Separation part and method of use thereof

Abstract

Provided herein are separation moieties suitable for use with various therapeutic payloads. The separation moieties function to generate conditionally active macromolecules, whereby the macromolecule has reduced or minimal biological activity until the separation moiety is modified under specific conditions. Provided herein are compositions and methods for generating and using highly efficient separation moieties and / or linkers. The linkers can impart site selectivity to the biological activity of the attached payload(s). In some embodiments, the separation moieties and / or linkers are used with therapeutic proteins to treat diseases or disorders, such as proliferative diseases, neoplastic diseases, inflammatory diseases, immune diseases, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, graft-versus-host disease, etc.

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 847,914, filed May 14, 2019, and U.S. Provisional Application No. 62 / 938,786, filed November 21, 2019, each of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy was created on May 14, 2020, is named 761146_000140_SL.txt, and is 896,815 bytes in size. [Background technology]

[0003] Recombinant fusion proteins containing two or more functional polypeptides have applications in many fields, including protein purification, imaging, therapeutics, and drug delivery. For example, protein drugs can be fused to the Fc domain of an antibody or a carrier protein (i.e., human serum albumin) for targeting, extending plasma half-life, and / or achieving therapeutic efficacy. (Chen et al., (2013), Adv Drug Deliv Rev. 65(10):1357-1369).

[0004] Direct fusion of functional polypeptides or domains without a linker can lead to many undesirable outcomes, including fusion protein misfolding (Zhao et al., (2008), Protein Expr. Purif., 61:73-77), low protein production yields (Amet et al., (2009), Pharm. Res. 26:523-528), or even inactive biological activities (Bai et al., (2006) Proc. Natl. Acad. Sci. USA, 102:7292-7296). One approach to overcome these problems is to use a linker sequence between the component polypeptides or domains of the fusion protein. However, selecting a suitable linker for linking protein domains can be complex and is often overlooked in fusion protein design. Chen et al., (2013), Adv Drug Deliv Rev. 65(10):1357-1369. Linker sequence characteristics (e.g., length, hydrophobicity, amino acid composition, secondary structure, and overall folding) can affect linker suitability and must be considered when designing and selecting an appropriate linker. In addition, linkers that can be cleaved under selected conditions or at selected biological locations (e.g., tumor microenvironment) to deliver active therapeutic agents (e.g., therapeutic polypeptides) can provide targeted pharmacological activity of the therapeutic agent and reduce undesired systemic effects. This further complicates the design of suitable linkers. There is a need for improved liner sequences that can be used to prepare stable fusion proteins, including linkers that can be cleaved under selected conditions. Accordingly, novel detachment moieties or linkers are disclosed herein. The detachment moieties or linkers disclosed herein can be used, for example, to specifically deliver prodrugs (e.g., conditionally active and / or targeted cytokines) to target sites where the linker is engineered to activate biological activity. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Chen et al.,(2013),Adv Drug Deliv Rev.65(10):1357-1369 [Non-patent document 2] Zhao et al,(2008),Protein Expr. Purif., 61:73-77 [Non-patent document 3] Amet et al.,(2009),Pharm.Res.26:523-528 Summary of the Invention [Means for solving the problem]

[0006] Provided herein are compositions and methods for producing and using highly efficient separation moieties and / or linkers. The linkers can confer site selectivity for the biological activity of the attached payload(s). In some embodiments, the separation moieties and / or linkers are used in conjunction with therapeutic proteins to treat diseases or disorders, such as proliferative diseases, neoplastic diseases, inflammatory diseases, immune diseases, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, graft-versus-host disease, etc.

[0007] Disclosed herein is a recombinant polypeptide comprising a cleavable portion, the cleavable portion comprising an amino acid sequence that is a substrate for an enzyme (specifically, a protease). The protease may be selected from the group consisting of fibroblast activation protein alpha (FAPα (also known as prolyl endopeptidase FAP)), cathepsin L (CTSL1), ADAM (selected from ADAM8, ADAM9, ADAM10, ADAM12, ADAM17, and ADAMTS1), and MMP (selected from MMP1, MMP2, MMP9, or MMP14). The cleavable portion may also be a substrate for a cathepsin (e.g., cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin K, and / or cathepsin L). Preferably, the cathepsin is cathepsin L. Preferably, the protease is MMP14 or cathepsin L. The cleavable portion can comprise an amino acid sequence that is a substrate for at least two proteases. In another embodiment, the separation portion comprises two or more cleavable portions, each of which is a substrate for a protease. The separation portion can comprise a first cleavable portion comprising a first amino acid sequence that is a substrate for a first protease and a second cleavable portion comprising a second amino acid sequence that is a substrate for a second protease. In embodiments, the present disclosure relates to a recombinant polypeptide comprising a separation portion that includes a protease cleavage motif disclosed herein. The recombinant polypeptide can comprise a separation portion comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 195-220, or an amino acid sequence having 90% or greater identity to SEQ ID NOs: 195-220. Preferred separation portions comprise the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198). The present disclosure also relates to functional variants of separation portions comprising SEQ ID NOs: 195-220. A functional variant of SEQ ID NO: 195 can include any of SEQ ID NOs: 258-331. A functional variant of SEQ ID NO: 198 can include SEQ ID NO: 199 or any of SEQ ID NOs: 332-408. A separating moiety disclosed herein can have the formula I: [D1]-[L1]-[D2], where D1 is the first domain of interest.L1 is a separate moiety that connects or joins D1 to D2, and the separate moiety comprises an amino acid sequence selected from SEQ ID NOs: 195 to 220, or an amino acid sequence having at least about 90% identity to SEQ ID NOs: 195 to 220. D2 is a second domain of interest.

[0008] In one embodiment, the recombinant polypeptide may further comprise a non-cleavable linker sequence. The recombinant polypeptide may comprise a therapeutic protein, such as a cytokine, chemokine, growth factor, soluble receptor, antigen-binding portion of an antibody (e.g., scFV, dAb), etc. In another embodiment, the recombinant polypeptide comprises a cytokine, chemokine, growth factor, soluble receptor, or any combination thereof. In another embodiment, the recombinant polypeptide comprises at least one of an extracellular domain, a transmembrane domain, and an intracellular domain. In one embodiment, the recombinant polypeptide comprises a cell surface receptor, a chimeric antigen receptor (CAR), or a T cell receptor (TCR) subunit. In one embodiment, the recombinant polypeptide comprises an antigen-binding polypeptide, an antibody, or an antigen-binding portion thereof.

[0009] In one embodiment, the cleavable moiety is cleaved by one or more proteases with (a) greater catalytic efficiency, (b) greater specificity, or (c) both (a) and (b) than the reference polypeptide sequence. In another embodiment, the one or more proteases are selected from the group consisting of FAPα, CTSL1, ADAM (selected from ADAM8, ADAM9, ADAM10, ADAM12, ADAM17, and ADAMTS1), and MMP (selected from MMP1, MMP2, MMP9, and MMP14). The one or more proteases can also include a protease selected from a cathepsin (e.g., cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin K, and / or cathepsin L). In another embodiment, the reference polypeptide sequence is present in a natural polypeptide substrate of FAPα, CTSL1, an ADAM (selected from ADAM8, ADAM9, ADAM10, ADAM12, ADAM17, and ADAMTS1), an MMP (selected from MMP1, MMP2, MMP9, and MMP14), a cathepsin (e.g., cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin K, and / or cathepsin L), or a combination thereof. In another embodiment, the cleavable moiety is cleaved by one or more proteases with reduced catalytic efficiency relative to the reference polypeptide sequence. In another embodiment, the cleavable moiety is cleaved by one or more serum proteases with reduced catalytic efficiency. In another embodiment, the cleavable moiety is cleaved by one or more liver proteases with reduced catalytic efficiency. In another embodiment, the cleavable moiety is cleaved by one or more factor Xa, hepsin, or thrombin with reduced catalytic efficiency.

[0010] In one embodiment, the recombinant polypeptide comprises two or more separate moieties, hi one embodiment, the recombinant polypeptide is operably linked to a moiety selected from the group consisting of a polypeptide moiety, a lipid moiety, a nucleic acid moiety, a detectable moiety, and a small molecule.

[0011] Provided herein is a recombinant proprotein, comprising: a recombinant polypeptide comprising a cleavable moiety that is a substrate for a protease, the cleavable moiety comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 195-220; and a biologically active polypeptide, wherein the biological activity of the proprotein is attenuated, and cleavage of the cleavable moiety by a protease produces a polypeptide with unattenuated biological activity. In one embodiment, the biologically active polypeptide comprises a cytokine, chemokine, growth factor, soluble receptor, or combinations thereof. In some preferred embodiments, the linker is a component of a therapeutically useful fusion protein, wherein the linker is not cleaved or is cleaved with low efficiency in the peripheral circulation but is cleaved with high efficiency in a desired location in the body, such as a tumor microenvironment or a site of inflammation. In another embodiment, the biologically active polypeptide comprises at least one of an extracellular domain, a transmembrane domain, and an intracellular domain. In another embodiment, the biologically active polypeptide comprises a cell surface receptor, a chimeric antigen receptor (CAR), or a T cell receptor (TCR) subunit. In another embodiment, the biologically active polypeptide comprises an antigen-binding polypeptide, an antibody or antigen-binding portion thereof.

[0012] The present disclosure also relates to a fusion protein comprising: a. a signaling protein or molecule; b. a blocking moiety selected from a steric-blocking moiety, a specificity-blocking moiety, and a combination thereof; and c. a peptide linker comprising a cleavable moiety having at least one protease-cleavable sequence (e.g., a cleavable moiety disclosed herein). In one embodiment, the fusion protein comprises (a) and (b) operably linked by (c). In another embodiment, the fusion protein comprises two or more copies of the same cleavable moiety. In another embodiment, the fusion protein comprises the steric-blocking moiety comprises human serum albumin (HSA) or an anti-HSA antibody. In another embodiment, the fusion protein comprises the signaling protein is an interleukin-2 amino acid sequence comprising (i) a non-native N-terminus and / or (ii) a non-native C-terminus. In another embodiment, the fusion protein comprises the signaling protein is an interleukin-2 amino acid sequence. In another embodiment, the fusion protein further comprises one or more half-life-extending domains, which are not also specificity blockers. In another embodiment, the fusion protein is generated by circular permutation of the non-native N-terminus and / or C-terminus.

[0013] The fusion polypeptides provided herein can include a first polypeptide fusion partner linked to a ligand by a protease-cleavable linker, where the catalytic efficiency of the cleavable linker is optimized, the ligand is optionally modified, and the first polypeptide fusion partner is a blocking moiety that prevents binding of the modified ligand to a target receptor or a subunit of a target receptor until cleavage of the protease-cleavable linker.

[0014] A fusion polypeptide provided herein can also be a fusion polypeptide comprising a first polypeptide fusion partner linked to a ligand by a protease-cleavable linker, wherein the catalytic efficiency of the cleavable linker is optimized; the ligand is optionally modified, including by cyclic permutation, to create a non-native N-terminus and a novel C-terminus relative to the native ligand; and at least one of the novel N-terminus or novel C-terminus of the modified ligand is operably linked to the first polypeptide fusion partner to form the fusion polypeptide; and the first polypeptide fusion partner is a blocking moiety that prevents binding of the modified ligand to a target receptor or a subunit of a target receptor until cleavage of the protease-cleavable linker.

[0015] In one embodiment, the first polypeptide fusion partner is selected from the group consisting of an antibody, an antibody fragment, and an albumin molecule. In another embodiment, the first polypeptide fusion partner further comprises a second polypeptide fusion partner comprising a second blocking moiety. In another embodiment, the second polypeptide fusion partner is a different type of blocking moiety from the first polypeptide fusion partner. In another embodiment, the first polypeptide fusion partner is albumin, and the second polypeptide fusion partner is a domain comprising a complementary amino acid sequence that blocks cytokine activity. In another embodiment, the first polypeptide fusion partner is a steric blocker, e.g., albumin, and the second polypeptide is a specific blocker, e.g., a cytokine receptor, a portion of a cytokine receptor, a novel affinity peptide specific for a cytokine, or an antibody or antibody fragment that specifically binds to the cytokine of the fusion polypeptide. In another embodiment, the second polypeptide fusion partner is the same type of blocking moiety as the first polypeptide fusion partner.

[0016] In one embodiment, the fusion protein further comprises a tumor antigen-binding moiety, hi another embodiment, the fusion protein further comprises a serum half-life extending domain. In another embodiment, the ligand is a helix bundle protein and cytokine (including but not limited to, growth hormone, IL-2, IL-4, IL-5, IL-6, IL-10, IL-22, IL-23p19, IL-11, IL-13, IL-15, IL-12p35, IL-21, IL-30 (IL27p28), IL-34, IL-35, IL-35p35, IFN-α, IFN-β, IFNγ, LIF, CNTF, oncostatin M, CLCF-1, GCSF, GM-CSF, EPO, ferritin, leptin, placental lactogen, prolactin, apolipoprotein e), b-trefoil protein (including but not limited to, IL-1α, IL-1β, IL-1Ra, IL18, IL-33, IL-36Ra), , IL-36a, IL-36b, IL-36g, IL-37, IL-38, IL1Hy2, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8a, FGF-8b, FGF-8e, FGF-8f, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23), α / β (TIM) barrel proteins (including but not limited to triosephosphate isomerase), beta sandwich proteins (including but not limited to galectin-1, galectin-3), TNF-beta, seven beta propeller proteins, class 1 In one embodiment, the ligand is selected from the group consisting of an MHC α1α2 domain, an integrin I domain, a GYF domain, a C1 domain, a C2 domain (e.g., cPLA2, PKC, synaptotagmin), a PDZ domain, C3d, and C5a. In another embodiment, the ligand comprises an IL-2 polypeptide or a fragment or fragments thereof.In another embodiment, the protease-cleavable linker polypeptide comprises a sequence cleavable by at least one protease selected from the group consisting of kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin G, elastase, FAP, ADAM (selected from ADAM8, ADAM9, ADAM10, ADAM12, ADAM17, and ADAMTS1), PR-3, granzyme M, calpain, matrix metalloproteinase (MMP), plasminogen activator, cathepsin, caspase, tryptase, and tumor cell surface protease. In another embodiment, the cytokine, or fragment or mutein thereof, is substantially dissociated from the cytokine blocking moiety after the protease-cleavable polypeptide linker is cleaved by the protease.

[0017] Disclosed herein is a fusion polypeptide comprising at least one of each of the following: a cytokine polypeptide or functional fragment or mutein thereof [A]; a cytokine blocking moiety [B]; and an optimized protease-cleavable polypeptide linker [L], wherein the blocking moiety is selected from the group consisting of an antibody, an antibody fragment, and albumin, and the cytokine comprises a circularly permuted cytokine. In some embodiments, the fusion protein further comprises a tumor antigen-binding moiety and / or a serum half-life extending domain.In some embodiments, the fusion protein is a fusion protein in which the cytokine peptide or functional fragment or mutein thereof is selected from the group consisting of a helical bundle protein and a cytokine (including but not limited to growth hormone, IL-2, IL-4, IL-5, IL-6, IL-10, IL-22, IL-23p19, IL-11, IL-13, IL-15, IL-12p35, IL-21, IL-30 (IL27p28), IL-34, IL-35, IL-35p35, IFN-α, IFN-β, IFNγ, LIF, CNTF, oncostatin M, CLCF-1, GCSF, GM-CSF, EPO, ferritin, leptin, placental lactogen, prolactin, apolipoprotein e), a FAP (e.g., Fapα), an ADAM (selected from ADAM8, ADAM9, ADAM10, ADAM12, ADAM17, and ADAMTS1), b-trefoil Proteins (including but not limited to IL-1α, IL-1β, IL-1Ra, IL18, IL-33, IL-36Ra, IL-36a, IL-36b, IL-36g, IL-37, IL-38, IL1Hy2, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8a, FGF-8b, FGF-8e, FGF-8f, FGF-9, FGF-10, FGF-11, FGF-12, In one embodiment, the cytokine peptide or functional fragment or mutein thereof is selected from the group consisting of FGF-13, FGF-14, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23), α / β (TIM) barrel proteins (including but not limited to triosephosphate isomerase), beta sandwich proteins (including but not limited to galectin-1, galectin-3), TNF-β, seven β propeller proteins, class 1 MHC α1α2 domain, integrin I domain, GYF domain, C1 domain, C2 domain (e.g., cPLA2, PKC, synaptotagmin), PDZ domain, C3d, and C5a. In one embodiment, the cytokine peptide or functional fragment or mutein thereof comprises IL-2.In another embodiment, the cytokine blocking moiety comprises a ligand binding domain or fragment or mutein of the cytokine's cognate receptor, a single domain antibody or scFv that binds to a cytokine polypeptide or a functional fragment or mutein thereof, or an antibody or antibody fragment that binds to the cytokine's receptor. In another embodiment, the antibody is a single domain antibody or scFv. In another embodiment, the blocking moiety extends the serum half-life of the cytokine or fragment thereof.

[0018] Disclosed herein are fusion polypeptides comprising a protease-cleavable moiety, the sequence of which is catalytically optimized for cleavage by a particular protease, such that protease cleavage renders the composition inducible within the tumor microenvironment. In one embodiment, the fusion protein further comprises a biologically inactive polypeptide, and cleavage of the cleavable moiety by the protease converts the biologically inactive polypeptide into a biologically active polypeptide. In one embodiment, the biologically inactive polypeptide comprises a cytokine, chemokine, growth factor, or soluble receptor. In another embodiment, the biologically inactive polypeptide comprises at least one of an extracellular domain, a transmembrane domain, and an intracellular domain. In another embodiment, the biologically inactive polypeptide comprises a cell surface receptor, a chimeric antigen receptor (CAR), or a T-cell receptor (TCR) subunit. In another embodiment, the biologically inactive polypeptide comprises an antigen-binding polypeptide, an antibody, or an antigen-binding portion thereof.

[0019] The present disclosure further relates to nucleic acids encoding any of the polypeptides disclosed herein, vectors comprising any of the nucleic acids encoding any of the polypeptides disclosed herein, and host cells comprising the vectors.

[0020] Provided herein are methods for producing pharmaceutical compositions, comprising culturing host cells containing a vector comprising a nucleic acid encoding any of the polypeptides disclosed herein under conditions suitable for expression and harvesting of the desired polypeptide. Methods for using any of the polypeptides disclosed herein are described, comprising administering an effective amount of a pharmaceutical composition comprising such a polypeptide to a subject in need thereof. For example, use for treating a subject with a disease or disorder disclosed herein is described.

[0021] Provided herein are pharmaceutical compositions comprising an effective amount of any recombinant polypeptide, any proprotein, any fusion protein, any fusion polypeptide, any nucleic acid, any vector, or any host cell containing such a vector disclosed herein, for example, for treating a subject having a disease or disorder disclosed herein.

