiments, the half-life extending moiety is an IgG4 Fc domain or fragment thereof and comprises amino acid substitutions: L235A, G237A and E318A; S228P, L234A and L235A; H268Q, V309L, A330S and P331S; and / or S228P and L235A, Numbering is according to the Kabat EU numbering system. In some embodiments, the half-life extending moiety is an IgG2 Fc domain or fragment thereof and comprises 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.

[0254] In some embodiments, the half-life extending moiety comprises an Fc domain or fragment thereof comprising one or more amino acid substitutions that enhance effector function. In some embodiments, the half-life extending moiety is an IgG1 Fc domain or fragment thereof and comprises amino acid substitutions: S298A, E333A and K334A; S239D and I332E; S239D, A330L and I332E; P247I and A339D or A339Q; D280H and K290S; D280H, K290S and S298D or S298V; F243L, R292P and Y300L; F243L, R292P, Y300L and P396L; F243L, R292P, Y300L, V305I and P396L; G236A, S239D and I332E; K326A and E3 33A; 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, E2 94L or S298T; E233L, Q311M and K334V; L234I, Q311M and K334V; K334V and S298T, A330M or A330F; K334V, Q311M and A330M or A330F; K334V, S298T and A330M or A330F; K334V, S239D and A330M or S298T; L234Y, Y2 96W and K290Y, F243V or E294L; Y296W and L234Y or K290Y; S239D, A330S and I332E, V264I; F243L and V264I; L328M; I332E; L328M and I332E; V264I and I332E; S239E and I332E; S239Q and I332E; 32D; L328I and I332E; L328Q and I332E; V264T; V240I; V266I; S239D; S239D and I332D; S239D and I332N; S239D and I332Q; S239E and I332D; 39Q 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 L3 28D, L328V, L328T or L328I; V264I, I332E and S239E or S239Q; S239E, V264I, A330Y and I332E; A330Y, I332E and S239D or S239N; A330L, I332E and S239D or S239N; V264I, S298A and I332E; S298A, I332E and S2 39D 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; 74Y; F275W; N276L; Y278T; V302I; E318R; S324D, S324I or S324V; K326I or K326T; T335D, T335R or T335Y; V240I and V266I; S239D, A330Y, I332E and L234I; 30Y, I332E and V240I; S239D, A330Y, I332E and V264T; and / or S239D, A330Y, I332E and K326E or K326T, numbered according to the Kabat EU numbering system. In some embodiments, the half-life extending moiety is an IgG1 Fc domain or fragment thereof and comprises one or more amino acid substitutions selected from the group consisting of: 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, 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.

[0255] In some embodiments, the half-life-extending moiety comprises one or more amino acid substitutions that enhance binding of the half-life-extending moiety to FcRn. In some embodiments, one or more amino acid substitutions increase the binding affinity of an Fc-containing polypeptide (eg, a heavy chain polypeptide or an Fc domain or fragment thereof) to FcRn at acidic pH. In some embodiments, the half-life extending moiety comprises 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; 34A; N434H; and V308P.

[0256] For manufacturing purposes, a signal peptide can be engineered upstream of the half-life domain to improve secretion of the protein. Signal peptides are selected according to cell line requirements as known in the art. It is understood that signal peptides do not appear to be part of the protein to be purified and formulated into a drug product.

[0257] 1.1.1 Heterodimerization Modifications The half-life-extending moieties described herein may include one or more modifications that promote heterodimerization of two different half-life-extending moieties. In some embodiments, it is desirable to promote heterodimerization of the first and second half-life-extending moieties to allow efficient production of the drug construct in its correct heterodimeric form. Thus, using any strategy available in the art, including any strategy as described in Klein et al. (2012), MAbs, 4(6): 653-663, the first half-life extending moiety can be subjected to one or more Amino acid modifications and one or more amino acid modifications can be made to the second half-life extending moiety. Exemplary strategies and modifications are described in detail below.

[0258] 1.1.2 Knob-in-the-hole pathway One strategy to promote heterodimerization of two different half-life-extending moieties is the so-called "knob-in-the-hole" pathway.

[0259] In some embodiments, the drug construct comprises a first half-life extending moiety and a second half-life extending moiety, each of which comprises a CH3 domain. In some embodiments, the half-life extending moiety comprises a CH3 domain that is a heavy chain polypeptide or fragment thereof (eg, an Fc domain or fragment thereof). The CH3 domains of the two half-life-extending moieties can be altered by the "knob-in-the-hole" technique, which is described in detail in several examples eg in WO 1996 / 027011; Ridgway, J.B. et al., Protein Eng. (1996) 9(7) : 617-621; Merchant, A.M. et al., Nat. Biotechnol. (1998) 16(7): 677-681. See also Klein et al. (2012), MAbs, 4(6): 653-663. Using the knob-in-hole approach, the interaction surface of the two CH3 domains was altered to increase the heterodimerization of the two half-life-extending moieties containing the two altered CH3 domains. This occurs by introducing a large residue into the CH3 domain of one of the half-life extending moieties to act as a "knob". Subsequently, to accommodate large residues, a "hole" that accommodates the pestle is formed in the other half-life extension section. Either of the altered CH3 domains can be a "knob", while the other can be a "hole". Introduction of disulfide bridges to further stabilize heterodimers (Merchant, A.M. et al., Nat. Biotechnol. (1998) 16(7); Atwell, S. et al., J. Mol. Biol. (1997) 270(1) : 26-35) and increased productivity.

[0260] It has been reported that a heterodimerization yield higher than 97% can be achieved by introducing the S354C and T366W mutations in the heavy chain to create a "knob" and by introducing the Y349C, T366S, L368A and Y407V mutations in the heavy chain to This was achieved by creating "holes" (residue numbering is according to the Kabat EU numbering system). Carter et al. (2001), J. Immunol. Methods, 248: 7-15; Klein et al. (2012), MAbs, 4(6): 653-663.

[0261] In some embodiments comprising a first half-life extending moiety and a second half-life extending moiety, the first half-life extending moiety comprises a heavy chain polypeptide or portion thereof (e.g., an Fc domain or fragment thereof) comprising an amino acid mutation S354C and T366W (numbering is according to the Kabat EU numbering system), and the second half-life extending portion comprises a heavy chain polypeptide or part thereof (such as an Fc domain or a fragment thereof) comprising amino acid mutations Y349C, T366S, L368A and Y407V ( Numbering is according to the Kabat EU numbering system). In some embodiments comprising a first half-life extending moiety and a second half-life extending moiety, the first half-life extending moiety comprises a heavy chain polypeptide or portion thereof (e.g., an Fc domain or fragment thereof) comprising the amino acid mutations Y349C, T366S , L368A and Y407V (numbering is according to the Kabat EU numbering system), and the second half-life extending portion comprises a heavy chain polypeptide or part thereof (such as an Fc domain or a fragment thereof) comprising amino acid mutations S354C and T366W (numbering is according to Kabat EU numbering system).

[0262] Additional examples of substitutions that may form the pestles and sockets include those described in US20140302037A1 (herein incorporated by reference). For example, in some embodiments, the following amines can be performed on a first half-life extending moiety ("first domain") and a paired second half-life extending moiety ("second domain") each containing an Fc domain Any of the amino acid substitutions: (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 the second domain T366Y in; (f) T366Y and F405A in the first domain and T394W and Y407T in the second domain; (g) T366W and F405 in the first domain and T394S and Y407A in the second domain; (h) F405W and Y407A in the first structural domain and T366W and T394S in the second structural domain; or (i) T366W in the first structural domain and T366S, L368A and Y407V in the second structural domain, the numbering system is according to Kabat EU numbering system.

[0263] In some embodiments, the following amine groups can be performed on the first half-life-extending moiety ("first domain") and paired second half-life-extending moiety ("second domain") each containing an Fc domain Any of the acid substitutions: (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 first domain; (d) F405W in the second domain and T394S in the first domain; (e) Y407T in the second domain and in the first domain (f) T366Y and F405A in the second domain and T394W and Y407T in the first domain; (g) T366W and F405 in the second domain and T394S and Y407A in the first domain; ( h) F405W and Y407A in the second domain and T366W and T394S in the first domain; or (i) T366W in the second domain and T366S, L368A and Y407V in the first domain, numbering according to Kabat EU numbering system.

[0264] In embodiments comprising a first half-life extending moiety and a second half-life extending moiety each comprising an Fc domain, any of the heterodimeric alterations described herein may be used in the Fc domain to facilitate the described herein. Heterodimers of any of the described drug constructs.

[0265] Therapeutic Moieties In some embodiments, provided herein is an interleukin prodrug, wherein the therapeutic moiety is an interleukin moiety. The masking moiety in the interleukin prodrug may comprise a domain of the extracellular domain of the interleukin receptor. Interleukin prodrugs can therefore be considered masked interleukins.

[0266] The cytokine moiety may comprise a wild-type cytokine moiety or a variant cytokine moiety.

[0267] Cytokines exemplified herein are IL-2, IL-12 and IL-15.

[0268] Interleukin Prodrugs Interleukins play a role in cell signaling, especially in cells of the immune system. Provided herein is an interleukin moiety comprising an interleukin (eg, IL-2, IL-15, or IL-12 interleukin) or a functional fragment thereof for use in a masked interleukin or cleavage product thereof.

[0269] 1.1 "Heterodimeric" masked interleukins In some embodiments, provided herein is a masked interleukin comprising a masked moiety in a first polypeptide chain and another masked moiety in a second polypeptide chain. Cytokines section. Such masked interleukins may be referred to as "heterodimeric" masked interleukins.

[0270] In some embodiments, the masked cytokine comprises a protein heterodimer comprising: c) a first polypeptide chain comprising a masking moiety linked to a first half-life extending moiety via a first linker; and d) a second polypeptide chain comprising its cytokine portion linked to a second half-life-extending moiety via a second linker, wherein the first half-life-extending moiety is associated with the second half-life-extending moiety, and wherein at least the first The linker or second linker is a proteolytic peptide linker comprising a protein cleavable peptide (CP) consisting of the amino acid sequence DLLAVVAAS (SEQ ID NO: 248) or ISSGLLSGRS (SEQ ID NO: 249).

[0271] In some embodiments, the first linker is a protein cleavable peptide comprising a protein cleavage peptide (CP) consisting of the amino acid sequence DLLAVVAAS (SEQ ID NO: 248) or ISSGLLSGRS (SEQ ID NO: 249) linker. The protein cleavable peptide linker can be as described anywhere herein. In some embodiments, the first linker is a proteolytic peptide linker comprising a proteolytic peptide (CP) consisting of the amino acid sequence DLLAVVAAS (SEQ ID NO: 248). In some embodiments, the first linker is a proteolytic peptide linker comprising a proteolytic peptide (CP) consisting of the amino acid sequence ISSGLLSGRS (SEQ ID NO: 249). In some embodiments, the first linker is a protein cleavable peptide linker and the second linker is a non-cleavable linker. A non-cleavable linker can be as described anywhere herein.

[0272] In some embodiments, the second linker is a protein cleavable peptide comprising a protein cleavable peptide (CP) consisting of the amino acid sequence DLLAVVAAS (SEQ ID NO: 248) or ISSGLLSGRS (SEQ ID NO: 249) linker. The protein cleavable peptide linker can be as described anywhere herein. In some embodiments, the second linker is a proteolytic peptide linker comprising a proteolytic peptide (CP) consisting of the amino acid sequence DLLAVVAAS (SEQ ID NO: 248). In some embodiments, the second linker is a proteolytic peptide linker comprising a proteolytic peptide (CP) consisting of the amino acid sequence ISSGLLSGRS (SEQ ID NO: 249). In some embodiments, the second linker is a proteolytic peptide linker and the first linker is non-cleavable. A non-cleavable linker can be as described anywhere herein.

[0273] The protein cleavable peptide linker can be as described anywhere herein.

[0274] The half-life extending moiety can be as described anywhere herein.

[0275] The combination of masking moieties and interleukin moieties can be as described anywhere herein.

[0276] In some embodiments, in the first polypeptide chain, the first half-life extending domain is linked to the amino terminus of the first linker and the carboxy terminus of the first linker is linked to the amino terminus of the masking moiety, And in the second polypeptide chain, the second half-life extending domain is linked to the amino terminus of the second linker and the carboxy terminus of the second linker is linked to the amino terminus of the interleukin moiety thereof.

[0277] In some embodiments, the first polypeptide chain comprises: N'HL1-L1-MMC' and the second polypeptide chain comprises: N'HL2-L2-CC' wherein HL1 is the first half-life extending domain, L1 is the first linker, MM is the masking moiety, HL2 is the second half-life extending domain, L2 is the second linker, and C is the cytokine moiety.

[0278] In some embodiments, the second linker is a protein cleavable linker and the first linker is a non-cleavable linker. This configuration is described herein as "Configuration A". In some embodiments, the first polypeptide chain comprises: N'HL1-non-cleavable L1-MM C' and the second polypeptide chain comprises: N'HL2-cleavable L2-CC'

[0279] In some embodiments, the first linker is a protein cleavable linker and the second is a non-cleavable linker. This configuration is described herein as "Structure B". In some embodiments, the first polypeptide chain comprises: N'HL1-cleavable L1-MM C' and the second polypeptide chain comprises: N'HL2-non-cleavable L2-CC'

[0280] 1.2 "Linear" masked interleukins In some embodiments, provided herein is a masked interleukin comprising a masked portion and the interleukin portion thereof linked in a single polypeptide chain. In some embodiments, the masked cytokine comprises a polypeptide chain comprising the following formula: N' HL-L2-C-L1-MM C' wherein HL is a half-life-extending domain, L1 is a first linker, and MM is Masked moieties, L2 is the second linker, and C is the cytokine moiety, wherein at least the first linker comprises a proteolytic peptide.

[0281] In some embodiments, the masked interleukin comprises a polypeptide chain comprising the formula: N' HL-L2-MM-L1-C C' wherein HL is the half-life extending domain and L1 is the first linker , MM is the masking moiety, L2 is the second linker, and C is its interleukin moiety, wherein at least the first linker comprises a proteolytic peptide. In some embodiments, the first linker is a cleavable linker as described anywhere herein. In some embodiments, the second linker is a non-cleavable linker as described anywhere herein. In some embodiments, the cytokine moiety is as described anywhere herein. In some embodiments, the half-life extending domain (HL) comprises the Fc region of an antibody (ie, the C-terminal region of an immunoglobulin heavy chain) or a fragment thereof comprising a dimeric Fc domain (HL1-HL2). Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position Cys226 or from Pro230 to its carboxy-terminus. In some embodiments, the dimeric Fc domain (HL1-HL2) of the antibody comprises a first half-life-extending domain and a second half-life-extending domain as described anywhere herein, wherein the first half-life-extending moiety comprises a first The Fc domain or fragment thereof and the second half-life extending moiety comprises a second Fc domain or fragment thereof. In some embodiments, HL2 is a component of the polypeptide chain and HL1 is dimerized with HL2.

[0282] 1.1 Interleukin Moieties and Masking Moieties The interleukin moieties and masking moieties disclosed herein (e.g., IL-2, IL-12, and IL-15 interleukin and masking moieties) can be used in any of the In the polypeptide drug construct.

[0283] The interleukin moieties and masking moieties disclosed herein can be used in heterodimeric masked interleukins of Structure A as disclosed herein.

[0284] The interleukin moieties and masking moieties disclosed herein can be used in a heterodimeric masked interleukin of Structure B as disclosed herein.

[0285] The interleukin moieties and masking moieties disclosed herein can be used with linear masked interleukins as disclosed herein.

[0286] 1.1.1 IL-2 cytokine part and IL-2 masking part (a) IL-2 cytokine part

[0287] In some embodiments, the therapeutic moiety comprises an IL-2 cytokine or a functional fragment thereof.

[0288] IL-2 is an interleukin, a type of interleukin signaling molecule that regulates the activity of white blood cells in the immune system.

[0289] In eukaryotic cells, naturally occurring IL-2 is synthesized as a 153 amino acid precursor polypeptide having SEQ ID NO: 1 which is subsequently processed by removing amino acid residues 1-20 into mature IL-2. This results in a mature form of IL-2 consisting of 133 amino acids (amino acid residues 21-153), which has SEQ ID NO:2. A "functional fragment" of an IL-2 interleukin comprises a portion of the full-length interleukin protein that retains or has an altered interleukin receptor binding ability (e.g., at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% active). Interleukin receptor binding ability can be demonstrated, for example, in the ability of an interleukin to bind to an interleukin cognate receptor or a component thereof (eg, one or more chains of a heterotrimeric receptor complex).

[0290] In some embodiments, the IL-2 interleukin or functional fragment thereof is any naturally occurring interleukin-2 (IL-2 ) protein or a modified variant thereof. In the case of IL-2 cytokine binding, the target protein can be IL-2R (including IL-2Rα, IL-2Rβ and IL-2Rγ chain), IL-2Rα chain, IL-2Rβ chain or IL-2Rα / β Dimeric complex. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises the amino acid sequence of amino acid residues 21-153 of SEQ ID NO: 1. In some embodiments, the IL-2 polypeptide or functional fragment thereof comprises the amino acid sequence of mature IL-2, SEQ ID NO: 2.

[0291] In some embodiments, the IL-2 cytokine or functional fragment thereof 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 can be any amino acid modification, such as substitution, insertion or deletion. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises at least 1, at least 2, at least 3, at least 4, at least 5 amino acid sequences compared with the amino acid sequence of SEQ ID NO: 2 , an amino acid sequence with at least 6, at least 7, at least 8, at least 9 or at least 10 amino acid substitutions. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 5 amino acid substitutions compared with the amino acid sequence of SEQ ID NO: 2.

[0292] In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amine having one or more amino acid substitutions compared to the amino acid sequence of wild-type IL-2 of SEQ ID NO: 2 An amino acid sequence that reduces the affinity of IL-2 peptides or functional fragments thereof for IL-2Rα (CD25). In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions compared with the amino acid sequence of SEQ ID NO: 2, such that the amino acid One or more of residues 38, 42, 45 and 62 are alanine (A). In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions compared with the amino acid sequence of SEQ ID NO: 2, such that the amino acid Residues 38, 42, 45 and 62 are alanine (A).

[0293] In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence substitution C125A compared to the amino acid sequence of SEQ ID NO:2.

[0294] In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 2, such that Amino acid residues 38, 42, 45 and 62 are alanine (A) and amino acid residue 125 is alanine (A). In some embodiments, the IL-2 cytokine or functional fragment thereof comprises amino acids having amino acid residues R38, F42, Y45 and E62 replacing alanine in the amino acid sequence of SEQ ID NO: 2 sequence. In some embodiments, IL-2 cytokines or functional fragments thereof comprise amino acid residues R38, F42, Y45 and E62 with substitutions for alanine (A) and substitutions in the amino acid sequence of SEQ ID NO: 2 The amino acid sequence of amino acid residue C125 of alanine (A).

[0295] In some embodiments, the IL-2 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90% of the amino acid sequence of SEQ ID NO: 3 An amino acid sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0296] In some embodiments, for the purpose of removing O-glycosylation sites, IL-2 cytokines or functional fragments thereof are compared with the amino acid sequence of mature IL-2 of SEQ ID 2 One or more amino acid residues are removed, for example residues 1-3. In some embodiments, for the purpose of removing O-glycosylation sites, IL-2 cytokines or functional fragments thereof are substituted with one or more amino acid sequences compared with the mature IL-2 amino acid sequence of SEQ ID 2 amino acid residues. In some embodiments, for the purpose of removing O-glycosylation sites, IL-2 cytokines or functional fragments thereof are compared with the amino acid sequence of mature IL-2 of SEQ ID 2, for example at residues One or more amino acid residues are inserted in the region 1-3. In some embodiments, the IL-2 cytokine or functional fragment thereof does not have an O-glycosylation site within residues 1-3.

[0297] (b) IL-2 Masking Moieties Provided herein is a masking moiety for masking a therapeutic moiety comprising the IL-2 cytokine or a functional fragment thereof.

[0298] It is understood that the masked moiety is cleaved from the masked cytokine to form its cleavage product. The masking part in the shielded interleukin shields the IL-2 cytokine or its functional fragment, thereby reducing or preventing the binding of the IL-2 cytokine or its functional fragment to its cognate receptor. In some embodiments, the masking moiety reduces or prevents the binding of IL-2 cytokine or functional fragment thereof to IL-2Rα (CD25). In some embodiments, a masking moiety as provided herein refers to an IL-2 cytokine capable of binding to or a functional fragment thereof (such as an anti-IL-2 antibody or IL-2 cognate receptor protein) or otherwise exhibiting part of affinity. Methods for determining the degree of binding of a protein (eg, an interleukin) to a cognate protein (eg, an interleukin receptor) are well known in the art.

[0299] In some embodiments, the masking moiety comprises an IL-2 interleukin receptor or a subunit or functional fragment thereof.

[0300] In some embodiments, the masking moiety comprises IL-2Rβ (also known as CD122) or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-2.

[0301] In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:4. In some embodiments, the masking portion comprises about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% of the amino acid sequence of SEQ ID NO: 4. %, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences. In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 4 with 1 to 4 amino acid substitutions. In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 4 with one or two amino acid substitutions.

[0302] In some embodiments, the IL-2Rβ or fragment, portion or variant thereof has a mutation at amino acid position C122 compared to IL-2Rβ of SEQ ID NO:4.

[0303] In some embodiments, the IL-2Rβ or fragment, portion or variant thereof has the mutation C122S at amino acid position 122 compared to IL-2Rβ of SEQ ID NO: 4.

[0304] In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO: 4 with a C122 mutation.

[0305] In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO: 4 with a C122S mutation.

[0306] In some embodiments, the IL-2Rβ or fragment, portion or variant thereof has a mutation at amino acid position C168 compared to IL-2Rβ of SEQ ID NO:4.

[0307] In some embodiments, the IL-2Rβ or fragment, portion or variant thereof has the mutation C168S at amino acid position 168 compared to IL-2Rβ of SEQ ID NO:4.

[0308] In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO: 4 with a C168 mutation.

[0309] In some embodiments, the masking portion comprises the amino acid sequence of SEQ ID NO: 4 with a C168S mutation.

[0310] In some embodiments, the IL-2Rβ or fragment, portion or variant thereof has mutations at amino acid positions C122 and C168 compared to IL-2Rβ of SEQ ID NO:4.

[0311] In some embodiments, the IL-2Rβ or fragment, portion or variant thereof has the mutations C122S and C168S compared to IL-2Rβ of SEQ ID NO:4.

[0312] In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO:5.

[0313] In some embodiments, when (i) the masked interleukin is a structure A heterodimeric masked interleukin and (ii) the interleukin moiety is an IL-2 interleukin moiety, then the protein The cleavage peptide linker does not have the amino acid sequence GGSGISSGLLSGRSSSGP (SEQ ID NO: 120) or GISSGLLSGRSSSGP (SEQ ID NO: 121).