[0022] Also disclosed is the use of a recombinant polypeptide, proprotein, fusion protein, fusion polypeptide, nucleic acid, vector, or host cell containing such a vector, as disclosed herein, for the manufacture of a medicament for the treatment of a disease or disorder disclosed herein. The present invention provides, for example, the following items. (Item 1) A polypeptide comprising a protease-cleavable amino acid sequence selected from the group consisting of SEQ ID NOs: 195 to 220, or an amino acid sequence having at least about 90% identity to SEQ ID NOs: 195 to 220. (Item 2) A polypeptide comprising SEQ ID NO: 195 or a functional variant of SEQ ID NO: 195, or SEQ ID NO: 198 or a functional variant of SEQ ID NO: 198. (Item 3) 3. The polypeptide according to item 2, wherein the functional variant of SEQ ID NO: 195 comprises any one of SEQ ID NOs: 258 to 331. (Item 4) 3. The polypeptide according to Item 2, wherein the functional variant of SEQ ID NO: 198 comprises any one of SEQ ID NO: 199 and SEQ ID NOs: 332 to 408. (Item 5) 3. The polypeptide of item 2, wherein the polypeptide comprises SEQ ID NO: 195. (Item 6) 3. The polypeptide of item 2, wherein the polypeptide comprises SEQ ID NO: 198. (Item 7) A polypeptide comprising formula I, [D1]-[L1]-[D2] wherein D1 is a first domain of interest; L1 is a separate moiety that connects or joins D1 to D2, and said separate moiety comprises an amino acid sequence selected from SEQ ID NOs: 195 to 220, or an amino acid sequence having at least about 90% identity to SEQ ID NOs: 195 to 220; The polypeptide wherein D2 is a second domain of interest. (Item 8) 8. The polypeptide according to item 1 or 7, wherein the separated portion comprises at least one proline residue adjacent to an amino acid sequence selected from the group consisting of SEQ ID NOs: 195 to 220 or an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 195 to 220. (Item 9) 9. The polypeptide according to any one of items 1 to 8, wherein the separating portion comprises a cleavable portion that is a substrate for two or more proteases. (Item 10) 10. The polypeptide according to any one of items 1 to 9, wherein the protease is selected from the group consisting of FAPa, CTSL1, ADAM (selected from ADAM8, ADAM9, ADAM10, ADAM12, ADAM17, and ADAMTS1), and MMP (selected from MMP1, MMP2, MMP9, and MMP14). (Item 11) 10. The polypeptide according to any one of items 1 to 9, wherein the protease is selected from MMP1, MMP2, MMP9, MMP4, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin K, or cathepsin L. (Item 12) 8. The polypeptide according to any one of items 1 to 7, wherein the detached portion comprises an amino acid sequence that is a substrate for at least one protease present in the tumor microenvironment of human tumors. (Item 13) 9. The polypeptide according to any one of items 1 to 8, wherein the separating moiety comprises two or more cleavable moieties, each of the cleavable moieties being a substrate for a protease. (Item 14) 9. The polypeptide according to any one of items 1 to 8, comprising a first cleavable moiety comprising a first amino acid sequence that is a substrate for a first protease, and a second cleavable moiety comprising a second amino acid sequence that is a substrate for a second protease. (Item 15) 9. The polypeptide according to any one of items 1 to 8, further comprising a non-cleavable linker sequence. (Item 16) 9. The polypeptide according to any one of items 1 to 8, wherein the polypeptide comprises a cytokine, a chemokine, a growth factor, a soluble receptor, or any combination thereof. (Item 17) 9. The polypeptide according to any one of items 1 to 8, wherein the polypeptide comprises at least one of an extracellular domain, a transmembrane domain, and an intracellular domain. (Item 18) 9. The polypeptide according to any one of items 1 to 8, wherein the polypeptide comprises a subunit of a cell surface receptor, a chimeric antigen receptor (CAR), or a T cell receptor (TCR). (Item 19) 9. The polypeptide according to any one of items 1 to 8, wherein the polypeptide comprises an antigen-binding polypeptide, an antibody or an antigen-binding portion thereof. (Item 20) 15. The polypeptide of any one of items 1 to 14, wherein the cleavable moiety is cleaved by one or more proteases with (a) greater catalytic efficiency, (b) greater specificity, or (c) both (a) and (b) than a reference polypeptide sequence. (Item 21) 21. The polypeptide of item 20, wherein the one or more proteases are selected from the group consisting of FAPa, CTSL1, ADAM (selected from ADAM8, ADAM9, ADAM10, ADAM12, ADAM17, and ADAMTS1), and MMP (selected from MMP1, MMP2, MMP9, and MMP14). (Item 22) 22. The polypeptide of item 21, wherein the protease is selected from MMP1, MMP2, MMP9, MMP4, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin K, or cathepsin L. (Item 23) 22. The polypeptide of item 21, wherein the reference polypeptide sequence is present in a natural polypeptide substrate of FAPa, CTSL1, ADAM (selected from ADAM8, ADAM9, ADAM10, ADAM12, ADAM17, and ADAMTS1), MMP (selected from MMP1, MMP2, MMP9, and MMP14), or a combination thereof. (Item 24) 24. The polypeptide of any one of items 1 to 23, wherein the cleavable moiety is cleaved by one or more proteases with reduced catalytic efficiency relative to a reference polypeptide sequence. (Item 25) 25. The polypeptide of item 24, wherein the cleavable moiety is cleaved with reduced catalytic efficiency by one or more serum proteases. (Item 26) 26. The polypeptide of item 25, wherein the cleavable moiety is cleaved with reduced catalytic efficiency by one or more liver proteases. (Item 27) 26. The polypeptide of claim 25, wherein the cleavable moiety is cleaved with reduced catalytic efficiency by one or more of factor Xa, hepsin, or thrombin. (Item 28) 26. The polypeptide of any one of items 1 to 25, wherein the polypeptide is operably linked to a moiety selected from the group consisting of a polypeptide moiety, a lipid moiety, a nucleic acid moiety, a detectable moiety, and a small molecule. (Item 29) A recombinant proprotein comprising: a. a recombinant polypeptide comprising a cleavable moiety that is a substrate for a protease, wherein the cleavable moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 195-220, or an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 195-220; b. and a polypeptide having biological activity, The recombinant proprotein, wherein the proprotein has an attenuated biological activity, and wherein cleavage of the cleavable moiety by the protease produces a polypeptide having non-attenuated biological activity. (Item 30) 30. The recombinant proprotein of item 29, wherein the biologically active polypeptide comprises a cytokine, a chemokine, a growth factor, a soluble receptor, or a combination thereof. (Item 31) 30. The recombinant proprotein of item 29, wherein the biologically active polypeptide comprises at least one of an extracellular domain, a transmembrane domain, and an intracellular domain. (Item 32) 30. The recombinant proprotein of item 29, wherein the biologically active polypeptide comprises a cell surface receptor, a chimeric antigen receptor (CAR), or a T cell receptor (TCR) subunit. (Item 33) 30. The recombinant proprotein of item 29, wherein the polypeptide having biological activity comprises an antigen-binding polypeptide, an antibody, or an antigen-binding portion thereof. (Item 34) A fusion protein comprising: a. a signaling protein or molecule; b. a blocking moiety selected from a steric blocking moiety, a specific blocking moiety, and combinations thereof; c. A peptide linker comprising a cleavable moiety as defined in item 29. (Item 35) 35. The fusion protein of item 34, wherein (a) and (b) are operably linked by (c). (Item 36) 35. The fusion protein of item 34, wherein the peptide linker comprises two or more copies of the same cleavable moiety. (Item 37) 35. The fusion protein of item 34, wherein the steric blocking moiety comprises human serum albumin (HSA) or an anti-HSA antibody. (Item 38) 35. The fusion protein of item 34, wherein the signaling protein is an interleukin-2 amino acid sequence comprising (i) a non-native N-terminus and / or (ii) a non-native C-terminus. (Item 39) 35. The fusion protein of item 34, wherein the signaling protein is an interleukin 2 amino acid sequence. (Item 40) 35. The fusion protein of item 34, further comprising one or more half-life prolonging domains, wherein said domains are not also specific blockers. (Item 41) 41. The fusion protein of item 40, wherein the non-native N-terminus and / or C-terminus is generated by circular permutation. (Item 42) 1. A fusion polypeptide comprising a first polypeptide fusion partner linked to a ligand by a protease-cleavable linker, wherein the cleavable linker has been optimized for catalytic efficiency, and wherein the ligand is optionally modified, and wherein the first polypeptide fusion partner is a blocking moiety that prevents binding of the modified ligand to a target receptor or a subunit of a target receptor until cleavage of the protease-cleavable linker. (Item 43) 1. A fusion polypeptide comprising a first polypeptide fusion partner linked to a ligand by a protease-cleavable linker, wherein the cleavable linker has been optimized for catalytic efficiency; the ligand has optionally been modified, including by cyclic permutation, to create a non-native N-terminus and a novel C-terminus compared to the native ligand; and at least one of the novel N-terminus or the novel C-terminus of the modified ligand is operably linked to a first polypeptide fusion partner to form a fusion polypeptide; and the first polypeptide fusion partner is a blocking moiety that prevents binding of the modified ligand to a target receptor or a subunit of a target receptor until cleavage of the protease-cleavable linker. (Item 44) 44. The fusion polypeptide of claim 42 or 43, wherein the first polypeptide fusion partner is selected from the group consisting of an antibody, an antibody fragment, and an albumin molecule. (Item 45) 44. The fusion polypeptide of item 42 or 43, further comprising a second polypeptide fusion partner comprising a second blocking moiety. (Item 46) 44. The fusion polypeptide of item 42 or 43, wherein the second polypeptide fusion partner is a different type of blocking moiety than the first polypeptide fusion partner. (Item 47) 44. The fusion polypeptide of item 42 or 43, wherein the first polypeptide fusion partner is albumin and the second polypeptide fusion partner is a domain comprising a complementary amino acid sequence that blocks the activity of a cytokine. (Item 48) 44. The fusion polypeptide of item 42 or 43, wherein the first polypeptide fusion partner is a steric blocker, such as albumin, and the second polypeptide is a specific blocker, such as a cytokine receptor, a part of a cytokine receptor, a novel affinity peptide specific for a cytokine, or an antibody or antibody fragment that specifically binds to the cytokine of the fusion polypeptide. (Item 49) 48. The fusion polypeptide of claim 47, wherein the second polypeptide fusion partner is the same type of blocking moiety as the first polypeptide fusion partner. (Item 50) 44. The fusion polypeptide of any one of items 34, 42, or 43, further comprising a tumor antigen-binding moiety. (Item 51) 44. The fusion polypeptide of any one of items 34, 42, or 43, further comprising a serum half-life prolonging domain. (Item 52) The ligand may be a helix bundle protein and a cytokine (including, but not limited to, growth hormone, IL-2, IL-4, IL-5, IL-6, IL-10, IL-22, IL-23p19, IL-11, IL-13, IL-15, IL-12p35, IL-12p40, IL-12p70, IL-21, IL-30 (IL27p28), IL-34, IL-35, IL-35p35, IFN-α, IFN-β, IFNγ, LIF, CNTF, oncostatin M, CLCF-1, GCSF, GM-CSF, EPO, ferritin, leptin, placental lactogen, prolactin, apolipoprotein e), a b-trefoil protein (including, but not limited to, IL-1α, IL-1β, IL-1Ra, IL18, IL-33, IL- 36Ra, IL-36a, IL-36b, IL-36g, IL-37, IL-38, IL1Hy2, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF -7, FGF-8a, FGF-8b, FGF-8e, FGF-8f, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-16, FGF-17 , FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23), alpha / beta (TIM) barrel proteins (including but not limited to triosephosphate isomerase), beta sandwich proteins (including but not limited to galectin-1, galectin-3), TNF-beta, seven beta propeller proteins, class 1 MHC alpha1alpha2 domain, integrin I domain, GYF domain, C1 domain, C2 domain (e.g., cPLA2, PKC, synaptotagmin), PDZ domain, C3d, C5a. (Item 53) 44. The fusion polypeptide of item 42 or 43, wherein the ligand comprises an IL-2 polypeptide or a fragment thereof. (Item 54) 44. The fusion polypeptide of any one of paragraphs 34, 42, or 43, wherein the protease-cleavable linker polypeptide comprises a sequence capable of being cleaved by at least one protease selected from the group consisting of kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin G, cathepsin L, elastase, FAP, ADAM (selected from ADAM8, ADAM9, ADAM10, ADAM12, ADAM17, and ADAMTS1), PR-3, granzyme M, calpain, matrix metalloproteinase (MMP), plasminogen activator, cathepsin, caspase, tryptase, and tumor cell surface protease. (Item 55) 44. The fusion polypeptide of any one of paragraphs 34, 42, or 43, wherein the cytokine or fragment or mutein thereof substantially dissociates from the cytokine blocking moiety after the protease-cleavable polypeptide linker is cleaved by a protease. (Item 56) 1. A fusion polypeptide comprising: a. a cytokine polypeptide or a functional fragment or mutein thereof [A], b. a cytokine blocking moiety [B], and c. Optimized protease-cleavable polypeptide linker [L] wherein the blocking moiety is selected from the group consisting of an antibody, an antibody fragment, and albumin, and the cytokine comprises a circularly permuted cytokine. (Item 57) 57. The fusion polypeptide of item 56, further comprising a tumor antigen-binding moiety. (Item 58) 57. The fusion polypeptide of item 56, further comprising a serum half-life prolonging domain. (Item 59) Cytokine peptides or functional fragments or muteins thereof are used in helix bundle proteins and cytokines (including but not limited to growth hormone, IL-2, IL-4, IL-5, IL-6, IL-10, IL-22, IL-23p19, IL-11, IL-13, IL-15, IL-12p35, IL-12p40, IL-17p70, IL-21, IL-30 (IL-27p28), IL-34, IL-40, IL-50, IL-60, IL-70, IL-80, IL-90, IL-100, IL-111, IL-112, IL-113, IL-114, IL-115, IL-116, IL-117, IL-118, IL-119 ... IL-35, IL-35p35, IFN-α, IFN-β, IFN-γ, LIF, CNTF, oncostatin M, CLCF-1, GCSF, GM-CSF, EPO, ferritin, leptin, placental lactogen, prolactin, apolipoprotein e), FAP, ADAM (selected from ADAM8, ADAM9, ADAM10, ADAM12, ADAM17, and ADAMTS1), b-trefoil protein (including but not limited to), Although not included, IL-1α, IL-1β, IL-1Ra, IL18, IL-33, IL-36Ra, IL-36a, IL-36b, IL-36g, IL-37, IL-38, IL1Hy2, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8a, FGF-8b, FGF-8e, FGF-8f, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13 , FGF-14, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23), alpha / beta (TIM) barrel proteins (including but not limited to triosephosphate isomerase), beta sandwich proteins (including but not limited to galectin-1, galectin-3), TNF-beta, seven beta propeller proteins, class 1 MHC alpha1alpha2 domain, integrin I domain, GYF domain, C1 domain, C2 domain (e.g., cPLA2, PKC, synaptotagmin), PDZ domain, C3d, C5a. (Item 60) 57. The fusion polypeptide of item 56, wherein the cytokine peptide or functional fragment or mutein thereof comprises IL-2. (Item 61) 57. The fusion polypeptide of claim 56, wherein the cytokine blocking moiety comprises a ligand binding domain or a fragment or mutein of the cytokine's cognate receptor, a single domain antibody or scFv that binds to the cytokine polypeptide or a functional fragment or mutein thereof, or an antibody or antibody fragment that binds to the receptor of the cytokine. (Item 62) 57. The fusion polypeptide of item 56, wherein the antibody is a single domain antibody or an scFv. (Item 63) 57. The fusion polypeptide of item 56, wherein the blocking moiety extends the serum half-life of the cytokine or fragment thereof. (Item 64) A fusion polypeptide comprising a protease-cleavable portion, the sequence of which is catalytically optimized for cleavage by a particular protease, wherein protease cleavage renders the composition inducible within the tumor microenvironment. (Item 65) 65. The fusion polypeptide of item 64, further comprising a biologically inactive polypeptide, wherein cleavage of the cleavable moiety by the protease converts the biologically inactive polypeptide into a biologically active polypeptide. (Item 66) 66. The fusion polypeptide of item 65, wherein the biologically inactive polypeptide comprises a cytokine, chemokine, growth factor, or soluble receptor. (Item 67) 66. The fusion polypeptide of item 65, wherein the biologically inactive polypeptide comprises at least one of an extracellular domain, a transmembrane domain, and an intracellular domain. (Item 68) 66. The fusion polypeptide of item 65, wherein the biologically inactive polypeptide comprises a cell surface receptor, a chimeric antigen receptor (CAR), or a T cell receptor (TCR) subunit. (Item 69) 66. The fusion polypeptide of item 65, wherein the biologically inactive polypeptide comprises an antigen-binding polypeptide, an antibody, or an antigen-binding portion thereof. (Item 70) 70. A nucleic acid encoding the polypeptide according to any one of items 7 to 69. (Item 71) 71. A vector comprising the nucleic acid of item 70. (Item 72) A host cell comprising the vector of item 71. (Item 73) 73. A method for producing a pharmaceutical composition, comprising culturing the host cell of item 72 under conditions suitable for the expression and harvesting of a desired polypeptide. (Item 74) 70. A method of using the polypeptide according to any one of items 1 to 69, comprising administering an effective amount of the pharmaceutical composition to a subject in need thereof. [Brief explanation of the drawings]

[0023] [Figure 1A]Schematic diagram showing a protease-activated cytokine or chemokine containing a blocking moiety, which may optionally function as a serum half-life extending domain. The diagram to the left of the arrow shows that the cytokine is connected to the blocking moiety via a protease-cleavable linker, thus blocking the cytokine's ability to bind to its receptor. The diagram to the right of the arrow shows that in an inflammatory or tumor environment, a protease cleaves at the protease cleavage site on the linker, releasing the blocking moiety and allowing the cytokine to bind to its receptor. [Figure 1B] Schematic diagram showing a protease-activated cytokine or chemokine, with HSA (the blocking moiety) directly linked to the cytokine or chemokine of interest and a protease cleavage site between the HSA and the cytokine or chemokine of interest. The diagram to the left of the arrow shows that the cytokine is connected to the blocking moiety via a protease-cleavable linker, thus blocking the cytokine's ability to bind to its receptor. The diagram to the right of the arrow shows that in an inflammatory or tumor environment, a protease cleaves at the protease cleavage site on the linker, releasing the blocking moiety and allowing the cytokine to bind to its receptor. [Figure 1C] Schematic diagram showing a protease-activated cytokine or chemokine with multiple HSAs (blocking moieties) directly attached to the molecule of interest. If desired, one or more HSAs may be attached to the cytokine or chemokine via a linker (e.g., a linker containing a protease cleavage site). The diagram to the left of the arrow shows that the cytokine is connected to the blocking moiety via a protease-cleavable linker, thus blocking the cytokine's ability to bind to its receptor. The diagram to the right of the arrow shows that in an inflammatory or tumor environment, a protease cleaves the linker at the protease cleavage site, releasing the blocking moiety and allowing the cytokine to bind to its receptor. This cytokine has similar pK characteristics (e.g., a short half-life) compared to the native cytokine. [Figure 1D] Schematic diagram showing protease-activated cytokines or chemokines, including multiple cytokines of the same or different types, each linked to a binding domain via a protease-cleavable linker. The diagram to the left of the arrow shows that the cytokine is connected to a blocking moiety via a protease-cleavable linker, thus blocking the cytokine's ability to bind to its receptor. The diagram to the right of the arrow shows that in an inflammatory or tumor environment, a protease cleaves at the protease cleavage site on the linker, releasing the blocking moiety and allowing the cytokine to bind to its receptor. [Figure 2] Schematic diagram showing a protease-activated cytokine or chemokine comprising a cytokine or chemokine polypeptide connected by at least one protease-cleavable linker, a blocking moiety, and a serum half-life extending domain. The diagram to the left of the arrow shows that the cytokine is connected to the blocking moiety via the protease-cleavable linker, thus blocking the cytokine's ability to bind to its receptor. The cytokine is also linked to a separate half-life extending element, which extends its serum half-life. The diagram to the right of the arrow shows that in an inflammatory or tumor environment, a protease cleaves at the protease cleavage site on the linker, releasing the serum half-life extending element and the blocking moiety, allowing the cytokine to bind to its receptor. The cytokine now has similar pK properties (e.g., a short half-life) compared to the native cytokine. [Figure 3]

[0023] Figure 1 is a schematic diagram showing a protease-activated cytokine or chemokine comprising a cytokine or chemokine polypeptide, a blocking moiety, and a targeting domain connected by at least one protease-cleavable linker. The diagram to the left of the arrow shows that the cytokine is connected to the blocking moiety and targeting domain via the protease-cleavable linker, thus blocking the cytokine's ability to bind to its receptor. The diagram to the right of the arrow shows that in an inflammatory or tumor microenvironment, a protease cleaves at the protease cleavage site in the linker, releasing the targeting domain and blocking moiety, allowing the cytokine to bind to its receptor. [Figure 4A] Schematic diagram showing a protease-activated cytokine or chemokine comprising a cytokine or chemokine polypeptide, a blocking moiety, a targeting domain, and a serum half-life extending domain connected by at least one protease-cleavable linker, where the cytokine polypeptide and targeting domain are connected by the protease-cleavable linker. The diagram to the left of the arrow shows that the cytokine or chemokine is connected to the targeting domain, blocking moiety, and half-life extending element via the protease-cleavable linker(s), thus blocking the cytokine's or chemokine's ability to bind to its receptor. The diagram to the right of the arrow shows that in an inflammatory or tumor microenvironment, a protease cleaves at the protease cleavage site on the linker(s), releasing the serum half-life extending element, targeting domain, and blocking moiety, allowing the cytokine to bind to its receptor. The cytokine then has similar pK characteristics (e.g., a short half-life) compared to the native cytokine. [Figure 4B]Schematic diagram showing a protease-activated cytokine or chemokine comprising a cytokine or chemokine polypeptide, a blocking moiety, a targeting domain, and a serum half-life extending domain connected by at least one protease-cleavable linker. The diagram to the left of the arrow shows that the cytokine is connected to the targeting domain, blocking moiety, and half-life extending element via the protease-cleavable linker(s), thus blocking the cytokine's ability to bind to its receptor. The diagram to the right of the arrow shows that in inflammation or the tumor microenvironment, proteases cleave the linker(s) at the protease cleavage site, releasing the serum half-life extending element and blocking moiety, allowing the cytokine to bind to its receptor. The targeting moiety remains bound, retaining the cytokine within the tumor microenvironment. The cytokine then has similar pK characteristics (e.g., a short half-life) compared to the native cytokine. [Figure 5] 1 shows a graph demonstrating that linker 2 (GPAGLYAQ, SEQ ID NO: 195) and linker 3 (ALFKSSFP, SEQ ID NO: 198) are minimally cleaved in lung, kidney, and liver cells. [Figure 6] Graphs A-B show that polypeptides containing recombinant human IL-2 and the sequence of linker 1 (GPAGMKGL, SEQ ID NO: 196), linker 2 (GPAGLYAQ, SEQ ID NO: 195), or linker 3 (ALFKSSFP, SEQ ID NO: 198) are not processed by healthy lung fibroblasts. [Figure 7A]

[0023] Figure 1 is a graph showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. The graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo® (Promega) luminescence-based cell viability assay. The proliferation assay was performed without HSA. Each fusion protein contained an anti-HSA binder, and both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in the assay. [Figure 7B]1 is a graph showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. The graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo® (Promega) luminescence-based cell viability assay. The proliferation assay was performed in the presence of HSA. Each fusion protein contained an anti-HSA binder, and both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in the assay. [Figure 7C]

[0023] Figure 1 is a graph showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. The graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo® (Promega) luminescence-based cell viability assay. The proliferation assay was performed without HSA. Each fusion protein contained an anti-HSA binder, and both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in the assay. [Figure 7D] 1 is a graph showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. The graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo® (Promega) luminescence-based cell viability assay. The proliferation assay was performed in the presence of HSA. Each fusion protein contained an anti-HSA binder, and both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in the assay. [Figure 7E]

[0023] Figure 1 is a graph showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. The graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo® (Promega) luminescence-based cell viability assay. The proliferation assay was performed without HSA. Each fusion protein contained an anti-HSA binder, and both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in the assay. [Figure 7F] 1 is a graph showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. The graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo® (Promega) luminescence-based cell viability assay. The proliferation assay was performed in the presence of HSA. Each fusion protein contained an anti-HSA binder, and both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in the assay. [Figure 7G]

[0023] Figure 1 is a graph showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. The graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo® (Promega) luminescence-based cell viability assay. The proliferation assay was performed without HSA. Each fusion protein contained an anti-HSA binder, and both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in the assay. [Figure 7H] 1 is a graph showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. The graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo® (Promega) luminescence-based cell viability assay. The proliferation assay was performed in the presence of HSA. Each fusion protein contained an anti-HSA binder, and both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in the assay. [Figure 8] (A-F) are a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in each assay. [Figure 9A]1 is a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in each assay. [Figure 9B] 1 is a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in each assay. [Figure 9C] 1 is a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in each assay. [Figure 9D] 1 is a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in each assay. [Figure 9E]1 is a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in each assay. [Figure 9F] 1 is a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in each assay. [Figure 9G] 1 is a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in each assay. [Figure 9H] 1 is a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in each assay. [Figure 9I]1 is a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in each assay. [Figure 9J] 1 is a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in each assay. [Figure 9K] 1 is a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in each assay. [Figure 9L] 1 is a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in each assay. [Figure 9M]1 is a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in each assay. [Figure 9N] 1 is a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in each assay. [Figure 9O] 1 is a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in each assay. [Figure 9P] 1 is a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in each assay. [Figure 9Q]1 is a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in each assay. [Figure 9R] 1 is a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in each assay. [Figure 9S] 1 is a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in each assay. [Figure 9T] 1 is a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in each assay. [Figure 9U]1 is a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in each assay. [Figure 9V] 1 is a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in each assay. [Figure 9W] 1 is a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in each assay. [Figure 9X] 1 is a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in each assay. [Figure 9Y]1 is a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in each assay. [Figure 9Z] 1 is a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay, quantified by the CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved versions of the fusion proteins were used in each assay. [Figure 10]

[0023] Figure 1 shows the results of a protein cleavage assay. The fusion protein ACP16 was run on an SDS-PAGE gel in both cleaved and uncleaved forms. As can be seen in the gel, cleavage was complete. [Figure 11] (A-B) are a series of graphs showing the results of a HEK-Blue IL-12 reporter assay performed with human p40 / mouse p35 IL-12 fusion protein and recombinant human IL-12 (Rec hIL-12). The analysis was based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results confirm that the IL-12 protein fusion protein is active. [Figure 12A] This graph shows the results of a HEK-blue assay of four IL-12 fusion proteins before and after cleavage by MMP9. The analysis was based on quantification of secreted alkaline phosphatase (SEAP) activity using QUANTI-Blue (InvivoGen) reagent. The data show that the cleaved IL12 is more active than the intact fusion protein. The construct tested was ACP06. [Figure 12B]Figure 1 shows the results of a HEK-blue assay of four IL-12 fusion proteins before and after cleavage by MMP9. The analysis was based on quantification of secreted alkaline phosphatase (SEAP) activity using QUANTI-Blue (InvivoGen) reagent. The data show that the cleaved IL12 is more active than the intact fusion protein. The construct tested was ACP08. [Figure 12C] Figure 1 shows the results of a HEK-blue assay of four IL-12 fusion proteins before and after cleavage by MMP9. The analysis was based on quantification of secreted alkaline phosphatase (SEAP) activity using QUANTI-Blue (InvivoGen) reagent. The data show that the cleaved IL12 is more active than the intact fusion protein. The construct tested was ACP07. [Figure 12D] Figure 1 shows the results of a HEK-blue assay of four IL-12 fusion proteins before and after cleavage by MMP9. The analysis was based on quantification of secreted alkaline phosphatase (SEAP) activity using QUANTI-Blue (InvivoGen) reagent. The data show that the cleaved IL12 is more active than the intact fusion protein. The construct tested was ACP09. [Figure 12E] This graph shows the results of a HEK-blue assay of four IL-12 fusion proteins before and after cleavage by MMP9. The analysis was based on quantification of secreted alkaline phosphatase (SEAP) activity using QUANTI-Blue (InvivoGen) reagent. The data show that the cleaved IL12 is more active than the intact fusion protein. The construct tested was ACP10. [Figure 12F]This graph shows the results of a HEK-blue assay of four IL-12 fusion proteins before and after cleavage by MMP9. The analysis was based on quantification of secreted alkaline phosphatase (SEAP) activity using QUANTI-Blue (InvivoGen) reagent. The data show that the cleaved IL12 is more active than the intact fusion protein. The construct tested was ACP11. [Figure 13]

[0023] Figure 1 shows the results of a protein cleavage assay. The fusion protein ACP11 was run on an SDS-PAGE gel in both cleaved and uncleaved forms. As can be seen in the gel, cleavage was complete. [Figure 14] Schematic diagram showing a non-limiting example of an inducible cytokine protein, where the construct is activated upon protease cleavage of the linker attached between the two subunits of the cytokine. [Figure 15A] Figure 1 shows the results of a HEK-Blue assay performed on human p40 / mouse p35 IL12 fusion protein and recombinant human IL12 (Rec hIL-12). The results confirm that the IL12 protein fusion protein is active. Each proliferation assay was performed with and without HSA. [Figure 15B] Figure 1 shows the results of a HEK-Blue assay performed on human p40 / mouse p35 IL12 fusion protein and recombinant human IL12 (Rec hIL-12). The results confirm that the IL12 protein fusion protein is active. Each proliferation assay was performed with and without HSA. [Figure 15C] Figure 1 shows the results of a HEK-Blue assay performed on human p40 / mouse p35 IL12 fusion protein and recombinant human IL12 (Rec hIL-12). The results confirm that the IL12 protein fusion protein is active. Each proliferation assay was performed with and without HSA. [Figure 15D]Figure 1 shows the results of a HEK-Blue assay performed on human p40 / mouse p35 IL12 fusion protein and recombinant human IL12 (Rec hIL-12). The results confirm that the IL12 protein fusion protein is active. Each proliferation assay was performed with and without HSA. [Figure 16] (A-F) are a series of graphs showing the activity of exemplary IFNγ fusion proteins compared to the activity of a murine IFNγ control using a WEHI 279 cell viability assay. Each assay was performed in medium containing HSA (+HSA) or without HSA (-HSA). Each fusion protein contained an anti-HSA binder, and both uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in the assay. [Figure 17] (A-F) are a series of graphs showing the activity of exemplary IFNγ fusion proteins compared to the activity of a mouse IFNγ control using a B16 reporter assay. Each assay was performed in medium containing HSA (+HSA) or without HSA (-HSA). Each fusion protein contained an anti-HSA binder, and both uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in the assay. [Figure 18] Figures A-B show the results of a protein cleavage assay. Two constructs, ACP31 (IFN-α fusion protein; A) and ACP55 (IFN-γ fusion protein; B), were run on an SDS-PAGE gel in both cleaved and uncleaved forms. As can be seen in the gel, cleavage was complete. [Figure 19] 1A-B are a series of graphs showing the activity of exemplary IFNγ fusion proteins compared to the activity of a mouse IFNγ control using a B16 reporter assay. Each assay was performed in culture medium containing HSA. Each fusion protein contains an anti-HSA binder. Both uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 20]1A-B are a series of graphs showing the activity of exemplary IFNα fusion proteins compared to the activity of a mouse IFNα A control using a B16 reporter assay. Each assay was performed in medium containing HSA. Each fusion protein contains an anti-HSA binder. Both uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 21] (A-D) are a series of graphs showing the results of tumor growth studies using the MC38 cell line. (A-C) show the effect of IFNγ and IFNγ fusion proteins on tumor growth when injected intraperitoneally (IP) using different dose levels and schedules (μg = microgram, BID = twice daily, BIW = twice weekly, QW = once weekly). (D) shows the effect of intratumoral (IT) injection of IFNγ and IL-2 on tumor growth. [Figure 22] (A-B) are a series of graphs showing the activity of exemplary IFNγ fusion proteins (ACP51 and ACP52) cleaved by MMP9 protease compared to the activity of the uncleaved fusion proteins using a B16 reporter assay. Each fusion protein contains an anti-HSA binder and a tumor-targeting domain. [Figure 23] (A-B) are a series of graphs showing the activity of exemplary IFNγ fusion proteins (ACP53 and ACP54) cleaved by MMP9 protease compared to the activity of the uncleaved fusion proteins using a B16 reporter assay. Each fusion protein contains IFNγ directly fused to albumin. [Figure 24]Figures 24A and 24B show two graphs showing the stability of IL-2 fusion proteins containing linker 1 (GPAGMKGL, SEQ ID NO: 196), linker 2 (GPAGLYAQ, SEQ ID NO: 195), or linker 3 (ALFKSSFP, SEQ ID NO: 198) in human serum from normal and cancer patients. Figure 24A shows the stability of IL-2 fusion proteins containing linker 1 (GPAGMKGL, SEQ ID NO: 196), linker 2 (GPAGLYAQ, SEQ ID NO: 195), or linker 3 (ALFKSSFP, SEQ ID NO: 198) at 24 hours. Figure 24B shows the stability of IL-2 fusion proteins containing linker 1 (GPAGMKGL, SEQ ID NO: 196), linker 2 (GPAGLYAQ, SEQ ID NO: 195), or linker 3 (ALFKSSFP, SEQ ID NO: 198) at 72 hours. [Figure 25A] 1 is a graph showing analysis of ACP16 in a HEKBlue IL-2 reporter assay in the presence of HSA. [Figure 25B] 1 is a graph showing the analysis of ACP124 in a HEKBlue IL-2 reporter assay in the presence of HSA. Circles indicate activity of the uncut polypeptide, squares indicate activity of the cut polypeptide, and triangles indicate IL-2 alone as a control. [Figure 25C] Figure 1 shows the results of a CTLL-2 proliferation assay. CTLL-2 cells (ATCC) were plated at 500,000 cells / well in culture medium with or without 40 mg / ml human serum albumin (HSA) and stimulated with serial dilutions of recombinant hIL2 or activatable hIL2 for 72 hours at 37°C and 5% CO2. The activity of uncleaved and cleaved activatable ACP16 was tested. Cleaved activatable hIL2 was generated by incubation with active MMP9. Cell viability was assessed using the CellTiter-Glo (Promega) luminescence-based cell viability assay. Triangles represent wild-type cytokines, circles represent intact fusion proteins, and squares represent protease-cleaved fusion proteins. [Figure 26](A-C) are a series of graphs showing the activity of fusion proteins in the HEKBlue IL-12 reporter assay. (A) is a graph showing the activity of truncated and uncut ACP11 (human p40 / mouse p35 IL-12 fusion protein). (B) is a graph showing the analysis of ACP91 (chimeric IL-12 fusion protein). Squares show the activity of the uncut ACP91 polypeptide, and triangles show the activity of the truncated polypeptide (ACP91 + MMP9). The EC50 values ​​for each are shown in the table. (C) is a graph showing the analysis of ACP136 (chimeric IL-12 fusion protein). Squares show the activity of the uncut ACP136 polypeptide, and triangles show the activity of the truncated polypeptide (ACP136 + MMP9). The EC50 values ​​for each are shown in the table insert. [Figure 27A] Figures 27A and 27B are graphs showing that truncated IL-12 polypeptides are active in the HEKBlue IL2 reporter assay. Fusion proteins were evaluated both uncut (circles) and cut (squares), with wild-type IL2 used as a control + HSA in Figure 27A. Data for ACP31 + HSA are shown. EC50 values ​​for each are shown in the table below each graph. [Figure 27B] Figures 27A and 27B are graphs showing that truncated IL-12 polypeptides are active in the HEKBlue IL2 reporter assay. Fusion proteins were evaluated both uncut (circles) and cut (squares), with wild-type IL2 used as a control + HSA in Figure 27B. Data for ACP125 + HSA are shown. EC50 values ​​for each are shown in the table below each graph. [Figure 27C] Figures 27A-27C are graphs showing that truncated IL-12 polypeptides are active in the HEKBlue IL2 reporter assay. Fusion proteins were evaluated both uncut (circles) and cut (squares), with wild-type IL2 used as a control + HSA in Figure 27C. Data for ACP126 + HSA are shown. EC50 values ​​for each are shown in the table below each graph. [Figure 27D]Figures 27A-27D are graphs showing that truncated IL-12 polypeptides are active in the HEKBlue IL2 reporter assay. Fusion proteins were evaluated both uncut (circles) and cut (squares), with ACP131 used as a control (triangles) in Figure 27D. Data for ACP127 is shown. EC50 values ​​for each are shown in the table below each graph. [Figure 27E] Figures 27A-27D are graphs showing that truncated IL-12 polypeptides are active in the HEKBlue IL2 reporter assay. Fusion proteins were evaluated both uncut (circles) and cut (squares), with ACP131 used as a control (triangles) in Figure 27E. Data for ACP128 is shown. EC50 values ​​for each are shown in the table below each graph. [Figure 27F] Figures 27A-27C are graphs showing that truncated IL-12 polypeptides are active in the HEKBlue IL2 reporter assay. Fusion proteins were evaluated both uncut (circles) and cut (squares), with ACP131 used as a control (triangles) in Figure 27F. Data for ACP129 is shown. EC50 values ​​for each are shown in the table below each graph. [Figure 28A] Figure 1 shows the activity of APC56 in a HEKBlue IFNα reporter assay. Each fusion was tested for activity with (squares) and without (circles). Analysis of mouse IFNγ is included in each graph for comparison. [Figure 28B] Figure 1 shows the activity of APC57 in a HEKBlue IFNα reporter assay. Each fusion was tested for activity with (squares) and without (circles). Analysis of mouse IFNγ is included in each graph for comparison. [Figure 28C] Figure 1 shows the activity of APC58 in a HEKBlue IFNα reporter assay. Each fusion was tested for activity with (squares) and without (circles). Analysis of mouse IFNγ is included in each graph for comparison. [Figure 28D]Figure 1 shows the activity of APC59 in a HEKBlue IFNα reporter assay. Each fusion was tested for activity with (squares) and without (circles). Analysis of mouse IFNγ is included in each graph for comparison. [Figure 28E] Figure 1 shows the activity of APC60 in a HEKBlue IFNα reporter assay. Each fusion was tested for activity with (squares) and without (circles). Analysis of mouse IFNγ is included in each graph for comparison. [Figure 28F] Figure 1 shows the activity of APC61 + HSA in a HEKBlue IFNα reporter assay. Each fusion was tested for activity with (squares) and without (circles). Analysis of mouse IFNγ is included in each graph for comparison. [Figure 28G] Figure 1 shows the activity of ACP30 + HSA in a HEKBlue IFNα reporter assay. Each fusion was tested for activity with (squares) and without (circles). Analysis of mouse IFNγ is included in each graph for comparison. [Figure 28H] Figure 1 shows the activity of ACP73 in a HEKBlue IFNα reporter assay. Each fusion was tested for activity with (squares) and without (circles). Analysis of mouse IFNγ is included in each graph as a comparison. [Figure 28I] Figure 1 shows the activity of ACP70 + HSA in a HEKBlue IFNα reporter assay. Each fusion was tested for activity with (squares) and without (circles). Analysis of mouse IFNγ is included in each graph for comparison. [Figure 28J] Figure 1 shows the activity of ACP71 in a HEKBlue IFNα reporter assay. Each fusion was tested for activity with (squares) and without (circles). Analysis of mouse IFNγ is included in each graph as a comparison. [Figure 28K]Figure 1 shows the activity of ACP72 in a HEKBlue IFNα reporter assay. Each fusion was tested for activity with (squares) and without (circles). Analysis of mouse IFNγ is included in each graph as a comparison. [Figure 28L] Figure 1 shows the activity of ACP73 in a HEKBlue IFNα reporter assay. Each fusion was tested for activity with (squares) and without (circles). Analysis of mouse IFNγ is included in each graph as a comparison. [Figure 28M] Figure 1 shows the activity of ACP74 in a HEKBlue IFNα reporter assay. Each fusion was tested for activity with (squares) and without (circles). Analysis of mouse IFNγ is included in each graph for comparison. [Figure 28N] Figure 1 shows the activity of ACP75 in a HEKBlue IFNα reporter assay. Each fusion was tested for activity with (squares) and without (circles). Analysis of mouse IFNγ is included in each graph as a comparison. [Figure 29](A-B) Two graphs show the results of analyzing ACP31 (a murine IFNα1 fusion protein) and ACP11 (a human p40 / murine p35 IL12 fusion protein) in a tumor xenograft model. (A) shows tumor volume over time in mice treated with 33 μg ACP31 (circles), 110 μg ACP31 (triangles), 330 μg ACP31 (diamonds), and as controls, 1 μg murine wild-type IFNα1 (dashed line, squares) and 10 μg mIFNα1 (dashed line, small circles). Vehicle alone is indicated by large open circles. The data show that tumor volume decreased over time in mice treated with ACP31 in a dose-dependent manner. (B) Tumor volume over time in mice treated with 17.5 μg ACP11 (squares), 175 μg ACP31 (triangles), 525 μg ACP31 (circles), and controls of 2 μg ACP04 (dashed line, triangles) and 10 μg ACP04 (dashed line, diamonds). Vehicle alone is indicated by a large open circle. The data demonstrate a dose-dependent reduction in tumor volume over time in mice treated with both ACP11 and ACP04 (human p40 / mouse p35 IL12 fusion protein). [Figure 30] (A-F) A series of spaghetti plots showing tumor volume over time in a mouse xenograft tumor model treated with vehicle alone (A), 2 μg ACP04 (B), 10 μg ACP04 (C), 17.5 μg ACP11 (D), 175 μg ACP11 (E), and 525 μg ACP11 (F). Each line represents an individual mouse. [Figure 31]Figures A-C show three graphs illustrating the analysis of ACP16 and ACP124 in tumor xenograft models. Figure A shows tumor volume over time in mice treated with 4.4 μg ACP16 (squares), 17 μg ACP16 (triangles), 70 μg ACP16 (inverted triangles), and 232 μg ACP16 (dark circles), as well as 12 μg wild-type IL-2 (dashed line, triangles) and 36 μg wild-type IL-2 (dashed line, diamonds) for comparison. Vehicle alone is indicated by a large open circle. The data show that tumor volume in mice treated with high concentrations of ACP16 decreased over time in a dose-dependent manner. (B) Tumor volume over time in mice treated with 17 μg ACP124 (squares), 70 μg ACP124 (triangles), 230 μg ACP124 (downward triangles), and 700 μg ACP124. Vehicle alone is indicated by a large open circle. (C) Tumor volume over time in mice treated with 17 μg ACP16 (triangles), 70 μg ACP16 (circles), 232 μg ACP16 (dark circles), and, for comparison, 17 μg ACP124 (dashed line, triangles), 70 μg ACP124 (dashed line, diamonds), and 230 μg ACP124 (dashed line, diamonds). Vehicle alone is indicated by a dark inverted triangle. The data show that tumor volume decreased over time in a dose-dependent manner in mice treated with ACP16, but not ACP124. [Figure 32A] 1 is a series of spaghetti plots showing the activity of fusion proteins in the MC38 mouse xenograft model, with each line in the plot representing a different mouse. [Figure 32B-1] 1 is a series of spaghetti plots showing the activity of fusion proteins in the MC38 mouse xenograft model, with each line in the plot representing a different mouse. [Figure 32B-2] Same as above. [Figure 32C-1] 1 is a series of spaghetti plots showing the activity of fusion proteins in the MC38 mouse xenograft model, with each line in the plot representing a different mouse. [Figure 32C-2] Same as above. [Figure 33]1 is a graph showing tumor volume over time in a mouse xenograft model showing tumor growth in control mice (open circles) and AP16-treated mice (squares). [Figure 34] 1 is a series of survival plots showing the survival of mice over time after treatment with cleavable fusion proteins. A shows data for mice treated with vehicle alone (gray line), 17 μg ACP16 (dark line), and 1 μg ACP124 (dashed line). B shows data for mice treated with vehicle alone (gray line), 70 μg ACP16 (dark line), and 70 μg ACP124 (dashed line). C shows data for mice treated with vehicle alone (gray line), 232 μg ACP16 (dark line), and 230 μg ACP124 (dashed line). D shows data for mice treated with vehicle alone (gray line), 232 μg ACP16 (dark line), and 700 μg ACP124 (dashed line). [Figure 35-1]

[0033] Figure 1 is a series of spaghetti plots showing the activity of fusion proteins in an MC38 mouse xenograft model. All groups of mice received a total of four doses, except for the highest three doses of ACP132, for which fatal toxicity was detected after 1 week / 2 doses. Shown are vehicle alone (top), 17, 55, 70, and 230 μg ACP16 (all top rows), 9, 28, 36, and 119 μg ACP132 (all middle rows), and 13, 42, 54, and 177 μg ACP21 (all bottom rows). Each line in the plot represents an individual animal. [Figure 35-2] Same as above. [Figure 36] 36 is a schematic diagram showing substrate cleavage activity in conditioned complete (+FBS) medium by FRET endpoint assay in four cell lines. The ratio of tumor vs. control activity was estimated by averaging the three tumor cell lines and comparing with a control myofibroblast cell line in which signal was detectable. SEQ ID NOs: 201, 198, 197, 196, and 195 are disclosed in order of appearance in Figure 36. [Figure 37] Figure 37 is a schematic diagram showing the dynamics of ADAM17_2 substrates in cell culture. Figure 37 discloses SEQ ID NO: 235. [Figure 38]Figure 38 is a schematic diagram showing the dynamics of FAPα_1 substrate in conditioned medium. Figure 38 discloses SEQ ID NO: 197. [Figure 39] Figure 39 is a schematic diagram showing the dynamics of FAPα_1 substrate in cell lysates. Figure 39 discloses SEQ ID NO: 197. [Figure 40] Figure 40 is a schematic diagram showing the dynamics of MMP9_1 substrates in cell lysates. Figure 40 discloses SEQ ID NO: 196. [Figure 41] 41 is a schematic diagram showing substrate cleavage activity in cell lysates by FRET endpoint assay. In Figure 41, SEQ ID NOS: 198 and 197 are disclosed in order of appearance, respectively. [Figure 42] Figure 42 is a schematic diagram showing the dynamics of CTSL1_1 substrates in cell lysates. Figure 42 discloses SEQ ID NO: 198. [Figure 43] Figure 43 is a schematic diagram showing the dynamics of MMP14_1 substrates in cell lysates. Figure 43 discloses SEQ ID NO: 195. [Figure 44] 44 is a schematic diagram showing the calculated concentration of enzyme equivalents per cell culture derived sample. Figure 44 discloses SEQ ID NOs: 201, 198, 197, 196, and 195, respectively, in order of appearance. [Figure 45] 45 is a schematic diagram showing the enzymatic progress curves of CTSL1 cleavage for CTSL1_2 vs. CTSL1_1. In Figure 45, SEQ ID NOs: 198, 199, and 236 are disclosed in order of appearance, respectively. [Figure 46] FIG. 1 is a schematic diagram showing 30-mer truncation of CTSL1_1 (ALFKSSFP, SEQ ID NO: 198) vs CTSL1_2 (ALFFSSPP, SEQ ID NO: 199). [Figure 47] 47 is a schematic diagram showing the sensitivity of CTSL1 FRET substrates to CTSK cleavage. Rates of product formation were measured as specific activities in pmol min-1 μg-1. The threshold for the reference substrate Z-LR-AMC is indicated by the dashed line. SEQ ID NOs: 198 and 199 are disclosed in order of appearance in Figure 47. [Figure 48]48 is a schematic diagram showing 30-mer substrate degradation by MMP9. Substrates are ranked by relative degradation rate with a "+" and uncleaved substrates are indicated with a "-". Figure 48 discloses SEQ ID NOs: 204, 205, 214, 216, 202, 217, 203, 211, 219, 207, 215, 212, 213, 206, 208, 209, 210, 218, and 220, respectively, in order of appearance. [Figure 49] Schematic diagram showing tandem MMP14_1 motif degradation by MMP9. Top: Substrate degradation trace, modeled with first-order kinetics. Bottom: Product formation trace showing complex kinetics. SEQ ID NOS: 202-205 are disclosed in order of appearance in Figure 49. [Figure 50] 50 is a schematic diagram showing 30-mer substrate degradation by FAPα. Substrates are ranked by relative degradation rate with a "+" and uncleaved substrates are indicated with a "-". Figure 50 discloses SEQ ID NOs: 205, 204, 206, 217, 203, 218, 219, 213, 216, 207, 214, 210, 202, 211, 208, 209, 212, 215, and 220, respectively, in order of appearance. [Figure 51] 51 is a schematic diagram showing 30-mer substrate degradation by CTSL1. Substrates are ranked by relative degradation rate with a "+" and uncleaved substrates are indicated with a "-". Figure 51 discloses SEQ ID NOs: 207, 208, 202, 218, 219, 212, 215, 217, 211, 209, 214, 206, 213, 210, 216, 203, 204, 205, and 220, respectively, in order of appearance. [Figure 52] 52 is a schematic diagram showing 30-mer substrate degradation by ADAM17. Substrates are ranked by relative degradation rate with a "+" and uncleaved substrates are indicated with a "-". Figure 52 discloses SEQ ID NOs: 208, 209, 211, 214, 217, 219, 213, 218, 215, 210, 212, 216, 207, 206, 202, 203, 204, 205, and 220, respectively, in order of appearance. [Figure 53]53 is a schematic diagram showing 30-mer substrate degradation by Factor Xa. Substrates are ranked by their relative rate of degradation with a "+" and uncleaved substrates are indicated with a "-". Figure 53 discloses SEQ ID NOs: 220, 206, 202, 214, 208, 209, 215, 210, 218, 217, 207, 213, 216, 211, 212, 219, 203, 204, and 205, respectively, in order of appearance. [Figure 54] 54 is a schematic diagram showing 30-mer substrate cleavage by thrombin. Substrates are ranked by relative cleavage rate with a "+" and uncleaved substrates are indicated with a "-". Figure 54 discloses SEQ ID NOS: 220, 204, 202, 207, 205, 211, 212, 215, 209, 218, 219, 217, 210, 213, 216, 214, 208, 206, and 203, respectively, in order of appearance. [Figure 55] 55 is a schematic diagram showing 30-mer substrate degradation by hepsin. Substrates are ranked by relative degradation rate with a "+" and uncleaved substrates are indicated with a "-". Figure 55 discloses SEQ ID NOs: 220, 209, 216, 215, 210, 213, 206, 214, 212, 207, 217, 208, 211, 218, 219, 202, 203, 204, and 205, respectively, in order of appearance. [Figure 56A] Western blots probed with IL-2 antibody are shown, demonstrating the stability of ACP16 in 90% serum. Serum was pooled from three human donors. The construct was incubated with PBS, serum, or MMP9 protease, and cleavage was assessed at T=0 and T=24 hours. [Figure 56B] Western blots probed with IL-2 antibody are shown, demonstrating the stability of ACP153 in 90% serum. Serum was pooled from three human donors. The construct was incubated with PBS, serum, or MMP9 protease, and cleavage was assessed at T=0 and T=24 hours. [Figure 56C]Western blots probed with IL-2 antibody are shown, demonstrating the stability of ACP157 in 90% serum. Serum was pooled from three human donors. The construct was incubated with PBS, serum, or MMP9 protease, and cleavage was assessed at T=0 and T=24 hours. [Figure 57A] Western blots using an IL-2 antibody are shown, demonstrating that ACP153, ACP155, ACP156, ACP16, and ACP372 are 90% stable in serum. Serum was pooled from three human donors. The constructs were incubated with PBS, serum, or MMP9 protease, and cleavage was assessed at T = 24 hours and T = 72 hours. Figure 57A shows the results using human serum. [Figure 57B] Western blots using IL-2 antibodies are shown, demonstrating that ACP153, ACP155, ACP156, ACP16, and ACP372 are 90% stable in serum. Serum was pooled from three human donors. The constructs were incubated with PBS, serum, or MMP9 protease, and cleavage was assessed at T = 24 hours and T = 72 hours. Figure 57B shows the results using mouse serum. [Figure 58A] A series of spaghetti plots showing the activity of fusion proteins in an MC38 mouse xenograft model are shown. Vehicle alone (FIG. 58A, top), 17, 55, and 230 μg ACP16 (FIG. 58A) are shown. Each line in the plot represents an individual animal. [Figure 58B]