[0314] 1.1.2 IL-12 interleukin part and IL-12 masking part (a) IL-12 interleukin part

[0315] In some embodiments, the therapeutic moiety comprises the IL-12 cytokine or a functional fragment thereof.

[0316] IL-12 is an interleukin, a type of interleukin signaling molecule that regulates the activity of white blood cells in the immune system.

[0317] Endogenous IL-12 exists as two distinct molecules, IL-12 p40 and IL-12 p35, which dimerize in cells during biosynthesis.

[0318] The full sequences of IL-12 p40 and IL-12 p35 are (peptides shown in bold before cleavage during biosynthesis): IL-12 p40 subunit (SEQ ID NO: 122): IL-12 p35 subunit (SEQ ID NO: 123):

[0319] The mature forms are as follows: IL-12 p40 subunit (SEQ ID NO: 124): IL-12 p35 subunit (SEQ ID NO: 125):

[0320] It appears as two chains that covalently dimerize during biosynthesis via a disulfide bond that binds between two subunits: cysteine ​​C199 of the p40 subunit and half of the p35 subunit Cystine C96 association.

[0321] A "functional fragment" of an IL-12 interleukin comprises a portion of the full-length interleukin protein that retains or has an altered interleukin receptor binding ability (e.g., at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% active). Interleukin receptor binding ability can be demonstrated, for example, by the ability of an interleukin to bind to a cognate receptor for an interleukin or a component thereof.

[0322] In some embodiments, the IL-12 interleukin or functional fragment thereof is any naturally occurring interleukin-2 (IL-12) protein or a modified protein thereof capable of binding to the interleukin-12 receptor variants.

[0323] In some embodiments, the IL-12 polypeptide or functional fragment thereof comprises an IL-12p40 polypeptide or functional fragment thereof covalently linked to an IL-12p35 polypeptide or functional fragment thereof.

[0324] An IL-12p40 polypeptide or functional fragment thereof may be attached to a first half-life extending domain such that the first polypeptide chain comprises the formula: N'HL1-L1-MMC' and the second polypeptide chain comprises the formula: N' HL2-L2-[IL-12p40-linker-IL-12p35] C' where "IL-12p40" is IL-12p40 polypeptide or its functional fragment and "IL-12p35" is IL-12p35 polypeptide or its functional fragment .

[0325] In some embodiments, the IL-12p40 polypeptide comprises SEQ ID NO: 204 as shown in the IL-12 Cytokines section table below. In some embodiments, the IL-12p40 polypeptide comprises an amino acid sequence with at least one amino acid modification compared to the amino acid sequence of SEQ ID NO: 204 shown in the IL-12 interleukins section table below. Each of the at least one amino acid modification can be any amino acid modification, such as substitution, insertion or deletion. In some embodiments, the IL-12 cytokine or functional fragment thereof comprises at least 1, at least 2 Amino acid sequences substituted with at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 amino acids. In some embodiments, the IL-12 cytokine or functional fragment thereof comprises at least 5 amino acids compared to the amino acid sequence of SEQ ID NO: 204 shown in the IL-12 interleukin section table below Substituted amino acid sequence.

[0326] The IL-12p40 polypeptide comprises a glycosaminoglycan (GAG)-binding domain (KSKREKKDRV (SEQ ID NO: 117)). GAGs such as heparan sulfate and heparan sulfate have been shown to bind a number of growth factors and cytokines, including IL-12. The physiological significance of this binding was twofold. First, GAGs can serve as coreceptors on the cell surface to maintain high local concentrations of cytokines. Second, GAGs can regulate the biological activity of growth factors and cytokines through multiple mechanisms, including dimerization and avoidance of proteolytic degradation.

[0327] The GAG ​​binding domain in the mature form of the IL-12 p40 subunit is shown below in bold (SEQ ID NO: 124):

[0328] Modifications of the GAG ​​binding domain (KSKREKKDRV (SEQ ID NO: 117)) have been shown herein to enhance the IL-12 interleukin comprising a mutated GAG binding domain without any reduction in interleukin activity. The PK characteristic curve of the construct. Accordingly, in some embodiments, the IL-12p40 polypeptide comprises at least one amino acid modification of the GAG ​​binding domain. In some embodiments, the GAG ​​binding domain is modified as a deletion mutation. In some embodiments, the GAG ​​binding domain is modified with a deletion mutation and at least one substitution mutation.

[0329] In some embodiments, the GAG ​​binding domain comprises the amino acid sequence KDNTERV (SEQ ID NO: 126). In some embodiments, the IL-12p40 polypeptide comprises the amino acid sequence SEQ ID NO: 205 shown in the following IL-12 cytokine section table. In some embodiments, the GAG ​​binding domain comprises the amino acid sequence KDNTEGRV (SEQ ID NO: 127). In some embodiments, the IL-12p40 polypeptide comprises the amino acid sequence SEQ ID NO: 206 shown in the following IL-12 cytokine section table.

[0330] In some embodiments, the GAG ​​binding domain consists of the amino acid sequence KDNTERV (SEQ ID NO: 126). In some embodiments, the IL-12p40 polypeptide comprises the amino acid sequence SEQ ID NO: 205 shown in the following IL-12 cytokine section table. In some embodiments, the GAG ​​binding domain consists of the amino acid sequence KDNTEGRV (SEQ ID NO: 127). In some embodiments, the IL-12p40 polypeptide comprises the amino acid sequence SEQ ID NO: 206 shown in the following IL-12 cytokine section table.

[0331] In some embodiments, the IL-12p40 polypeptide comprises one or more cysteine ​​substitutions compared to the amino acid sequence of SEQ ID NO: 204 shown in the following IL-12 cytokine section table the amino acid sequence. In some embodiments, the IL-12p40 polypeptide comprises an amino acid having an amino acid substitution at position C252 compared to the amino acid sequence of SEQ ID NO: 204 shown in the IL-12 interleukin section table below sequence. 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: 207 shown in the following IL-12 cytokine section table. In some embodiments, the IL-12p40 polypeptide comprises about or at least about 85%, 86%, 87%, 88% of the amino acid sequence of SEQ ID NO: 207 shown in the following IL-12 cytokine part table %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity of amino acid sequences. In some embodiments, the IL-12p40 polypeptide consists of the amino acid sequence of SEQ ID NO: 207 shown in the following IL-12 cytokine section table.

[0332] In some embodiments, the IL-12p40 polypeptide comprises one or more cysteine ​​substitutions compared to the amino acid sequence of SEQ ID NO: 204 shown in the IL-12 interleukin section table below and at least one amino acid modified amino acid sequence of 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: 204 shown in the IL-12 interleukin section table below, and the GAG ​​binding structure The domain comprises the amino acid sequence KDNTERV (SEQ ID NO: 126). 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: 204 shown in the IL-12 interleukin section table below, and the GAG ​​binding structure The domain comprises the amino acid sequence KDNTEGRV (SEQ ID NO: 127). In some embodiments, the IL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO: 208 shown in the following IL-12 cytokine section table. In some embodiments, the IL-12p40 polypeptide comprises about or at least about 85%, 86%, 87%, 88% of the amino acid sequence of SEQ ID NO: 208 shown in the following IL-12 cytokine part table %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity of amino acid sequences. In some embodiments, the IL-12p40 polypeptide consists of the amino acid sequence of SEQ ID NO: 208 shown in the following IL-12 cytokine section table.

[0333] In some embodiments, the IL-12p35 polypeptide comprises SEQ ID NO: 209 as shown in the IL-12 Cytokines section table below. In some embodiments, the IL-12p35 polypeptide comprises an amino acid sequence with at least one amino acid modification compared to the amino acid sequence of SEQ ID NO: 209 shown in the IL-12 interleukins section table below. Each of the at least one amino acid modification can be any amino acid modification, such as substitution, insertion or deletion. In some embodiments, the IL-12 cytokine or functional fragment thereof comprises at least 1, at least 2 Amino acid sequences substituted with at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 amino acids. In some embodiments, the IL-12 cytokine or functional fragment thereof comprises at least 5 amino acids compared to the amino acid sequence of SEQ ID NO: 209 shown in the IL-12 interleukin section table below Substituted amino acid sequence.

[0334] In some embodiments, the IL-12p40-IL-12p35 linker is between 5 and 20 amino acids in length.

[0335] In some embodiments, the IL-12p40-IL-12p35 linker is enriched in amino acid residues G and S.

[0336] In some embodiments, the IL-12p40-IL-12p35 linker includes only types of amino acid residues selected from the group consisting of G and S.

[0337] In some embodiments, the IL-12p40-IL-12p35 linker comprises [(G)nS], wherein n=4 or 5 (SEQ ID NO: 435).

[0338] In some embodiments, the IL-12p40-IL-12p35 linker comprises a (GGGGS (SEQ ID NO: 277)) repeat sequence.

[0339] In some embodiments, the IL-12p40-IL-12p35 linker comprises SEQ ID NO:116. (GGGGSGGGGSGGGGS)

[0340] In some embodiments, the IL-12 interleukin or functional fragment thereof comprises SEQ ID NO: 210 as shown in the IL-12 interleukin section table below. In some embodiments, the IL-12 cytokine or functional fragment thereof comprises at least one amine group compared to the amino acid sequence of SEQ ID NO: 204 and 209 shown in the IL-12 interleukin section table below Acid-modified amino acid sequences. Each of the at least one amino acid modification can be any amino acid modification, such as substitution, insertion or deletion. In some embodiments, the IL-12 cytokine or functional fragment thereof comprises at least 1, An amino acid sequence in which at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 amino acids are substituted. In some embodiments, the IL-12 cytokine or functional fragment thereof comprises at least 5 amines compared to the amino acid sequence of SEQ ID NO: 204 and 209 shown in the IL-12 interleukin section table below A sequence of amino acid substitutions.

[0341] In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 210 shown in the IL-12 interleukin section table below. In some embodiments, the IL-12 cytokine or functional fragment thereof comprises about or at least about 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity of amino acid sequences.

[0342] In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 211 shown in the IL-12 interleukin section table below. In some embodiments, the IL-12 cytokine or functional fragment thereof comprises about or at least about 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity of amino acid sequences.

[0343] In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 212. In some embodiments, the IL-12 cytokine or functional fragment thereof comprises about or at least about 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity of amino acid sequences.

[0344] In some embodiments, the IL-12 cytokine or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 213 shown in the IL-12 interleukin section table below. In some embodiments, the IL-12 cytokine or functional fragment thereof comprises about or at least about 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity of amino acid sequences.

[0345] In some embodiments, the IL-12 interleukin or functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 214 shown in the IL-12 interleukin section table below. In some embodiments, the IL-12 cytokine or functional fragment thereof comprises about or at least about 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity of amino acid sequences. Table - IL-12 Cytokines Section: components SEQ ID NO sequence hIL12B IL-12 p40 subunit 204 IL-12 p40 Subunit [KDNTERV] IL-12 p40 Subunit [KDNTEGRV] 205 IL-12 p40 Subunit [C252S] 206 207 p40 Subunit [KDNTE GRV] + [C252S] 208 hIL12A IL-12 p35 Subunit 209 interleukin domain hIL12B-hIL12A 210 hIL12B-hIL12A [KDNTERV] 211 hIL12B-hIL12A [KDNTEGRV] 212 hIL12B-hIL12A [C252S] 213 hIL12B-hIL12A [KDNTE GRV] + [C252S] 214

[0346] In some embodiments, the IL-12 interleukin moiety has an amino acid sequence as set forth in one of the sequences in the table above.

[0347] (b) IL-12 Masking Moieties Provided herein is a masking moiety for masking a therapeutic moiety comprising the IL-12 cytokine or a functional fragment thereof.

[0348] It is understood that the masked moiety is cleaved from the masked cytokine to form its cleavage product. In the masked interleukin, the masking part masks the IL-12 cytokine or its functional fragment, thereby reducing or preventing the binding of IL-12 or its functional fragment to its cognate receptor.

[0349] IL-12 receptor β1 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) with low affinity. This protein forms disulfide-linked oligomers, which are required for its IL-12 binding activity. IL-12 receptor β2 or IL-12Rβ2 is a subunit of the IL-12 receptor complex. Co-expression of IL-12Rβ1 and IL-12Rβ2 proteins has been shown to result in the formation of high affinity IL-12 binding sites.

[0350] Methods for determining the degree of binding of a protein (eg, an interleukin) to a cognate protein (eg, an interleukin receptor) are well known in the art.

[0351] In some embodiments, the masking moiety comprises the extracellular domain of an IL-12 interleukin receptor or subunit or functional fragment thereof.

[0352] Interleukin-12 receptor subunit beta-1 (also known as CD212) has the following sequence: (SEQ ID NO: 128)

[0353] Interleukin-12 receptor subunit beta-2 has the following sequence: (SEQ ID NO: 129)

[0354] Bold indicates propeptide, underlined italics indicates extracellular domain, italics indicates transmembrane domain and underlined bold indicates cytoplasmic domain.

[0355] In some embodiments, the masking moiety comprises the extracellular domain of human IL-12Rβ1 or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-12.

[0356] In some embodiments, the masking moiety comprises 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 moiety comprises an amino acid sequence having the amino acid sequence of human IL-12Rβ1 with one or two amino acid substitutions.

[0357] In some embodiments, the masked moiety comprises residues 24 to 237 of human IL-12Rβ1 that retain or otherwise exhibit affinity for IL-12, i.e., have The sequence of SEQ ID NO: 215 or a fragment, part or variant thereof. In some embodiments, the masking moiety comprises IL-12Rβ1 having SEQ ID NO: 215 as shown in the IL-12 Masking Moiety Table below. In some embodiments, the masking moiety comprises about or at least about 85%, 86%, 87% of any of the amino acid sequences of SEQ ID NO: 215 as shown in the IL-12 masking moiety table below An amino acid sequence having 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the masking moiety comprises an amino acid sequence having an amino acid sequence of SEQ ID NO: 215 as shown in the IL-12 Masking Moiety Table below, with 1 to 4 amino acid substitutions. In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 215 as shown in the IL-12 Masking Moieties Table below, an amino acid sequence having one or two amino acid substitutions.

[0358] In some embodiments, the masking moiety comprises residues 24 to 545 of human IL-12Rβ1 that retain or otherwise exhibit affinity for IL-12, i.e., have The sequence of SEQ ID NO: 216 or a fragment, part or variant thereof. In some embodiments, the masking moiety comprises IL-12Rβ1 having SEQ ID NO: 216 as shown in the IL-12 Masking Moiety Table below. In some embodiments, the masking moiety comprises about or at least about 85%, 86%, 87% of any of the amino acid sequences of SEQ ID NO: 216 as shown in the IL-12 masking moiety table below An amino acid sequence having 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the masking moiety comprises an amino acid sequence having an amino acid sequence of SEQ ID NO: 216, having 1 to 4 amino acid substitutions, as shown in the IL-12 Masking Moieties Table below. In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 216 as shown in the IL-12 Masking Moieties Table below, an amino acid sequence having one or two amino acid substitutions.

[0359] In some embodiments, the masking moiety comprises the extracellular domain of human IL-12Rβ2, or a fragment, portion or variant thereof, that retains or otherwise exhibits affinity for IL-12. In some embodiments, the masking moiety comprises 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 comprises an amino acid sequence having the amino acid sequence of human IL-12Rβ2 with one or two amino acid substitutions.

[0360] In some embodiments, the masked portion comprises residues 24 to 212 of human IL-12Rβ2, ie, has the sequence of SEQ ID NO: 217 as shown in the IL-12 Blocked Moieties Table below. In some embodiments, the masking moiety comprises about or at least about 85%, 86%, 87% of any of the amino acid sequences of SEQ ID NO: 217 as shown in the IL-12 masking moiety table below An amino acid sequence having 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the masking moiety comprises an amino acid sequence having an amino acid sequence of SEQ ID NO: 217, with 1 to 4 amino acid substitutions, as shown in the IL-12 Masking Moieties Table below. In some embodiments, the masking moiety comprises an amino acid sequence having an amino acid sequence of SEQ ID NO: 217 as shown in the IL-12 Masking Moieties Table below, an amino acid sequence having one or two amino acid substitutions.

[0361] In some embodiments, the masked portion comprises residues 24 to 222 of human IL-12Rβ2, ie, having the sequence of SEQ ID NO: 218 as shown in the IL-12 Blocked Moieties Table below. In some embodiments, the masking moiety comprises about or at least about 85%, 86%, 87% of any of the amino acid sequences of SEQ ID NO: 218 as shown in the IL-12 Masking Moiety Table below An amino acid sequence having 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the masking moiety comprises an amino acid sequence having an amino acid sequence of SEQ ID NO: 218 as shown in the IL-12 Masking Moiety Table below, with 1 to 4 amino acid substitutions. In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 218 as shown in the IL-12 Masking Moieties Table below, an amino acid sequence having one or two amino acid substitutions.

[0362] In some embodiments, the masked portion comprises residues 24 to 319 of human IL-12Rβ2, ie, has the sequence of SEQ ID NO: 219 as shown in the IL-12 Blocked Moieties Table below. In some embodiments, the masking moiety comprises an amino acid sequence that is about or at least about 85%, 86%, 87% identical to any of the amino acid sequences of SEQ ID NO: 219 as shown in the IL-12 Masking Moiety Table below. An amino acid sequence having 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the masking moiety comprises an amino acid sequence having an amino acid sequence of SEQ ID NO: 219 as shown in the IL-12 Masking Moieties Table below, with 1 to 4 amino acid substitutions. In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 219 as shown in the IL-12 Masking Moieties Table below, an amino acid sequence having one or two amino acid substitutions.

[0363] In some embodiments, the masked moiety comprises residues 24 to 319 of human IL-12Rβ2, i.e., having SEQ ID NO: 219, having one or more half Cystine substitution sequence. In some embodiments, the masked moiety comprises residues 24 to 319 of human IL-12Rβ2, i.e., having SEQ ID NO: 219 with an amino acid substitution at position C242 as shown in the IL-12 masked moiety table below the sequence of. In some embodiments, the amino acid substitution is C242 at position C242S. In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 220 as shown in the IL-12 Masking Moieties Table below. In some embodiments, the masking moiety comprises about or at least about 85%, 86%, 87%, 88%, 89% of the amino acid sequence of SEQ ID NO: 220 as shown in the IL-12 Masking Moiety Table below. %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity of amino acid sequences. In some embodiments, the masking moiety consists of the amino acid sequence of SEQ ID NO: 220 as shown in the IL-12 Masking Moieties Table below. In some embodiments, the masked portion comprises residues 24 to 622 of human IL-12Rβ2, ie, having the sequence of SEQ ID NO: 221 as shown in the IL-12 Blocked Moieties Table below. In some embodiments, the masking moiety comprises about or at least about 85%, 86%, 87% of any of the amino acid sequences of SEQ ID NO: 221 as shown in the IL-12 masking moiety table below An amino acid sequence having 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the masking moiety comprises an amino acid sequence having an amino acid sequence of SEQ ID NO: 221 as shown in the IL-12 Masking Moieties Table below, with 1 to 4 amino acid substitutions. In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 221 as shown in the IL-12 Masking Moieties Table below, an amino acid sequence having one or two amino acid substitutions.

[0364] In some embodiments, the masked portion comprises residues 24 to 227 of human IL-12Rβ2, ie, having the sequence of SEQ ID NO: 222 as shown in the IL-12 Blocked Moieties Table below. In some embodiments, the masking moiety comprises about or at least about 85%, 86%, 87% of any of the amino acid sequences of SEQ ID NO: 222 as shown in the IL-12 masking moiety table below An amino acid sequence having 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the masking moiety comprises an amino acid sequence having an amino acid sequence of SEQ ID NO: 222, having 1 to 4 amino acid substitutions, as shown in the IL-12 Masking Moieties Table below. In some embodiments, the masking moiety comprises an amino acid sequence having an amino acid sequence of SEQ ID NO: 222 as shown in the IL-12 Masking Moieties Table below, an amino acid sequence having one or two amino acid substitutions. Form-IL-12 Masked Section: components SEQ ID NO sequence shaded part (MM) hCD212 (24-237) 215 hCD212 (24-545) 216 IL12RB 2 (24-212) 217 IL12RB 2 (24-222) 218 IL12RB 2(24-319) 219 IL12RB 2(24-319) [C242S] 220 IL12RB 2(24-622) 221 IL12RB 2 (24-227) 222

[0365] In some embodiments, the IL-12 masking moiety has an amino acid sequence as set forth in one of the sequences in the table above.

[0366] 1.1.3 IL-15 interleukin part and IL-15 masking part (a) IL-15 interleukin part

[0367] In some embodiments, the therapeutic moiety comprises IL-15 cytokine or a functional fragment thereof.

[0368] IL-15 is an interleukin, a type of interleukin signaling molecule that regulates the activity of white blood cells in the immune system.

[0369] In eukaryotic cells, IL-15 is synthesized as a 162 amino acid precursor polypeptide that is subsequently processed to mature IL-15 by removal of amino acid residues 1-48. This results in a mature form of IL-15 consisting of 114 amino acids (amino acid residues 49-162), which is secreted in the mature active form.

[0370] IL-15 precursor polypeptide (SEQ ID NO: 130):

[0371] IL-15 mature polypeptide (SEQ ID NO: 131):

[0372] As used herein, the term "IL-15" or "IL-15 polypeptide" refers to any interleukin-15 (IL-15) protein or functional fragment or variant thereof. The term encompasses any native IL15 from any vertebrate source, including mammals, such as primates (eg, humans) and rodents (eg, rats and mice). The term encompasses unprocessed IL-15 (e.g., the full-length precursor form of IL-15 consisting of amino acid residues 1-162) as well as any form of IL-15 produced by processing in the cell (e.g., the mature form of IL-15 consisting of acid residues 49-162). Thus, the term encompasses the protein encoded by the amino acid sequence of SEQ ID NO: 224 as shown in the IL-15 interleukin section table below, as well as sequence variants thereof. The term also encompasses naturally occurring variants of IL-15. The term also encompasses non-naturally occurring variants of IL-15, such as truncated, deleted forms of IL-15 linked to another molecule, and at least one amino acid change (e.g., substitution, addition, or deletion) from the amino acid sequence. ) variants produced. In some aspects, with a naturally occurring IL-15 polypeptide, such as the IL-15 polypeptide encoded by the amino acid sequence of SEQ ID NO: 223 or 224 as shown in the IL-15 interleukin section table below Compared, variants or homologues have at least 90%, 95%, 96%, 97%, 98%, 99% of the entire complete sequence or a portion of the sequence (for example, a portion of 50, 100 or 114 consecutive amino acids) Or 100% amino acid sequence identity. Thus, the term "IL-15" or "IL-15 polypeptide" includes an IL-15 protein comprising the amino acid sequence of SEQ ID NO: 223 or 224 as shown in the IL-15 interleukin section table below, including Variants thereof, such as variants produced by one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 223 or 224 as shown in the IL-15 interleukin section table below.