[0023] Figure 1 shows a series of spaghetti plots demonstrating the activity of fusion proteins in an MC38 mouse xenograft model. 55 and 230 μg ACP153 are shown. Each line in the plot represents an individual animal. [Figure 58C]

[0023] Figure 1 shows a series of spaghetti plots demonstrating the activity of fusion proteins in an MC38 mouse xenograft model. 55 and 230 μg ACP155 are shown. Each line in the plot represents an individual animal. [Figure 58D]

[0023] Figure 1 shows a series of spaghetti plots demonstrating the activity of fusion proteins in an MC38 mouse xenograft model. 55 and 230 μg ACP156 are shown. Each line in the plot represents an individual animal. [Figure 59] Graph showing the results of a STAT activation reporter assay performed on IL-2 fusion proteins and recombinant human IL-2 (Rec hIL-2). The analysis was based on quantification of secreted alkaline phosphatase (SEAP) activity using QUANTI-Blue (InvivoGen) reagent. [Figure 60] Graph showing the results of a STAT activation reporter assay performed on IL-2 fusion proteins and recombinant human IL-2 (Rec hIL-2). The analysis was based on quantification of secreted alkaline phosphatase (SEAP) activity using QUANTI-Blue (InvivoGen) reagent. [Figure 61] Graph showing the results of a STAT activation reporter assay performed on IL-2 fusion proteins and recombinant human IL-2 (Rec hIL-2). The analysis was based on quantification of secreted alkaline phosphatase (SEAP) activity using QUANTI-Blue (InvivoGen) reagent. [Figure 62-1] Graph showing the results of a STAT activation reporter assay performed on IL-2 fusion proteins and recombinant human IL-2 (Rec hIL-2). The analysis was based on quantification of secreted alkaline phosphatase (SEAP) activity using QUANTI-Blue (InvivoGen) reagent. [Figure 62-2] Same as above. [Figure 63-1]

[0049] Figure 63 shows a table reporting the extent of cleavage observed using the engineered cleavage substrates described herein and associated proteases. Flanking sequences are shown in lowercase, first cleavable sequences are underlined, second cleavable sequences are in bold font, and third cleavable sequences are in italics. In some cases, overlap exists between cleavable sequences and is indicated accordingly. Figure 63 discloses SEQ ID NOS: 202-220, respectively, in order of appearance. [Figure 63-2] Same as above. [Figure 64] Schematic diagram of an inducible tetravalent antibody format. [Figure 65] A to B show that multivalent 4-1BB antibodies can inducibly stimulate 4-1BB. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present disclosure relates to novel separation moieties or linkers and polypeptides (e.g., fusion proteins) containing the linkers, which are preferably protease-cleavable and which connect a first amino acid sequence of interest (e.g., a first domain of interest) to a second amino acid sequence of interest (e.g., a second domain of interest).

[0025] The separation moieties of the present disclosure confer site selectivity for the action of the attached payload(s). The payload can be a therapeutic agent, a half-life extender, a blocking agent, etc., or any combination thereof. The separation moieties can be used to attach any desired payload (including, for example, cytokines, antibodies, cell-based therapeutics, etc.). The separation moieties can be used individually or in tandem, triple, quadruple, etc., so long as the separation moieties are less than about 100 amino acids. The individual separation moieties can be directly linked to each other or interspersed with non-cleavable linkers, all of which promote high efficiency and site specificity.

[0026] Various embodiments of the present disclosure are described in further detail in the following paragraphs.

[0027] Unless otherwise defined, all technical terms, notations, and other scientific terms used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a deviation from what is commonly understood in the art. The techniques and procedures described or referenced herein are generally well understood by those skilled in the art and commonly employed using conventional methodology (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Where appropriate, procedures involving the use of commercially available kits and reagents are generally carried out according to manufacturer-defined protocols and conditions unless otherwise specified.

[0028] "Cytokine" is a well-known term of art and refers to any class of immunomodulatory proteins (e.g., interleukins or interferons) that are secreted by cells, particularly cells of the immune system, and that are immune system regulators. Cytokine polypeptides that can be used in the fusion proteins disclosed herein include, but are not limited to, transforming growth factors, such as TGF-α and TGF-β (e.g., TGFbeta1, TGFbeta2, TGFbeta3); interferons, such as interferon-α, interferon-β, interferon-γ, interferon-kappa, and interferon-omega; interleukins, such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49, IL-50, IL-51, IL-52, IL-53, IL-54, IL-55, IL-56, IL- tumor necrosis factors, such as tumor necrosis factor alpha and lymphotoxin; transforming growth factor beta (TGF beta) family proteins, chemokines (e.g., C-X-C motif chemokine 10 (CXCL10), CCL19, CCL20, CCL21), and granulocyte-macrophage colony-stimulating factor (GM-CS), as well as fragments of such polypeptides (i.e., functional fragments of the foregoing) that activate the cytokine's cognate receptor. "Chemokine" is a term of art that refers to any of a family of small cytokines capable of inducing directed chemotaxis of nearby responsive cells.

[0029] Cytokines are well known to have a short serum half-life, often only a few minutes.Even cytokine forms with amino acid sequence modifications intended to prolong serum half-life while maintaining receptor agonist activity typically have similarly short serum half-lives.As used herein, "short-half-life cytokine" refers to cytokines with a substantially short half-life circulating in the serum of subjects, for example, cytokines with a serum half-life of less than 10 minutes, less than 15 minutes, less than 30 minutes, less than 60 minutes, less than 90 minutes, less than 120 minutes, less than 240 minutes, or less than 480 minutes.As used herein, short-half-life cytokines include cytokines whose sequence has not been modified to achieve a longer-than-normal half-life in the subject's body, and polypeptides with amino acid sequence modifications intended to prolong serum half-life while maintaining receptor agonist activity.This latter case is not intended to include the addition of heterologous protein domains, such as bona fide half-life-extending elements, such as serum albumin.

[0030] As used herein, "conservative" amino acid substitutions generally refer to the substitution of one amino acid residue with another amino acid within a recognized group, which may alter the structure of the peptide but substantially maintain the biological activity of the peptide. Conservative amino acid substitutions are known to those skilled in the art. Conservative amino acid substitutions include, but are not limited to, substitutions made between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. For example, one skilled in the art would reasonably expect that the substitution of leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similarly, the substitution of an amino acid with a structurally related amino acid, alone, would not significantly affect the biological activity of the resulting molecule.

[0031] A "sortase" is a transpeptidase that modifies proteins by recognizing and cleaving carboxyl-terminal sorting signals embedded in or terminally attached to target proteins or peptides. Sortase A catalyzes the cleavage of an LPXTG motif (where X is any standard amino acid) (SEQ ID NO: 237) between a Thr and a Gly residue on the target protein, forming an enzyme-thioacyl intermediate by temporary attachment of the Thr residue to an active site Cys residue on the enzyme. To complete transpeptidation and generate a peptide-monomer conjugate, a biomolecule bearing an N-terminal nucleophilic group (typically an oligoglycine motif) attacks the intermediate, displacing sortase A and linking the two molecules.

[0032] As used herein, the term "steric blocker" refers to a polypeptide or polypeptide moiety that can be covalently attached to a cytokine polypeptide, directly or indirectly, via other moieties such as linkers, e.g., in the form of a chimeric polypeptide (fusion protein), but that is not otherwise covalently attached to the cytokine polypeptide. A steric blocker can be non-covalently attached to a cytokine polypeptide, e.g., by electrostatic, hydrophobic, ionic, or hydrogen bonding. A steric blocker typically inhibits or blocks the activity of the cytokine moiety due to its proximity to the cytokine site and its comparative size.

[0033] As used herein, a "half-life extending element" is a portion of a chimeric polypeptide that increases serum half-life and improves pK, for example, by altering size (e.g., above the renal filtration cutoff), shape, hydrodynamic radius, charge, or parameters of absorption, biodistribution, metabolism, and elimination.

[0034] As used herein, the term "separating moiety" or "linker" refers to an amino acid sequence, typically less than about 100 amino acids, that connects or joins a first amino acid sequence of interest (e.g., an amino acid sequence that folds to form a first protein domain) to a second amino acid sequence of interest (e.g., an amino acid sequence that folds to form a second protein domain) within a continuous polypeptide chain. Separating moieties or linkers typically contain one or more protease cleavage sites and are therefore protease-cleavable. A "tandem linker" refers to a linker that contains two or more protease cleavage sites that can be cleaved by the same or different proteases. Tandem linkers can be oriented in any desired orientation, e.g., one cleavage site can be adjacent to another cleavage site, one cleavage site can overlap the other cleavage site, or one cleavage site can be followed by another cleavage site with intervening amino acids between the two cleavage sites.

[0035] As used herein, the terms "activatable," "activate," "induce," and "inducible" refer to the ability of a protein that is part of a conjugate, i.e., a cytokine, to bind to a receptor and exert an activity when additional elements are cleaved from the conjugate.

[0036] As used herein, a "plasmid" or "viral vector" is an agent that transports a nucleic acid of the present disclosure into a cell without degradation, and includes a promoter that drives expression of the nucleic acid molecule and / or polypeptide in the delivered cell.

[0037] As used herein, the terms "peptide," "polypeptide," or "protein" are used loosely to refer to two or more amino acids joined by a peptide bond. Protein, peptide, and polypeptide are also used interchangeably herein to refer to an amino acid sequence. It should be recognized that the term polypeptide is not used herein to imply a particular size or number of amino acids comprising the molecule, and that the peptides of the present invention may contain up to a few amino acid residues or more.

[0038] As used throughout, a "subject" can be a vertebrate, more specifically a mammal (e.g., a human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig), bird, reptile, amphibian, fish, and any other animal. The term does not denote a particular age or sex. Thus, it is intended to cover adult and newborn subjects, whether male or female.

[0039] As used herein, "patient" or "subject" may be used interchangeably and may refer to a subject having a disease or disorder (e.g., cancer). The term patient or subject includes human and veterinary subjects.

[0040] As used herein, the terms "treatment," "treat," "treating," or grammatically related terms refer to a method of reducing the effects of a disease or condition, or the symptoms of a disease or condition. Thus, in the methods of the present disclosure, treatment can refer to a reduction in the severity of an established disease or condition, or the symptoms of a disease or condition, by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or substantially completely. For example, a method for treating a disease is considered therapeutic if one or more symptoms of the disease in a subject are reduced by 10% compared to a control. Thus, this reduction can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage reduction between 10% and 100% compared to native or control levels. It is well understood in the art that treatment does not necessarily refer to a cure or complete elimination of a disease, condition, or symptoms of a disease or condition. Desirable therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

[0041] As used herein, the terms "preventing," "preventing," and "prevention" of a disease or disorder refer to an act of inhibiting or delaying the onset or progression of one or more symptoms of a disease or disorder, occurring before or at about the same time that a subject begins to exhibit one or more symptoms of the disease or disorder, e.g., administration of a chimeric polypeptide or a nucleic acid sequence encoding a chimeric polypeptide.

[0042] As used herein, references to "decrease," "reducing," or "inhibiting" include an alteration of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more compared to a suitable control level. Such terms may, but do not necessarily, include complete loss of function or property (e.g., agonist activity).

[0043] A "reduced cytokine receptor agonist" is a cytokine receptor agonist that has reduced receptor agonist activity compared to a naturally occurring agonist for the cytokine receptor. A reduced cytokine agonist may have agonist activity that is at least about 10x, at least about 50x, at least about 100x, at least about 250x, at least about 500x, at least about 1000x, or less compared to the naturally occurring agonist for the receptor. When a fusion protein comprising a cytokine polypeptide described herein is described as being "reduced" or having "reduced activity," it means that the fusion protein is a reduced cytokine receptor agonist.

[0044] An "intact fusion protein" is one in which a domain has not been removed from the fusion protein, for example, by cleavage with a protease. Domains can be removed by protease cleavage or other enzymatic activity, but this has not occurred when a fusion protein is "intact."

[0045] As used herein, "moiety" refers to a portion of a molecule that has a characteristic function within the molecule, which function can be performed by the moiety in the context of another molecule. A moiety can be a chemical entity with a specific function, or a portion of a biological molecule with a specific function. For example, a "blocking moiety" in a fusion protein is a portion of the fusion protein that can block the activity of some or all of the fusion polypeptide. It can be a protein domain (e.g., serum albumin).

[0046] A. Separation Moiety or Linker The present disclosure relates to novel protease-cleavable separation moieties. As described herein, the protease-cleavable separation moieties are designed to be cleaved with high efficiency by proteases at desired locations (e.g., proteases selectively expressed or expressed at high levels in the tumor microenvironment) but are stable and not cleaved or cleaved with low efficiency at other locations (e.g., the periphery, e.g., healthy tissue or serum).

[0047] The protease-cleavable separation moiety was designed using a process that included prioritizing suitable proteases for cleaving the separation moiety based on expression in the target indication (e.g., expression in specific tumor types (e.g., colon cancer, lung cancer, breast cancer, melanoma)). Multiple data sources (including mRNA, proteomics, and tissue staining data) on upregulated or differential expression of proteases in the target indication were used. Proteases were also prioritized based on their specific activity and inherent specificity, with high specific activity and high intrinsic activity being preferred. Serum stability was an important design consideration, and proteases that do not rely on arginine in the substrate were selected to avoid potential off-target cleavage of the separation moiety by serum proteases. This is because many off-target enzymes are active against arginine residues.

[0048] Starting sequences in the design process were selected using diverse peptide libraries as protease substrates, and protease cleavage products were detected by mass spectrometry to identify sequence motifs preferred by each candidate protease. For the selected initial motifs, new peptide libraries tailored to the sequence motifs preferred by the candidate proteases were designed, created, and analyzed. The peptide motifs were also counterscreened for cleavage by serum proteases thrombin and factor Xa, as well as by the liver / kidney protease hepsin. This process yielded peptides containing sequence motifs that are highly cleaved by certain tumor-associated proteases (e.g., matrix metalloproteinase 9 (MMP9), MMP14, and / or cathepsin L) but are stable (not cleaved or cleaved with low efficiency) in serum or normal healthy tissues (e.g., by thrombin, factor Xa, and hepsin). The isolated moieties disclosed herein are efficiently cleaved by human tumors and minimally cleaved by normal tissues or serum.

[0049] The present disclosure relates to a separating moiety or linker that connects a first amino acid sequence of interest (e.g., a first domain of interest) to a second amino acid sequence of interest (e.g., a second domain of interest). Typically, the first amino acid sequence of interest and the second amino acid sequence of interest are not found together in a natural protein. For example, a separating moiety can connect or link the first domain of interest and the second domain of interest of a fusion protein. A separating moiety is an amino acid sequence that can be of any suitable length and is preferably cleavable by a protease.

[0050] The separating moieties disclosed herein can confer functionality, including flexibility and the ability to be cleaved. Flexible linkers are typically applied when the linked domains require some degree of movement or interaction. Cleavable linkers are introduced to release the free functional domain to a target site in vivo. The separating moieties disclosed herein function to connect at least two domains of interest. The separating moiety can maintain cooperative interdomain interactions or retention of biological activity. The separating moiety can link functional domains (e.g., payloads and half-life extension elements) that are released from the separating moiety at a target site (e.g., tumor microenvironment).

[0051] In a preferred embodiment, the separating portion is cleavable by a cleavage agent, such as an enzyme. Preferably, the separating portion comprises a protease cleavage site. In some cases, the separating portion comprises one or more cleavage sites. The separating portion may comprise a single protease cleavage site. Alternatively, the separating portion may comprise two or more protease cleavage sites. For example, the separating portion may comprise two, three, four, five, or more cleavage sites. When the separating portion comprises two or more protease cleavage sites, the cleavage sites may be cleaved by the same protease or by different proteases. A separating portion comprising two or more cleavage sites is referred to as a "tandem linker." The two or more cleavage sites may be arranged in any desired orientation, including, but not limited to, when one cleavage site is adjacent to another cleavage site, when one cleavage site overlaps another cleavage site, or when one cleavage site follows another cleavage site with intervening amino acids between the two cleavage sites.

[0052] Of particular interest in the present invention are disease-specific protease-cleavable linkers. Furthermore, the protease-cleavable linker is preferably preferentially cleaved in a desired location in the body, such as the tumor microenvironment, relative to the peripheral circulation. For example, the rate at which the protease-cleavable linker is cleaved in the tumor microenvironment may be at least about 10 times, at least about 100 times, at least about 1000 times, or at least about 10,000 times faster in the desired location in the body (e.g., the tumor microenvironment) than in the peripheral circulation (e.g., in plasma).

[0053] Proteases known to be associated with diseased cells or tissues include, but are not limited to, serine proteases, cysteine ​​proteases, aspartic acid proteases, threonine proteases, glutamic acid proteases, metalloproteinases, asparagine peptide lyases, serum proteases, cathepsin, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin K, cathepsin L, kallikrein, hKl, hK10, hK15, plasmin, collagenase, type IV collagenase, stromelysin, factor Xa, chymotrypsin-like proteases, trypsin-like proteases, elastase-like proteases, and subtilisin-like proteases. Actinidain, bromelain, calpain, caspase, caspase-3, Mirl-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidase, metalloendopeptidase, matrix metalloproteinase (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, urokinase plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin-1β converting enzyme, thrombin, FAP (FAPα), dipeptidyl peptidase, meprin, granzyme, and dipeptidyl peptidase IV (DPPIV / CD26). The protease capable of cleaving the linker amino acid sequence (which may be encoded by the chimeric nucleic acid sequence provided herein) may be selected from the group consisting of prostate-specific antigen (PSA), matrix metalloproteinase (MMP), a disintegrin-metalloproteinase (ADAM), plasminogen activator, cathepsin, caspase, tumor cell surface protease, and elastase. The MMP may be, for example, matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), or matrix metalloproteinase 14 (MMP14).Additionally or alternatively, the linker can be cleaved by a cathepsin (e.g., cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin K, and / or cathepsin L). Preferably, the linker can be cleaved by MMP14 or cathepsin L.

[0054] Proteases useful for cleaving linkers and for use in the methods disclosed herein are listed in Table 1, and exemplary proteases and their cleavage sites are listed in Table 1a. [Table 1-1] [Table 1-2] [Table 1A-1] [Table 1A-2]

[0055] Exemplary protease linkers include, but are not limited to, kallikrein-cleavable linkers, thrombin-cleavable linkers, chymase-cleavable linkers, carboxypeptidase A-cleavable linkers, cathepsin-cleavable linkers, elastase-cleavable linkers, FAP-cleavable linkers, ADAM-cleavable linkers, PR-3-cleavable linkers, granzyme M-cleavable linkers, calpain-cleavable linkers, matrix metalloproteinase (MMP)-cleavable linkers, plasminogen activator-cleavable linkers, caspase-cleavable linkers, tryptase-cleavable linkers, or tumor cell surface proteases. Specific examples include MMP9-cleavable linkers, ADAM-cleavable linkers, CTSL1-cleavable linkers, FAPα-cleavable linkers, and cathepsin-cleavable linkers. Preferred protease-cleavable linkers are cleaved by MMPs and / or cathepsins.

[0056] Separating moieties disclosed herein are typically less than 100 amino acids. Such separating moieties can vary in length, for example, from 1 amino acid (e.g., Gly) to 30 amino acids, 1 to 40 amino acids, 1 to 50 amino acids, 1 to 60 amino acids, 1 to 70 amino acids, 1 to 80 amino acids, 1 to 90 amino acids, or 1 to 100 amino acids. In some embodiments, the linker length is at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids. Preferred linkers are typically about 5 to 30 amino acids.

[0057] Preferably, the length of the linker varies from 2 to 30 amino acids and is optimized for each condition so as not to impose any constraints on the conformation or interactions of the domains to which the linker is attached.

[0058] In some embodiments, the separating portion has the sequence GPAGLYAQ (SEQ ID NO: 195); GPAGMKGL (SEQ ID NO: 196); PGGPAGIG (SEQ ID NO: 197); ALFKSSFP (SEQ ID NO: 198); ALFFSSPP (SEQ ID NO: 199); LAQRLRSS (SEQ ID NO: 200); LAQKLKSS (SEQ ID NO: 201); GALFKSSFPSGGGPAGLYAQGGSGKGGSGK (SEQ ID NO: 202); RGSGGGPAGLYAQGSGGGPAGLYAQGGSGK (SEQ ID NO: 203); KGGGPAGLYAQGPAGLYAQGPAGLYAQGSR (SEQ ID NO: 204); RGGPAGLYAQGGPAGLYAQGGGPAGLYAQK (SEQ ID NO: 205); KGGALFKSSFPGGPAGIGPLAQKLKSSGGS (SEQ ID NO: 206); SGGPGGPAGIGALFKSSFPLAQKLKSSGGG (SEQ ID NO: 207); RGPLAQKLKSSALFKSSFPGGPAGIGGGGK (SEQ ID NO: 208); GGGALFKSSFPLAQKLKSSPGGPAGIGGG R (SEQ ID NO: 209); RGPGGPAGIGPLAQKLKSSALFKSSFPGGG (SEQ ID NO: 210); RGGPLAQKLKSSPGGPAGIGALFKSSFPGK (SEQ ID NO: 211); RSGGPAGLYAQALFKSSFPLAQKLKSSGGG (SEQ ID NO: 212); GGPLAQKLKSSALFKSSFPGPAGLYAQGGR (SEQ ID NO: 213); GGALFKSSFPGPAGLYAQPLAQKLKSSGGK (SEQ ID NO: 214); RGGALFKSSFPLAQKLK SSGPAGLYAQGGK (SEQ ID NO: 215); RGGGPAGLYAQPLAQKLKSSALFKSSFPGG (SEQ ID NO: 216); SGPLAQKLKSSGPAGLYAQALFKSSFPGSK (SEQ ID NO: 217); KGGPGGPAGIGPLAQRLRSSALFKSSFPGR (SEQ ID NO: 218); KSGPGGPAGIGALFFSSPPLAQKLKSSGGR (SEQ ID NO: 219); or SGGFPRSGGSFNPRTFGSKRKRRGSRGGGG (SEQ ID NO: 220).

[0059] Certain preferred separating portions comprise the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198). The separating portions disclosed herein can contain one or more cleavage motifs or functional variants, which may be the same or different. A separating portion can contain one, two, three, four, five, or more cleavage motifs or functional variants. A separating portion comprising 30 amino acids can contain two cleavage motifs or functional variants, three cleavage motifs or functional variants, or more. A "functional variant" of a separating portion retains the ability to be cleaved with high efficiency at the target site (e.g., a tumor microenvironment expressing high levels of proteases) and is not cleaved or is cleaved with low efficiency in the periphery (e.g., serum). For example, a functional variant retains at least about 50%, about 55%, about 60%, about 70%, about 80%, about 85%, about 95%, or more of the cleavage efficiency of a separating portion comprising any one of SEQ ID NOs: 195-220.

[0060] A separation portion comprising multiple cleavage motifs may be selected from SEQ ID NOs: 195 to 201 and combinations thereof. Preferred separation portions comprising multiple cleavage motifs comprise amino acids selected from SEQ ID NOs: 202 to 220.

[0061] The separating portion may comprise both ALFKSSFP (SEQ ID NO: 198) and GPAGLYAQ (SEQ ID NO: 195). The separating portion may comprise two cleavage motifs, each having the sequence GPAGLYAQ (SEQ ID NO: 195). Alternatively or additionally, the separating portion may comprise two cleavage motifs, each having the sequence ALFKSSFP (SEQ ID NO: 198). The separating portion may comprise a third cleavage motif, which may be the same or different.

[0062] In some embodiments, the separated portion comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99% identical to SEQ ID NOs: 195-220 over the entire length of SEQ ID NOs: 195-220.

[0063] The present disclosure also relates to functional variants of isolated portions comprising SEQ ID NOs: 195-220. Functional variants of isolated portions comprising SEQ ID NOs: 195-220 generally differ from SEQ ID NOs: 195-220 by one or several amino acids (including substitutions, deletions, insertions, or any combination thereof) and substantially retain the ability to be cleaved by a protease.

[0064] A functional variant may include at least one or more amino acid substitutions, deletions, or insertions relative to a separate portion comprising SEQ ID NOs: 195-220. A functional variant may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes compared to a separate portion comprising SEQ ID NOs: 195-220. In some preferred embodiments, a functional variant differs from a separate portion comprising SEQ ID NOs: 195-220 by fewer than 10, fewer than 8, fewer than 5, fewer than 4, fewer than 3, fewer than 2, or even one amino acid change (e.g., amino acid substitution or deletion). In other embodiments, a functional variant may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions relative to a separate portion comprising SEQ ID NOs: 195-220. Amino acid substitutions may be conservative or non-conservative, but are preferably conservative.

[0065] In other embodiments, functional variants of the separating portion may contain one, two, three, four, five, or more non-conservative amino acid substitutions compared to the separating portion comprising SEQ ID NOs: 195-220. Non-conservative amino acid substitutions would be recognizable to one of skill in the art. Functional variants of the separating portion preferably contain no more than one, two, three, four, or five amino acid deletions.

[0066] The amino acid sequences disclosed in the separation portion can be described by their relative linear position within the separation portion relative to the scissile bond. As will be appreciated by those skilled in the art, a separation portion containing an eight amino acid protease substrate (e.g., SEQ ID NOS: 195-201) will contain amino acids at positions P4, P3, P2, P1, P1', P2', P3', and P4', with the scissile bond being between P1 and P1'. For example, the amino acid positions of a separation portion containing the sequence GPAGLYAQ (SEQ ID NO: 195) can be described as follows: [Table 1B]

[0067] The amino acid positions of the isolated portion comprising the sequence ALFKSSFP (SEQ ID NO: 198) can be written as follows: [Table 1C]

[0068] Preferably, the amino acids surrounding the cleavage site (eg, positions P1 and P1' in SEQ ID NOs: 195-201) are not substituted.

[0069] In embodiments, the separated portion comprises the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198), or a functional variant of SEQ ID NO: 195 or a functional variant of SEQ ID NO: 198. As described herein, functional variants of PAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) can contain one or more amino acid substitutions and substantially retain the ability to be cleaved by a protease. Specifically, functional variants of GPAGLYAQ (SEQ ID NO: 195) are cleaved by MMP14, and functional variants of ALFKSSFP (SEQ ID NO: 198) are cleaved by cathepsin L (CTSL1). The functional variants also retain the ability to be cleaved with high efficiency at target sites (e.g., tumor microenvironments expressing high levels of proteases). For example, a functional variant of GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) retains at least about 50%, about 55%, about 60%, about 70%, about 80%, about 85%, about 95%, or more cleavage efficiency of a separation portion comprising the amino acid sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198), respectively.

[0070] Preferably, a functional variant of GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) contains no more than 1, 2, 3, 4, or 5 conservative amino acid substitutions compared to GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198). Preferably, the amino acids at positions P1 and P1' are not substituted. The amino acids at positions P1 and P1' in SEQ ID NO: 195 are G and L, and the amino acids at positions P1 and P1' in SEQ ID NO: 198 are K and S.

[0071] Functional variants of GPAGLYAQ (SEQ ID NO: 195) may preferably include one or more of the following: a) an amino acid substitution of arginine at position P4; b) an amino acid substitution of leucine, valine, asparagine, or proline at position P3; c) an amino acid substitution of asparagine at position P2; d) an amino acid substitution of histidine, asparagine, or glycine at position P1; e) an amino acid substitution of asparagine, isoleucine, or leucine at position P1'; f) an amino acid substitution of tyrosine or arginine at position P2'; g) an amino acid substitution of glycine, arginine, or alanine at position P3'; or h) an amino acid substitution of serine, glutamine, or lysine at position P4'. In functional variants of GPAGLYAQ (SEQ ID NO: 195), the following amino acid substitutions are disfavored: a) arginine or isoleucine at position P3, b) alanine at position P2, c) valine at position P1, d) arginine, glycine, asparagine, or threonine at position P1', e) aspartic acid or glutamic acid at position P2', f) isoleucine at position P3', g) valine at position P4'. In some embodiments, functional variants of GPAGLYAQ (SEQ ID NO: 195) do not include amino acid substitutions at positions P1 and / or P1'.

[0072] The amino acid substitutions of functional variants of GPAGLYAQ (SEQ ID NO: 195) preferably include amino acid substitutions at positions P4 and / or P4'. For example, functional variants of GPAGLYAQ (SEQ ID NO: 195) may include a leucine at position P4, or a serine, glutamine, lysine, or phenylalanine at position P4. Alternatively or additionally, functional variants of GPAGLYAQ (SEQ ID NO: 195) may include a glycine, phenylalanine, or proline at position P4'.

[0073] In some embodiments, amino acid substitutions at positions P2 or P2' of GPAGLYAQ (SEQ ID NO: 195) are not preferred.

[0074] In some embodiments, a functional variant of GPAGLYAQ (SEQ ID NO: 195) comprises an amino acid sequence selected from SEQ ID NOs: 258 to 331. Specific functional variants of GPAGLYAQ (SEQ ID NO: 195) include GPLGLYAQ (SEQ ID NO: 295) and GPAGLKGA (SEQ ID NO: 285).

[0075] Functional variants of LFKSSFP (SEQ ID NO: 198) preferably contain hydrophobic amino acid substitutions. Functional variants of LFKSSFP (SEQ ID NO: 198) may preferably contain one or more of the following: (a) lysine, histidine, serine, glutamine, leucine, proline, or phenylalanine at position P4; (b) lysine, histidine, glycine, proline, asparagine, or phenylalanine at position P3; (c) arginine, leucine, alanine, glutamine, or histatine at position P2; (d) phenylalanine, histidine, threonine, alanine, or glutamine at position P1; (e) phenylalanine, histidine, threonine, alanine, or glutamine at position P1 (f) histidine, leucine, lysine, alanine, isoleucine, arginine, phenylalanine, asparagine, glutamic acid, or glycine at position P2'; (g) phenylalanine, leucine, isoleucine, lysine, alanine, glutamine, or proline at position P3'; and phenylalanine, histidine, glycine, alanine, serine, valine, glutamine, lysine, or leucine.

[0076] Inclusion of aspartic acid and / or glutamic acid in functional variants of SEQ ID NO: 198 is generally disfavored and avoided. The following amino acid substitutions are also disfavored in functional variants of LFKSSFP (SEQ ID NO: 198): (a) alanine, serine, or glutamic acid at position P3; (b) proline, threonine, glycine, or aspartic acid at position P2; (c) proline at position P1; (d) proline at position P1'; (e) glycine at position P2'; (f) lysine or glutamic acid at position P3', (g) aspartic acid at position P4'.

[0077] Amino acid substitutions in functional variants of LFKSSFP (SEQ ID NO: 198) preferably include amino acid substitutions at positions P4 and / or P1. In some embodiments, amino acid substitutions at position P4' in functional variants of LFKSSFP (SEQ ID NO: 198) are not preferred.

[0078] In some embodiments, a functional variant of LFKSSFP (SEQ ID NO: 198) comprises an amino acid sequence selected from SEQ ID NOs: 332 to 408. Specific functional variants of LFKSSFP (SEQ ID NO: 198) include ALFFSSPP (SEQ ID NO: 199), ALFKSFPP (SEQ ID NO: 381), ALFKSLPP (SEQ ID NO: 382), ALFKHSPP (SEQ ID NO: 370), ALFKSIPP (SEQ ID NO: 383), ALFKSSLP (SEQ ID NO: 390), or SPFRSSRQ (SEQ ID NO: 333).

[0079] The separating moieties disclosed herein can form stable complexes under physiological conditions with the amino acid sequence (e.g., domain) to which they are attached, while still being cleavable by proteases. For example, the separating moieties are stable in circulation (e.g., not cleaved or cleaved with low efficiency) and are cleaved with high efficiency at the target site (i.e., tumor microenvironment). Thus, fusion polypeptides comprising the linkers disclosed herein can, if desired, have an extended circulating half-life and / or reduced biological activity in circulation compared to the components of the fusion polypeptide as separate molecular entities. Furthermore, when in the desired location (e.g., tumor microenvironment), the linker can be efficiently cleaved to release the components linked by the linker, restoring or nearly restoring the half-life and biological activity of the components as separate molecular entities.

[0080] The isolated portion desirably remains stable in the circulation for at least 2 hours, at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 24 hours, at least 30 hours, at least 35 hours, at least 40 hours, at least 45 hours, at least 50 hours, at least 60 hours, at least 65 hours, at least 70 hours, at least 80 hours, at least 90 hours, or more.

[0081] In some embodiments, the separation moiety is cleaved in circulation at less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 20%, 5%, or 1% relative to the target location. The separation moiety is also stable in the absence of an enzyme capable of cleaving the linker. However, upon exposure to a suitable enzyme (i.e., a protease), the separation moiety is cleaved, resulting in separation of the linked domains.

[0082] B. Polypeptides and Compositions Comprising Isolated Moieties The separation moieties disclosed herein can be used in a wide range of applications. They are also suitable for, but not limited to, fusion proteins. As further described herein, separation moieties are particularly useful for preparing therapeutic fusion proteins in which the therapeutic biological activity of the fusion protein is attenuated and eliminated by cleavage of the separation moiety. Separation moieties can also be used to conjugate various payloads, such as therapeutic and / or diagnostic agents, to carriers or targeting agents (e.g., antibodies and antibody fragments, nanoparticles). Suitable methods for preparing such conjugates are well known in the art; see, for example, Bioconjugate Techniques, Third Ed., G.T. Hermanson (Ed.) Academic Press 2013. Exemplary payloads include, but are not limited to, cytokines, antibodies, cell-based therapeutics, antibiotics, cytotoxic drugs, or other recombinant polypeptide conjugates. Of particular interest are separation moieties suitable for use with payloads that target or are targeted to the tumor microenvironment.

[0083] The present disclosure relates to recombinant polypeptides, wherein a separating moiety as disclosed herein links a first amino acid sequence of interest (e.g., a first domain of interest) to a second amino acid sequence of interest (e.g., a second domain of interest). Typically, the first amino acid sequence of interest and the second amino acid sequence of interest are not found together in a naturally occurring protein. Preferred linkers are SEQ ID NOS: 195-220. In embodiments, at least one of the first amino acid sequence of interest and the second amino acid sequence of interest is the amino acid sequence of a therapeutic polypeptide. In some embodiments where at least one of the first amino acid sequence of interest and the second amino acid sequence of interest is the amino acid sequence of a therapeutic polypeptide, the other amino acid sequence of interest can be the amino acid sequence of a targeting polypeptide, a half-life extending polypeptide, and / or a blocking polypeptide.

[0084] A polypeptide comprising a detachment moiety can be represented by Formula I: [D1]-[L1]-[D2], where D1 is a first amino acid sequence of interest (e.g., a domain of interest), L1 is a detachment moiety that connects or joins D1 to D2, and D2 is a second amino acid sequence of interest (e.g., a second domain of interest). Preferably, L1 is a protease-cleavable detachment moiety, and more preferably, L1 comprises or consists of any of SEQ ID NOs: 195-220.

[0085] The polypeptide can also be represented by Formula II: [D1]-[L1]-[D2]-[L2]-[D3], where D1 is a first amino acid of interest (e.g., a domain of interest), L1 and L2 are each independently a linker, D2 is a second amino acid sequence of interest (e.g., a domain of interest), D3 is a third amino acid of interest (e.g., a domain of interest), and at least one of L1 and L2 is a protease-cleavable detachable moiety, preferably comprising or consisting of any of SEQ ID NOs: 195-220.

[0086] The polypeptide can also be represented by Formula III: [D1]-[L1]-[D2]-[L2]-[D3]-[L3]-[D4], where D1 is a first amino acid of interest (e.g., a domain of interest), L1, L2, and L3 are each independently a linker, D2 is a second amino acid sequence of interest (e.g., a domain of interest), D3 is a third amino acid of interest (e.g., a domain of interest), D4 is a fourth amino acid of interest (e.g., a domain of interest), and at least one of L1, L2, and L3 is a protease-cleavable detachable moiety, preferably comprising or consisting of any of SEQ ID NOs: 195-220.

[0087] Further specific uses of the separation moiety are described in further detail herein.

[0088] i. Payload Delivery The separate moieties described herein can be used to attach therapeutic drug moieties. In this approach, a therapeutic drug moiety is attached to the separate moiety to create a therapeutic drug moiety conjugate. The individual drug moiety conjugate can be a prodrug that is inactive until a target protease cleaves the prodrug, releasing the free drug.

[0089] ii. Antibody-drug conjugates Another example of the use of detached moieties is in the field of antibody-drug conjugates (ADCs), primarily targeted at the treatment of cancer. ADCs are typically antibodies linked to a cytotoxic moiety (e.g., a cytotoxic drug). ADCs distinguish between healthy and diseased cells and provide targeted delivery of drugs (e.g., a cytotoxic drug) to diseased cells. ADCs typically contain an antibody targeting a tumor marker specific to tumor cells. When the antibody attaches itself to the tumor cells, the ADC is internalized, releasing the cytotoxic moiety to kill the tumor cells. An important aspect of ADCs is providing a stable linker between the antibody component and the cytotoxic drug. In such applications, the linker can be cleavable or non-cleavable. In the case of a non-cleavable linker, the antibody, linker, and cytotoxic unit are internalized within the tumor cells. The nature of the linker typically determines the release profile of the cytotoxic drug. For example, a cleavable linker between the antibody and the cytotoxic agent is typically catalyzed by an enzyme within the tumor cell or tumor microenvironment, where the antibody and cytotoxic agent are cleaved to release the cytotoxic agent.

[0090] In certain embodiments, the separation moieties disclosed herein bind or connect the drug moiety to the antibody moiety.

[0091] iii. Peptide-drug conjugates The detachment moieties disclosed herein are suitable for use in peptide-drug conjugates. Such compounds typically include a cytotoxic payload and a linker, but peptide-drug conjugates are equipped with a peptide capable of penetrating tumors instead of an antibody, allowing for intratumoral delivery of the cargo. In some embodiments, the detachment moiety binds or connects the cytotoxic payload to the peptide. The peptide-drug conjugate remains stable and has no biological activity until a target protease cleaves the detachment moiety.

[0092] iv. Inducible adoptive cell therapy The isolated moieties disclosed herein are suitable for use in constructs designed for use in adoptive cell transfer (ACT) therapy. Currently, the field of adoptive cell transfer (ACT) consists of chimeric antigen receptor (CAR) engineered T cells (and next generation therapies) that target T cells to cell surface-expressed targets (e.g., tumor cells that express surface targets), and T cell receptor (TCR) engineered T cells that can target intracellular antigens.

[0093] In one embodiment, a separation moiety is used to tether the targeting moiety to the CAR construct. CARs replace the endogenous TCR complex with a new receptor that uses a fragment of a human or mouse antibody to bind to a target outside the cancer cell. The antibody fragment is linked to various signaling proteins in the T cell that mediate receptor activation when the CAR binds to its target. Porter et al., (2011) NEJM, 365:725-733; Grupp et al., (2013) NEJM, 368:1509-1518; US10221245 / WO / 2014 / 153270, Treatment of cancer using humanized anti-CD19 chimeric antigen receptor.

[0094] In another embodiment, a separation moiety is used to tether the targeting moiety to the TCR construct. TCRs are based on genes for protein receptors that already naturally exist in T cells. The gene for a desired TCR can be discovered within a single patient, for example, a patient that can mount an effective immune response against a certain type of cancer. This gene can then be incorporated into a TCR T cell construct and introduced into other patients, or re-engineered to improve binding interactions with MHC targets. Guy et al., (2013) Nat Immunol., 14(3):262-70; Kuhns et al., (2012) Front Immunol., 25;3:159; Fesnak et al., (2016) Nat Rev Cancer, 16(9):566-581.

[0095] These types of engineered T cells, whether autologous or allogeneic, contain an engineered T cell receptor component that includes a targeting agent, such as an isolated human or humanized antibody. In CAR-T cells and TCR-T cells, the binding affinity of the targeting moiety can be influenced by the steric, chemical, or flexibility properties of the detachment moiety that tethers the targeting moiety to the construct and the rest of the T cell. The detachment moieties disclosed herein are suitable for use with engineered constructs to generate CAR T cells and TCR T cells.

[0096] v. antigen-binding protein The isolating moieties disclosed herein are suitable for use in antigen-binding proteins. An "antigen-binding protein" (ABP) is a protein containing one or more antigen-binding domains that specifically bind to an antigen or epitope. In some embodiments, the antigen-binding domain binds to an antigen or epitope with similar specificity and affinity as a natural antibody. Typically, the isolating moiety binds a polypeptide that blocks the antigen-binding site of the ABP from binding to its cognate antigen. However, upon cleavage of the isolating moiety, the blocking polypeptide diffuses away from the ABP antigen-binding site, allowing the ABP to bind to its cognate antigen. Exemplary binding polypeptides that can block the antigen-binding site of an ABP include steric blockers (e.g., human serum albumin) and peptides that interact with one or more complementarity-determining regions (CDRs) within the antigen-binding site of the ABP. Such blocking peptides can be obtained by screening libraries or by screening peptide fragments of the cognate antigen of the ABP of interest. Typically, when the ABP comprises an antibody antigen-binding site, the separating moiety and blocker are attached to the amino terminus of the antibody light chain or the amino terminus of the antibody heavy chain, such that the blocker is tethered near the antigen-binding site and readily blocks the antigen-binding site. Suitable variations of this approach are used when the ABP comprises an alternative scaffold for the binding site. Similarly, when a single-chain antibody binding site (e.g., an scFV of a dAb) is used, the blocker separating moiety is typically attached amino-terminally near the antigen-binding site. In certain embodiments, the ABP comprises an antibody binding site comprising a VH and a VL, and the blocker separating moiety is attached amino-terminally to the VL.

[0097] The ABP may be an antibody (e.g., the first and second antigen-binding domains take the form of an antibody). Preferably, at least one antigen-binding domain of the ABP takes the form of an antibody. In another preferred embodiment, the first or second antigen-binding domain takes the form of an antibody, and the first or second antigen-binding domain takes the form of an antigen-binding fragment (e.g., the first antigen-binding domain is an antibody and the second antigen-binding domain is an antigen-binding fragment. Alternatively, the first antigen-binding domain is an antigen-binding fragment and the second antigen-binding domain is an antibody).

[0098] In some embodiments, the ABP comprises an antibody. In some embodiments, the ABP consists essentially of an antibody. In some embodiments, the ABP comprises an alternative scaffold. In some embodiments, the ABP consists essentially of an alternative scaffold. In some embodiments, the ABP comprises an antibody fragment. In some embodiments, the ABP consists essentially of an antibody fragment. In some embodiments, the ABP consists essentially of an antibody fragment.

[0099] In some embodiments, the isolated moieties disclosed herein are suitable for use with antibodies. The term "antibody" is used in the broadest sense herein and includes a specific type of immunoglobulin molecule that contains one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies specifically include intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multispecific antibodies. An antibody is one type of ABP.

[0100] In some embodiments, the isolated moieties disclosed herein are suitable for use with antigen binding proteins comprising alternative scaffolds. "Alternative scaffolds" refers to molecules in which one or more regions have been diversified to produce one or more antigen binding domains that specifically bind to an antigen or epitope.

[0101] In some embodiments, the antigen-binding domain binds to an antigen or epitope with similar specificity and affinity as an antibody. Exemplary alternative scaffolds include fibronectin (e.g., Adnectin™), beta-sandwich (e.g., iMab), lipocalin (e.g., Anticalin®), EETI-II / AGRP, BPTI / LACI-D1 / ITI-D2 (e.g., Kunitz domain), thioredoxin peptide aptamer, Protein A (e.g., Affibody®), ankyrin repeat (e.g., DARPin), gamma-B-crystallin / ubiquitin (e.g., Affilin), CTLD3 (e.g., Tetranectin), finomer, and LDLR-A module (e.g., Avimer). Further information regarding alternative scaffolds is provided in Binz et al., Nat. Biotechnol., 2005 23:1257-1268; Skerra, Current Opin. in Biotech., 2007 18:295-304; and Silacci et al., J. Biol. Chem., 2014, 289:14392-14398, each of which is incorporated by reference in its entirety. An alternative scaffold is a type of ABP.

[0102] In some embodiments, the isolated moieties disclosed herein are suitable for use with antibody fragments. An "antibody fragment" comprises a portion of an intact antibody, such as the antigen-binding or variable region of the intact antibody. Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv (sFv) fragments, and scFv-Fc fragments.

[0103] In some embodiments, the isolated portions disclosed herein are suitable for use with one or more Fv, Fab, or F(ab')2 fragments. An "Fv" fragment comprises a noncovalently bound dimer of one heavy chain variable domain and one light chain variable domain. A "Fab" fragment comprises the heavy and light chain variable domains as well as the light chain constant domain and the first heavy chain constant domain (CH1). Fab fragments can be produced, for example, by recombinant methods or by papain digestion of a full-length antibody. An "F(ab')2" fragment comprises two Fab' fragments linked near the hinge region by a disulfide bond. F(ab')2 fragments can be produced, for example, by recombinant methods or by pepsin digestion of an intact antibody. F(ab')2 fragments can be dissociated, for example, by treatment with 1-mercaptoethanol.