[0373] "Functional fragments" of the IL-15 interleukin comprise a portion of the full-length interleukin protein that retains or has an altered interleukin receptor binding ability (e.g., at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% active). Interleukin receptor binding ability can be demonstrated, for example, in the ability of an interleukin to bind to an interleukin cognate receptor or a component thereof (eg, one or more chains of a heterotrimeric receptor complex).

[0374] In some embodiments, the IL-15 interleukin or functional fragment thereof is any naturally occurring interleukin-2 (IL-15 ) protein or a modified variant thereof.

[0375] In some embodiments, the IL-15 interleukin or fragment thereof comprises SEQ ID NO: 224 or a functional fragment thereof as shown in the IL-15 interleukin section table below.

[0376] In some embodiments, the IL-15 interleukin or functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 224 as shown in the IL-15 interleukin section table below.

[0377] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises at least one Amino acid modified amino acid sequence. Each of the at least one amino acid modification can be any amino acid modification, such as substitution, insertion or deletion. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises at least 1, at least Amino acid sequences substituted with 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 amino acids. In some embodiments, the IL-15 interleukin or functional fragment thereof comprises at least 5 amine groups compared to the amino acid sequence of SEQ ID NO: 224 as shown in the IL-15 interleukin section table below Acid-substituted amino acid sequence. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises about or at least about 85% of the amino acid sequence of SEQ ID NO: 224 as shown in the IL-15 interleukin section table below, An amino acid sequence having 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0378] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having one or An amino acid sequence in which multiple amino acids are substituted.

[0379] In some embodiments, the IL-15 interleukin or functional fragment thereof comprises an amino acid sequence at position D22 compared to the amino acid sequence of SEQ ID NO: 224 as shown in the IL-15 interleukin section table below , E46, and E53 have one or more amino acid substituted amino acid sequences. In some embodiments, IL-15 cytokines or functional fragments thereof comprise amino acids at positions D22, E46, Amino acid sequences with one or more amino acid substitutions at E53, N71, N79, and N112.

[0380] In some embodiments, the IL-15 interleukin or functional fragment thereof comprises an amino acid sequence at position D22 compared to the amino acid sequence of SEQ ID NO: 224 as shown in the IL-15 interleukin section table below Amino acid sequences with amino acid substitutions.

[0381] In some embodiments, the IL-15 interleukin or functional fragment thereof comprises an amino acid sequence at position E46 compared to the amino acid sequence of SEQ ID NO: 224 as shown in the IL-15 interleukin section table below Amino acid sequences with amino acid substitutions.

[0382] In some embodiments, the IL-15 interleukin or functional fragment thereof comprises an amino acid sequence at position E53 compared to the amino acid sequence of SEQ ID NO: 224 as shown in the IL-15 interleukin section table below Amino acid sequences with amino acid substitutions.

[0383] In some embodiments, the IL-15 interleukin or functional fragment thereof comprises an amino acid sequence at position N71 compared to the amino acid sequence of SEQ ID NO: 224 as shown in the IL-15 interleukin section table below Amino acid sequences with amino acid substitutions.

[0384] In some embodiments, the IL-15 interleukin or functional fragment thereof comprises an amino acid sequence at position N79 compared to the amino acid sequence of SEQ ID NO: 224 as shown in the IL-15 interleukin section table below Amino acid sequences with amino acid substitutions.

[0385] In some embodiments, the IL-15 interleukin or functional fragment thereof comprises an amino acid sequence at position N112 compared to the amino acid sequence of SEQ ID NO: 224 as shown in the IL-15 interleukin section table below Amino acid sequences with amino acid substitutions.

[0386] In some embodiments, the IL-15 interleukin or functional fragment thereof comprises an amino acid sequence at position E46 compared to the amino acid sequence of SEQ ID NO: 224 as shown in the IL-15 interleukin section table below and an amino acid sequence having an amino acid substitution at E53.

[0387] In some embodiments, the IL-15 interleukin or functional fragment thereof comprises an amino acid sequence at position N71 compared to the amino acid sequence of SEQ ID NO: 224 as shown in the IL-15 interleukin section table below and amino acid sequences with amino acid substitutions at N79.

[0388] In some embodiments, the IL-15 interleukin or functional fragment thereof comprises an amino acid sequence at position N71 compared to the amino acid sequence of SEQ ID NO: 224 as shown in the IL-15 interleukin section table below and amino acid sequences with amino acid substitutions at N112.

[0389] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence at position N79 compared to the amino acid sequence of SEQ ID NO: 224 as shown in the IL-15 interleukin section table below and amino acid sequences with amino acid substitutions at N112.

[0390] In some embodiments, the IL-15 interleukin or functional fragment thereof comprises an amino acid sequence at position N71 compared to the amino acid sequence of SEQ ID NO: 224 as shown in the IL-15 interleukin section table below , N79 and N112 have amino acid substitution amino acid sequences.

[0391] In some embodiments, the amino acid substitution at position D22 is D22A.

[0392] In some embodiments, the amino acid substitution at position E46 is E46A.

[0393] In some embodiments, the amino acid substitution at position E46 is E46R.

[0394] In some embodiments, the amino acid substitution at position E46 is E46S.

[0395] In some embodiments, the amino acid substitution at position E53 is E53A.

[0396] In some embodiments, the amino acid substitution at position E53 is E53R.

[0397] In some embodiments, the amino acid substitution at position E53 is E53S.

[0398] In some embodiments, the amino acid substitution at position N71 is N71Q.

[0399] In some embodiments, the amino acid substitution at position N79 is N79Q.

[0400] In some embodiments, the amino acid substitution at position N112 is N112Q.

[0401] In some embodiments, the IL-15 interleukin or functional fragment thereof comprises an amine group compared to the amino acid sequence of SEQ ID NO: 224 as shown in the IL-15 interleukin section table below Acid substitution in the amino acid sequence of D22A.

[0402] In some embodiments, the IL-15 interleukin or functional fragment thereof comprises an amine group compared to the amino acid sequence of SEQ ID NO: 224 as shown in the following IL-15 interleukin section table Acid substitution in the amino acid sequence of E46A.

[0403] In some embodiments, the IL-15 interleukin or functional fragment thereof comprises an amine group compared to the amino acid sequence of SEQ ID NO: 224 as shown in the following IL-15 interleukin section table Acid substitutions in the amino acid sequences of E46A and E53A.

[0404] In some embodiments, the IL-15 interleukin or functional fragment thereof comprises an amine group compared to the amino acid sequence of SEQ ID NO: 224 as shown in the following IL-15 interleukin section table Acid substitutions in the amino acid sequences of E46R and E53R.

[0405] In some embodiments, the IL-15 interleukin or functional fragment thereof comprises an amine group compared to the amino acid sequence of SEQ ID NO: 224 as shown in the following IL-15 interleukin section table Acid substitutions in the amino acid sequences of E46S and E53S.

[0406] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amine group compared to the amino acid sequence of SEQ ID NO: 224 as shown in the IL-15 interleukin section table below The acid substituted the amino acid sequence of E53A.

[0407] In some embodiments, the IL-15 interleukin or functional fragment thereof comprises an amine group compared to the amino acid sequence of SEQ ID NO: 224 as shown in the following IL-15 interleukin section table Acid substitution in the amino acid sequence of N71Q.

[0408] In some embodiments, the IL-15 interleukin or functional fragment thereof comprises an amine group compared to the amino acid sequence of SEQ ID NO: 224 as shown in the following IL-15 interleukin section table The amino acid sequence of acid substitution N79Q.

[0409] In some embodiments, the IL-15 interleukin or functional fragment thereof comprises an amine group compared to the amino acid sequence of SEQ ID NO: 224 as shown in the following IL-15 interleukin section table Amino acid sequence of acid substitution N112Q.

[0410] In some embodiments, the IL-15 interleukin or functional fragment thereof comprises an amine group compared to the amino acid sequence of SEQ ID NO: 224 as shown in the IL-15 interleukin section table below Acid substitutions in the amino acid sequences of N71Q and N79Q.

[0411] In some embodiments, the IL-15 interleukin or functional fragment thereof comprises an amine group compared to the amino acid sequence of SEQ ID NO: 224 as shown in the IL-15 interleukin section table below Acid substitutions in the amino acid sequences of N71Q and N112Q.

[0412] In some embodiments, the IL-15 interleukin or functional fragment thereof comprises an amine group compared to the amino acid sequence of SEQ ID NO: 224 as shown in the IL-15 interleukin section table below Acid substitutions in the amino acid sequences of N79Q and N112Q.

[0413] In some embodiments, the IL-15 interleukin or functional fragment thereof comprises an amine group compared to the amino acid sequence of SEQ ID NO: 224 as shown in the IL-15 interleukin section table below Acid substitutions in the amino acid sequences of N71Q, N79Q and N112Q. Table - IL-15 Cytokines Section: components SEQ ID NO sequence DC hIL-15 (Precursor) 223 hIL15 224 AK401 AK402 AK403 AK481 AK482 AK483 AK478 AK479 AK480 AK242 AK243 AK247 AK248 AK245 AK250 AK419 AK245 AK251 AK419 AK421 AK457 AK399 AK404 AK405 AK400 AK243 AK249 AK418 AK507 AK564 hIL15(D22A) 225 AK458 hIL15(E46A) 226 AK459 hIL15 (E46A, E53A) 227 AK461 AK527 AK506 hIL15 (E46R, E53R) 228 Not yet named - 2 hIL15 (E46S, E53S) 229 Not yet named - 1 hIL15(E53A) 230 AK4 60 hIL-15 (N-terminal) 231 unnamed-3 unnamed-4 hIL15 (C-terminal) 232 unnamed-3 unnamed-4 hIL-15 (N71Q) 233 AK595 AK596 hIL-15 (N79Q) 234 AK901 AK907 hIL-15 (N112Q) 235 AK900 AK906 hIL-15 (N71Q, N79Q) 236 AK904 AK910 AK929 AK935 AK931 AK937 AK934 AK940 AK933 AK939 hIL-15 (N71Q, N112Q) 237 AK903 AK909 hIL-15 (N79Q, N112Q) 238 AK902 AK908 hIL-15 (N71Q, N79Q, N112Q) 239 AK905 AK911

[0414] In some embodiments, the IL-15 interleukin moiety has an amino acid sequence as set forth in one of the sequences in the table above.

[0415] In some embodiments, an additional mutation may be included at position N71 in any of the above sequences. In some embodiments, the mutation is N71A, N71R, N71W, N71F, N71P, N71M, N71L, N71T, N71S or N71Y.

[0416] In some embodiments, an additional mutation may be included at position S73 in any of the above sequences. In some embodiments, the mutation is S73A, S73W, S73V or S73M.

[0417] In some embodiments, additional mutations may be included at one or more of amino acid positions N72, N79, V80, T81 and N112 in any of the above sequences. In some embodiments, one or more additional mutations selected from N72A, N79A, V80A, T81A, and N112R may be included in any of the above sequences.

[0418] In some embodiments, additional mutations may be included at one or more of amino acid positions N72, S73, N79, V80, T81 and N112 in any of the above sequences. In some embodiments, one or more additional mutations N72A, S73A, N79A, V80A, T81A, and N112 may be included in any of the above sequences.

[0419] In some embodiments, for the purpose of removing O-glycosylation sites, IL-15 cytokines or functional fragments thereof have one or more amino acid residues, for example with IL-15 as above 15 Residues 1-3 were removed compared to the amino acid sequence of mature IL-15 of SEQ ID 224 shown in the Table 15 Cytokines section. In some embodiments, for the purpose of removing the O-glycosylation site, the IL-15 cytokine or a functional fragment thereof has the sequence of SEQ ID 224 as shown in the above IL-15 cytokine section table One or more amino acid residues are substituted compared to the amino acid sequence of mature IL-15. In some embodiments, for the purpose of removing the O-glycosylation site, the IL-15 interleukin or functional fragment thereof is matured with SEQ ID 224 as shown in the above IL-15 interleukin section table The amino acid sequence of IL-15 has one or more intervening amino acid residues compared, eg, in the region of residues 1-3. In some embodiments, the IL-15 interleukin or functional fragment thereof does not have an O-glycosylation site within residues 1-3.

[0420] In some embodiments, the masked IL-15 interleukin further comprises a domain comprising an IL-15Rα subunit or a functional fragment thereof ("IL-15Rα domain"). It has been demonstrated that the incorporation of the "IL-15Rα domain" into the masked IL-15 interleukin construct enhances the efficacy of the interleukin in activating CD8 T cells and NK cells.

[0421] The IL-15Rα subunit (also known as CD215) is structurally similar to IL-2Rα; the extracellular domain of IL-15Rα consists of a single protein-binding sushi domain, a juxtamembrane rich proline-threonine (PT) region and the linker / hinge region connecting the sushi domain and the PT region. The IL-15Rα subunit specifically binds IL-15 with extremely high affinity and is capable of binding IL-15 independently of the β and γ subunits.

[0422] Interleukin (IL)-15 is a gene that acts on multiple cell types but is optimal for the development, constancy and function of specific immune cell populations including CD8 T cells, NK cells, NKT cells and CD8αα intraepithelial lymphocytes key cytokines. IL-15 signaling is transmitted through the IL-2 / 15Rβ and common gamma (γC) chains; however, delivery of IL-15 to these signaling components is quite unique. In contrast to other secreted cytokines, IL-15 exists primarily in association with high-affinity IL-15Rα. When the IL-15 / IL-15Rα complex is shuttled to the cell surface, it can stimulate opposing cells via the β / γC receptor complex. This novel mechanism of IL-15 delivery has been called trans-presentation (S. W. Stonier and K. S. Schluns, "Trans-presentation: a novel mechanism regulating IL-15 delivery and responses", Immunol Lett 2010 Jan 4; 127(2 ): 85-92, the contents of which are incorporated herein by reference).

[0423] The IL-15Rα subunit contains a conserved protein binding motif known as the sushi domain. The sushi domain of sIL-15Rα, comprising amino acids 31 to 95 of the IL-15Rα subunit, is responsible for the interaction with IL-15 and is critical for IL-15 / IL-15Rα function (Wei X et al. "The Sushi Domain of Soluble IL-15 Receptor α Is Essential for Binding IL-15 and Inhibiting Inflammatory and Allogenic Responses In Vitro and In Vivo”, J Immunol 2001 Jul 1; 167(1) 277-282, the contents of which are cited in incorporated into this article).

[0424] The sequence of the wild-type IL-15Rα subunit is shown below, along with a breakdown of the main domain: (SEQ ID NO: 132) >sp|Q13261|1-30 (signal peptide) (SEQ ID NO: 133) >sp |Q13261|31-205 (extracellular domain) [Note: 31-95 is a typical "sushi domain"] (SEQ ID NO: 134) > sp|Q13261|206-228 (transmembrane domain) (SEQ ID NO: 135) >sp|Q13261|229-267 (cytoplasmic domain) (SEQ ID NO: 136)

[0425] The "IL-15Rα domain" herein may comprise the sequence of the extracellular domain of a wild-type IL-15Rα subunit or a variant thereof, such as having one or more (for example 1, 2, 3 or 4 ) The sequence of the extracellular domain of the amino acid substituted wild-type IL-15Rα subunit.

[0426] The "IL-15Rα domain" herein may comprise the sequence of the wild-type sushi domain sIL-15Rα or a variant thereof, such as having one or more (eg 1, 2, 3 or 4) amino acids Sequence of substituted wild-type sushi domain sIL-15Rα.

[0427] The "IL-15Rα domain" herein may be substituted by the sequence of the wild-type sushi domain sIL-15Rα or a variant thereof, such as having one or more (eg 1, 2, 3 or 4) amino acids Sequence composition of the wild-type sushi domain sIL-15Rα.

[0428] In some embodiments, the IL-15Rα domain comprises an amino acid substitution at position R26. In some embodiments, the IL-15Rα domain comprises the amino acid substitution R26N. In some embodiments, the IL-15Rα domain comprises the amino acid substitution R26S. In some embodiments, the IL-15Rα domain comprises an amino acid substitution at position R35. In some embodiments, the IL-15Rα domain comprises the amino acid substitution R35Q. In some embodiments, the IL-15Rα domain comprises the amino acid substitution R35S. In some embodiments, the IL-15Ra domain comprises amino acid substitutions at positions R26 and R35. In some embodiments, the IL-15Rα domain comprises the amino acid substitutions R26S or R26N and R35Q or R35S. In some embodiments, the IL-15Rα domain comprises the amino acid substitutions R26N and R35Q.

[0429] An exemplary sequence of the IL-15Rα domain is shown below: components sequence hCD215 (SEQ ID NO: 134) hCD215 (1 to 66) (SEQ ID NO: 137) hCD215(1 to 66) R26N (SEQ ID NO: 138) hCD215(1 to 66) R35Q (SEQ ID NO: 139) hCD215(1 to 66) R26S (SEQ ID NO: 140) hCD215(1 to 66) R35S (SEQ ID NO: 141) hCD215(1 to 66) R26N; R35Q (SEQ ID NO: 142) hCD215 (sushi) (SEQ ID NO: 143) hCD215 (truncated) (SEQ ID NO: 144)

[0430] In some embodiments, the IL-15Rα domain has an amino acid sequence as set forth in one of the sequences in the table above.

[0431] (b) IL-15 Masking Moieties Provided herein is a masking moiety for masking a therapeutic moiety comprising the IL-15 cytokine or a functional fragment thereof.

[0432] It is understood that the masked moiety is cleaved from the masked cytokine to form its cleavage product. The shielding part in the shielded interleukin shields the IL-15 cytokine or a functional fragment thereof, thereby reducing or preventing the binding of the IL-interleukin or a functional fragment thereof to its cognate receptor. In some embodiments, the masking moiety reduces or prevents the binding of IL-15 interleukin or functional fragment thereof to IL-15Rα. In some embodiments, a masking moiety as provided herein refers to an IL-15 cytokine capable of binding to or functional fragment thereof (such as an anti-IL-15 antibody or IL-15 cognate receptor protein) or otherwise exhibiting part of affinity. Methods for determining the degree of binding of a protein (eg, an interleukin) to a cognate protein (eg, an interleukin receptor) are well known in the art.

[0433] In some embodiments, the masking moiety comprises an IL-15 cytokine receptor or subunit or functional fragment thereof.

[0434] In some embodiments, the masking moiety comprises IL-15Rβ (also known as CD122) or a fragment, portion or variant thereof that retains or otherwise exhibits affinity for IL-15.

[0435] The wild-type sequence of IL-15Rβ is shown in SEQ ID NO: 240 in the IL-15 masked portion table below.

[0436] In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 240 in the IL-15 Masking Moieties Table below. In some embodiments, the masking moiety comprises about or at least about 85%, 86%, 87%, 88%, 89%, 90% of the amino acid sequence of SEQ ID NO: 240 in the IL-15 masking moiety table below %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity of amino acid sequences. In some embodiments, the masking moiety comprises an amino acid sequence having SEQ ID NO: 240 in the IL-15 masking moiety table below, an amino acid sequence having 1 to 4 amino acid substitutions. In some embodiments, the masking moiety comprises an amino acid sequence having SEQ ID NO: 240 in the IL-15 masking moiety table below, an amino acid sequence having one or two amino acid substitutions.

[0437] In some embodiments, the masking moiety comprises IL-15Rβ (or a functional fragment, portion or variant thereof), wherein IL-15Rβ has an amino acid substitution at position C122.

[0438] In some embodiments, the masking moiety comprises IL-15Rβ (or a functional fragment, portion or variant thereof), wherein IL-15Rβ has the amino acid substitution C122S.

[0439] In some embodiments, the IL-15Rβ or fragment, portion or variant thereof has an amino acid substitution at position C122 compared to IL-15Rβ of SEQ ID NO: 240 in the IL-15 Masked Moiety Table below .

[0440] In some embodiments, the IL-15Rβ or fragment, portion or variant thereof has a mutation at amino acid position 122 compared to IL-15Rβ of SEQ ID NO: 240 in the IL-15 Masked Moiety Table below C122S.

[0441] In some embodiments, the masked moiety comprises the amino acid sequence of SEQ ID NO: 240 in the IL-15 Masked Moieties Table below with a C122 mutation.

[0442] In some embodiments, the masked moiety comprises the amino acid sequence of SEQ ID NO: 240 in the IL-15 Masked Moieties Table below with a C122S mutation.

[0443] In some embodiments, the masking moiety comprises IL-15Rβ (or a functional fragment, portion or variant thereof), wherein IL-15Rβ has an amino acid substitution at position C168.

[0444] In some embodiments, the masking moiety comprises IL-15Rβ (or a functional fragment, portion or variant thereof), wherein IL-15Rβ has the amino acid substitution C168S.

[0445] In some embodiments, the IL-15Rβ or fragment, portion or variant thereof has a mutation at amino acid position C168 compared to IL-15Rβ of SEQ ID NO: 240 in the IL-15 Masked Moiety Table below .

[0446] In some embodiments, the IL-15Rβ or fragment, portion or variant thereof has a mutation at amino acid position 168 compared to IL-15Rβ of SEQ ID NO: 240 in the IL-15 Masked Moiety Table below C168S.

[0447] In some embodiments, the masked moiety comprises the amino acid sequence of SEQ ID NO: 240 in the IL-15 Masked Moieties Table below with a C168 mutation.

[0448] In some embodiments, the masked moiety comprises the amino acid sequence of SEQ ID NO: 240 in the IL-15 Masked Moieties Table below with a C168S mutation.

[0449] In some embodiments, the IL-15Rβ or fragment, portion or variant thereof is at amino acid positions C122 and C168 compared to IL-15Rβ of SEQ ID NO: 240 in the IL-15 Masked Moiety Table below have mutations.

[0450] In some embodiments, the IL-15Rβ or fragment, portion or variant thereof has the mutations C122S and C168S compared to IL-15Rβ of SEQ ID NO: 240 in the IL-15 Masked Portion Table below.

[0451] In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 241 in the IL-15 Masking Moieties Table below.

[0452] Form-IL-15 Covered Section: components SEQ ID NO sequence DC shaded part (MM) hCD122 240 AK247 AK248 AK421 AK457 AK249 AK418 AK250 AK251 AK399 AK400 AK404 AK405 AK419 AK420 AK401 AK402 AK403 AK458 AK459 AK460 AK461 AK478 AK479 AK480 AK481 AK482 AK483 AK527 unnamed-3 unnamed-4 hCD122 (C122S, C168S) 241 AK564 AK596 AK900 AK901 AK902 AK903 AK904 AK905 AK906 AK907 AK908 AK909 AK910 AK911 AK929 AK935 AK931 AK937 AK934 AK940 AK933 AK939

[0453] In some embodiments, the IL-15 masking moiety has an amino acid sequence as set forth in one of the sequences in the table above.