[0104] In some embodiments, the separated moieties disclosed herein are suitable for use with scFv or scFv-Fc. "Single-chain Fv" or "sFv" or "scFv" antibody fragments comprise a VH domain and a VL domain in a single polypeptide chain. The VH and VL are generally connected by a peptide linker. See Pluckthun A. (1994). Any suitable linker can be used.

[0105] In some embodiments, the linker is (GGGGS)n (SEQ ID NO: 231). In some embodiments, n=1, 2, 3, 4, 5, or 6. See Antibodies from Escherichia coli. In Rosenberg M. & Moore GP (Eds.), The Pharmacology of Monoclonal Antibodies vol. 113 (pp. 269-315). Springer-Verlag, New York (incorporated by reference in its entirety). An "scFv-Fc" fragment comprises an scFv attached to an Fc domain. For example, the Fc domain may be attached to the C-terminus of the scFv. The Fc domain follows VH or VL, depending on the orientation of the variable domains of the scFv (i.e., VH-VL or VL-VH). Any suitable Fc domain known in the art or described herein can be used. In some cases, the Fc domain comprises an IgG4 Fc domain.

[0106] In some embodiments, the isolated moieties disclosed herein are suitable for use with single-domain antibodies. The term "single-domain antibody" refers to a molecule in which one variable domain of an antibody specifically binds to an antigen without the presence of another variable domain. Single-domain antibodies and fragments thereof are described in Arabi Ghahroudi et al., FEBS Letters, 1998, 414:521-526 and Muyldermans et al., Trends in Biochem. Sci., 2001, 26:230-245 (each of which is incorporated herein by reference in its entirety). Single-domain antibodies are also known as sdAbs or nanobodies.

[0107] In some embodiments, the separating moieties disclosed herein are suitable for use with monospecific ABPs. A "monospecific ABP" is an ABP that contains one or more binding sites that specifically bind to the same epitope. An example of a monospecific ABP is a natural IgG molecule. This molecule is bivalent (having two antigen-binding domains), but each of the two antigen-binding domains recognizes the same epitope. The binding specificity can be present in any suitable valency.

[0108] In some embodiments, the isolated moieties disclosed herein are suitable for use with monoclonal antibodies. The term "monoclonal antibody" refers to an antibody from a substantially homogeneous population of antibodies. A substantially homogeneous population of antibodies contains antibodies that are substantially similar and bind to the same epitope(s), except for variants that may normally arise during monoclonal antibody production. Such variants are generally present in small amounts. Monoclonal antibodies are typically obtained by a process that includes selecting a single antibody from a plurality of antibodies. For example, the selection process can be the selection of a unique clone from a plurality of clones (e.g., a pool of hybridoma clones, phage clones, or recombinant DNA clones). The selected antibody can be further modified, for example, to improve affinity for a TNFR superfamily member protein ("affinity maturation"), humanize the antibody, improve yield in cell culture, and / or reduce immunogenicity in a subject.

[0109] In some embodiments, the isolated moieties disclosed herein are suitable for use with chimeric antibodies. The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0110] In some embodiments, the isolated moieties disclosed herein are suitable for use with humanized antibodies. "Humanized" forms of non-human antibodies are chimeric antibodies containing minimal sequence derived from the non-human antibody. Humanized antibodies are generally human antibodies (recipient antibodies) in which residues from one or more CDRs have been replaced with residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody (e.g., mouse, rat, rabbit, chicken, or non-human primate antibody) with the desired specificity, affinity, or biological effect. In some cases, selected framework region residues of the recipient antibody are replaced with corresponding framework region residues from the donor antibody. Humanized antibodies may also contain residues not found in either the recipient antibody or the donor antibody. Such modifications may be made to further refine antibody function. Further details are described, for example, in Jones et al., Nature, 1986:321-522(525); Riechmann et al., Nature, 1988, 332:323(329); and Presta, Curr. Op. Struct. Biol., 1992, 2:593-596 (each of which is incorporated herein by reference in its entirety).

[0111] In some embodiments, the isolated moieties disclosed herein are suitable for use with human antibodies. A "human antibody" refers to one having an amino acid sequence corresponding to an antibody produced by a human or human cell, or one derived from a non-human source that utilizes the human antibody repertoire or human antibody coding sequences (e.g., obtained from a human source or designed de novo). Human antibodies specifically exclude humanized antibodies.

[0112] In some embodiments, the ABP provided herein specifically binds to the extracellular domain of a TNFR superfamily protein. In some embodiments, the TNFR superfamily protein is CD27, CD137, CD40, GITR, LT-betaR, CD30, HVEM, TNFR1, TNFR2, or OX-40. The TNFR superfamily protein can be expressed on the surface of any suitable target cell. In some embodiments, the target cell is a T cell. In some embodiments, the target cell is an effector T cell. In some embodiments, the target cell is a regulatory T cell. In some embodiments, the target cell is a natural killer (NK) cell. In some embodiments, the target cell is a natural killer T (NKT) cell. In some embodiments, the target cell is a B cell. In some embodiments, the target cell is a bone marrow-derived cell. In some embodiments, the target cell is a myeloid-derived suppressor cell. In some embodiments, the target cell is a dendritic cell.

[0113] In some embodiments, the ABPs provided herein are antibodies. In some embodiments, the ABPs provided herein are antibody fragments. In some embodiments, the ABPs provided herein are alternative scaffolds.

[0114] In some embodiments, the ABPs provided herein comprise immunoglobulin molecules. In some embodiments, the ABPs provided herein consist of immunoglobulin molecules. In some embodiments, the ABPs provided herein consist essentially of immunoglobulin molecules. In some embodiments, the immunoglobulin molecules comprise antibodies. In some embodiments, the immunoglobulin molecules consist of antibodies. In some embodiments, the immunoglobulin molecules consist essentially of antibodies.

[0115] In some embodiments, the ABP provided herein comprises a light chain. In some embodiments, the light chain is a kappa light chain. In some embodiments, the light chain is a lambda light chain.

[0116] In some embodiments, the ABP provided herein comprises a heavy chain. In some embodiments, the heavy chain is IgA. In some embodiments, the heavy chain is IgD. In some embodiments, the heavy chain is IgE. In some embodiments, the heavy chain is IgG. In some embodiments, the heavy chain is IgM. In some embodiments, the heavy chain is IgG1. In some embodiments, the heavy chain is IgG2. In some embodiments, the heavy chain is IgG3. In some embodiments, the heavy chain is IgG4. In some embodiments, the heavy chain is IgA1. In some embodiments, the heavy chain is IgA2.

[0117] In some embodiments, the separation moiety is connected to an additional blocking moiety.

[0118] In some embodiments, the separating moiety is positioned such that movement of the antigen-binding protein subunit is restricted.

[0119] a. Multispecific and monospecific multivalent antigen-binding proteins In some embodiments, the separating moieties disclosed herein are suitable for use with multispecific antigen-binding proteins (ABPs). The multispecific ABPs provided herein bind to multiple antigens. For example, a multispecific antibody can bind to two, three, four, five, or more antigens. Alternatively, the multispecific ABP can bind to two or more different epitopes. When an ABP binds to two or more epitopes, the two or more different epitopes may be epitopes on the same antigen (e.g., a single TNFR superfamily protein molecule expressed by a single cell) or on different antigens (e.g., different TNFR superfamily protein molecules expressed by the same cell, or a TNFR superfamily protein molecule and a non-TNFR superfamily protein molecule). In some embodiments, the multispecific ABP binds to two different epitopes (i.e., a "bispecific ABP"). In some embodiments, the multispecific ABP binds to three different epitopes (i.e., a "trispecific ABP"). In some embodiments, the multispecific ABP binds four different epitopes (i.e., a "tetraspecific ABP"). In some embodiments, the multispecific ABP binds five different epitopes (i.e., a "pentaspecific ABP"). In some embodiments, the multispecific ABP binds six, seven, eight, or more different epitopes. Each binding specificity can be present in any suitable valency.

[0120] In various embodiments, the antigen-binding protein comprises a blocking domain. The separating moieties disclosed herein bind the blocking domain to the first antigen-binding domain such that the blocking domain can inhibit (a) the binding affinity or avidity of the antigen-binding protein for the epitope and / or (b) the agonist or antagonist activity of the antigen-binding protein for the epitope. Preferably, the separating moiety comprises a cleavage site. Cleavage of the separating moiety by a disease-specific enzyme (i.e., a protease) increases (a) the binding affinity or avidity of the antigen-binding protein for the epitope and / or (b) the agonist or antagonist activity of the antigen-binding protein for the epitope. In some embodiments, the antigen-binding protein comprises an additional separating moiety that binds or connects the additional blocking domain to the antigen-binding domain. The additional separating moiety comprises a cleavage site.

[0121] In various embodiments, the antigen binding protein comprises an additional blocking domain (e.g., a second, third, or fourth blocking domain) operably linked to the antigen binding domain (e.g., a second, third, or fourth antigen binding domain) by a cleavable separation moiety as disclosed herein. The blocking domain may be a steric blocker or a specific blocker. In some embodiments, the steric blocker is independently selected from the group consisting of a fragment of the extracellular portion of an antibody binding protein, serum albumin, a fragment of serum albumin, and an antibody or antigen-binding fragment thereof that binds serum albumin. In some embodiments, the specific blocker is independently selected from an antibody or antigen-binding fragment thereof that binds to the first, second, third, or fourth antigen-binding domain of an antigen binding protein described herein. Preferably, the blocking domain is serum albumin or a fragment thereof, or an antibody or antigen-binding fragment thereof that binds serum albumin.

[0122] The antigen-binding domain may further comprise a third antigen-binding domain and a fourth antigen-binding domain, each having binding specificity for a target antigen. TNFR superfamily target antigens are well known in the art and are encompassed by the present disclosure. Exemplary TNFR superfamily members include, but are not limited to, CD27, CD30, CD137 (4-1BB), TNFR1 (CD120a), TNFR2 (CD120b), CD40, CD95 (Fas / Apo-1), HVEM, LT-betaR, GITR, nerve growth factor receptor, or OX-40 (CD34). Preferred TNFR superfamily target antigens are CD27, CD137, and OX40.

[0123] Antigen-binding domains specific for an antigen may have binding specificity for the same epitope on the same antigen or for different epitopes on the same antigen. For example, the first, second, and third antigen-binding domains may have binding specificity for the same epitope or for different epitopes, or two of the antigen-binding domains may have binding specificity for the same epitope or three of the antigen-binding domains may have binding specificity for the same epitope. In some embodiments, the antigen-binding protein may further comprise a fifth antigen-binding domain specific for a tumor antigen.

[0124] In some embodiments, the antigen-binding polypeptide comprises an Fc region. In one format, two antigen-binding domains are located at both ends of the Fc region. In another format, two antibody arms are attached to the N-terminus of the Fc region, each arm comprising two antigen-binding domains. The antigen-binding domain may also comprise a half-life prolonging domain. The half-life prolonging domain may be albumin, an antigen-binding domain that recruits albumin, or an immunoglobulin Fc or a fragment thereof. In some embodiments, the half-life prolonging domain is operably linked to the antigen-binding polypeptide by a cleavable linker.

[0125] The present disclosure also relates to antigen-binding proteins comprising at least a first polypeptide and a second polypeptide. The first polypeptide comprises an antibody heavy chain constant region and at least a portion of an antibody heavy chain variable region (VH). The second polypeptide comprises an antibody light chain constant region and at least a portion of an antibody light chain variable region (VL). At least one of the first and second polypeptides further comprises a blocking domain operably linked to the VH or VL via a protease-cleavable linker. The first polypeptide associates with the second polypeptide, and the VH and VL form an antigen-binding site having binding specificity for a target antigen (e.g., 4-1BB or CS27). The blocking domain inhibits the antigen-binding site from binding to the target antigen (e.g., 4-1BB or CD27). In some embodiments, the first polypeptide further comprises a second VH, and the first polypeptide associates with two of the second polypeptides to form two VH / VL antigen-binding sites, each having specificity for human 4-1BB. In some embodiments, the antigen binding protein is a dimer of a first polypeptide and an associated second polypeptide. The antigen binding protein may further comprise a third, fourth, fifth, or sixth polypeptide.

[0126] In some embodiments, the ABP is a monospecific multivalent ABP. Such formats can have a variety of structures and can be prepared using suitable antibody engineering techniques. For example, a double-Fab antibody can be prepared containing a heavy chain with the structure VH-CH1-non-cleavable liner-VH-CH1-CH2-CH3. Such a heavy chain can be expressed and paired with two light chains. The heavy chains can dimerize via conventional interchain disulfide bonds to form an antibody format containing four Fab antigen-binding sites. In such formats, a separating moiety as described herein can be attached to the amino terminus of the light chain polypeptide, allowing a blocker to be attached to each of the Fab antigen-binding sites. Other suitable monospecific multivalent ABP formats can be readily envisioned by those skilled in the art. In one such example, a heavy chain with the structure VH-CH1-CH2-CH3-VH-CH1 is prepared. Two such heavy chains dimerize and associate with four light chains to form a monospecific tetravalent ABP format. In such a format, a separation moiety as described herein can be attached to the amino terminus of the light chain polypeptide, allowing a blocker to be attached to each of the Fab antigen-binding sites. The binding activity of such a monospecific tetravalent ABP is masked by the blocking domain, and this masking is removed upon cleavage of the separation domain (e.g., in the tumor microenvironment), allowing the ABP to bind to its cognate antigen.

[0127] The monospecific multivalent ABP format can have binding specificity for any desired antigen. In some embodiments, the monospecific multivalent ABP format specifically binds to the extracellular domain of a TNFR superfamily protein (e.g., CD27, CD30, CD137 (4-1BB), TNFR1 (CD120a), TNFR2 (CD120b), CD40, CD95 (Fas / Apo-1), HVEM, LT-betaR, GITR, nerve growth factor receptor, or OX-40 (CD34)).

[0128] In embodiments, a multivalent antigen-binding protein may comprise a first antigen-binding site having specificity for a target antigen (e.g., CD27 or TNFR1), a second antigen-binding site having specificity for the target antigen, a blocking polypeptide, at least one protease-cleavable linker, and an optional half-life extending element. In such embodiments, the first blocking polypeptide is operably linked to the first antigen-binding site by a protease-cleavable linker, and optionally the second blocking polypeptide is operably linked to the second antigen-binding site by a protease-cleavable linker. Preferably, the blocking polypeptide is operably linked to the second antigen-binding site by a protease-cleavable linker. The blocking polypeptide inhibits (a) the binding affinity or avidity of the antigen binding protein for the target antigen and / or (b) the agonist or antagonist activity of the antigen binding protein for the target antigen, and cleavage of the protease-cleavable linker increases (a) the binding affinity or avidity of the antigen binding protein for the target antigen and / or (b) the agonist activity of the antigen binding protein for the target antigen. An optional half-life extending element may be operably linked to the first antigen-binding site and / or the second antigen-binding site via an optional protease-cleavable linker. In such embodiments, the first and second binding sites may have specificity for the same epitope or different epitopes.

[0129] In embodiments, the antigen binding protein further comprises a third antigen binding site specific for the same target antigen. The antigen binding protein may further comprise a fourth antigen binding site having specificity for the same target antigen as the first, second, and third antigen binding sites. The third and fourth antigen binding sites may each further comprise a blocking polypeptide operably linked to the antigen binding site via a protease-cleavable linker.

[0130] The present disclosure also relates to tetravalent antigen-binding proteins, which may comprise a first polypeptide comprising at least a portion of an antibody heavy chain constant region and an antibody heavy chain variable region (VH) and a second polypeptide comprising at least a portion of an antibody light chain constant region and an antibody light chain variable region (VL). The first polypeptide associates with the second polypeptide, and the VH and VL form an antigen-binding site with binding specificity for a target antigen. The tetravalent antigen-binding protein further comprises a blocking polypeptide operably linked to the VH or VL via a protease-cleavable linker. The blocking polypeptide inhibits binding of the antigen-binding site to the target antigen. In some embodiments, the first polypeptide may further comprise a second VH. The first polypeptide associates with two second polypeptides to form two VH / VL antigen-binding sites, each with specificity for a target antigen.

[0131] The tetravalent antigen-binding protein may further comprise a third polypeptide, a fourth polypeptide, a fifth polypeptide, a sixth polypeptide, a seventh polypeptide, and an eighth polypeptide. The third polypeptide may comprise an antibody heavy chain constant region and at least a portion of an antibody heavy chain variable region (VH). The fourth polypeptide may comprise an antibody light chain constant region and at least a portion of an antibody light chain variable region (VL). The fifth polypeptide may comprise an antibody heavy chain constant region and at least a portion of an antibody heavy chain variable region (VH). The sixth polypeptide may comprise an antibody light chain constant region and at least a portion of an antibody light chain variable region (VL). The seventh polypeptide may comprise an antibody heavy chain constant region and at least a portion of an antibody heavy chain variable region (VH). The eighth polypeptide may comprise an antibody light chain constant region and at least a portion of an antibody light chain variable region (VL).

[0132] In some embodiments, at least one of the third polypeptide and the fourth polypeptide further comprises a blocking polypeptide operably linked to the VH or VL via a protease-cleavable linker, wherein the third polypeptide associates with the fourth polypeptide, the VH and VL form an antigen-binding site having binding specificity for a target antigen, and the blocking domain inhibits the antigen-binding site from binding to the target antigen.

[0133] In some embodiments, the fifth and sixth polypeptides further comprise a blocking polypeptide operably linked to the VH or VL via a protease-cleavable linker, wherein the fifth polypeptide associates with the sixth polypeptide, the VH and VL form an antigen-binding site having binding specificity for a target antigen, and the blocking domain inhibits the antigen-binding site from binding to the target antigen.

[0134] In some embodiments, the seventh and eighth polypeptides further comprise a blocking polypeptide operably linked to the VH or VL via a protease-cleavable linker, wherein the seventh polypeptide associates with the eighth polypeptide, the VH and VL form an antigen-binding site having binding specificity for a target antigen, and the blocking domain inhibits the antigen-binding site from binding to the target antigen.

[0135] vi. Inducible cytokines Disclosed herein are methods and compositions for engineering and using constructs containing inducible cytokines. Cytokines are potent immune agonists and are therefore considered promising therapeutic agents in oncology. However, cytokines have a very narrow therapeutic window. Cytokines are also considered to have short serum half-lives and high potency. As a result, therapeutic administration of cytokines results in undesirable systemic effects and toxicity. This effect and toxicity is further exacerbated by the need to administer large amounts of cytokines to achieve desired levels of cytokine at the intended site of cytokine action (e.g., tumor). Unfortunately, due to the biological properties of cytokines and the inability to effectively target and control their activity, cytokines have not achieved the expected clinical benefits in tumor treatment.

[0136] Disclosed herein are fusion proteins that overcome the problems of toxicity and short half-life that have severely limited the clinical use of cytokines in oncology. The fusion proteins contain cytokine polypeptides with receptor agonist activity. However, in the context of the fusion protein, the cytokine receptor agonist activity is attenuated and the circulating half-life is extended. The fusion protein contains a protease cleavage site that is cleaved by a protease associated with the desired cytokine activity site (e.g., tumor) and is typically enriched or preferentially present at the desired activity site. In this way, the fusion protein is preferentially (or selectively) and efficiently cleaved at the desired activity site, substantially restricting cytokine activity to the desired activity site, such as the tumor microenvironment. Upon protease cleavage at the desired activity site (e.g., tumor microenvironment), a form of cytokine is released from the fusion protein that has much greater cytokine receptor agonist activity than the fusion protein (typically at least about 100-fold more active than the fusion protein). The form of cytokine released upon cleavage of the fusion protein typically has a short half-life, often substantially similar to that of the natural cytokine, further limiting cytokine activity to the tumor microenvironment.Even if the half-life of the fusion protein is extended, toxicity is dramatically reduced or eliminated because circulating fusion protein is attenuated and active cytokine is targeted to the tumor microenvironment.The fusion proteins described herein substantially limit cytokine activity to the tumor microenvironment, making it possible for the first time to administer effective therapeutic doses of cytokines for tumor treatment, dramatically reducing or eliminating the undesirable systemic effects and toxicity of cytokines.

[0137] In general, the therapeutic use of cytokines is severely limited by their systemic toxicity. For example, TNF was initially discovered for its ability to induce hemorrhagic necrosis in some tumors and its in vitro cytotoxic effects on various tumor lines. However, it has since been found to have potent pro-inflammatory activity and, under conditions of overproduction, may have dangerous effects on the human body. Because systemic toxicity is a fundamental problem when using pharmacologically active doses of cytokines in humans, novel derivatives and therapeutic strategies are currently being evaluated that aim to reduce the toxic effects of this class of biological effectors while maintaining their therapeutic efficacy.

[0138] IL-2 exerts stimulatory and regulatory functions in the immune system and, together with other members of the common gamma chain (γc) cytokine family, is central to immune homeostasis. IL-2 mediates its effects by binding to the IL-2 receptor (IL-2R). This receptor consists of either a trimeric receptor composed of the IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (γc, CD132) chains, or the dimeric βγ IL-2R (1, 3). Both IL-2R variants can transduce signals upon IL-2 binding. However, the trimeric αβγ IL-2R has approximately 10- to 100-fold higher affinity for IL-2 than the dimeric βγ IL-2R (3). This suggests that CD25, although conferring high-affinity binding of IL-2 to the receptor, is not essential for signal transduction. Trimeric IL-2R is found on activated T cells and CD4+ forkhead box P3 (FoxP3)+ T regulatory cells (Tregs), which are sensitive to IL-2 in vitro and in vivo. Conversely, antigen-experienced (memory) CD8+, CD44-high memory phenotype (MP) CD8+, natural killer (NK) cells harbor high levels of dimeric βγIL-2R, and these cells also respond vigorously to IL-2 in vitro and in vivo.

[0139] Expression of the high-affinity IL-2R is essential for T cells to respond to transiently available low concentrations of IL-2 in vivo. IL-2Rα expression is absent on naive and memory T cells but is induced after antigen activation. IL-2Rβ is constitutively expressed by NK, NKT, and memory CD8+ T cells, but is also induced on naive T cells after antigen activation. γc is much less tightly regulated and is constitutively expressed by all lymphoid cells. Upon induction of the high-affinity IL-2R by antigen, IL-2R signaling upregulates IL-2Rα expression, in part through Stat5-dependent regulation of Il2ra transcription (Kim et al., 2001). This process represents a mechanism for maintaining high-affinity IL-2R expression and IL-2 signaling while a source of IL-2 remains available.

[0140] IL-2 is captured by IL-2Rα through a large hydrophobic binding surface surrounded by a polar periphery, resulting in a relatively weak interaction (Kd 10-8 M) and rapid on-off binding kinetics. However, the IL-2Rα-IL-2 binary complex induces a very small conformational change in IL-2, which facilitates its association with IL-2Rβ through a distinct polar interaction between IL-2 and IL-2Rβ. The pseudo-high affinity of the IL2 / α / β trimeric complex (i.e., Kd ∼300 pM) clearly indicates that the trimeric complex is more stable than IL2 bound to either the α chain alone (Kd = 10 nM) or the β chain alone (Kd = 450 nM). In either case, the IL2 / α / β trimer then recruits the γ chain into a signaling-competent tetramer. This is facilitated by the large, complex binding site for the γ chain on the IL2-bound β chain.

[0141] In other words, the ternary complex of IL-2Rα-IL-2Rβ-IL-2 recruits γc through weak interactions with IL-2 and strong interactions with IL-2Rβ, generating a stable, high-affinity quaternary IL-2R (Kd 10-11 M, i.e., 10 pM). Formation of the high-affinity quaternary IL-2-IL-2R complex results in signal transduction via the tyrosine kinases Jak1 and Jak3 (associated with IL-2Rβ and γc, respectively) (Nelson and Willerford, 1998). The quaternary IL-2-IL-2R complex is rapidly internalized, and IL-2, IL-2Rβ, and γc are rapidly degraded, whereas IL-2Rα is recycled to the cell surface (Hemar et al., 1995; Yu and Malek, 2001). Thus, such functional activity, which requires sustained IL-2R signaling, requires a continuous source of IL-2 to engage with IL-2Rα and form additional IL-2-IL-2R signaling complexes.

[0142] Interleukin-15 (IL-15), another member of the four-alpha-helical bundle family of cytokines, has also emerged as an immunomodulator for cancer therapy. IL-15 is initially captured via IL-15Rα, which is expressed on antigen-presenting dendritic cells, monocytes, and macrophages. IL-15 exhibits a broad range of activities, inducing the differentiation and proliferation of T cells, B cells, and natural killer (NK) cells by signaling through the IL-15 / IL-2-R-β (CD122) and common γ chain (CD132). IL-15 also mediates the differentiation and proliferation of CD8 + It also enhances the cytolytic activity of T cells and promotes long-lasting antigen-experienced CD8 +IL-15 induces CD44 memory T cells. IL-15 promotes B cell differentiation and immunoglobulin synthesis and induces dendritic cell maturation. It does not stimulate immunosuppressive T regulatory cells (Tregs). Therefore, selectively boosting IL-15 activity in the tumor microenvironment may enhance innate and specific immunity to combat tumors (Waldmann et al., 2012). IL-15 was initially identified for its ability to stimulate T cell proliferation in a manner similar to IL-2, via a shared receptor component (IL-2R / 15Rβ-γc) and signaling through JAK1 / JAK3 and STAT3 / STAT5. Similar to IL-2, IL-15 has been shown to stimulate the proliferation of activated CD4-CD8-, CD4+CD8+, CD4+, and CD8+ T cells, as well as induce cytotoxic T lymphocytes and promote the generation, proliferation, and activation of NK cells (Waldmann et al., 1999). However, unlike IL-2, which is required for the maintenance of forkhead box P3 (FOXP3)-expressing CD4+CD25+ Treg cells and their peripheral retention, IL-15 has little effect on Tregs (Berger et al., 2009). This is important because FOXP3-expressing CD4+CD25+ Tregs inhibit effector T cells, thereby inhibiting immune responses, including those against tumors. IL-2 also plays an essential role in initiating activation-induced cell death (AICD), a process that leads to the elimination of autoreactive T cells, whereas IL-15 is an anti-apoptotic factor for T cells (Marks-Konczalik et al., 2000). Co-delivery of IL-15 and HIV peptide vaccines has been shown to overcome CD4+ T cell deficiency by promoting the longevity of antigen-specific CD8+ T cells and blocking TRAIL-mediated apoptosis (Oh et al., 2008). Furthermore, IL-15 promotes the long-term maintenance of CD8+CD44hi memory T cells (Kanegane et al., 1996).

[0143] The importance of IL-15 and IL-15Rα for the development of T cells and NK cells is- / - and IL-15 - / - This finding is further emphasized by the phenotype of these mice. Knockout mice have been demonstrated to have reduced total CD8+ T cell numbers and to lack memory CD8+ T cells, NK cells, NK / T cells, and several subsets of intestinal intraepithelial lymphocytes, indicating that IL-15 confers essential positive homeostatic functions to these cell subsets (Lodolce et al., 1996; Kennedy et al., 1998). The phenotypic similarities between these two strains of knockout mice suggest that IL-15Rα is important for maintaining physiologically appropriate IL-15 signaling.

[0144] IL-15 is trans-presented by the IL-15 receptor alpha chain to the IL-15Rβγc complex, which is displayed on the surface of T cells and natural killer (NK) cells (Han et al., 2011). The IL-15Ra chain acts as a chaperone protein, stabilizing and increasing IL-15 activity (Desbois et al., 2016). Exogenous IL-15 may have limited effect on cancer patients due to its dependency on IL-15Ra, which is frequently downregulated in cancer patients. Therefore, the fusion protein RLI, consisting of the sushi domain of IL15Ra linked to IL-15 via a linker, has been proposed as an alternative approach for IL15 therapy (Bessard et al., 2009). Administration of soluble IL-15 / IL-15Rα complexes significantly enhanced the serum half-life and bioavailability of IL-15 in vivo (Stoklasek et al., 2010).

[0145] In addition to its effects on T cells and NK cells, IL-15 also exerts several effects on other immune system components. IL-15 protects neutrophils from apoptosis, regulates phagocytosis, and stimulates the secretion of IL-8 and IL-1R antagonists. IL-15 functions by activating JAK2, p38 and ERK1 / 2 MAPKs, Syk kinase, and NF-kB transcription factors (Pelletier et al., 2002). IL-15 functions as a growth factor and apoptosis inhibitor in mast cells. In these cells, IL-15 activates the JAK2 / STAT5 pathway without requiring γc binding (Tagaya et al., 1996). IL-15 also induces the proliferation and differentiation of B lymphocytes and increases immunoglobulin secretion (Armitage et al., 1995). It also prevents Fas-mediated apoptosis and allows the induction of antibody responses partially independent of CD4 help (Demerci et al., 2004; Steel et al., 2010). Monocytes, macrophages, and dendritic cells efficiently transcribe and translate IL-15 and respond to IL-15 stimulation. Macrophages respond by increasing phagocytosis, inducing IL-8, IL-12, and MCP-1 expression, and secreting IL-6, IL-8, and TNFα (Budagian et al., 2006). Dendritic cells incubated with IL-15 show maturation with increased expression of CD83, CD86, CD40, and MHC class II, are resistant to apoptosis, and exhibit enhanced interferon-γ secretion (Anguille et al., 2009).

[0146] IL-15 has also been shown to affect non-blood cells, including myocytes, adipocytes, endothelial cells, and neurons. IL-15 exerts anabolic effects on muscle and can direct muscle cell differentiation (Quinn et al., 1995). IL-15 stimulates muscle cells and muscle fibers to accumulate contractile proteins and can delay muscle wasting in rats with cancer-associated cachexia (Figueras et al., 2004). IL-15 has also been shown to stimulate angiogenesis (Angiolillo et al., 1997) and induce the growth and survival of microglia (Hanisch et al., 1997).

[0147] Interleukin-7 (IL-7), a member of the IL-2 / IL-15 family, is a well-characterized pleiotropic cytokine expressed by stromal cells, epithelial cells, endothelial cells, fibroblasts, smooth muscle cells, and keratinocytes, and also by dendritic cells after activation (Alpdogan et al., 2005). IL-7 was initially described as a growth and differentiation factor for precursor B lymphocytes, but subsequent studies have shown that it is critically involved in the development and differentiation of T lymphocytes. IL-7 signaling is essential for optimal CD8 T cell function, homeostasis, and memory establishment (Schluns et al., 2000), is required for the survival of most T cell subsets, and its expression has been proposed to be important for regulating T cell numbers.

[0148] IL-7 is expressed by IL-7Rα and γ cIL-7Rα binds to a dimeric receptor containing IL-7Rα, forming a ternary complex that plays a fundamental role in extracellular matrix remodeling and T and B cell development and homeostasis (Mazzucchelli and Durum, 2007). IL-7Rα also cross-reacts with thymic stromal lymphopoietin (TSLP) and its receptor (TSLPR) to form a ternary complex, activating the TSLP pathway, resulting in the proliferation of T cells and dendritic cells in humans and the further development of B cells in mice (Leonard, 2002). Therefore, tight regulation of the signaling cascade activated by this complex is essential for normal cellular function. Insufficient stimulation of the IL-7 pathway, caused by mutations in the IL-7Rα ectodomain, inhibits T and B cell development, resulting in patients with severe combined immunodeficiency (SCID) (Giliani et al., 2005; Puel et al., 1998).

[0149] IL-7 may play a role in enhancing immune reconstitution in cancer patients after cytotoxic chemotherapy. IL-7 therapy can enhance immune reconstitution and augment even limited thymic function by promoting the peripheral expansion of even a small number of thymic emissive cells. Therefore, IL-7 therapy has the potential to restore the immune system of patients depleted by cytotoxic chemotherapy (Capitini et al., 2010).

[0150] Interleukin-12 (IL-12) is a disulfide-linked heterodimer of two separately encoded subunits (p35 and p40), which covalently link to form the so-called biologically active heterodimeric (p70) molecule (Lieschke et al., 1997; Jana et al., 2014). In addition to the formation of heterodimers (IL-12 and IL-23), the p40 subunit is also secreted as a monomer (p40) and a homodimer (p402). It is known in the art that the full biological activity of the heterodimer is maintained when the heterodimer is synthesized as a single chain with a linker connecting the p35 and p40 subunits. IL-12 plays an essential role in the early inflammatory response to infection and in the generation of Th1 cells (which favor cell-mediated immunity). IL-12 overproduction is known to be dangerous to the host, as it is involved in the pathogenesis of several autoimmune inflammatory diseases (e.g., MS, arthritis, type 1 diabetes).

[0151] The IL-12 receptor (IL-12R) is a heterodimeric complex consisting of the IL-12Rβ1 and IL-12Rβ2 chains expressed on the surface of activated T cells and natural killer cells (Trinchieri et al., 2003). The IL-12Rβ1 chain binds to the IL-12p40 subunit, while IL-12p35 associates with IL-12Rβ2 to confer intracellular signaling capabilities (Benson et al., 2011). Signaling through IL-12R induces the phosphorylation of Janus kinase (Jak2) and tyrosine kinase (Tyk2), which phosphorylate and activate signal transducer and activator of transcription (STAT) 1, STAT3, STAT4, and STAT5. The specific cellular effects of IL-12 are primarily due to the activation of STAT4. IL-12 induces natural killer cells and T cells to produce cytokines, specifically interferon (IFN)γ, which mediates many of its pro-inflammatory activities, including the differentiation of CD4+ T cells toward a Th1 phenotype (Montepaone et al., 2014).

[0152] Treg cells actively suppress immune system activation, preventing pathological self-reactivity and resulting autoimmune disease. The development of drugs and methods to selectively activate regulatory T cells for the treatment of autoimmune disease has been the subject of intense research, largely unsuccessful until the development of the present invention, which can selectively deliver active interleukins to sites of inflammation. Tregs are a class of CD4+CD25+ T cells that suppress the activity of other immune cells. Tregs are central to immune system homeostasis, playing a major role in maintaining tolerance to self-antigens and regulating immune responses to foreign antigens. Several autoimmune and inflammatory diseases, including type 1 diabetes (T1D), systemic lupus erythematosus (SLE), and graft-versus-host disease (GVHD), have been shown to be deficient in Treg cell numbers and function.

[0153] Therefore, there is considerable interest in developing therapies to boost the number and / or function of Treg cells. One therapeutic approach being investigated for autoimmune diseases is the transplantation of ex vivo expanded autologous Treg cells (Tang, Q., et al., 2013, Cold Spring Harb. Perspect. Med., 3:1-15). While this approach has shown promise in animal disease models and in several early-stage human clinical trials, it requires personalized therapy using the patient's own T cells, is invasive, and is technically complex. Another approach is treatment with low-dose interleukin-2 (IL-2). Treg cells are characterized by constitutive expression of high levels of the high-affinity IL-2 receptor IL2Rαβγ, which is composed of the subunits IL2Rα (CD25), IL2Rβ (CD122), and IL2Rγ (CD132), and Treg cell development has been shown to be dependent on IL-2 (Malek, TR, et al., 2010, Immunity, 33:153-65).

[0154] Conversely, immune activation has also been achieved using IL-2, and recombinant IL-2 (Proleukin®) has been approved for the treatment of certain cancers. High-dose IL-2 is used to treat patients with metastatic melanoma and metastatic renal cell carcinoma, with long-term impact on overall survival.

[0155] Clinical trials of low-dose IL-2 treatment in patients with chronic GVHD (Koreth, J., et al., 2011, N Engl J Med., 365:2055-66) and HCV-associated autoimmune vasculitis (Saadoun, D., et al., 2011, N Engl J Med., 365:2067-77) have demonstrated increased Treg levels and signs of clinical efficacy. New clinical trials are beginning to explore the efficacy of IL-2 in several other autoimmune and inflammatory diseases. The rationale for using so-called low-dose IL-2 was to take advantage of the high IL-2 affinity of the trimeric IL-2 receptor constitutively expressed on Tregs while leaving other T cells that do not express the high-affinity receptor in an inactivated state. The recombinant form of IL-2 used in these trials, aldesleukin (sold as Proleukin® by Prometheus Laboratories, San Diego, CA), is associated with high toxicity. Aldesleukin is approved for the treatment of metastatic melanoma and metastatic renal cancer, but its side effects are so severe that it is only recommended for use in hospital settings with access to intensive care (web address: www.proleukin.com / assets / pdf / proleukin.pdf).

[0156] Clinical trials of IL-2 in autoimmune diseases have used low doses of IL-2 to target Treg cells. This is because Treg cells, by expressing IL2Ralpha, respond to lower concentrations of IL-2 than many other immune cell types (Klatzmann D, 2015 Nat Rev Immunol. 15:283-94). However, even at these low doses, safety and tolerability issues have arisen, and treatments have involved chronic daily subcutaneous infusions or intermittent 5-day treatment courses. Therefore, there is a need for autoimmune disease therapies that enhance the number and function of Treg cells, specifically target Treg cells more specifically than IL-2, and are safer, more tolerable, and require less frequent dosing.

[0157] One proposed approach to improving the therapeutic index of IL-2-based therapy is to use IL-2 variants that are selective for Treg cells over other immune cells. IL-2 receptors are expressed on a variety of different immune cell types, including T cells, NK cells, eosinophils, and monocytes, and this broad expression pattern may contribute to its pleiotropic effects on the immune system and its high systemic toxicity. Specifically, activated T effector cells, like lung epithelial cells, express IL2Rαβγ. However, activation of T effector cells directly contradicts the goal of downregulating and controlling immune responses, and activation of lung epithelial cells leads to known dose-limiting side effects of IL-2, including pulmonary edema. Indeed, the main side effect of high-dose IL-2 immunotherapy is vascular leak syndrome (VLS), which is the accumulation of intravascular fluid in organs such as the lungs and liver, subsequently causing pulmonary edema and hepatocellular injury. There is no treatment for VLS other than discontinuing IL-2. Low-dose IL-2 regimens are being tested in patients to avoid VLS, but at the cost of suboptimal treatment outcomes.

[0158] Literature suggests that VLS is caused by the release of proinflammatory cytokines from IL-2-activated NK cells. However, strong evidence suggests that pulmonary edema results from the direct binding of IL-2 to pulmonary endothelial cells that express low to moderate levels of functional αβγ IL-2R. Furthermore, the pulmonary edema associated with the interaction of IL-2 with pulmonary endothelial cells was abrogated in CD25-deficient host mice by blocking CD25 binding with an anti-CD25 monoclonal antibody (mAb) or by using a CD122-specific IL-2 / anti-IL-2 mAb (IL-2 / mAb) complex, thereby preventing VLS.

[0159] Treatments using interleukin cytokines other than IL-2 are more limited. IL-15 exhibits immune cell stimulatory activity similar to IL-2 but without the same inhibitory effects, making it a promising immunotherapy candidate. Clinical trials using recombinant human IL-15 to treat metastatic melanoma or renal cell carcinoma demonstrated significant changes in immune cell distribution, proliferation, and activation, suggesting potential antitumor activity (Conlon et al., 2014). IL-15 is currently undergoing clinical trials for the treatment of various forms of cancer. However, IL-15 therapy is known to be associated with undesirable toxic effects, such as progression of certain leukemias, graft-versus-host disease, hypotension, thrombocytopenia, and liver injury. (Mishra A.,et al.,Cancer Cell,2012,22(5):645-55;Alpdogan O.et al.,Blood,2005,105(2):866-73;Conlon KC et al.,J Clin Oncol,2015,33(1):74-82)

[0160] IL-7 promotes lymphocyte development in the thymus and maintains the homeostatic survival of naive and memory T cells in the periphery. Furthermore, IL-7 is important for lymph node (LN) organogenesis and the maintenance of activated T cells recruited to secondary lymphoid organs (SLOs) (Gao et al., 2015). In clinical trials of IL-7, patients treated with IL-7 showed increases in both CD4+ and CD8+ T cells, but the number of regulatory T cells, as monitored by FoxP3 expression, did not increase significantly (Sportes et al., 2008). In clinical trials reported in 2006, 2008, and 2010, patients with different types of cancer, such as metastatic melanoma or sarcoma, received subcutaneous injections of different doses of IL-7. Little toxicity was observed, except for transient fever and mild erythema. Circulating levels of CD4+ and CD8+ T cells were significantly increased, while the number of Tregs was reduced. TCR repertoire diversity increased after IL-7 therapy. However, the antitumor activity of IL-7 has not been fully evaluated (Gao et al., 2015). Results suggest that IL-7 therapy can enhance and broaden immune responses.

[0161] IL-12 is a pleiotropic cytokine whose actions interconnect innate and adaptive immunity. IL-12 was initially reported as a factor secreted by PMA-induced EBV-transformed B cell lines. Based on its actions, IL-12 has been called a cytotoxic lymphocyte maturation factor and natural killer cell-stimulating factor. IL-12 appears to be an ideal candidate for human tumor immunotherapy because it bridges innate and adaptive immunity and potently stimulates the production of IFNγ, a cytokine that orchestrates natural mechanisms of anti-cancer defense. However, clinical studies have shown severe side effects associated with systemic administration of IL-12, and the cytokine's very narrow therapeutic index has significantly diminished interest in its use in cancer patients (Lasek et al., 2014). Targeting IL-12 delivery to tumors may address past issues with IL-12 therapy and is currently being studied in cancer clinical trials.

[0162] The direct use of IL-2 as an agonist to bind to IL-2R and modulate immune responses is problematic due to well-documented therapeutic risks (e.g., its short half-life and high toxicity). These risks also limit the therapeutic development and use of other cytokines. New forms of cytokines that reduce these risks are needed. Disclosed herein are compositions and methods comprising IL-2 and IL-15 as well as other cytokines, functional fragments and muteins of cytokines, and conditionally active cytokines designed to address these risks and provide needed immunomodulatory therapeutics.

[0163] The present invention is designed to address the shortcomings of direct IL-2 therapy and other cytokine-based therapies, for example, by using cytokine-blocking moieties, such as steric-blocking polypeptides, serum half-life extending polypeptides, targeting polypeptides, binding polypeptides (including protease-cleavable linkers), and combinations thereof. Cytokines, such as interleukins (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23), interferons (IFNs, including IFNα, IFNβ, and IFNγ), tumor necrosis factors (e.g., TNFα, lymphotoxin), transforming growth factors (e.g., TGFβ1, TGFβ2, TGFβ3), chemokines (C-X-C motif chemokine 10 (CXCL10), CCL19, CCL20, CCL21), and granulocyte-macrophage colony-stimulating factor (GM-CS), are highly potent when administered to patients. As used herein, "chemokine" refers to a family of small molecule cytokines capable of inducing directed chemotaxis in nearby responsive cells. While cytokines can provide powerful therapeutics, their clinical use has been limited by undesirable effects that are difficult to clinically control. The present disclosure relates to novel forms of cytokines that can be used in patients with reduced or eliminated undesirable effects. Specifically, the present disclosure relates to pharmaceutical compositions comprising a chimeric polypeptide (fusion protein), a nucleic acid encoding the fusion protein, and a pharmaceutical formulation as described above that includes a cytokine or an active fragment or mutein of a cytokine that has reduced cytokine receptor activation activity compared to the corresponding cytokine. However, under selected conditions or in a selected biological environment, the chimeric polypeptide activates its cognate receptor with the same or greater potency than the corresponding native cytokine. As described herein, this is typically achieved using a cytokine-blocking moiety that blocks or inhibits the receptor activation function of the cytokine, its active fragment, or mutein under prevailing conditions, but does not block or inhibit said function under selected conditions, such as those present at the desired site of cytokine activity (e.g., a site of inflammation or a tumor).

[0164] Chimeric polypeptides and nucleic acids encoding chimeric polypeptides can be produced using any suitable method. For example, nucleic acids encoding chimeric polypeptides can be produced using recombinant DNA techniques, synthetic chemistry, or a combination of these techniques, and expressed in a suitable expression system, such as CHO cells. Chimeric polypeptides can be produced similarly, for example, by expressing suitable nucleic acids using synthetic or semi-synthetic chemical techniques. In some embodiments, a blocking moiety can be attached to a cytokine polypeptide by sortase-mediated conjugation. A "sortase" is a transpeptidase that modifies proteins by recognizing and cleaving carboxyl-terminal sorting signals embedded in or attached to the terminus of a target protein or peptide. Sortase A catalyzes the cleavage of an LPXTG motif (where X is any standard amino acid) (SEQ ID NO: 237) between a Thr and a Gly residue on a target protein, forming an enzyme-thioacyl intermediate through the temporary attachment of the Thr residue to an active site Cys residue on the enzyme. To complete the transpeptidation and create the peptide-monomer conjugate, a biomolecule bearing an N-terminal nucleophilic group (typically an oligoglycine motif) attacks the intermediate, displacing sortase A and linking the two molecules.

[0165] To form a cytokine blocking moiety conjugate, a cytokine polypeptide is first tagged at the N-terminus with a polyglycine sequence or at the C-terminus with an LPXTG (SEQ ID NO: 237) motif. The blocking moiety or other element has attached to it a respective peptide that serves as the acceptor site for the tagged polypeptide. For conjugation to a domain bearing the LPXTG (SEQ ID NO: 237) acceptor peptide attached via its N-terminus, the polypeptide is tagged at the N-terminus with a polyglycine stretch. For conjugation to a domain bearing the polyglycine peptide attached via its C-terminus, the polypeptide is tagged at the C-terminus with the LPXTG (SEQ ID NO: 237) sortase recognition sequence. Sortase, recognizing the polyglycine and LPXTG (SEQ ID NO: 237) sequences, forms a peptide bond between the polymer peptide and the tagged polypeptide. The sortase reaction cleaves at a glycine residue as an intermediate and is carried out at room temperature.

[0166] Various mechanisms can be utilized to remove or reduce the inhibition caused by the blocking moiety. For example, a pharmaceutical composition can include a cytokine moiety and a blocking moiety (e.g., a steric blocking moiety), and the protease-cleavable linker includes a protease cleavage site located between the cytokine and the cytokine blocking moiety or within the cytokine blocking moiety. When the protease cleavage site is cleaved, the blocking moiety dissociates from the cytokine, allowing the cytokine to then activate the cytokine receptor.

[0167] Any suitable linker can be used, for example, the linker can be glycine-glycine, a sortase recognition motif, or a sortase recognition motif and a peptide sequence (Gly4Ser). n (SEQ ID NO: 238) or (Gly3Ser) n(SEQ ID NO:239) (n is 1, 2, 3, 4, or 5). Typically, the sortase recognition motif comprises the peptide sequence LPXTG (SEQ ID NO:237) (X is any amino acid). In some embodiments, the covalent bond is between a reactive lysine residue attached to the C-terminus of the cytokine polypeptide and a reactive aspartic acid attached to the N-terminus of a blocker or other domain. In other embodiments, the covalent bond is between a reactive aspartic acid residue attached to the N-terminus of the cytokine polypeptide and a reactive lysine residue attached to the C-terminus of a blocker or another domain.