[0454] 1.2 Non-cleavable peptide linkers Provided herein are non-cleavable peptide linkers for use in a drug construct as described herein, or a cleavage product thereof. A linker as provided herein refers to a peptide of two or more amino acids used to link together two functional components in a masked interleukin described herein.

[0455] The masked cytokine comprises a first linker and a second linker, wherein at least the first linker or the second linker comprises a proteolytic peptide.

[0456] In some embodiments, the second linker comprises a proteolytic peptide (linker herein referred to as a "protein cleavable linker"), and the first linker does not comprise a protein cleavage peptide (linker herein called the "non-cleavable linker"). This configuration is described herein as "Configuration A". In some embodiments, the first polypeptide chain comprises the formula: N'HL1-non-cleavable L1-MM C' and the second polypeptide chain comprises the formula: N'HL2-cleavable L2-CC'

[0457] In some embodiments, the first linker comprises a proteolytic peptide (linkers referred to herein as "protein cleavable linker" or "cleavable linker"), and the second linker does not comprise a protein cleavable peptide Peptide (herein the linker is referred to as "non-cleavable linker"). This configuration is described herein as "Structure B". In some embodiments, the first polypeptide chain comprises the formula: N'HL1-cleavable L1-MM C' and the second polypeptide chain comprises the formula: N'HL2-non-cleavable L2-CC'

[0458] The non-cleavable linkers and cleavable linkers of some embodiments are described in more detail below.

[0459] In some embodiments, the non-cleavable linker is between 3 and 25 amino acids in length.

[0460] In some embodiments, the non-cleavable linker is between 3 and 18 amino acids in length.

[0461] In some embodiments, the non-cleavable linker is between 3 and 8 amino acids in length.

[0462] In some embodiments, the non-cleavable linker is between 4 and 6 amino acids in length.

[0463] In some embodiments, the non-cleavable linker is enriched in amino acid residues G, S, and P.

[0464] In some embodiments, the non-cleavable linker includes only types of amino acid residues selected from the group consisting of G, S, and P.

[0465] In some embodiments, the non-cleavable linker comprises a "GS" repeat sequence.

[0466] In some embodiments, the non-cleavable linker includes an N' terminal "P" residue.

[0467] In some embodiments, the non-cleavable linker comprises the amino acid sequence PGSGS (SEQ ID NO: 14). In some embodiments, the non-cleavable linker consists of the amino acid sequence PGSGS (SEQ ID NO: 14).

[0468] In some embodiments, the non-cleavable linker comprises the amino acid sequence GGSSPPGGGSSGGGSGP (SEQ ID NO: 23).

[0469] In some embodiments, the non-cleavable linker consists of the amino acid sequence GGSSPPGGGSSGGGSGP (SEQ ID NO: 23).

[0470] In some embodiments, wherein the second linker is a protein cleavable linker and the first linker is a non-cleavable linker, the non-cleavable linker comprises PGSGS (SEQ ID NO: 14). In some embodiments, wherein the second linker is a protein cleavable linker and the first linker is a non-cleavable linker, the non-cleavable linker consists of the amino acid sequence PGSGS (SEQ ID NO: 14).

[0471] In some embodiments, wherein the first linker is a protein cleavable linker and the second linker is a non-cleavable linker, the non-cleavable linker comprises GGSSPPGGGSSGGGSGP (SEQ ID NO: 23). In some embodiments, wherein the first linker is a protein cleavable linker and the second linker is a non-cleavable linker, the non-cleavable linker consists of the amino acid sequence GGSSPPGGGSSGGGSGP (SEQ ID NO: 23).

[0472] In some embodiments, wherein the second linker is a protein cleavable linker and the first linker is a non-cleavable linker, the non-cleavable linker is between 3 and 8 amino acids in length. In some embodiments, the non-cleavable linker is between 4 and 6 amino acids in length. In some embodiments, the non-cleavable linker comprises the amino acid sequence set forth in SEQ ID NO: 14 (PGSGS).

[0473] In some embodiments, wherein the first linker is a protein cleavable linker and the second linker is a non-cleavable linker, the non-cleavable linker is between 3 and 18 amino acids in length. In some embodiments, wherein the first linker is a protein cleavable linker and the second linker is a non-cleavable linker, the non-cleavable linker is between 10 and 18 amino acids in length. In some embodiments, the non-cleavable linker comprises the amino acid sequence set forth in SEQ ID NO: 23 (GGSSPPGGGSSGGGSGP).

[0474] In some embodiments, it is desirable for the first and second polypeptide chains to be the same or similar in length in order to facilitate association of the first half-life-extending domain with the second half-life-extending domain and masking of the moiety in the assembled construct Cytokines or their functional fragments. Thus, in case the masked part is an amino acid sequence shorter than that of the interleukin or its functional fragment, the difference in length can be fully or partly compensated by using a longer linker L1.

[0475] In some embodiments, the first polypeptide chain comprises the formula: N'HL1-non-cleavable L1-MM C' and the second polypeptide chain comprises the formula: N'HL2-SD1-CP-SD2-CC '

[0476] In some embodiments, the first polypeptide chain comprises the following formula: N'HL1-SD1-CP-SD2-MM C' and the second polypeptide chain comprises the following formula: N'HL2-non-cleavable L2-C C'

[0477] The linker combinations disclosed in the exemplary AK molecules can be used with any of the cytokine moieties disclosed herein. The linker combinations disclosed in the exemplary AK molecules can be used with any of the masking moieties disclosed herein. The linker combinations disclosed in the exemplary AK molecules can be used with any half-life extending moiety. In other words, the linkers disclosed in the exemplary AK molecules can be used in combination with any of the cytokine moieties disclosed herein, the masking moieties disclosed herein, and / or the half-life extending moieties disclosed herein.

[0478] 2. Cleavage Products Provided herein is a cleavage product capable of comprising an active therapeutic moiety prepared by protein cleavage of a protein cleavable linker in a polypeptide drug construct as described anywhere herein .

[0479] Provided herein is a cleavage product of a "heterodimeric" masked cytokine described anywhere herein.

[0480] The masked cytokines described herein comprise a cleavable linker. Following proteolytic cleavage of the cleavable linker at the cleavage site, a cleavage product comprising an interleukin moiety is formed. The cytokines in the lysate are partially activated because they are no longer masked by the masking moiety. The interleukin moiety in the lysate is thus capable of binding to the protein of interest.

[0481] The tumor cell environment is complex and may contain many different proteases. Thus, enabling the precise site of cleavage of a given cleavable peptide within the masked interleukin in the tumor cell environment can result in cleavage between tumor types, between patients with the same tumor type, and even within the same tumor vary between products. Furthermore, even after cleavage, further modification of the initial cleavage product, for example by removal of one or two terminal amino acids, can be performed by further action of proteases in the tumor cell environment. Thus, following administration of a masked cytokine as described herein, a distribution of cleavage products can be expected to form in the patient's tumor cell environment.

[0482] It will be understood that a cleavage site as referred to herein refers to a site within a cleavable peptide between two specific amino acid residues known to be associated with the tumor cell environment. target of the protease. In this sense, there may be more than one cleavage site in a cleavable peptide as described herein, wherein different proteases cleave the cleavable peptide at different cleavage sites. More than one protease can also act on the same cleavage site within a cleavable peptide. A discussion of protease cleavage sites can be found in the art.

[0483] Accordingly, the cleavable peptides disclosed herein can be cleaved by one or more proteases.

[0484] Provided herein is a cleavage product comprising a portion of a cytokine capable of binding to its cognate receptor by proteolytic cleavage in a masked cytokine as described anywhere herein Protein cleavage of linkers can be prepared.

[0485] Also provided herein is a distribution of cleavage products obtained or obtainable from a single structure of a masked cytokine, wherein within the cleavage product distribution each cleavage product (i) is capable of binding to the protein of interest and (ii) comprises as herein An interleukin (eg IL-2, IL-15 or IL-12 interleukin) moiety as defined anywhere.

[0486] Also provided herein is a cleavage product of a masked cytokine, wherein the cleavage product is capable of binding to a protein of interest, the cleavage product comprising a polypeptide comprising the formula: PCP-SD-C wherein PCP is a protein cleavage peptide part; SD is the spacer domain; and C is the cytokine part.

[0487] Further provided herein is a cleavage product of a masked cytokine, wherein the cleavage product is capable of binding to a protein of interest, the cleavage product comprising a protein heterodimer comprising: a) a first polypeptide chain , comprising a first half-life extending moiety; and b) a second polypeptide chain comprising a polypeptide comprising the formula: HL2-L2-C wherein HL2 is a second half-life extending moiety; L2 is a non-cleavable linker; and C is a cytokine moiety; and wherein the first half-life-extending moiety is associated with the second half-life-extending moiety. Also provided herein is a distribution of cleavage products obtained or obtainable from a single structure of a masked cytokine, wherein within the cleavage distribution each cleavage product (i) is capable of binding to a protein of interest and (ii) comprises a protein heterodimer, It comprises: a) a first polypeptide chain comprising a first half-life extending moiety; and b) a second polypeptide chain comprising a polypeptide comprising the formula: HL2-L2-C wherein HL2 is a second half-life extending moiety moiety; L2 is a non-cleavable linker; and C is a cytokine moiety; and wherein the first half-life-extending moiety is associated with the second half-life-extending moiety.

[0488] Further provided herein is a cleavage product of a masked cytokine, wherein the cleavage product is capable of binding to a protein of interest, the cleavage product comprising a protein heterodimer comprising: a) a first polypeptide chain , comprising a polypeptide comprising the formula: HL1-SD-PCP wherein HL1 is a first half-life extending moiety; SD is a spacer domain; and PCP is part of a proteolytic peptide; and b) a second polypeptide chain, It comprises a polypeptide comprising the formula: HL2-L2-C wherein HL2 is a second half-life extending moiety; L2 is a non-cleavable linker; and C is a cytokine moiety; and wherein the first half-life extending moiety and the second half-life extending moiety Extended partial association.

[0489] Within the cleavage product, the masking moiety, half-life extending moiety, interleukin moiety, linker, steric domain can be any of those described herein and any of them described herein combination.

[0490] The position of the cleavable peptide determines the structure of the resulting cleavage product comprising the interleukin moiety.

[0491] "Part of a protein cleavage peptide" refers to a portion of the original protein cleavage peptide sequence after cleavage at the cleavage site. After cleavage, further modification of the initial cleavage product, for example by removal of one or two terminal amino acids, can also be performed by further action of the protease in the tumor cell environment. Thus, cleavage products within the distribution of cleavage products that may form in the patient's tumor cell environment following administration of a masked interleukin may not contain any portion of the proteolytic peptide.

[0492] In some embodiments, "portion" refers to 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, or 6 amino acids. In some embodiments, "portion" refers to 2 amino acids of the original protein cleavage peptide sequence. In some embodiments, "portion" refers to 3 amino acids of the original protein cleavage peptide sequence. In some embodiments, "portion" refers to the 4 amino acids of the original protein cleavage peptide sequence.

[0493] In some embodiments, the "portion" of the proteolytic peptide is 3 to 6 amino acids in length. In some embodiments, the "portion" of the proteolytic peptide is 3 or 4 amino acids in length.

[0494] Exemplary cleavage sites for the cleavable linkers disclosed herein are disclosed below (* indicates known or observed protease cleavage sites within the cleavable peptide): DLLA*VVAAS (SEQ ID NO: 248) ISSGLL*SG*RS (SEQ ID NO: 249)

[0495] Thus, disclosed herein are cleavage products of any of the polypeptide drug constructs or masked cytokines disclosed herein. 3. Binding assays such as immunological binding interactions between cytokines or functional fragments thereof and binding partners (e.g. proteins of interest such as interleukin receptors) specifically targeted by cytokines or functional fragments thereof Strength or affinity can be expressed in terms of the dissociation constant (Kd) of the interaction, where the smaller the Kd, the greater the affinity. Binding of an interleukin to an interleukin 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 protein of interest (such as an interleukin receptor). In the case of IL-2 cytokine binding, the target protein can be IL-2R (including IL-2Rα, IL-2Rβ and IL-2Rγ chain), IL-2Rα chain, IL-2Rβ chain or IL-2Rα / β Dimeric complex. The immunological binding properties of proteins can be quantified using methods well known in the art. For example, one method involves measuring the rates of formation and dissociation of interleukin receptor (e.g., IL-2R) / interleukin (e.g., IL-2) complexes, wherein the rates depend on the concentration of the complexed partner , the affinity of the interaction, and the geometric parameters that affect the velocity in both directions equally. The "Association Rate Constant" (Kon) and "Dissociation Rate Constant" (Koff) can be determined by calculating the concentration and the actual rates of association and dissociation. The ratio Koff / Kon cancels out all parameters not related to affinity and is equal to the dissociation constant Kd. See Davies et al., Annual Rev Biochem. 59:439-473, (1990).

[0497] In some aspects, a masked interleukin described herein binds with about the same or higher affinity to target protein. The target protein can be any cytokine receptor. In some embodiments, the target protein is IL-2R (comprising IL-2Rα, IL-2Rβ and IL-2Rγ chains). In some embodiments, the protein of interest is IL-2Rα. In some embodiments, the protein of interest is IL-2Rβ. In some embodiments, the protein of interest is IL-2Rα / β dimeric complex.

[0498] In some embodiments, the dissociation constant (Kd) of a masked interleukin provided herein that does not contain a cleavable peptide and a protein of interest is ≤ 1M, ≤ 150 nM, ≤ 100 nM, ≤ 50 nM, ≤ 10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤ 0.001 nM (e.g. 10-8 M or less, e.g. 10-8 M to 10-13 M, e.g. 10-9 M to 10-13 M). In some embodiments, a masked interleukin provided herein comprising a cleavable peptide in a linker has a dissociation constant (Kd) ≤ 1 M, ≤ 150 nM, ≤ 100 nM with the protein of interest prior to cleavage by a protease , ≤50 nM, ≤ 10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or 0.001 nM (eg 10-8 M or less, eg 10-8 M to 10-13 M, eg 10-9 M to 10-13 M). In some embodiments, a masked interleukin provided herein comprising a cleavable peptide in a linker has a dissociation constant (Kd) ≤ 1 M, ≤ 150 nM, ≤ 100 nM for a protein of interest after cleavage by a protease , ≤50 nM, ≤ 10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or 0.001 nM (eg 10-8 M or less, eg 10-8 M to 10-13 M, eg 10-9 M to 10-13 M). In some embodiments, the dissociation constant (Kd) of the masked interleukin or functional fragment thereof and the masked portion of the masked interleukin provided herein is ≥ 500 M, ≥ 250 M, ≥ 200 M , ≥ 150 M, ≥ 100 M, ≥ 50 M, ≥ 10 M, ≥ 1 M, ≥ 500 nM, ≥ 250 nM, ≥ 150 nM, ≥ 100 nM, ≥ 50 nM, ≥ 10 nM, ≥ 1 nM, ≥ 0.1 nM, ≥ 0.01 nM, or ≥ 0.001 nM. In some embodiments, the dissociation constant (Kd) of the interleukin or functional fragment thereof of the masked interleukin provided herein is between about 200 M and about 50 nM, such as about or at least about 175 M, about or at least about 150 M, about or at least about 125 M, about or at least about 100 M, about or at least about 75 M, about or at least about 50 M, about or at least about 25 M, about or at least about 5 M, about or At least about 1 M about or at least about 750 nM, about or at least about 500 nM, about or at least about 250 nM, about or at least about 150 nM, about or at least about 100 nM, about or at least about 75 nM or about or at least about 50 nM. Assays for assessing binding affinity are well known in the art.

[0499] In some aspects, a masked cytokine exhibiting a desired occlusion rate is provided. As used herein, the term "occlusion rate" refers to the ratio of (a) the maximum detection level of a parameter under a first set of conditions to (b) the minimum detection value of a parameter under a second set of conditions. In the case of a masked IL-2 polypeptide, occlusion refers to (a) the binding of the target protein to the masked IL-2 polypeptide in the presence of at least one protease capable of cleaving the cleavable peptide of the masked IL-2 polypeptide The ratio of the maximum level of detection (eg, IL-2R protein) to (b) the minimum level of detection of a target protein (eg, IL-2R protein) that binds to the masked IL-2 polypeptide in the absence of the protease. Thus, the occlusion rate of a masked interleukin can be calculated by dividing the EC50 of the masked interleukin before cleavage by the EC50 of the masked interleukin after cleavage. The occlusion rate of a masked interleukin can also be calculated as the ratio of the dissociation constant of the masked interleukin before cleavage to the dissociation constant of the masked interleukin after cleavage with a protease. In some embodiments, a greater occlusion rate of the masked interleukin indicates that binding of the masked interleukin to the target protein occurs to a greater extent in the presence of a protease capable of cleaving the cleavable peptide of the masked interleukin than in the absence of Large (eg occurs predominantly) in the presence of proteases.

[0500] In some embodiments, provided herein are masked interleukins with optimal occlusion. In some embodiments, an optimal occlusion rate of a masked interleukin indicates that the masked interleukin has desirable properties for use in the methods or compositions contemplated herein. In some embodiments, the masked cytokines provided herein exhibit an optimal occlusion ratio of about 2 to about 10,000, eg, about 80 to about 100. In another embodiment of any of the masked cytokines provided herein, the occlusion rate is from about 2 to about 7,500, from about 2 to about 5,000, from about 2 to about 2,500, from 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, about 2 to About 200, about 2 to about 100, about 2 to about 50, about 2 to about 25, about 2 to about 15, about 2 to about 10, about 5 to about 10, about 5 to about 15, about 5 to about 20 , about 10 to about 100, about 20 to about 100, about 30 to about 100, about 40 to about 100, about 50 to about 100, about 60 to about 100, about 70 to about 100, about 80 to about 100, or About 100 to about 1,000. In some embodiments, the masked cytokines provided herein exhibit an optimal occlusion ratio of about 2 to about 1,000. Binding of the masked cytokine to the protein of interest before and / or after cleavage with a protease can be determined using techniques well known in the art, such as by ELISA.

[0501] In some embodiments, a masking moiety described herein binds to a cell as described herein with an affinity lower than the affinity between an interleukin or a functional fragment thereof and a protein of interest (e.g., an interleukin receptor) interferons or their functional fragments. In certain embodiments, the masking moieties provided herein are ≥ 500 M, ≥ 250 M, ≥ 200 M, ≥ 150 M, ≥ 100 M, ≥ 50 M, ≥ 10 M, ≥ 1 M, ≥ 500 nM, A dissociation constant (Kd) of ≥ 250 nM, ≥ 150 nM, ≥ 100 nM, ≥ 50 nM, ≥ 10 nM, ≥ 1 nM, ≥ 0.1 nM, ≥ 0.01 nM, or ≥ 0.001 nM binds to cells as described herein interferons or their functional fragments.

[0502] 4. Production of Masked Interleukins The masked interleukins described herein are prepared using techniques available in the art, and exemplary methods of such techniques are described.

[0503] 4.1 Antibody Production Some embodiments of masked cytokines comprise antibodies or fragments thereof. The following sections provide additional details regarding the generation of antibodies and antibody fragments, variants and derivatives thereof useful in some embodiments of the masked interleukins provided herein. In some embodiments, the masked interleukin is in the form of a dimer resulting from two copies of the masked interleukin associated via disulfide bonds.

[0504] 1. Antibody Fragments In some embodiments, the invention encompasses antibody fragments. An antibody fragment may be any antibody fragment, such as an Fc domain, a portion of a heavy chain, a portion of a light chain, Fab, Fv or scFv, among other fragments. Antibody fragments can be produced by traditional means, such as enzymatic digestion, or by recombinant techniques. In certain instances, there are advantages to linking antibody fragments rather than full antibodies to the masked interleukins described herein. For a review of certain antibody fragments, see Hudson et al. (2003) Nat. Med. 9:129134.

[0505] Various techniques have been developed for the production of antibody fragments. Traditionally, such fragments have been obtained by 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, such fragments can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted by E. coli and other cell types such as HEK293 and CHO cells, thus allowing easy production of large quantities of these fragments. Alternatively, Fab-SH fragments can be recovered directly from the culture medium and chemically coupled to form F(ab)2 fragments (Carter et al., Bio / Technology 10: 163-167 (1992)). According to another approach, F(ab)2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab)2 fragments with increased half-life in vivo and comprising FcRN / salvage receptor binding epitope residues are described in US Patent No. 5,869,046. Other techniques for generating antibody fragments for use in masked interleukins will be apparent to those skilled in the art. In certain embodiments, the masked cytokine comprises a single chain Fv fragment (scFv). See WO 93 / 16185; US Patent Nos. 5,571,894; and 5,587,458. scFv fusion proteins can be constructed to produce fusions of effector proteins at the amino or carboxyl termini of the scFv. See Antibody Engineering, ed. Borrebaeck, supra. Additionally, in some embodiments, dual scFvs comprising two scFvs linked via a polypeptide linker can be used with masked cytokines.

[0506] In some embodiments, the invention includes linear antibodies (eg, as described in US Patent No. 5,641,870) or single chain immunoglobulins comprising the heavy and light chain sequences of the antibody linked via appropriate linkers. Such linear antibodies or immunoglobulins may be monospecific or bispecific. Such single chain immunoglobulins can dimerize thereby retaining structure and activity similar to antibodies (which are originally tetramers). Furthermore, in some embodiments, an antibody or fragment thereof may be an antibody having a single heavy chain variable region and no light chain sequences. Such antibodies are called single domain antibodies (sdAbs) or nanobodies. Such antibodies are also encompassed within the meaning of functional fragments of the antibodies of the present invention. Antibody fragments can be linked to the masked cytokines described herein according to the guidelines provided herein.

[0507] 2. Humanized Antibodies In some embodiments, the invention encompasses humanized antibodies or antibody fragments thereof. In some embodiments, a humanized antibody can be any antibody, including any antibody fragment. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a source that is non-human. Such non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can essentially follow 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), This is done by substituting hypervariable region sequences for the corresponding sequences of human antibodies. Thus, such "humanized" antibodies are chimeric antibodies (US Patent No. 4,816,567) in which substantially less than an intact human variable domain has been substituted with the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Humanized antibodies can be linked to the masked cytokines described herein according to the guidelines provided herein.

[0508] 3. Human Antibodies Human antibodies of some embodiments of the present invention can be constructed by combining Fv clonal variable domain sequences selected from human-derived phage display libraries with known human constant domain sequences. Alternatively, human monoclonal antibodies of some embodiments of the present invention can be produced by the fusionoma method, eg, by using mouse, rat, bovine (eg, cow) or rabbit cells, eg, 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, the human monoclonal antibodies of the invention can be obtained by immunizing a non-human animal comprising a human immunoglobulin locus with an antigen of interest, and can be obtained from the immunized animal or derived from the immunized animal. Animal cells are isolated to produce antibodies. Examples of suitable non-human animals include transgenic or transchromosomal animals, such as HuMAb Mouse® (Medarex, Inc.), KM Mouse®, "TC mouse" and Xenomouse™. See, eg, 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, Nos. 6,150,584, 6,162,963; and Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727. Human myeloma and murine-human fusion 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 described by Boerner et al., J. Immunol., 147: 86 (1991). Human antibodies can be linked to the masked cytokines described herein according to the guidelines provided herein.

[0509] 4. Bispecific Antibodies Bispecific antibodies are monoclonal antibodies that have binding specificities for at least two different antigens. In certain embodiments, bispecific antibodies are human or humanized antibodies. In some embodiments, one of the binding specificities is for a first antigen and the other binding specificity is for a second antigen, which can be two different epitopes on the same target protein or two different epitopes. Two different epitopes on the target protein. 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 certain cells, such as cancer cells. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (eg, F(ab')2 bispecific antibodies). Bispecific antibodies can be linked to the masked cytokines described herein according to the guidelines provided herein.

[0510] Methods for making bispecific antibodies are known in the art. See Milstein and Cuello, Nature, 305: 537 (1983); WO 93 / 08829 published May 13, 1993; Traunecker et al., EMBO J., 10: 3655 (1991); Kontermann and Brinkmann, Drug Discovery Today, 20(7):838-847. For additional details on generating bispecific antibodies, see, eg, Suresh et al., Methods in Enzymology, 121:210 (1986). Bispecific antibodies include cross-linked or "heteroconjugated" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. Heterobinding antibodies can be prepared using any suitable cross-linking method. Suitable crosslinking agents are well known in the art, along with a number of crosslinking techniques, and are disclosed in US Patent No. 4,676,980.

[0511] 5. Single Domain Antibodies In some embodiments, single domain antibodies are linked to masked cytokines according to the guidelines provided herein. A single domain antibody can be any antibody. A single domain antibody is a single polypeptide chain comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see eg, US Patent No. 6,248,516 Bl ). In some embodiments, a single domain antibody consists of all or a portion of the heavy chain variable domain of an antibody. In some embodiments, the single domain antibody is a camelid-derived antibody obtained by immunizing camelids with an antigen of interest. In some embodiments, the single domain antibody is a shark-derived antibody obtained by immunizing a shark with an antigen of interest. In some embodiments, the single domain antibody is a Nanobody (see eg WO 2004041865A2 and US20070269422A1).

[0512] 6. Antibody Variants In some embodiments, amino acid sequence modifications of the antibodies or fragments thereof described herein are contemplated. For example, it may be desirable to improve the FcRn binding affinity and / or pH-dependent FcRn binding affinity of an antibody. It may also be desirable to facilitate heterodimerization of antibody heavy chains by introducing certain amino acid modifications. Methods for promoting heterodimerization of antibody chains, including certain modifications to promote heterodimerization, are described by Klein et al. (2012), MAbs, 4(6): 653-663.

[0513] Amino acid sequence variants of antibodies can be prepared by introducing appropriate changes into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics. Amino acid changes can be introduced in the amino acid sequence of the subject antibody at the time the sequence is made.

[0514] A suitable method for identifying certain residues or regions of an antibody as preferred positions for mutagenesis is called "alanine scanning mutagenesis", as in Cunningham and Wells (1989) Science, 244:1081 - 1085 described. Here, a residue or group of target residues (e.g. charged residues such as arg, asp, his, lys, and glu) are identified and replaced by neutral or negatively charged amino acids (e.g. alanine or polyalanine) , to affect the interaction between amino acids and antigens. Those amino acid positions exhibiting functional sensitivity to the substitution are then optimized by introducing additional or other variants at or for the site of substitution. Thus, while the sites for introducing amino acid sequence variations are predetermined, the nature of the mutation itself need not be predetermined. For example, to analyze mutational efficacy at a given site, a random mutagenesis ala scan is performed at the codon or region of interest and the expressed immunoglobulins are screened for the desired activity.

[0515] Amino acid sequence insertions include amino and / or carboxyl terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, and Intrasequence insertion of residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusions of the N-terminus or C-terminus of the antibody to enzymes or polypeptides, which prolong the serum half-life of the antibody.

[0516] In some embodiments, the masked interleukin is modified to eliminate, reduce or otherwise impede proteolytic cleavage near the hinge region. The "hinge region" of IgG is usually defined as including E216 and ending at P230 of human IgG1 (according to the EU index as in Kabat), but functionally, the flexible part of the chain can be considered to include what is called the upper hinge region and Additional residues of the lower hinge region, such as from E216 to G237 (Roux et al., 1998 J Immunol 161:4083), and the lower hinge are referred to as residues 233 to 239 of the Fc region, where FcγR binding is usually attributed. Any of the masked interleukins described herein can be modified, for example, according to the methods described in US 20150139984A1, incorporated herein by reference, and by incorporating any of the modifications described therein.

[0517] 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, V3 08P. In some embodiments, such mutations enhance antibody binding to FcRn at low pH, but do not alter antibody affinity at neutral pH.

[0518] In certain embodiments, the antibody or fragment thereof is altered to increase or decrease the degree of glycosylation of the antibody. Glycosylation of polypeptides is typically either N-linked or O-linked. N-linkage refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are the enzymatic attachment of the carbohydrate moiety to asparagine Recognition sequence for amide side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactamine, galactose, or xylose to a hydroxylamine acid, most commonly serine or threonine, although it can also Use 5-hydroxyproline or 5-hydroxylysine.

[0519] Addition or deletion of glycosylation sites for masked cytokines is preferably accomplished by altering the amino acid sequence such that one or more of the tripeptide sequences described above are produced or removed (for N-linked glycosylation sites). Changes may also be effected by addition, deletion or substitution of one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0520] Where the antibody or fragment thereof comprises an Fc region, the carbohydrate attached thereto may be altered. For example, antibodies with mature carbohydrate structures that do not have trehalose attached to the Fc region of the antibody are described in US Patent Application No. US 2003 / 0157108 (Presta, L.). See also US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibodies with bisected N-acetylglucosamine (GlcNAc) in carbohydrates attached to the Fc region of the antibody are proposed in WO 2003 / 011878 by Jean-Mairet et al. and U.S. Patent No. 6,602,684 by Umana et al. and. Antibodies having at least one galactose residue in the oligosaccharide attached to the Fc region of the antibody are reported in WO 1997 / 30087 by Patel et al. See also WO 1998 / 58964 (Raju, S.) and WO 1999 / 22764 (Raju, S.) for antibodies with altered carbohydrates attached to their Fc region. See also US 2005 / 0123546 (Umana et al.) for antigen binding molecules with modified glycosylation.

[0521] In certain embodiments, the glycosylation variant comprises an Fc region, wherein the carbohydrate structure attached to the Fc region has no or reduced fuhalose. Such variants have improved ADCC function. Optionally, the Fc region further comprises one or more amino acid substitutions that further improve ADCC, such as substitutions at positions 298, 333 and / or 334 (Eu numbering of residues) of the Fc region. Examples of publications related to "de-fucosylated" or "trehalose-deficient" antibodies include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; 78; WO2005 / 053742; Okazaki et al. J . Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines producing afucosylated antibodies include protein fucosylation-deficient Lee 13 CHO cells (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US 2003 / 0157108 Al, Presta, L; and WO 2004 / 056312 Al, Adams et al., especially in Example 11), and gene knockout cell lines, such as the alpha-1,6-fucosyltransferase gene (FUT8) knockout CHO cells (Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)), and overexpression (31,4-N-acetylglycosaminoglycanyltransferase III (GnT-III) and Golgi p- Mannosidase II (Manll) cells.

[0522] In any of the embodiments herein, masked cytokines can be engineered to improve antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In some embodiments, masked cytokines can be produced in alpha 1,6-fucosyltransferase (Fut8) knockout cell lines. In some embodiments, the host cell has been modified to have reduced intrinsic α1,6-fucosylation activity. Examples of methods for altering 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 by reference In this article. Examples of methods and compositions for partially or completely inactivating the expression of the FUT8 gene can be found in, for example, US Publication No. 20160194665A 1, WO2006133148A2, the contents of which are incorporated herein by reference. In some embodiments, Lecl3 variants in CHO cells (see, e.g., Shields et al., J. Biol. Chem., 277(30):26733-40 (2002)) or YB2 / 0 cells with reduced FUT8 activity Masked cytokines are produced in strains (see eg Shinkawa et al., J. Biol. Chem., 278(5): 3466-73 (2003)). In some embodiments, small interfering RNA (siRNA) directed against genes involved in α1,6-fucosylation can be introduced (see, e.g., Mori et al., Biotechnol. Bioeng. 88(7): 901-908 (2004) ; Imai-Nishiya et al., BMC Biotechnol. 7: 84 (2007); Omasa et al., J. Biosci. Bioeng., 106(2): 168-173 (2008)). In some other embodiments, masked cytokines can be produced in cell lines that overexpress |31,4-N-acetylglycosaminoglycanyltransferase III (GnT-III). In other embodiments, the cell line additionally overexpresses high-basic p-mannosidase II (Manll). In some embodiments herein, the masked interleukin can comprise at least one amino acid substitution in the Fc region that improves ADCC activity.

[0523] In some embodiments, masked cytokines are altered to prolong their serum half-life. To prolong the serum half-life of the cytokines, FcRN / salvage receptor binding epitopes can be incorporated into linked antibodies (particularly antibody fragments), eg, as described in US 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 (eg IgGl, IgG2, IgG3 or IgG4) that is responsible for prolonging the serum half-life of the IgG molecule in vivo (US 2003 / 0190311, U.S. Patent No. 6,821,505; U.S. Patent No. 6,165,745; U.S. Patent No. 5,624,821; U.S. Patent No. 5,648,260; U.S. Patent No. 6,165,745; U.S. Patent No. 5,834,597).

[0524] Another type of variant is an amino acid substitution variant. Such variants have at least one amino acid residue in the antibody molecule replaced by a different residue. Relevant sites for substitutional mutagenesis include hypervariable regions, but also encompass FR changes. Conservative substitutions are shown in Table 2 under the heading "Preferred Substitutions". If such substitutions result in the desired change in biological activity, further substantial changes, termed "exemplary substitutions" in Table 2 or as further described below with reference to amino acid classes, can be introduced, and the products screened. Table 2: initial residue Exemplary substitution better replacement Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gin; Asn Lys Asn (N) Gin; His; Asp, Lys; Arg Gln Asp (D) Glu;Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser(S) Thr Thr Thr (T) Val; Ser Trp (W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

[0525] Substantial modification of the biological properties of an antibody is achieved by selected substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone, e.g., sheet or helical conformation, in the region of substitution, (b) the charge or hydrophobicity of the molecule at the target site, or c) the volume of the side chain. Amino acids can be grouped according to the similarity of the properties of their side chains (A. L. Lehninger, Biochemistry, 2nd edition, pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala ( A ), Val (V), Leu (L), lie (I), Pro (P), Phe (F), Trp (W), Met (M) (2) Uncharged polarities: 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) Alternatively, naturally occurring residues can be grouped based on common side chain properties: (1) Hydrophobicity: Norleucine, Met, Ala, Val, Leu, he; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromaticity: Trp, Tyr, Phe.

[0526] Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues may also introduce conservative substitution sites or introduce remaining (non-conservative) sites.

[0527] Another type of substitutional variant involves the substitution of a non-naturally occurring amino acid residue for a naturally occurring amino acid residue. Non-naturally occurring amino acid residues can be incorporated eg via tRNA recoding or via any of the methods as described eg in WO 2016154675A1 which is incorporated herein by reference.

[0528] One type of substitutional variant involves substituting one or more hypervariable region residues of a parental antibody (eg, a humanized or human antibody). Typically, the resulting variant selected for further development has modified (eg, improved) biological properties relative to the parent antibody from which it was generated. A suitable means for generating such substitutional variants involves affinity maturation using phage display, yeast display or mammalian display. Briefly, several hypervariable region positions (eg, 6-7 positions) are mutated to generate all possible amino acid substitutions at each position. The antibodies thus generated are presented from filamentous phage particles as fusions to at least part of a phage sheath protein (eg, the gene III product of M13) encapsulated within each particle. The phage-displayed variants are then screened for biological activity (eg, binding affinity). To identify candidate hypervariable region sites for modification, scanning mutagenesis (eg, alanine scanning) can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or additionally, it may be advantageous to analyze the crystal structure of the antigen-antibody complex to identify contact points between the antibody and the antigen. Such contact residues and neighboring residues are candidates for substitution according to techniques known in the art, including those described in detail herein. Once such variants have been generated, the panel of variants is screened using techniques known in the art, including those described herein, and antibodies with superior properties in one or more relevant assays may be selected for further development.

[0529] Nucleic acid molecules encoding amino acid sequence variants of masked cytokines are prepared by various methods known in the art. Such methods include, but are not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or oligosaccharide-mediated, e.g., variant or non-variant versions by earlier preparation of antibodies ( or site-directed) mutagenesis, PCR mutagenesis and cassette mutagenesis.

[0530] It may be desirable to introduce one or more amino acid modifications in the Fc region of an antibody of the invention, thereby generating Fc region variants. Fc region variants may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amine at one or more amino acid positions, including the amino acid position of a hinge cysteine. Amino Acid Modifications (e.g. Substitutions).

[0531] In some embodiments, the masked cytokine provided herein comprises an antibody or fragment thereof of an IgGl, IgG2, IgG3 or IgG4 isotype with enhanced effector function. In some embodiments, the masked cytokine provided herein comprises an antibody or fragment thereof of an IgGl isotype with enhanced effector function. In some embodiments, the masked cytokines provided herein have an IgGl isotype with enhanced effector function. In some embodiments, the masked interleukin is afucosylated. In some embodiments, the masked interleukin has an increased content of mannose moieties. In some embodiments, the masked interleukin has an increased content of bisected glycan moieties. In some embodiments, IgG1 comprises amino acid mutations.

[0532] In some embodiments, a masked cytokine provided herein comprises an antibody having an IgGl isotype, eg, a human IgGl isotype. In some embodiments, IgG1 comprises one or more amino acid substitutions that enhance effector function. In one embodiment, IgGl comprises amino acid substitutions S298A, E333A and K334A, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgG1 comprises amino acid substitutions S239D and I332E, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgG1 comprises amino acid substitutions S239D, A330L, and I332E, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgG1 comprises amino acid substitutions P247I and A339D or A339Q, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgG1 comprises amino acid substitutions D280H, K290S (with or without S298D or S298V), wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgG1 comprises amino acid substitutions F243L, R292P, and Y300L, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgGl comprises amino acid substitutions F243L, R292P, Y300L, and P396L, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgGl comprises amino acid substitutions F243L, R292P, Y300L, V305I and P396L, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgGl comprises amino acid substitutions G236A, S239D, and I332E, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgGl comprises amino acid substitutions K326A and E333A, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgG1 comprises amino acid substitutions K326 and E333S, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgG1 comprises amino acid substitutions K290E, S298G, T299A (with or without K326E), wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgG1 comprises amino acid substitutions K290N, S298G, T299A (with or without K326E), wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgG1 comprises the amino acid substitution K334V, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgGl comprises amino acid substitutions L235S, S239D and K334V, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgGl comprises amino acid substitutions K334V and Q331M, S239D, F243V, E294L or S298T, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgGl comprises amino acid substitutions E233L, Q311M, and K334V, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgG1 comprises amino acid substitutions L234I, Q311M, and K334V, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgGl comprises amino acid substitutions K334V and S298T, A330M or A330F, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgGl comprises amino acid substitutions K334V, Q311M and A330M or A330F, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgGl comprises amino acid substitutions K334V, S298T and A330M or A330F, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgG1 comprises amino acid substitutions K334V, S239D and A330M or S298T, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgGl comprises amino acid substitutions L234Y, Y296, and K290Y, F243V or E294L, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgG1 comprises amino acid substitutions Y296 and L234Y or K290Y, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgGl comprises amino acid substitutions S239D, A330S, and I332E, wherein the amino acid residues are numbered according to the EU index as in Kabat.

[0533] In some embodiments, IgGl comprises one or more amino acid substitutions that reduce or inhibit effector function. In one embodiment, IgGl comprises amino acid substitution N297A, N297G or N297Q, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, the IgGl comprises the amino acid substitution L234A or L235A, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgGl comprises amino acid substitutions C220S, C226S, C229S and P238S, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgGl comprises amino acid substitutions C226S, C229S, E233P, L234V, and L235A, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgGl comprises amino acid substitutions L234F, L235E and P331S, wherein the amino acid residues are numbered according to the EU index as in Kabat. In one embodiment, IgG1 comprises amino acid substitutions S267E and L328F, wherein the amino acid residues are numbered according to the EU index as in Kabat.

[0534] In light of the present specification and the teachings of the art, it is contemplated that in some embodiments, interleukin-masked antibodies or fragments thereof may, for example, comprise one or more Change. For example, it is believed that certain changes can be made in the Fc region which will result in altered (ie increased or decreased) Clq binding and / or complement dependent cytotoxicity (CDC), eg as described in WO99 / 51642. See also Duncan and Winter, Nature 322:738-40 (1988); US Patent No. 5,648,260; US Patent No. 5,624,821; and WO94 / 29351 for additional examples of Fc region variants. WO 00 / 42072 (Presta) and WO 2004 / 056312 (Lowman) describe antibody variants with improved or reduced binding to FcRs. The contents of these patent publications are specifically incorporated herein by reference. See also Shields et al. J. Biol. Chem. 9(2): 6591-6604 (2001). Prolonged half-life and interaction with the neonatal Fc receptor (FcRn) responsible for 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) ) binding improved antibodies are described in US 2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions that improve the binding of the Fc region to FcRn. Polypeptide variants with altered Fc region amino acid sequences and increased or decreased C1q binding ability are described in US Pat. No. 6,194,551 B1, WO99 / 51642. The contents of those patent publications are specifically incorporated herein by reference. See also Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0535] 4.2 Masked Interleukin-Drug Conjugates The present invention also provides masked interleukin-drug conjugates (MCDCs) comprising a masked interleukin provided herein conjugated to one or more pharmaceutical agents, The cytokine can be any of the masked cytokines disclosed herein. In some embodiments, the one or more agents are cytotoxic agents, such as chemotherapeutics or drugs, growth inhibitors, toxins (e.g., protein toxins, bacterial, fungal, enzymatically active toxins of plant or animal origin or fragments thereof) or radioactive isotopes. In some embodiments, one or more agents are immunostimulants.

[0536] In some embodiments, the one or more drugs that bind to the masked cytokine include, but are not limited to, maytansinoids (see U.S. Patent No. 5,208,020, No. 5,416,064 and European Patent EP 0 425 235 B1); auristatins such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin; calicheamicin or its derivatives (see U.S. Patent Nos. No. 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); anthracyclines such as Daunorubicum Daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006) ; Torgov et al., Bioconj. Chem. 16:717721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate (methotrexate); vindesine (vindesine) ; taxanes; such as docetaxel, paclitaxel, larotaxel, tesetaxel and ortataxel; trichothecene and CC1065.

[0537] In another embodiment, one or more drugs that bind to masked cytokines include, but are not limited to, tubulin polymerization inhibitors (e.g., maytansinoids and auristatins), DNA damaging agents (such as pyrrolobenzodiazepine (PBD) dimer, calicheamicin, duocarmycin, and indo-linobenzodiazepine dimer) and DNA synthesis inhibitors (eg exatecan derivative Dxd).

[0538] In another embodiment, the masked cytokine-drug conjugate comprises a masked cytokine as described herein conjugated to an enzymatically active toxin or fragment thereof comprising (but Not limited to) diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, acacia abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, pokeweed (Phytolaca americana) protein (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin ), mitogellin, restrictocin, phenomycin, enomycin and mycothecenes.

[0539] In another embodiment, a masked interleukin-drug conjugate comprises a masked interleukin as described herein bonded to a radioactive atom to form a radioconjugate. Various radioactive isotopes can be used to make radioconjugates. Examples include radioisotopes of At211, 1131, 1125, Y90, Rel86, Rel88, Sml53, B1212, P32, Pb212, and Lu. When radioconjugates are used for detection, they may contain radioactive atoms such as tc99m or 1123 for scintigraphic studies; or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri) , and such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

[0540] In some embodiments, a masked interleukin-drug conjugate comprises a masked interleukin as described herein in combination with one or more immunostimulatory agents. In some embodiments, the immunostimulatory agent is a stimulating protein of interferon genes (STING) agonist or a toll-like receptor (TLR) agonist.

[0541] The STING agonist can be any agonist of STING. In some embodiments, the STING agonist is a cyclic dinucleotide (CDN). A CDN may 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][l,4]thiathiol-6-formamide, or a derivative or variant thereof. See eg Sali et al. (2015) PloS Pathog., 11(12): e!005324.

[0542] The TLR agonist may 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 surface of a cell, such as TLR1, TLR2, TLR4, or TLR5. In some embodiments, the TLR agonist is an agonist of a TLR expressed intracellularly, such as TLR3, TLR7, TLR8, TLR9, or TLR10.

[0543] A variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2- Pyridyldithio)propionate (SPDP), succimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), Aminothiolane (IT), bifunctional derivatives of imide esters (such as diiminoadipate HCl), active esters (such as disuccinimidyl suberate ), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexamethylenediamine), dinitrogen derivatives (such as bis(p-diazobenzoyl)- ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-reactive fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon 14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See W094 / 11026. A linker may be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or linkers containing disulfide bonds can be used (Chari et al., Cancer Res. 52:127- 131 (1992); US Patent No. 5,208,020).

[0544] MCDC herein expressly contemplates (but is not limited to) such conjugates prepared with cross-linking agents including (but not limited to) BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, Sulfo-EMCS, Sulfo-GMBS, Sulfo-KMUS, Sulfo-MBS, Sulfo-SIAB, Sulfo-SMCC, and Sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), such crosslinkers are commercially available (eg, from Pierce Biotechnology, Inc., Rockford, IL., U.S.A).

[0545] 4.3 Vectors, host cells and recombination methods For recombinant production of the masked cytokines of the present invention, one or more nucleic acids encoding them are isolated and inserted into replicable vectors for further breeding (DNA amplification) or expression . DNA encoding the masked interleukin, including components thereof, is readily isolated and sequenced using known procedures. Many vectors are available. The choice of vector will depend in part on the host cell used. Typically, host cells are of prokaryotic or eukaryotic (usually mammalian) origin. It will be appreciated that constant regions of any isotype of antibodies or fragments thereof may be used for this purpose, including IgG, IgM, IgA, IgD and IgE constant regions, as appropriate, and that such constant regions may be obtained from any human or animal species. In some embodiments, a vector is used to encode a masked cytokine. In some embodiments, more than one vector is used to encode the masked cytokine. 1. Production of masked cytokines using prokaryotic host cells a. Vector construction

[0547] Polynucleotide sequences encoding the polypeptide components of the masked interleukins of the invention can be obtained using standard recombinant techniques. The desired polynucleotide sequence of an antibody or antibody fragment thereof can be isolated and sequenced from antibody producing cells such as fusionoma cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesizer or PGR techniques, or obtained from other sources. After harvesting, the sequence encoding the components of the masked interleukin is inserted into a recombinant vector capable of replicating and expressing the heterologous polynucleotide in a prokaryotic host. Many vectors available and known in the art can be used for the purposes of the present invention. Selection of an appropriate vector will primarily depend on the size of the nucleic acid inserted into the vector and the particular host cell transformed with the vector. Each vector contains various components depending on its function (amplification or expression of a heterologous polynucleotide, or both) and compatibility with the particular host cell in which it resides. Vector components typically include, but are not limited to, an origin of replication, a selectable marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, a heterologous nucleic acid insert, and a transcription terminator sequence.

[0548] In general, plastid vectors containing replicon and control sequences derived from a species compatible with the host cell are used in conjunction with such hosts. Vectors usually carry replication sites as well as marker sequences capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322 (a plasmid derived from E. coli species). pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance and thus provides a means of easily identifying transformed cells. pBR322, derivatives thereof, or other microbial plastids or phages may also contain or be modified to contain promoters that can be used by the microorganism to express endogenous proteins. Examples of pBR322 derivatives useful for expressing specific antibodies are described in detail in US Patent No. 5,648,237 to Carter et al.

[0549] Alternatively, phage vectors containing replicons and control sequences compatible with the host microorganism can be used as transformation vectors in conjunction with such hosts. For example, phage such as 7GEM.TM.-11 can be used to generate recombinant vectors that can be used to transform sensitive host cells such as E. coli LE392.

[0550] Expression vectors of the invention may comprise two or more promoter-cistronic pairs encoding each of the polypeptide components. A promoter is an untranslated regulatory sequence located upstream (5') of a cistron that regulates its expression. Prokaryotic promoters typically fall into two categories: inducible and constitutive. An inducible promoter is one under whose control the level of transcription of the initiating cistron increases in response to a change in culture conditions, such as the presence or absence of nutrients or a change in temperature.

[0551] A number of promoters recognized by a variety of potential host cells are well known. The promoter of choice can be operably linked to the gene encoding either strand of the masked interleukin by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the invention. Cistronic DNA. Both the native promoter sequence and many heterologous promoters can be used to direct the amplification and / or expression of the gene of interest.

[0552] In some embodiments, heterologous promoters are used because they generally achieve greater transcription and higher yields of expressed target genes than native target polypeptide promoters. Promoters suitable for use with prokaryotic hosts include the PhoA promoter, the [3-galactase and lactose promoter systems, the tryptophan (trp) promoter system, and hybrid promoters such as the tac or trc promoters . However, other promoters that function in bacteria, such as other known bacterial or phage promoters, are also suitable. Its nucleotide sequence has been published, thereby allowing the skilled worker to use linkers or adapters to operably join it to the cis of light and heavy chains of interest encoding, for example, masked cytokines comprising light and heavy chains. (Siebenlist et al., (1980) Cell 20:269) to supply any desired restriction sites.

[0554] In one aspect of the invention, each cistron within the recombinant vector comprises a secretory signal sequence component that directs the translocation of the expressed polypeptide across the membrane. In general, the signal sequence may be a component of the vector, or it may be part of the DNA of the polypeptide of interest inserted into the vector. The signal sequence selected for the purposes of the present invention should be one that is recognized and processed (ie, cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native signal sequence of the heterologous polypeptide, the signal sequence is replaced by a prokaryotic signal sequence selected from, for example, the group consisting of: alkaline phosphatase, penicillinase, Ipp, or thermostable enterotoxin II (STII) leader, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of the present invention, the signal sequences used in the two cistrons of the expression system are STII signal sequences or variants thereof.

[0555] In another aspect, the production of the polypeptide components according to the invention can take place in the cytoplasm of the host cell, and thus does not require the presence of a secretion signal sequence within each cistron. In that regard, with respect to embodiments comprising immunoglobulin light and heavy chains, for example, the light and heavy chains are expressed with or without sequences of masking moieties, linker sequences, etc., folded and assembled to function within the cytoplasm sex immunoglobulin. Certain host strains (eg, E. coli trxB-strain) provide cytoplasmic conditions that favor disulfide bond formation, thereby enabling correct folding and assembly of expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

[0556] The masked interleukins of the invention can also be produced by using an expression system in which the quantitative ratios of expressed polypeptide components can be adjusted in order to maximize the yield of secreted and properly assembled antibodies of the invention. Such modulation is accomplished, at least in part, by simultaneously modulating the translational strength of the polypeptide components.

[0557] Prokaryotic host cells suitable for expressing the masked interleukins of the present invention include Archaebacteria and Eubacteria, such as Gram-negative or Gram-positive )organism. Examples of suitable bacteria include Escherichia (e.g. E. coli), bacilli (e.g. B. subtilis), enterobacteria, Pseudomonas species (e.g. Pseudomonas aeruginosa (P. aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobium Rhizobia, Vitreoscilla or Paracoccus. In one embodiment, Gram-negative cells are used. In one embodiment, E. coli cells are used as hosts for the present invention. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, Vol. 2 (Washington, D. C.: American Society for Microbiology, 1987), pp. 1190-1219); ATCC Deposit No. 27,325) and Derivatives thereof include strain 33D3 with genotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 AompTA (nmpc-fepE) degP41 kanR (US Pat. No. 5,639,635). Other strains and derivatives thereof, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli k1776 (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 above-mentioned bacteria with defined genotypes are known in the art and described, for example, in Bass et al., Proteins, 8:309-314 (1990). Considering the replicability of the replicon in bacterial cells, it is often necessary to select suitable bacteria. For example, when a well-known plasmid such as pBR322, pBR325, pACYC177 or pKN410 is used to supply the replicon, E. coli, Serratia or Salmonella species are preferably used as hosts. Typically, host cells should secrete minimal amounts of proteolytic enzymes, and other protease inhibitors should preferably be incorporated into the cell culture. b. Masked cytokine production host cells are transformed with the expression vectors described above and cultured in conventional nutrient media conditioned as appropriate to induce promoters, select for transformants, or amplify encoding Gene of the desired sequence.

[0559] Transformation means the introduction of DNA into a prokaryotic host such that the DNA can be replicated as an extrachromosomal element or as a chromosomal integrant. Depending on the host cell used, transformation is performed using standard techniques appropriate for such cells. Calcium treatment with calcium chloride is typically used for bacterial cells that contain a substantial cell wall barrier. Another transformation method uses polyethylene glycol / DMSO. Yet another technique used is electroporation.

[0560] The prokaryotic cells used to produce the masked cytokines of the invention are grown in media known in the art and suitable for culturing the host cell of choice. Examples of suitable media include luria broth (LB) plus necessary nutritional supplements. In some embodiments, the medium also contains a selection agent selected based on the construction of the expression vector to selectively allow the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the culture medium for the growth of cells expressing the ampicillin resistance gene.

[0561] In addition to carbon, nitrogen, and inorganic phosphate sources, any necessary supplements may also be included at appropriate concentrations, introduced alone or mixed with other supplements or media such as complex nitrogen sources. Optionally, the medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, dithioerythritol, and dithiothreitol.

[0562] Prokaryotic host cells are cultured at a suitable temperature. In certain embodiments, for E. coli growth, the growth temperature ranges from about 20°C to about 39°C, from about 25°C to about 37°C, or is about 30°C. The pH of the medium can be anywhere in the range of about 5 to about 9, depending primarily on the host organism. In certain embodiments, for E. coli, the pH is from about 6.8 to about 7.4, or about 7.0.

[0563] If an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for activating the promoter. In one aspect of the invention, the PhoA promoter is used to control the transcription of the polypeptide. Accordingly, transformed host cells are cultured in phosphate-limited medium for induction. In certain embodiments, the phosphate-limiting medium is C.R.A.P. medium (see, eg, Simmons et al., J. Immunol. Methods (2002), 263:133-147). Depending on the vector construct used, a variety of other inducers may be used, as known in the art.

[0564] In one embodiment, masked cytokines of the present invention expressed are secreted into and recovered from the periplasm of the host cell. Protein recovery typically involves disruption of microorganisms, usually by means such as osmotic shock, sonication, or lysis. After cell disruption, cell debris or whole cells can be removed by centrifugation or filtration. Proteins can be further purified, for example, by affinity resin chromatography. Alternatively, the protein can be transferred to and isolated from the culture medium. Cells can be removed from the culture, and the culture supernatant filtered and concentrated to further purify the protein produced. The expressed polypeptides can be further isolated and identified using generally known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot analysis.

[0565] In one aspect of the invention, masked cytokines are produced in large quantities by fermentation methods. Various large-scale batch-fed fermentation procedures can be used to produce recombinant proteins. Large scale fermentations have a capacity of at least 1000 liters and in certain embodiments, have a capacity of about 1,000 to 100,000 liters. These fermenters use an agitator impeller to distribute oxygen and nutrients, especially glucose. Small-scale fermentation generally refers to fermentation in fermenters with a volume capacity not exceeding about 100 liters and which may range from about 1 liter to about 100 liters.

[0566] During fermentation, induction of protein expression typically begins after the cells have grown under suitable conditions to the desired density (eg, an OD550 of about 180-220), at which stage the cells are in the early stationary phase. Depending on the vector construct used, a variety of inducers can be used, as known in the art and described above. Cells can be grown for a shorter period of time prior to induction. Cells are typically induced for about 12-50 hours, but longer or shorter induction times can be used.

[0567] In order to improve the yield and quality of the polypeptide of the present invention, various fermentation conditions can be modified. For example, to improve the correct assembly and folding of e.g. secreted antibody polypeptides, other vectors that overexpress chaperonins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (with chaperonin activity) can be used peptidyl prolinyl cis, trans-isomerase) to co-transform host prokaryotic cells. Chaperones have been shown to promote proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. (1999) J. Biol. Chem. 274:19601-19605; Georgiou et al., U.S. Patent No. 6,083,715; Georgiou et al., U.S. 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 al. (2001) Mol. Microbiol. 39:199210.

[0568] To minimize proteolysis of expressed heterologous proteins, especially those that are proteolytically sensitive, certain proteolytic enzyme deficient host strains may be used in the present invention. For example, host cell strains can be modified to effect genetic mutations in genes encoding known bacterial proteases such as Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI, and combinations thereof. Some protease-deficient E. coli strains are available and described, for example, in Joly et al. (1998), supra; Georgiou et al., U.S. Patent No. 5,264,365; Georgiou et al., U.S. Patent No. 5,508,192; Kara et al., Microbial Drug Resistance, 2:63-72 (1996). In some embodiments, E. coli strains lacking proteolytic enzymes and undergoing plastid transformation to overexpress one or more accompanying proteins are used as host cells in the expression systems of the present invention.

[0569] c. Purification of Masked Interleukins In some embodiments, masked interleukins produced herein are further purified to obtain substantially homogeneous preparations for other assays and uses. Standard protein purification methods known in the art can be used. The following procedures illustrate suitable purification procedures: fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or cation exchange resins such as DEAE, chromatofocusing, SDS - PAGE, ammonium sulfate precipitation, and gel filtration using eg Sephadex G-75.

[0570] In some embodiments, the masked cytokines of the invention are immunoaffinity purified using Protein A immobilized on a solid phase. Protein A is a 41 kD cell wall protein 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 for immobilizing protein A can be a column with a glass or silica surface, or a glass column with controlled micropores or a silicic acid column. In some applications, the column is coated with a reagent, such as glycerol, to possibly prevent non-specific attachment of contaminants.

[0571] As a first step of purification, cell culture-derived preparations as described above can be applied to a solid phase immobilizing Protein A to achieve specific binding of the relevant masked cytokine to Protein A. The solid phase will then be washed to remove contaminants that are non-specifically bound to the solid phase. Finally, the relevant masked cytokines are recovered from the solid phase by elution.

[0572] Other purification methods that provide high affinity binding to components of the masked interleukin can be employed according to standard protein purification methods known in the art. 2. Production of masked cytokines using eukaryotic host cells Vectors for eukaryotic host cells typically include one or more of the following non-limiting components: signal sequence, origin of replication, one or more markers Genes, enhancer elements, promoters and transcription termination sequences.

[0574] a. Signal Sequence Components Vectors for use in eukaryotic host cells may also contain a signal sequence or other polypeptide with a specific cleavage site at the N-terminus of the relevant mature protein or polypeptide. The heterologous signal sequence of choice can be one that is recognized and processed (ie, cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, such as the herpes simplex gD signal, can be used.

[0575] The DNA of such precursor regions is joined in-frame to the DNA encoding the masked interleukin.

[0576] b. Origin of Replication Generally, mammalian expression vectors do not require an origin of replication component. For example, since the SV40 origin contains an early promoter, the SV40 origin can typically be used.

[0577] c. Selectable Gene Components Expression vectors and cloning vectors may contain selectable genes, also known as selectable markers. Typical selection genes encode proteins that: (a) confer resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate, or tetracycline, (b) auxotrophic deficiency of complement (if relevant), Or (c) provide important nutrients that cannot be obtained from complex media.

[0578] One example of a selection procedure utilizes drugs to arrest the growth of host cells. Those cells successfully transformed with a heterologous gene produce a protein that confers drug resistance and thus survive the selection therapy. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.

[0579] Another example of a selectable marker suitable for use in mammalian cells is a marker that enables the identification of cells capable of taking up nucleic acid encoding a masked interleukin, such as DHFR, thymidine kinase, metallothionein-I, and metal Thiolin-II, primate metallothionein gene, adenosine deaminase, ornithine decarboxylase, etc.

[0580] For example, in some embodiments, DHFR-selected gene-transformants are first identified by culturing all transformants in media containing methotrexate (Mtx), a competitive antagonist of DHFR. cell. In some embodiments, when wild-type DHFR is used, a suitable host cell is a Chinese hamster ovary (CHO) cell line lacking DHFR activity (eg, ATCC CRL-9096).

[0581] Alternatively, host cells transformed or co-transformed with DNA sequences encoding masked cytokines, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3'-phosphotransferase (APH) (especially wild-type hosts containing endogenous DHFR) can be detected by adding a selection agent for a selectable marker (such as an aminoglycoside antibiotic, such as kanamycin, neomycin or G418). for cell growth selection. See US Patent No. 4,965,199. Host cells can include NSOs, including glutamine synthetase (GS)-deficient cell lines. Methods for using GS as a selectable marker for mammalian cells are described in US Patent No. 5,122,464 and US Patent No. 5,891,693.

[0582] d. Promoter Component Expression and Colonization Vectors typically contain a promoter recognized by the host organism and operably linked to a masked cytokine encoding the relevant masked interleukin (which can be any masked cell described herein). Interleukin) nucleic acid. The promoter sequences of eukaryotes are known. For example, almost all eukaryotic genes have an AT-rich region located about 25 to 30 bases upstream from the transcription initiation site. Another sequence found 70 to 80 bases upstream from the start of transcription 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 signal the addition of the poly A tail to the 3' end of the coding sequence. In certain embodiments, any or all of these sequences may be suitably inserted into eukaryotic expression vectors.

[0583] Transcription from vectors in mammalian host cells is controlled, for example, by promoters such as polyoma virus, fowl pox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cell Promoters derived from viral genomes of giant viruses, retroviruses, hepatitis B virus, and Simian virus 40 (SV40); heterologous mammalian promoters, such as actin promoters or immunoglobulin promoters; thermal Shock promoters, provided that such promoters are compatible with the host cell system.

[0584] The early and late promoters of the SV40 virus are preferably obtained as an SV40 restriction fragment that also contains the SV40 virus origin of replication. The immediate early promoter of human cytomegalovirus is preferably obtained as a HindllllE restriction fragment. A system for expressing DNA in a mammalian host using bovine papilloma virus as a vector is disclosed in US Patent No. 4,419,446. Modifications of this system are described in US Patent No. 4,601,978. See also Reyes et al., Nature 297:598601 (1982) describing the expression of human [3-interferon cDNA in murine cells under the control of the thymidine kinase promoter from herpes simplex virus. Alternatively, the Rous Sarcoma Virus long terminal repeat can be used as a promoter.

[0585] e. Enhancer Element Components Transcription by higher eukaryotes of DNA encoding a masked interleukin of the invention is typically increased by inserting enhancer sequences into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein and insulin). Typically, however, enhancers from eukaryotic viruses will be used. Examples include SV40 enhancer on the late side of the source of replication (bp 100-270), human cytomegalovirus early promoter enhancer, murine cytomegalovirus early promoter enhancer, polyomavirus on the late side of the source of replication Enhancers and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) (describing enhancer elements for activation of eukaryotic promoters). The enhancer can be spliced ​​into the vector at a position 5' or 3' to the masked interleukin coding sequence, but is usually at a position 5' relative to the promoter.

[0586] f. Transcription Termination Components Expression vectors used in eukaryotic host cells may also contain sequences required for transcription termination and mRNA stabilization. Such sequences are usually obtained from the 5' and occasionally 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the masked interleukin. One suitable transcription termination component is the bovine growth hormone polyadenylation region. See WO94 / 11026 and the expression vehicles disclosed therein.

[0587] g. Selection and Transformation of Host Cells Suitable host cells for use herein for cloning or expressing DNA in vectors include higher eukaryotic cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become routine procedure. Examples of suitable mammalian host cell lines are the SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651); the human embryonic kidney cell line (293 or 293 cells sub-selected for growth in suspension culture , Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); Murine sertoli cell (murine sertoli cell) (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); Monkey kidney cell (CV1 ATCC CCL 70 ); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (buffalo rat liver cell) (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); murine breast tumors (MMT 060562, ATCC CCL51); TRI cells (Mather et al. Human, Annals N. Y Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human liver tumor cell line (Hep G2).

[0588] Host cells are transformed with the expression or cloning vectors described above for the production of masked cytokines and cultured, where appropriate, in conventional nutrient media modified to induce promoters, select for transformants or The gene encoding the desired sequence is amplified.

[0589] h. Culturing Host Cells The host cells used to produce the masked interleukins of the invention can be cultured in a variety of media. Commercially available media (such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma)) Suitable for culturing host cells. In addition, any one of the media described in the following documents can be used as a culture medium for the host cell: Ham et al., Meth. Enz. 58:44 (1979); Barnes et al., 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 Reexamination 30,985. Any of these media may be supplemented with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium salts, magnesium salts, and phosphate salts), Buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range) and Glucose or equivalent energy source. Any other supplements may also be included at suitable concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, and the like, are those previously used for host cells selected for expression and will be apparent to those of ordinary skill in the art.

[0590] i. Purification of Masked Interleukins Using recombinant techniques, masked interleukins can be produced intracellularly or secreted directly into the culture medium. If the masked cytokine is produced intracellularly, particulate debris (host cells or lysed fragments) can be removed as a first step, for example by centrifugation or ultrafiltration. In cases where secretion of cytokines into the medium is masked, supernatants from such expression systems can first be concentrated using commercially available protein concentration filters such as Amicon or Millipore Pellicon ultrafiltration units. Protease inhibitors such as PMSF may be included in any previous steps to inhibit proteolysis, and antibiotics may be included to prevent growth of adventitious contaminants.

[0591] Masked interleukin compositions produced from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a convenient technique. The suitability of protein A as an affinity ligand depends on the type and isotype, if any, of any immunoglobulin Fc domain present in the masked interleukin. Protein A can be used to purify antibodies based on human IgGl, IgG2, or IgG4 heavy chains (Lindmark et al., J. Immunol. Methods 62:1-13 (1983)). Protein G is recommended for all murine isotypes as well as human y3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached can be agarose, but other matrices can be used. Mechanically stable matrices such as controlled micropore glass or poly(styrene divinyl)benzene enable faster flow rates and shorter processing times than agarose can achieve. In cases where the masked interleukin contains a CH3 domain, Bakerbond ABX™ resin (J.T. Baker, Phillipsburg, N.J.) is suitable for purification.

[0592] Other protein purification techniques can also be utilized, such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, silica chromatography, on anion or cation exchange resins (such as polyaspartic acid columns) Chromatography, chromatofocusing, SDS-PAGE and ammonium sulfate precipitation of heparin SEPHAROSE™ chromatography, depending on the masked cytokines to be recovered.

[0593] After any initial purification steps, the mixture containing the relevant masked cytokines and contaminants can be removed, for example, by using a low pH hydrophobic interaction layer with an elution buffer at a pH between about 2.5-4.5. Further purification is carried out at low salt concentrations (eg, about 0-0.25 M salt).

[0594] In general, various methods for the preparation of masked cytokines for research, testing and clinical use are recognized in the art, conform to the methods described above and / or are familiar with the art The skilled person deems applicable to the particular masked interleukin concerned.

[0595] 5. Compositions In some aspects, also provided herein are compositions comprising any of the masked cytokines described herein. In some embodiments, the composition comprises any of the exemplary embodiments of masked interleukins described herein. In some embodiments, the composition comprises a dimer of any of the masked interleukins described herein. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a masked cytokine and further comprises one or more of the components as described in detail below. For example, in some embodiments, the composition comprises one or more pharmaceutically acceptable carriers, excipients, stabilizers, buffers, preservatives, tonicity agents, nonionic surfactants, or cleansing agents. agent, or other therapeutic agent or active compound, or a combination thereof. Various embodiments of compositions are sometimes referred to herein as formulations.

[0596] Therapeutic formulations for storage are prepared by mixing the active ingredient having the desired degree of purity with optionally a pharmaceutically acceptable carrier, excipient or stabilizer (Remington: The Science and Practice of Pharmacy, 20th ed., published by Lippincott Williams & Wiklins, edited by Gennaro, Philadelphia, Pa. 2000). Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants (including ascorbic acid, methionine, vitamin E, sodium metabisulfite); Preservatives, isotonic agents, stabilizers, metal complexes (eg Zn-protein complexes); chelating agents such as EDTA and / or non-ionic surfactants.

[0597] Buffers may be used to control the pH within a range that optimizes therapeutic effectiveness, especially where stability is pH dependent. The buffer may be present at a concentration ranging from about 50 mM to about 250 mM. Buffers suitable for use in the present invention include organic and inorganic acids and salts thereof. For example, citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. Alternatively, the buffer may consist of histidine and trimethylamine salts, such as Tris.

[0598] Preservatives may be added to prevent microbial growth and are typically present in the range of about 0.2%-1.0% (w / v). Examples of suitable preservatives commonly used with therapeutic agents include stearyldimethylbenzyl ammonium chloride; hexahydroxyl quaternary ammonium chloride; benzalkonium halides (e.g. chloride, bromide, iodide), Benzethonium Chloride; Thimerosal, Phenol, Butanol, or Benzyl Alcohol; Alkylparabens, such as Methylparaben or Propylparaben; Catechol; Resorcinol; Cyclohexanol , 3-pentanol, m-cresol, o-cresol, p-cresol, methylparaben, propylparaben, 2-phenoxyethanol, butylparaben, 2-phenyl Alcohol, ethanol, chlorobutanol, thimerosal, bronopol, benzoic acid, imidazolidinyl urea, chlorhexidine, anhydrous sodium acetate, chlorocresol, ethylparaben, and chlorphenesin (3p-chlorobutane phenoxypropane-1,2-diol).

[0599] Tonicity agents (sometimes referred to as "stabilizers") may be present to adjust or maintain the tonicity of the liquids in the composition. When used with large charged biomolecules such as proteins and antibodies, they are often referred to as "stabilizers" because they interact with the charged groups on the side chains of amino acids, thereby reducing the intermolecular and intramolecular possibility of interaction.

[0600] Considering the relative amounts of the other ingredients, the tonicity agent may be present in any amount between about 0.1% to about 25% by weight or between about 1 to about 5% by weight. In some embodiments, tonicity agents include polyhydric sugar alcohols, trihydric alcohols, or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0601] Additional excipients include agents that can act as one or more of: (1) bulking agents, (2) solubility enhancers, (3) stabilizers, and (4) and prevent denaturation or sticking to container walls medicine. Such excipients include: polyhydric sugar alcohols (listed above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine acid, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols, such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose , xylose, ribose, ribitol, myoinisitose, myoinositol, galactose, galactitol, glycerol, cyclic alcohols (such as inositol), polyethylene glycol; sulfur-containing reducing agents, Such as urea, glutathione, lipoic acid, sodium thioacetate, thioglycerol, a-monothioglycerol, and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin, or others 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 polydextrose.

[0602] Non-ionic 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 and Does not cause denaturation of active therapeutic proteins or antibodies. Nonionic surfactants are present in the range of about 0.05 mg / ml to about 1.0 mg / ml or about 0.07 mg / ml to about 0.2 mg / ml. In some embodiments, the nonionic surfactant is present in the range of about 0.001% to about 0.1% w / v, or about 0.01% to about 0.1% w / v, or about 0.01% to about 0.025% w / v.

[0603] Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), polyoxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, poly Oxyethylene sorbitan monoether (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, macrogol 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol mono Stearate, sucrose fatty acid ester, methylcellulose and carboxymethylcellulose. Anionic detergents that may be used include sodium lauryl sulfate, sodium dioctyl sulfosuccinate and sodium dioctyl sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0604] In order for a formulation to be useful for in vivo administration, it must be sterile. Formulations can be rendered sterile by filtration through sterile filtration membranes. Therapeutic compositions herein are typically presented in a container with a sterile access port, such as an intravenous solution bag or bottle with a stopper piercable by a hypodermic needle.

[0605] The route of administration is according to known and accepted methods, such as by single or multiple bolus injections or infusions over a prolonged period of time in a suitable manner, for example by subcutaneous, intravenous, intraperitoneal, intramuscular Injection or infusion by intra-, intra-arterial, intralesional or intra-articular routes, topical administration, inhalation or by sustained or sustained release.

[0606] Any of the masked cytokines described herein can be used alone or in combination with other therapeutic agents, as can the methods described herein. The term "combination" encompasses two or more therapeutic agents (eg, a masked cytokine and a therapeutic agent) included in the same or separate formulations. In some embodiments, "combination" refers to "simultaneous" administration, in which case the administration of a masked interleukin of the invention is administered simultaneously with the administration of one or more additional therapeutic agents (e.g., at the same time or over a period of time). The interval between the administration of the masked cytokine and the administration of the one or more additional therapeutic agents is within one hour). In some embodiments, "combination" refers to sequential administration, in which case the administration of the masked cytokines of the invention occurs before and / or after the administration of one or more additional therapeutic agents (e.g., masked The interval between the administration of the cytokine and the administration of the one or more additional therapeutic agents is greater than one hour). Agents contemplated herein include, but are not limited to, cytotoxic agents, cytokines, agents targeting immune checkpoint molecules, agents targeting immunostimulatory molecules, growth inhibitory agents, immunostimulatory agents, anti-inflammatory agents, or anticancer agents .

[0607] The formulations herein may also contain more than one active compound as necessary for the particular indication being treated, preferably active compounds with complementary activities that do not adversely affect each other. Alternatively or additionally, the composition may comprise a cytotoxic agent, a cytokine, an agent targeting an immune checkpoint molecule or a stimulatory molecule, a growth inhibitory agent, an immunostimulatory agent, an anti-inflammatory agent, or an anticancer agent. Such molecules are preferably present in combination in amounts effective to achieve the desired purpose.

[0608] The formulations may be presented in any suitable state, such as liquid, solid (lyophilized) or frozen formulations. Routes for the preparation of each of these types of formulations for therapeutic use are well known in the art.

[0609] 6. Methods of Treatment Provided herein are methods for treating or preventing a disease in a subject comprising administering to the individual an effective amount of any of the masked cytokines described herein or a composition thereof. In some embodiments, methods for treating or preventing a disease in a subject comprising administering to the subject any of the compositions described herein are provided. In some embodiments, the subject (eg, a human patient) has been diagnosed with cancer or is at risk of developing such a disorder. In some embodiments, there is provided a method for treating or preventing a disease in a subject, comprising administering to the subject an effective amount of any masked cytokine described herein or a composition thereof, wherein the masked Cytokines are activated upon cleavage by enzymes. In some embodiments, the masked interleukin is activated in the tumor microenvironment. Masked cytokines are therapeutically active after their cleavage. Thus, in some embodiments, the active agent is a cleavage product.

[0610] For the prophylaxis or treatment of disease, as defined above, the appropriate dosage of the active agent will depend on the type of disease being treated, the severity and course of the disease, whether the agent is being administered for prophylactic or therapeutic purposes, previous therapy, the individual's Depending on the clinical history and response to the drug and the judgment of the attending physician. The agent is preferably administered to the subject at one time or over a series of treatments.

[0611] In some embodiments, the protease that acts to cleave the protein cleavage peptide is an MMP.

[0612] In some embodiments of the methods described herein, the time interval between administrations of the masked cytokines described herein is about one week or more. In some embodiments of the methods described herein, the time interval between administrations of the masked cytokines described herein is about two days or more, about three days or more, about four days or more A long time, about five days or more, or about six days or more. In some embodiments of the methods described herein, the time interval between administrations of the masked cytokines described herein is about one week or more, about two weeks or more, about three weeks or more time or about four weeks or more. In some embodiments of the methods described herein, the time interval between administrations of the masked cytokines described herein is about one month or more, about two months or more, or about three months or longer. As used herein, the time interval between administrations refers to the period of time between one administration of a masked interleukin and the next administration of a masked interleukin. As used herein, an interval of about one month includes four weeks. In some embodiments, treatment comprises multiple administrations of a masked cytokine, wherein the time interval between administrations can vary. For example, the time interval between the first administration and the second administration is about one week, and the time interval between subsequent administrations is about two weeks. In some embodiments, the time interval between the first administration and the second administration is about two, three, four, or five or six days, and the time interval between subsequent administrations is about one week.

[0613] In some embodiments, the masked cytokine is administered at multiple times over a period of time. In some embodiments, the dose administered to the subject at multiple times may be the same dose at each administration, or in some embodiments, the masked interleukin may be administered to the subject in two or more different doses. For example, in some embodiments, a masked interleukin is initially administered at one dose at one or more time points and is later administered at a second dose at one or more time points beginning at a later time point.

[0614] In some embodiments, a masked polypeptide described herein is administered in a uniform dose. In some embodiments, a masked polypeptide described herein is administered to a subject at a dose of about 25 mg to about 500 mg per dose. In some embodiments, at 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, about 225 mg to about 250 mg, about 250 mg to about 275 mg, about 275 mg to about 300 mg, about 300 mg to about 325 mg, about 325 mg to about 350 mg, about 350 mg to about 375 mg, about 375 mg to about 400 mg, about 400 mg to about 425 mg, about 425 mg to about 450 mg, about 450 mg to about A dose of 475 mg, or about 475 mg to about 500 mg, of the masked polypeptide is administered to the subject.

[0615] In some embodiments, a masked polypeptide described herein is administered to a subject at a dose based on the subject's body weight or body surface area (BSA). Depending on the type and severity of the disease, about 1 µg / kg to 15 mg / kg (e.g. 0.1 mg / kg-10 mg / kg), whether for example by one or more separate administrations or by continuous infusion A masked polypeptide can be an initial candidate dose to be administered to a patient. A typical daily dosage might range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, as appropriate, treatment will generally be continued until the desired suppression of disease symptoms occurs. An exemplary dosage of masked polypeptide will be in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the patient. In some embodiments, a masked polypeptide described herein is administered to a subject at a dose of about 0.1 mg / kg to about 10 mg / kg, or about 1.0 mg / kg to about 10 mg / kg. In some embodiments, a masked polypeptide described herein is present at about 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 3.5 mg / kg, 4.0 mg / kg, 4.5 mg / kg, 5.0 mg / kg, 5.5 mg / kg, 6.0 mg / kg, 6.5 mg / kg, 7.0 mg / kg, 7.5 mg / kg, 8.0 mg / kg, Doses of any of 8.5 mg / kg, 9.0 mg / kg, 9.5 mg / kg, or 10.0 mg / kg are administered to the subject. In some embodiments, a masked polypeptide described herein is present at about or at least about 0.1 mg / kg, about or at least about 0.5 mg / kg, about or at least about 1.0 mg / kg, about or at least about 1.5 mg / kg, About or at least about 2.0 mg / kg, about or at least about 2.5 mg / kg, about or at least about 3.0 mg / kg, about or at least about 3.5 mg / kg, about or at least about 4.0 mg / kg, about or at least about 4.5 mg / kg, about or at least about 5.0 mg / kg, about or at least about 5.5 mg / kg, about or at least about 6.0 mg / kg, about or at least about 6.5 mg / kg, about or at least about 7.0 mg / kg, about or at least about 7.5 mg / kg, about or at least about 8.0 mg / kg, about or at least about 8.5 mg / kg, about or at least about 9.0 mg / kg, about or at least about 9.5 mg / kg, about or at least about 10.0 mg / kg, about or at least about 15.0 mg / kg, about or at least about 20 mg / kg, about or at least about 30 mg / kg, about or at least about 40 mg / kg, about or at least about 50 mg / kg, about or A dose of at least about 60 mg / kg, about or at least about 70 mg / kg, about or at least about 80 mg / kg, about or at least about 90 mg / kg, or about or at least about 100 mg / kg is administered to the subject. Any of the dosing frequencies described above can be used.

[0616] One method contemplated herein is the treatment of a disorder or disease, such as cancer, with any of the masked interleukins or compositions described herein. Conditions or diseases treatable with the formulations of the 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 (such as Merkel cell carcinoma), or testicular cancer.

[0617] In some embodiments, provided herein is a method of treating or preventing cancer by administering any of the masked cytokines or compositions described herein. In some embodiments, provided herein is a method of treating or preventing cancer by administering any of the masked interleukins or compositions described herein and an anticancer agent. An anticancer agent can be any agent capable of reducing cancer growth, interfering with cancer cell replication, killing cancer cells directly or indirectly, reducing cancer metastasis, reducing tumor blood supply, or reducing cell survival. In some embodiments, the anticancer agent is selected from the group consisting of: PD-1 inhibitors, EGFR inhibitors, HER2 inhibitors, VEGFR inhibitors, CTLA-4 inhibitors, BTLA inhibitors, B7H4 inhibitors, B7H3 inhibitors Agents, CSFIR inhibitors, HVEM inhibitors, CD27 inhibitors, KIR inhibitors, NKG2A inhibitors, NKG2D agonists, TWEAK inhibitors, ALK inhibitors, CD52 targeting antibodies, CCR4 targeting antibodies, PD-L1 inhibitors , KIT inhibitors, PDGFR inhibitors, BAFF inhibitors, HD AC inhibitors, VEGF ligand inhibitors, CD19 targeting molecules, FOFR1 targeting molecules, DFF3 targeting molecules, DKK1 targeting molecules, MUC1 targeting molecules, MUG 16 targeting molecule, PSMA targeting molecule, MSFN targeting molecule, NY-ES0-1 targeting molecule, B7H3 targeting molecule, B7H4 targeting molecule, BCMA targeting molecule, CD29 targeting molecule, CD151 targeting molecule, CD 123 targeting molecules, CD33 targeting molecules, CD37 targeting molecules, CDH19 targeting molecules, CEA targeting molecules, tight junction protein (Claudin) 18.2 targeting molecules, CFEC12A targeting molecules, EGFRVIII targeting molecules, EPCAM targeting molecules molecules, EPHA2 targeting molecules, FCRH5 targeting molecules, FLT3 targeting molecules, GD2 targeting molecules, Glypican 3 targeting molecules, gpA33 targeting molecules, GPRC5D targeting molecules, IL-23R targeting molecules, 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 inhibition Agents, ABE inhibitors, CD30 inhibitors, FLT3 inhibitors, MET inhibitors, RET inhibitors, IL-1 (3 inhibitors, MEK inhibitors, ROS1 inhibitors, BRAE inhibitors, CD38 inhibitors, RANKE inhibitors, B4GALNT1 inhibitor, SLAMF7 inhibitor, IDH2 inhibitor, mTOR inhibitor, CD20 targeting antibody, BTK inhibitor, PI3K inhibitor, FLT3 inhibitor, PARP inhibitor, CDK4 inhibitor, CDK6 inhibitor, EGFR inhibitor, RAF Inhibitors, JAK1 Inhibitors, JAK2 Inhibitors, JAK3 Inhibitors, IL-6 Inhibitors, IL-17 Inhibitors, Smoothened Inhibitors, IL-6R Inhibitors, BCL2 Inhibitors, PTCH Inhibitors, PIGF Inhibitors agonist, TGFB inhibitor, CD28 agonist, CD3 agonist, CD40 agonist, GITR agonist, OX40 agonist, VISTA agonist, CD137 agonist, LAG3 inhibitor, TIM3 inhibitor, TIGIT inhibitors and IL-2R inhibitors.

[0618] In some embodiments, provided herein is a method of treating or preventing cancer by administering any of the masked cytokines and anti-inflammatory agents described herein. An anti-inflammatory agent can be any agent capable of preventing, counteracting, inhibiting or otherwise reducing inflammation.

[0619] 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 (ie, the COX inhibitor inhibits the activity of COX-1 more than it inhibits the activity of COX-2). In some embodiments, the COX inhibitor selectively inhibits COX-2 (ie, the COX inhibitor inhibits the activity of COX-2 more than it inhibits the activity of COX-1). In some embodiments, the COX inhibitor inhibits both COX-1 and COX-2.

[0620] 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 ketone The group consisting of ketoprofen. In some embodiments, the COX inhibitor is a selective COX-2 inhibitor and is selected from the group consisting of celecoxib, rofecoxib, meloxicam, pyridoxine Piroxicam, deracoxib, parecoxib, valdecoxib, etoricoxib, ketone derivatives, chroman derivatives, N-( 2-cyclohexyloxynitrophenyl)methanesulfonamide, parecoxib, lumiracoxib, RS 57067, T-614, BMS-347070, JTE-522, S-2474, SVT- 2016, CT-3, ABT-963, SC-58125, nimesulide, flosulide, NS-398, L-745337, RWJ-63556, L-784512, darbufelone ), 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, tolmetin, piroxicam, and meclofenamate. In some embodiments, the COX inhibitor is selected from the group consisting of SC-560, FR122047, P6, mobendazole, TFAP, flurbiprofen, ketoprofen, celecoxib, rofexib Cloth, meloxicam, piroxicam, delacoxib, parecoxib, valdecoxib, etacoxib, ketone derivatives, chroman derivatives, N-(2-cyclohexyloxynitrate phenyl)methanesulfonamide, parecoxib, lumirocco, RS 57067, T-614, BMS-347070, JTE-522, S-2474, SVT-2016, CT-3, ABT-963, SC-58125, Nimesulide, Fluxamide, NS-398, L-745337, RWJ-63556, L-784512, Dabufeilong, CS-502, LAS-34475, LAS-34555, S-33516, Diclofenac , mefenamic acid, SD-8381, ibuprofen, naproxen, ketorolac, indomethacin, aspirin, naproxen, tolmetin, piroxicam and meclofenamic acid.

[0621] In some embodiments, the anti-inflammatory agent is an NF-kB inhibitor. A NF-kB inhibitor can be any agent that inhibits the activity of the NF-kB pathway. In some embodiments, the NF-kB inhibitor is selected from the group consisting of: IKK complex inhibitors, IkB degradation inhibitors, NF-kB nuclear translocation inhibitors, p65 acetylation inhibitors, NF-kB DNA binding Inhibitors, NF-kB transactivation inhibitors and p53 induction inhibitors.

[0622] In some embodiments, the IKK complex inhibitor is selected from the group consisting of TPCA-1, NF-KB Activation Inhibitor VI (BOT-64), BMS-345541, amlexanox, SC-514 (GK-01140), IMD-0354 and IKK-16. In some embodiments, the IkB degradation inhibitor is selected from the group consisting of BAY-11-7082, MG-115, MG-132, lactacystin, epoxomicin, feverfew Lactone (parthenolide), carfilzomib (carfilzomib) and MLN-4924 (pevonedistat). In some embodiments, the NF-kB 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-kB DNA binding inhibitor is selected from the group consisting of GYY-4137, p-XSC, CV-3988, and prostaglandin E2 (PGE2). In some embodiments, the NF-kB transactivation inhibitor is selected from the group consisting of LY-294002, wortmannin, and mesalamine. In some embodiments, the p53 induction inhibitor is selected from the group consisting of quinacrine and flavopiridol. In some embodiments, the NF-kB inhibitor is selected from the group consisting of TPCA-1, NF-kB activation inhibitor VI (BOT-64), BMS-345541, amlexanox, SC-514 (GK- 01140), IMD-0354, IKK-16, BAY-11-7082, MG-115, MG-132, Rectaxetine, Epthycin, Parthenolide, Carfilzomib, MLN-4924 ( pavorestat), JSH-23 rolipram, gallic acid, anacardic acid, GYY-4137, p-XSC, CV-3988, prostaglandin E2 (PGE2), LY-294002, wortmannin, meselah Ming, quinacrine, and frapindus.

[0623] In some embodiments, provided herein is a method of treating or preventing cancer by administering any of the masked cytokines or compositions described herein and an anti-cancer therapeutic protein. An anticancer therapeutic protein can be any therapeutic protein capable of reducing cancer growth, interfering with cancer cell replication, directly or indirectly killing cancer cells, reducing cancer metastasis, reducing tumor blood supply, or reducing cell survival. Exemplary anticancer therapeutic proteins can be 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, provided herein is a method of treating or preventing cancer by administering any of the masked cytokines or compositions described herein and CAR-NK (natural killer) cells.

[0624] 7. Articles of manufacture or kits In another aspect, there is provided an article of manufacture or kit comprising any of the masked cytokines described herein. The article of manufacture or kit may further comprise instructions for the use of the cytokine in the method of the present invention. Accordingly, in certain embodiments, the article of manufacture or kit comprises instructions for use of the masked interleukin in a method of treating or preventing a disorder (eg, cancer) in an individual, the methods comprising administering to the individual an effective amount of the interleukin Masked cytokines. For example, in certain embodiments, an article of manufacture or a kit comprises instructions for using a masked polypeptide in a method of treating or preventing a disorder (e.g., cancer) in an individual, the methods comprising administering to the individual an effective amount of the masked polypeptide. Masking peptides. In certain embodiments, the individual is 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, Cancer of the ovary, osteosarcoma, bladder, cervix, liver, kidney, skin, or testicles.

[0625] The article or kit may further comprise a container. Suitable containers include, for example, bottles, vials (eg, dual-chambered vials), syringes (such as single-chambered syringes or dual-chambered syringes), test tubes, and intravenous (IV) bags. The container can 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 formulations are liquid formulations.

[0626] The article of manufacture or kit may further comprise a label or package insert located on or associated with the container, which may indicate instructions for reconstitution and / or use of the formulation. The label or package insert may further indicate that the formulation is suitable or intended for subcutaneous, intravenous or other modes of administration for the treatment or prevention of a condition (eg, cancer) in an individual. The container in which the formulation is stored can be a single-use vial or a multiple-use vial, which allows repeated administration of the reconstituted formulation. The article or kit may further comprise a second container comprising a suitable diluent. The article or kit may further comprise other materials as desired from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes and packaging inserts with instructions for use.

[0627] In certain embodiments, the invention provides kits of administration units for single dose administration. Such kits include containers of aqueous formulations of therapeutic interleukins, including single or multi-chambered pre-filled syringes. Exemplary prefilled syringes are available from Vetter GmbH, Ravensburg, Germany.

[0628] The product or kit herein optionally further comprises a container comprising a second drug, wherein the masked interleukin is the first drug, and the product or kit further comprises information on the label or package insert about Instructions for treating the subject with an effective amount of the second medicament.

[0629] In another embodiment, provided herein is an article or kit comprising a formulation described herein for administration in an autoinjector device. An auto-injector can be described as an injection device which, when activated, will deliver its contents without other necessary action from the patient or the administerer. It is especially useful for self-administration of therapeutic formulations when the delivery rate must be constant and the delivery time needs to be sustained over a period of time.

[0630] 8. Definitions Unless otherwise defined, all technical terms, notations and other technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which claimed subject matter pertains. In some instances, terms having commonly understood meanings are defined herein for clarity and / or for ease of reference, and the inclusion of such definitions herein should not necessarily be construed to represent substantive differences from what is commonly understood in the art. difference.

[0631] It is to be understood that this invention is not limited to particular compositions or biological systems, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a / an" and "the" include plural references unless the context clearly dictates otherwise.

[0632] As used herein, the term "about" refers to the usual error range for the respective value readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments directed to that value or parameter per se.

[0633] It is to be understood that aspects and embodiments of the invention described herein include "comprising" aspects and embodiments, "consisting" and "consisting essentially of" aspects and embodiments.

[0634] As used herein, the term "and / or" refers to any one of the items associated with this term, any combination of such items, or all of the items. For example, the phrase "A, B, and / or C" is intended to cover 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).

[0635] The term "antibody" includes polyclonal antibodies, monoclonal antibodies (including full-length antibodies with an immunoglobulin Fc region), antibody compositions with multiple epitope specificities, multispecific antibodies (e.g. bispecific Antibodies, diabodies, and single-chain molecules) and antibody fragments (such as Fab, F(ab')2, and Fv). The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein.

[0636] The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable (VL) domain linked in the same polypeptide chain (VH-VL). chain variable (VH) domain.

[0637] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two consensus light (L) chains and two consensus heavy (H) chains. IgM antibodies consist of 5 basic heterotetrameric units plus other polypeptides called J chains and contain 10 antigen-binding sites, while IgA antibodies contain 2-5 basic 4-chain units that can polymerize to combine with the J chain Multivalent aggregates are formed. In the case of IgG, the 4-chain unit is typically about 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 bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) of each a and y chain and four CH domains of the p and s isotypes. Each L chain has a variable domain (VL) at its N-terminus followed by a constant domain at its other end. VL is aligned with VH and CL is aligned with the first constant domain (CHI) of the heavy chain. Certain amino acid residues are believed to form the interface between the light and heavy chain variable domains. VH and VL are paired together to form a single antigen-binding site. For the structure 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, pp. 71 pages and Chapter 6.

[0638] L chains from any vertebrate species can be assigned to one of two distinct types, termed kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domain of the heavy chain (CH) of an immunoglobulin, they can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains called 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, eg humans exhibit the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. IgGl antibodies can exist in a number of polymorphic variants called allotypes (reviewed in Jefferis and Lefranc 2009. mAbs Vol 1, Issue 4 1-7), any of which are suitable for use in the present invention. Common allotypic variants in the human population are variants designated by the letters a, f, n, z.

[0639] An "isolated" antibody is one that has been identified, isolated and / or recovered (eg, naturally or recombinantly) from a component of the environment in which it was produced. In some embodiments, an isolated polypeptide is free from all other components from the environment in which it was produced. Contaminant components of the environment in which they are produced, such as those produced by recombinantly transfected cells, are substances that would normally interfere with the research, diagnostic or therapeutic use of antibodies, and may include enzymes, hormones and other proteinaceous or nonproteinaceous solutes. In some embodiments, the polypeptide is purified to (1) greater than 95% by weight of the antibody, as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (1) Sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using, for example, a rotary cup sequencer; or (3) homogeneous, which uses Coomassie blue (Coomassie blue) or silver dyes in the Realized by SDS-PAGE under non-reducing or reducing conditions. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated polypeptide or antibody will be prepared by at least one purification step.

[0640] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are free from mutations that may occur naturally and / or may be present in small amounts. The same except for the presence of post-translational modifications (eg, isomerization, amidation). In some embodiments, the monoclonal antibody has a C-terminal cleavage at the heavy and / or light chain. For example, 1, 2, 3, 4 or 5 amino acid residues are cleaved at the C-terminus of the heavy and / or light chain. In some embodiments, the C-terminal cleavage removes the C-terminal lysine from the heavy chain. In some embodiments, monoclonal antibodies have N-terminal cleavages at the heavy and / or light chains. For example, 1, 2, 3, 4 or 5 amino acid residues are cleaved at the N-terminus of the heavy and / or light chain. In some embodiments, truncated forms of monoclonal antibodies can be produced by recombinant techniques. In some embodiments, monoclonal antibodies are highly specific for a single antigenic site. In some embodiments, monoclonal antibodies are highly specific for multiple antigenic sites (such as bispecific or multispecific antibodies). The modifier "monoclonal" indicates that the antibody is characterized as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be produced by a variety of techniques including, for example, the fusionoma method, recombinant DNA methods, phage display technology, and use in human immune A technique for producing human antibodies or human-like antibodies in animals based on the human immunoglobulin loci or genes of the globulin sequences.

[0641] The terms "full-length antibody", "intact antibody" or "whole antibody" are used interchangeably and refer to an antibody in substantially intact form, rather than an antibody fragment. In particular, full antibodies include antibodies having both heavy and light chains including the Fc region. The constant domain can be a native sequence constant domain (eg, a human native sequence constant domain) or an amino acid sequence variant thereof. In some cases, an intact antibody may have one or more effector functions.

[0642] An "antibody fragment" comprises a portion of an intact antibody, such as the antigen-binding and / or variable regions of an intact antibody, and / or the constant regions of an intact antibody. Examples of antibody fragments include the Fc region of an antibody, a portion of an Fc region, or a portion of an antibody comprising an Fc region. Examples of antigen-binding antibody fragments include domain antibody (dAb), Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S. Patent No. 5,641,870, Example 2; Zapata et al., 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 engineered or, in the case of heavy chains, isolated from camels, sharks, libraries or mice engineered to produce single heavy chain molecules.

[0643] Papain digestion of antibodies yields two identical antigen-binding fragments, termed the "Fab" fragment, and a residual "Fc" fragment, a designation that reflects the ability to readily crystallize. The Fab fragment consists of the entire L chain and the variable region domain (VH) of the H chain and the first constant domain (CHI) of a heavy chain. Each Fab fragment is monovalent in antigen binding, ie it has a single antigen binding site. Pepsin treatment of antibodies yields a single large F(ab')2 fragment that roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activities and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments in that Fab' fragments have several additional residues at the carboxy-terminus of the CHI domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue of the constant domain has a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known. The Fc fragment comprises the carboxy-terminal portion of two H chains held together by a disulfide bond. The effector functions of antibodies are determined by sequences and glycans in the Fc region, which is also recognized by Fc receptors (FcRs) found on certain types of cells.

[0644] "Amino acid sequence identity percentage (%)" relative to the reference polypeptide sequence is defined as after aligning the reference polypeptide sequence with the candidate sequence and introducing gaps if necessary to achieve the maximum sequence identity percentage, and without Where conservative substitutions are considered part of the sequence identity, the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a reference polypeptide sequence. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in various ways within the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, the % amino acid sequence identity of a given amino acid sequence A relative to, with or compared to a given amino acid sequence B (alternatively, it can be expressed as having Or a given amino acid sequence A) comprising a certain % amino acid sequence identity is calculated as follows: 100 multiplied by the score X / Y where X is the amino group scored as a consensus match by the sequence in the programmed alignment of A and B The number of acid residues, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of the amino acid sequence A is not equal to the length of the amino acid sequence B, the % amino acid sequence identity of A with respect to B is the same as the % amino acid sequence identity of B with respect to A will not be equal.

[0645] An antibody "effector function" refers to the biological activity attributable to the Fc region of an antibody (eg, a native sequence Fc region or an amino acid sequence variant Fc region) and varies with 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; and B cell activation.

[0646] As used herein, "binding affinity" refers to the strength of the non-covalent interaction between a single binding site of a molecule (eg, an interleukin) and its binding partner (eg, an interleukin receptor). In some embodiments, the affinity of a binding protein (eg, a cytokine) can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein.

[0647] An "isolated" nucleic acid molecule encoding an interleukin polypeptide described herein is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule with which it is ordinarily associated in the environment in which it is produced. In some embodiments, isolated nucleic acids are not associated with all components associated with the environment in which they were produced. Isolated nucleic acid molecules encoding the polypeptides and interleukin polypeptides herein are in a form or environment other than that in which they are found in nature. Isolated nucleic acid molecules thus differ from nucleic acids encoding the polypeptides and interleukin polypeptides herein that occur naturally in cells.

[0648] The term "pharmaceutical formulation" refers to a preparation that is in a form that permits the biological activity of the active ingredients to be effective, and that contains no other components that would be unacceptably toxic to the subject to which the formulation will be administered.

[0649] Such formulations are sterile.

[0650] As used herein, "carrier" includes a pharmaceutically acceptable carrier, excipient or stabilizer that is nontoxic to cells or mammals to which it is exposed at the dosages and concentrations employed. Usually a physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin , gelatin or immunoglobulin; 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 agents such as TWEEN™, polyethylene glycol (PEG) and PLURONICS™.

[0651] As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable therapeutic effects include decreased rate of disease progression, amelioration or palliation of disease condition and remission or improved prognosis. For example, a subject is successfully "treated" if one or more symptoms associated with a disorder (eg, a neoplastic disease) is alleviated or eliminated. For example, if the treatment results in improving the quality of life of the individual with the disease, reducing the dose of other drugs needed to treat the disease, reducing the rate of recurrence of the disease, reducing the severity of the disease, delaying the development or progression of the disease, and / or prolonging the Survival period, the individual is successfully "healed".

[0652] As used herein, "in conjunction with" or "in combination with" refers to the administration of one treatment modality in addition to another treatment modality. Thus, "in conjunction with" or "in combination with" refers to the administration of one treatment modality to a subject before, during or after administration of the other treatment modality.

[0653] As used herein, the term "prevention" includes providing control over the occurrence or recurrence of a disease in an individual. An individual may be predisposed to, sensitive to, or at risk of developing a disorder, but not yet diagnosed with the disorder. In some embodiments, the masked cytokines described herein are used to delay the development of a disorder.

[0654] As used herein, an individual "at risk of developing a disorder" may or may not have detectable disease or disease symptoms, and may or may not have shown detectable disease prior to the methods of treatment described herein disease or disease symptoms. "At risk" means that an individual has one or more risk factors, which are measurable parameters associated with the development of a disease, as known in the art. Individuals with one or more of these risk factors have a higher chance of developing the disorder than individuals without one or more of these risk factors.

[0655] "Effective amount" means an amount effective at least to achieve the desired or indicated effect (including therapeutic or prophylactic results) at the required dose and time period.

[0656] An effective amount can be provided in one or more administrations. A "therapeutically effective amount" is at least the minimum concentration required to achieve a measurable improvement in a particular condition. Herein, a therapeutically effective amount may vary depending on factors such as the disease state, age, sex and weight of the patient, and the ability of the antibody to elicit the desired response in the individual. A therapeutically effective amount can also be one in which any toxic or detrimental effects of the masked cytokine are outweighed by the therapeutically beneficial effects. "Prophylactically effective amount" refers to the amount that is effective in achieving the desired prophylactic effect at the necessary dosage and time period. Typically, but not necessarily, a prophylactically effective amount may be less than a therapeutically effective amount due to the prophylactic dose being used by the subject prior to or in the early stages of the disease.

[0657] "Chronic" administration refers to administration of an agent in a sustained mode, as opposed to the acute mode, so as to maintain the initial therapeutic effect (activity) over a prolonged period. "Intermittent" administration is therapy that is not administered continuously without interruption, but rather in cycles.

[0658] As used herein, an "individual" or "subject" is a mammal. "Mammal" for therapeutic purposes includes humans; domestic and farm animals; and zoo, sport, or pet animals such as dogs, horses, rabbits, cows, pigs, hamsters, gerbils, mice, ferrets, mouse, cat, etc. In some embodiments, the individual or subject is human.

[0659] 9. EXAMPLES The present invention will be more fully understood by reference to the following examples. However, it should not be construed as limiting the scope of the invention. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or variations therefrom will be suggested by those skilled in the art and are included within the spirit and scope of this application and the accompanying patent application Within the scope of the range.

[0660] While some examples describe the engineering, manufacture and / or testing of "masked" versions of polypeptide constructs, some examples also employ parental "unmasked" versions of polypeptide constructs, such as for comparison, or include as Other constructs of one or more components described herein are tested as comparative controls. Thus, for example, a description of testing performed on a masked polypeptide construct does not necessarily mean nor test the unmasked version of the construct.

[0661] Example 1: Engineering of Masked IL-2 Polypeptides Masked IL-2 polypeptide constructs were generated according to the teachings herein. In subsequent examples, some experiments involved the use of masked IL-2 polypeptide constructs in monomeric form, and some experiments involved the use of masked IL-2 constructs in dimeric form, such as by linking the same masked polypeptide constructs. A dimer formed by two copies of a disulfide bond (homodimer) or a heterodimer formed by two different polypeptides (see, eg, Table 5).

[0662] Masked IL-2 polypeptide constructs comprising IL-2 polypeptides or functional fragments thereof, masking moieties and half-life extending moieties such as antibodies or fragments thereof (eg Fc region, heavy chain and / or light chain) are produced. Some IL-2 polypeptide constructs comprising an IL-2 polypeptide or functional fragment thereof linked to a half-life extending moiety were also produced, which also did not include a masking moiety. Some constructs also include a linker comprising a cleavable peptide and linking the masking moiety to the IL-2 polypeptide or a functional fragment thereof, thereby creating an activatable masked IL-2 polypeptide construct. Some constructs also include a linker linking the IL-2 polypeptide or functional fragment thereof to the half-life extending domain. Some constructs also include a linker linking the IL-2 polypeptide or functional fragment thereof to the masking moiety. Masked IL-2 polypeptide constructs that do not include a cleavable peptide in the linker linking the IL-2 polypeptide or functional fragment thereof to the masking moiety are also referred to as non-activatable masked IL-2 polypeptide constructs or non-activatable IL-2 polypeptide construct because it does not include a cleavable peptide. The structures and compositions of exemplary IL-2 polypeptide constructs are provided in Table 3. table 3 Construct ID Cytokines or their functional fragments (C) Linker (L1) Shaded part (MM) Linker (L2) Half-life-extending domain (H) Structure (N to C terminal direction) amino acid sequence AK032 SEQ ID NO: 62 - - - SEQ ID NO: 65 H-C SEQ ID NO: 67 AK035 SEQ ID NO: 3 - - - SEQ ID NO: 65 H-C SEQ ID NO: 68

[0663] Masked IL-2 polypeptide constructs are also produced comprising IL-2 polypeptides or functional fragments thereof, first masking moieties, second masking moieties and half-life extending moieties, such as albumin, antibodies or fragments thereof (e.g. Fc region, heavy chain and / or light chain), albumin binding peptide, IgG binding peptide or polyamino acid sequence. Some constructs also include a linker linking the first masking moiety to the IL-2 polypeptide or functional fragment thereof. Some constructs also include a linker linking the second masking moiety to the IL-2 polypeptide or functional fragment thereof. Some constructs include a cleavable peptide in the linker linking the first masking moiety to the IL-2 polypeptide or functional fragment thereof and / or the linker linking the second masking moiety to the IL-2 polypeptide or functional fragment thereof, by This results in an activatable masked IL-2 polypeptide construct. Some constructs also include a linker linking the second masking moiety to the half-life extending moiety. Masked IL-2 polypeptide constructs that do not include a cleavable peptide in either linker linking the IL-2 polypeptide or a functional fragment thereof to the first masking moiety or the second masking moiety are also referred to as non-activatable masked ILs -2 polypeptide construct or non-activatable IL-2 polypeptide construct because it does not include a cleavable peptide. The structures and compositions of exemplary IL-2 polypeptide constructs are provided in Table 4. Table 4 Construct ID Shaded part (MM1) Linker (L1) Cytokines or their functional fragments (C) Linker (L2) Shaded part (MM2) Linker (L3) Half-life extension part (H) Structure (N to C terminal direction) amino acid sequence AK041 SEQ ID NO: 60 SEQ ID NO: 61 SEQ ID NO: 62 SEQ ID NO: 63 SEQ ID NO: 64 SEQ ID NO: 17 SEQ ID NO: 65 H-L1-MM1-L2-C-L3-MM2 SEQ ID NO: 66

[0664] Masked IL-2 polypeptide constructs are also generated comprising IL-2 polypeptides or functional fragments thereof, masking moieties, first half-life extending moieties and second half-life extending moieties, antibodies or fragments thereof (e.g., Fc regions, heavy chain and / or light chain). The shielding part is connected to the first half-life extension part, the IL-2 polypeptide or its functional fragment is connected to the second half-life extension part, and the first half-life extension part and the second half-life extension part contain the first half-life extension part and the second half-life extension part Modification of partial association. In an exemplary embodiment, the masking moiety is linked to the first half-life extending part and comprises the amino acid sequence of SEQ ID NO: 38, and the IL-2 polypeptide or a functional fragment thereof is linked to the second half-life extending part and comprises SEQ ID NO: The amino acid sequence of 48, and the first half-life extending part and the second half-life extending part contain modifications that promote the association of the first half-life extending part and the second half-life extending part. In an exemplary embodiment of an unmasked IL-2 polypeptide construct, the embodiment comprises an IL-2 polypeptide or functional fragment thereof linked to a first half-life-extending moiety, and comprises a second half-life-extending moiety, wherein the IL- 2 The polypeptide or its functional fragment is linked to the first half-life extending part and includes the amino acid sequence of SEQ ID NO: 48, and the second half-life extending part includes the amino acid sequence of SEQ ID NO: 79. Some constructs also include a linker linking the masking moiety to the first half-life-extending moiety, and / or a linker linking the IL-2 polypeptide or functional fragment thereof to the second half-life-extending moiety. The first half-life extending portion and the second half-life extending portion of some constructs were also linked. In some constructs, the first and second half-life extending moieties of some constructs are linked by a linker. Some constructs include a cleavable peptide in the linker that links the masking moiety to the first half-life-extending moiety and / or the linker that links the IL-2 polypeptide or functional fragment thereof to the second half-life-extending moiety, thereby generating an activatable The masked IL-2 polypeptide construct. Masked IL-2 polypeptide constructs that do not include a cleavable peptide in the linker linking the IL-2 polypeptide or functional fragment thereof to the second half-life extending moiety or the linker linking the masking moiety to the first half-life extending moiety also It is called a non-activatable masked IL-2 polypeptide construct or non-activatable IL-2 polypeptide construct because it does not include a cleavable peptide. The structures and compositions of exemplary IL-2 polypeptide constructs are provided in Table 5. table 5 Construct ID Cytokines or their functional fragments (C) Linker (L1) Shaded part (MM) Linker (L2) Half-life extension part (H) Structure (N to C terminal direction) amino acid...

Claims

1. A masked IL-12 cytokine comprising: a first polypeptide chain including a first Fc domain, a first linker, and a masked portion including the amino acid sequence of SEQ ID NO: 220; and a second polypeptide chain including a second Fc domain, a second linker including a protein-cleavable peptide of SEQ ID NO: 249, and an IL-12 cytokine including an IL-12p40 polypeptide linked to an IL-12p35 polypeptide; wherein the IL-12p40 polypeptide includes the amino acid sequence of SEQ ID NO: 208, and the IL-12p35 polypeptide includes the amino acid sequence of SEQ ID NO:

209.

2. The masked IL-12 cytokine of claim 1, wherein the IL-12p40 polypeptide is linked to the IL-12p35 polypeptide by a linker comprising the amino acid sequence of SEQ ID NO:

116.

3. A masked IL-12 cytokine comprising: a first polypeptide chain comprising a first Fc domain, a first linker, and a masked portion comprising an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 220; and a second polypeptide chain comprising a second Fc domain, a second linker comprising a protein-cleavable peptide of SEQ ID NO: 249, and an IL-12 cytokine or a functional fragment thereof comprising an amino acid sequence that is at least 95% identical to that of SEQ ID NO:

214.

4. The masked IL-12 cytokine of any one of claims 1 to 3, wherein the first linker comprises the non-cleavable linker of SEQ ID NO:

35.

5. The masked IL-12 cytokine of any one of claims 1 to 3, wherein the second linker comprises the amino acid sequence of SEQ ID NO:

115.

6. A masked IL-12 cytokine as claimed in any of claims 1 to 3, wherein the first Fc domain contains the Y349C, T366S, L368A and Y407V mutations relative to SEQ ID NO:7, numbered according to the Kabat EU numbering system.

7. The masked IL-12 cytokine of claim 6, wherein the second Fc domain contains the S354C and T366W mutations relative to SEQ ID NO:7, numbered according to the Kabat EU numbering system.

8. The masked IL-12 cytokine of any one of claims 1 to 3, wherein the first Fc domain comprises the amino acid sequence of SEQ ID NO:

9.

9. The masked IL-12 cytokine of claim 8, wherein the second Fc domain comprises the amino acid sequence of SEQ ID NO:

12.

10. The masked IL-12 cytokine as claimed in claim 3, wherein the masked portion comprises a sequence having at least 98% sequence identity with SEQ ID NO:

220.

11. The masked IL-12 cytokine of claim 10, wherein the IL-12 cytokine or a functional fragment thereof comprises a sequence having at least 98% sequence identity with SEQ ID NO:

214.

12. The masked IL-12 cytokine as claimed in claim 3, wherein the masked portion comprises a sequence having at least 99% sequence identity with SEQ ID NO:

220.

13. The masked IL-12 cytokine of claim 12, wherein the IL-12 cytokine or a functional fragment thereof comprises a sequence having at least 99% sequence identity with SEQ ID NO:

214.

14. The masked IL-12 cytokine of any one of claims 1 to 3, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:

192.

15. The masked IL-12 cytokine of any one of claims 1 to 3, wherein the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:

195.

16. A masked IL-12 cytokine comprising: a first polypeptide chain containing the amino acid sequence of SEQ ID NO: 192; and a second polypeptide chain containing the amino acid sequence of SEQ ID NO:

195.

17. A masked IL-12 cytokine comprising: a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 178; and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:

181.

18. A masked IL-12 cytokine comprising: a first polypeptide chain containing the amino acid sequence of SEQ ID NO: 172; and a second polypeptide chain containing the amino acid sequence of SEQ ID NO:

191.

19. A nucleic acid encoding either a masked IL-12 cytokine as claimed in any one of claims 1 to 18 or a polypeptide chain of a masked IL-12 cytokine as claimed in any one of claims 1 to 18.

20. A vector comprising the nucleic acid as claimed in claim 19.

21. A host cell comprising nucleic acid as claimed in claim 19 or a vector as claimed in claim 20.

22. The host cell of claim 21, wherein the host cell is an HEK cell or a CHO cell.

23. A composition comprising a masked IL-12 cytokine as claimed in any one of claims 1 to 18.

24. A pharmaceutical composition comprising a masked IL-12 cytokine as claimed in any one of claims 1 to 18 and a pharmaceutically acceptable carrier.

25. The pharmaceutical composition of claim 24, wherein the pharmaceutical composition is in a single unit dosage form.

26. The pharmaceutical composition of claim 25, wherein the pharmaceutical composition is formulated for intravenous administration.

27. The pharmaceutical composition of claim 25, wherein the pharmaceutical composition is formulated for injection.

28. The pharmaceutical composition of any one of claims 24 to 27, wherein the pharmaceutical composition is a liquid.

29. Use of a masked IL-12 cytokine as claimed in any one of claims 1 to 18, a composition as claimed in claim 23, or a pharmaceutical composition as claimed in any one of claims 23 to 28, for the preparation of a medicament for the treatment or prevention of cancer.

30. As used in claim 29, wherein the cancer is a solid tumor.

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