[0168] Thus, as described in detail herein, the cytokine blocking moiety used may be a steric blocker. As used herein, a "steric blocker" refers to a polypeptide or polypeptide moiety that can be covalently linked to a cytokine polypeptide, directly or indirectly, via another moiety such as a linker, e.g., in the form of a chimeric polypeptide (fusion protein), but is otherwise not covalently linked to the cytokine polypeptide. A steric blocker can be non-covalently bound to a cytokine polypeptide, e.g., by electrostatic, hydrophobic, ionic, or hydrogen bonding. A steric blocker typically inhibits or blocks the activity of the cytokine moiety due to its proximity to the cytokine moiety and its relative size. The steric hindrance of the cytokine moiety can be removed by spatially separating the cytokine moiety from the steric blocker, which can be achieved, for example, by enzymatically cleaving a fusion protein comprising the steric blocker and cytokine polypeptide at a site between the steric blocker and the cytokine polypeptide.

[0169] As described in detail herein, the blocking function can be combined with or result from the presence of additional functional components in the pharmaceutical composition (e.g., targeting domains, serum half-life extending elements, and protease-cleavable binding polypeptides). For example, a serum half-life extending polypeptide can also be a steric blocker.

[0170] To provide a concise disclosure of the full scope of the invention, embodiments of the invention will be described in detail using IL-2 as an exemplary cytokine. However, the invention and disclosure are not limited to IL-2. It will be apparent to those skilled in the art that the present disclosure, including the disclosed methods, polypeptides, and nucleic acids, adequately describes and enables the use of other cytokines, fragments, and muteins (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, IFNα, IFNβ, IFNγ, TNFα, lymphotoxin, TGF-β1, TGFβ2, TGFβ3, GM-CSF, CXCL10, CCL19, CCL20, CCL21, and functional fragments or muteins of any of the foregoing).

[0171] Various elements ensure delivery and activity of IL-2 preferentially at the desired IL-2 site of activity and ensure severely limited systemic exposure to the interleukin through a blocking and / or targeting strategy preferentially coupled with a serum half-life extension strategy in which a blocked version of the interleukin circulates for an extended period of time (preferentially 1-2 weeks or more) while the activated version has the typical serum half-life of the interleukin.

[0172] In comparison with serum half-life-extending versions, the serum half-life of intravenously administered IL-2 is only about 10 minutes. This is due to distribution within the large systemic extracellular space, approximately 15 L in an average-sized adult. IL-2 is then metabolized by the kidney with a half-life of approximately 2.5 hours (see Smith, K. "Interleukin 2 immunotherapy," Therapeutic Immunology 240 (2001)). Other measurements have shown that the plasma half-life of IL-2 is very short, at 85 minutes for intravenous administration and 3.3 hours for subcutaneous administration (Kirchner, GI, et al., 1998, Br J Clin Pharmacol. 46:5-10). In some embodiments of the present invention, the half-life-extending element is attached to the interleukin via a linker that is cleaved at the site of action (e.g., by an inflammation-specific protease) to release the full activity of the interleukin at the desired site and separate it from the uncleaved half-life-extending element. In such embodiments, the fully active, free form of the interleukin would have significantly different pharmacokinetic (pK) characteristics, with a half-life of hours rather than weeks. In addition, exposure to the active cytokine would be limited to the site of desired cytokine activity (e.g., site of inflammation or tumor), reducing systemic exposure to the active cytokine and associated toxicity and side effects.

[0173] Other cytokines contemplated by the present invention have similar pharmacology to IL-2 (e.g., IL-15 (reported in Blood 2011 117:4787-4795; doi:doi.org / 10.1182 / blood-2010-10-311456)), and therefore the present design addresses the shortcomings of using these agents directly, providing chimeric polypeptides that have extended half-lives and / or can be targeted to a desired site of activity (e.g., a site of inflammation or a tumor).

[0174] If desired, IL-2 can be engineered to bind generally to the IL-2R complex or specifically to one of the three IL-2R subunits with an affinity different from that of the corresponding wild-type IL-2, e.g., to selectively activate Tregs or Teffs (effector T cells). For example, an IL-2 polypeptide that is intended to have a higher affinity in its trimeric form for the dimeric beta / gamma form of the IL-2 receptor compared to wild-type IL-2 can have an amino acid sequence with respect to SEQ ID NO: 1 (the mature IL-2 protein comprising amino acids 21-153 of human IL-2 having UniProt Accession No. P60568-1) that includes one of the following sets of mutations: (a) K64R, V69A, and Q74P; (b) V69A, Q74P; (c) V69A, Q74P, and I128T; (d) N30D, V69A, Q74P, and F103S; (e) K49E, V69A, A73V, and K76E; (f) V69A, Q74P, T101A, and T133N; (g) N30S, V69A, Q74P, and I128A; (h) V69A, Q74P, N88D, and S99P; (i) N30S, V69A, Q74P, and I128T; (j) K9T, Q11R, K35R, V 69A, and Q74P; (k) A1T, M46L, K49R, E61D, V69A, and H79R; (l) K48E, E68D, N71T, N90H, F103S, and I114V; (m) S4P, T10A, Q11R, V69A, Q74P, N88D, and T133A; (n) E15K, N30S, Y31H, K35R, K48E, V69A, Q74P, and I92T; (o) N30S, E68D, V69A, N71A, Q74P, S75P, K76R, and N90H; (p) N30S, Y31C, T37A, V69A, A73V, Q74P, H79R, and I128T; (q) N26D, N29S, N30S, K54R, E67G, V69A, Q74P, and I92T; (r) K8R, Q13R, N26D, N30T, K35R, T37R, V69A, Q74P, and I92T; and (s) N29S, Y31H, K35R, T37A, K48E, V69A, N71R, Q74P, N88D, and I89V.This approach can also be applied to the preparation of muteins of other cytokines, including interleukins (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-23), interferons (IFNs, including IFN-alpha, IFN-beta, and IFN-gamma), tumor necrosis factors (e.g., TNF-alpha, lymphotoxin), transforming growth factors (e.g., TGF-beta 1, TGF-beta 2, TGF-beta 3), and granulocyte-macrophage-colony stimulating factor (GM-CS). For example, muteins can be prepared that have a desired binding affinity for their cognate receptors.

[0175] As noted above, any variant IL-2 polypeptide disclosed herein can include, or be limited to, the recited sequence, but can otherwise be identical to SEQ ID NO: 1. Additionally, any variant IL-2 polypeptide disclosed herein can optionally include a substitution of the cysteine ​​residue at position 125 with another residue (e.g., serine) and / or can optionally include a deletion of the alanine residue at position 1 of SEQ ID NO: 1.

[0176] Another approach to improving the therapeutic index of IL-2-based therapy is to optimize the pharmacokinetics of the molecule to maximize Treg cell activation. Early studies of IL-2 activity demonstrated that IL-2 stimulation of human T cell proliferation in vitro required a minimum of 5–6 hours of exposure to effective concentrations of IL-2 (Cantrell, DA, et al., 1984, Science, 224:1312–1316). When administered to human patients, the plasma half-life of IL-2 is very short: 85 minutes after intravenous administration and 3.3 hours after subcutaneous administration (Kirchner, GI, et al., 1998, Br J Clin Pharmacol. 46:5–10). Due to the short half-life of IL-2, maintaining circulating IL-2 at levels sufficient to stimulate T cell proliferation for the required period requires high doses or frequent administration to produce peak IL-2 levels significantly above the EC50 for Treg cells. Such high IL-2 peak levels may activate the IL2Rβγ receptor and result in other unintended or adverse effects, such as the VLS described above. IL-2 analogs, i.e., multifunctional proteins in which IL-2 is attached to a domain that enables binding to the FcRn receptor, have a longer circulating half-life than IL-2 and can achieve target drug concentrations for a specified period of time at lower doses and lower peak levels than IL-2. Therefore, such IL-2 analogs require lower doses or less frequent administration than IL-2 to effectively stimulate Treg cells. Less frequent subcutaneous administration of IL-2 drugs also improves patient tolerability. Clinically, therapeutic agents with these characteristics could lead to improved pharmacological efficacy, reduced toxicity, and improved patient compliance with therapy. Alternatively, IL-2 or a mutein of IL-2 (referred to herein as "IL-2*") may be selectively targeted to the intended site of action (e.g., a site of inflammation). This targeting can be achieved by one of several strategies, including adding a domain containing a cleaved blocker of IL-2 (or a mutein) to the administered agent, targeting domains, or a combination of the two.

[0177] In some embodiments, IL-2 * Partial agonists can be tailored to bind with higher or lower affinity depending on the desired target. For example, IL-2 * can be engineered to bind with high affinity to one receptor subunit but not to the other. Unlike full agonists or full antagonists, these types of partial agonists can be tailored to elicit desirable functional properties without exceeding the threshold for undesirable properties. Given the differential activity of partial agonists, it is conceivable that the IL-2 repertoire can be engineered to exhibit even finer degrees of distinct signaling activity, ranging from near-full agonism to partial agonism to full antagonism.

[0178] In some embodiments, IL-2 * In some embodiments, the IL-2Rα has an altered affinity for IL-2Rα. * has a higher affinity for IL-2Rα than wild-type IL-2. * In one embodiment, the IL-2 * In another embodiment, an IL-2Rα-binding protein is provided that has enhanced binding affinity to IL-2Rβ (e.g., the N-terminus of IL-2Rβ), thereby eliminating the functional requirement for IL-2Rα. In another embodiment, an IL-2Rα-binding protein is provided that has increased binding affinity to IL-2Rβ but exhibits reduced binding to IL-2Rγ, thereby resulting in defective IL-2Rβγ heterodimerization and signaling. * is generated.

[0179] Blocking moieties, described in more detail below, may also be used to favor binding to or activation of one or more receptors. In one embodiment, a blocking moiety is added to block IL-2Rβγ binding or activation but not IL-2Rα binding or activation. In another embodiment, a blocking moiety is added to attenuate IL-2Rα binding or activation. In another embodiment, a blocking moiety is added to inhibit binding to and activation of all three receptors. This blocking may be relievable under certain circumstances by removing the blocking moiety, for example, by proteolytic cleavage of a linker connecting one or more blocking moieties to the cytokine.

[0180] Similar approaches can be applied to improving other cytokines, particularly those used as immunostimulants, e.g., to treat cancer. For example, in this embodiment, the pharmacokinetics and / or pharmacodynamics of cytokines (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, IFNα, IFNβ, and IFNγ, TNFα, lymphotoxin, TGF-β1, TGFβ2, TGFβ3, GM-CSF, CXCL10, CCL19, CCL20, and CCL21) can be tailored to maximize activation of effector cells (e.g., T cells, NK cells) and / or cytotoxic immune response-promoting cells (e.g., inducing dendritic cell maturation) at the desired site of activity (e.g., within the tumor, but preferably not systemically).

[0181] Thus, provided herein are pharmaceutical compositions comprising at least one cytokine polypeptide, such as an interleukin (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23), an interferon (IFN, including IFNα, IFNβ, and IFNγ), a tumor necrosis factor (e.g., TNF, lymphotoxin), a transforming growth factor (e.g., TGF-β1, TGFβ2, TGFβ3), a chemokine (e.g., CXCL10, CCL19, CCL20, CCL21), and granulocyte-macrophage-colony-stimulating factor (GM-CS), or a functional fragment or mutein of any of the foregoing. The polypeptide also typically comprises at least one linker amino acid sequence, which, in certain embodiments, is cleavable by an endogenous protease. In one embodiment, the linker comprises an amino acid sequence comprising HSSKLQ (SEQ ID NO: 25), GPLGVRG (SEQ ID NO: 221), IPVSLRSG (SEQ ID NO: 222), VPLSLYSG (SEQ ID NO: 223), or SGESPAYYTA (SEQ ID NO: 224). In other embodiments, the chimeric polypeptide further comprises a blocking moiety (e.g., a steric-blocking polypeptide moiety) capable of blocking the activity of the interleukin polypeptide. The blocking moiety may comprise, for example, a human serum albumin (HSA)-binding domain or, optionally, a branched or multi-armed polyethylene glycol (PEG). Alternatively, the pharmaceutical composition comprises a first cytokine polypeptide or a fragment thereof and a blocking moiety (e.g., a steric-blocking polypeptide moiety), wherein the blocking moiety blocks the activity of the cytokine polypeptide on a cytokine receptor, and in certain embodiments, the blocking moiety comprises a protease-cleavable domain. In some embodiments, blocking or reducing cytokine activity is achieved simply by attaching additional domains with very short linkers to the N- or C-terminus of the interleukin domain.In such embodiments, protease digestion of the blocking moiety or of the short linker tethering the blocker to the interleukin is expected to relieve blocking: once the domain is clipped or released, it is no longer capable of blocking cytokine activity.

[0182] A pharmaceutical composition, such as a chimeric polypeptide, can contain two or more cytokines, which may be the same or different cytokine polypeptides. For example, the two or more different types of cytokines have complementary functions. In some examples, the first cytokine is IL-2 and the second cytokine is IL-12. In some embodiments, each of the two or more different types of cytokine polypeptides has an activity that regulates the activity of the other cytokine polypeptide. In some examples of a chimeric polypeptide containing two cytokine polypeptides, the first cytokine polypeptide is T cell activating and the second cytokine polypeptide is non-T cell activating. In some examples of a chimeric polypeptide containing two cytokine polypeptides, the first cytokine is a chemoattractant, such as CXCL10, and the second cytokine is an immune cell activator.

[0183] Preferably, the cytokine polypeptides (including functional fragments) included in the fusion proteins disclosed herein have not been mutated or engineered to alter the properties of the native cytokine, including receptor binding affinity and specificity or serum half-life, although amino acid sequence changes from the native (including wild-type) cytokine to facilitate cloning and achieve desired expression levels are acceptable.

[0184] a. Blocking part A blocking moiety can be any moiety that inhibits the ability of a cytokine to bind to and / or activate its receptor. A blocking moiety can inhibit the ability of a cytokine to bind to and / or activate its receptor by sterically blocking the cytokine and / or by non-covalently binding to the cytokine. Examples of suitable blocking moieties include full-length or cytokine-binding fragments or muteins of the cytokine's cognate receptor. Antibodies and fragments thereof can also be used, including polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-chain variable fragments (scFv), single-domain antibodies, such as heavy chain variable domains (VH), light chain variable domains (VL), and variable domains of camelid-type nanobodies (VHH), sdAbs that bind cytokines, and the like. Other suitable antigen-binding domains that bind to cytokines may also be used, including antibody binding and / or structurally mimicking non-immunoglobulin proteins, such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, finomers, Kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds, such as SpA, GroEL, fibronectin, lipocalin, and CTLA4 scaffolds. Further examples of suitable blocking polypeptides include polypeptides that sterically inhibit or block cytokines from binding to their cognate receptors. Advantageously, such moieties can also function as half-life extending elements. For example, peptides modified by conjugation with water-soluble polymers such as PEG can sterically inhibit or prevent cytokines from binding to their receptors. Polypeptides or fragments thereof with long serum half-lives, such as serum albumin (human serum albumin), immunoglobulin Fc, transferrin, etc., as well as fragments and muteins of such polypeptides, may also be used.Antibodies and antigen-binding domains that bind to proteins with long serum half-lives (e.g., HSA, immunoglobulins, or transferrin) or receptors that recycle to the plasma membrane (e.g., FcRn or transferrin receptor) can also inhibit cytokines, particularly when bound to their antigens. Examples of such antigen-binding polypeptides include single-chain variable fragments (scFv), single-domain antibodies, such as heavy chain variable domains (VH), light chain variable domains (VL), and variable domains of camelid-type nanobodies (VHH), sdAbs, etc. Other suitable antigen-binding domains that bind cytokines may also be used, including antibody binding and / or structurally mimicking non-immunoglobulin proteins, such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, finomers, Kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds, such as SpA, GroEL, fibronectin, lipocalin, and CTLA4 scaffolds.

[0185] In illustrative examples, when IL-2 is the chimeric polypeptide cytokine, the blocking moiety can be the full-length or fragment or mutein of the alpha chain of the IL-2 receptor (IL-2Rα), or the beta (IL-2Rβ) or gamma chain of the IL-2 receptor (IL-2Rγ), an anti-IL-2 single domain antibody (sdAb) or scFv, an anti-CD25 antibody or fragment thereof, and an anti-HSA sdAb or scFv, etc.

[0186] b. In vivo half-life extending element Preferably, the chimeric polypeptide contains an in vivo half-life-extending element. Increasing the in vivo half-life of a therapeutic molecule with a short natural half-life allows for more tolerable and manageable dosing regimens without sacrificing efficacy. As used herein, a "half-life-extending element" refers to a portion of a chimeric polypeptide that extends its in vivo half-life and improves its pK, for example, by altering its size (e.g., to be above the renal filtration cutoff), shape, hydrodynamic radius, charge, or parameters of absorption, biodistribution, metabolism, and excretion. An exemplary method for improving the pK of a polypeptide is to express elements within the polypeptide chain that bind to receptors that are recycled to the plasma membrane of cells rather than being degraded in lysosomes (e.g., FcRn receptors and transferrin receptors on endothelial cells). Three types of proteins, such as human IgG, HSA (or fragments thereof), and transferrin, persist in human serum much longer than would be predicted by their size, a function of their ability to bind to receptors that are recycled rather than degraded in lysosomes. These proteins or fragments thereof that retain FcRn binding typically bind to other polypeptides to extend serum half-life. In one embodiment, the half-life extending element is a human serum albumin (HSA) binding domain. HSA (SEQ ID NO: 2) can be directly bound to the pharmaceutical composition or can be bound via a short linker. Fragments of HSA can also be used. HSA and its fragments can function as both a blocking moiety and a half-life extending element. Human IgG can also perform a similar function.

[0187] The serum half-life extending element may be an antigen-binding polypeptide that binds to a protein with a long serum half-life, such as serum albumin or transferrin. Examples of such polypeptides include antibodies and fragments thereof, including polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-chain variable fragments (scFv), single-domain antibodies, such as heavy chain variable domains (VH), light chain variable domains (VL), and variable domains of camelid-type nanobodies (VHH), cytokine-binding sdAbs, etc. Other suitable antigen-binding domains include non-immunoglobulin proteins that mimic antibody binding and / or structure, such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, finomers, Kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds, such as SpA, GroEL, fibronectin, lipocalin, and CTLA4 scaffolds. Further examples of antigen-binding polypeptides include ligands of a desired receptor, ligand-binding portions of a receptor, lectins, and peptides that bind to or associate with one or more target antigens.

[0188] Some preferred serum half-life extending elements include polypeptides comprising complementarity-determining regions (CDRs) and, optionally, non-CDR loops. Advantageously, such serum half-life extending elements can extend the serum half-life of a cytokine and also function as cytokine inhibitors (e.g., via steric blocking, non-covalent interactions, or a combination thereof) and / or targeting domains. In some cases, the serum half-life extending element is a domain derived from an immunoglobulin molecule (Ig molecule) or an engineered protein scaffold mimicking antibody structure and / or binding activity. Ig may be of any class or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, etc.). The polypeptide chain of an Ig molecule folds into a series of parallel beta strands connected by loops. Within the variable region, three loops constitute the "complementarity-determining regions" (CDRs), which determine the antigen-binding specificity of the molecule. An IgG molecule comprises at least two heavy (H) chains and two light (L) chains, or antigen-binding fragments thereof, interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, termed complementarity-determining regions (CDRs), which are highly variable in sequence and / or involved in antigen recognition and / or usually form structurally defined loops, interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments of the present disclosure, at least a portion or all of the amino acid sequences of FR1, FR2, FR3, and FR4 are part of the "non-CDR loops" of the binding moieties described herein.The variable domain of an immunoglobulin molecule has several β-strands arranged in two sheets. The variable domains of both immunoglobulin light and heavy chains contain three hypervariable loops, or complementarity-determining regions (CDRs). The three CDRs of a V domain (CDR1, CDR2, CDR3) are clustered at one end of a β-barrel. The CDRs are the loops connecting β-strands BC, C'-C", and FG of the immunoglobulin fold, while the bottom loop connecting β-strands AB, CC', C"-D, and EF of the immunoglobulin fold and the top loop connecting the DE strand of the immunoglobulin fold are non-CDR loops. In some embodiments of the present disclosure, at least some amino acid residues of the constant domain CH1, CH2, or CH3 are part of the "non-CDR loops" of the binding moiety described herein. In some embodiments, the non-CDR loops include one or more of the AB, CD, EF, and DE loops of a C1 set domain of an Ig or Ig-like molecule; the AB, CC', EF, FG, BC, and EC' loops of a C2 set domain of an Ig or Ig-like molecule; and the DE, BD, GF, A(A1A2)B, and EF loops of an I (intermediate) set domain of an Ig or Ig-like molecule.

[0189] In the variable domain, CDRs are considered to be responsible for antigen recognition and binding, while FR residues are considered to be the scaffold of the CDRs. However, in certain cases, some FR residues play an important role in antigen recognition and binding. Framework region residues that affect Ag binding are divided into two categories. First, there are FR residues that contact the antigen and are part of the binding site, and some of these residues are close to the CDRs in terms of sequence. Other residues are far from the CDRs in terms of sequence, but close to the CDRs (e.g., heavy chain loops) in the 3D structure of the molecule.

[0190] The binding moiety can be any type of polypeptide. For example, in certain instances, the binding moiety is a natural peptide, a synthetic peptide, or a fibronectin scaffold, or an engineered bulk serum protein. Bulk serum proteins include, for example, albumin, fibrinogen, or globulin. In some embodiments, the binding moiety is an engineered scaffold. Engineered scaffolds include, for example, sdAb, scFv, Fab, VHH, fibronectin type III domain, immunoglobulin-like scaffold (as suggested in Halaby et al., 1999. Prot Eng 12(7):563-571), DARPin, cystine-knot peptide, lipocalin, three-helix bundle scaffold, protein G-related albumin binding module, or DNA or RNA aptamer scaffold.

[0191] In some cases, the serum half-life extending element comprises a binding site for a bulk serum protein. In some embodiments, the CDRs provide the binding site for the bulk serum protein. The bulk serum protein is, in some examples, globulin, albumin, transferrin, IgG1, IgG2, IgG4, IgG3, IgA monomer, Factor XIII, fibrinogen, IgE, or pentameric IgM. In some embodiments, the CDRs form the binding site for an immunoglobulin light chain, such as an Igκ free light chain or an Igλ free light chain.

[0192] The serum half-life extending element can be any type of binding domain, including, but not limited to, a domain from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, or a humanized antibody. In some embodiments, the binding moiety is a single-chain variable fragment (scFv), a single-domain antibody, such as a heavy chain variable domain (VH), a light chain variable domain (VL), or a variable domain of a camelid nanobody (VHH). In other embodiments, the binding moiety is a non-Ig binding domain, i.e., an antibody mimetic, such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, finomers, Kunitz domain peptides, or monobodies.

[0193] In other embodiments, the serum half-life extending element may be a water-soluble polymer or a peptide conjugated to a water-soluble polymer such as PEG. As used herein, "PEG," "polyethylene glycol," and "poly(ethylene glycol)" are interchangeable and include any non-peptidic, water-soluble poly(ethylene oxide). The term "PEG" also refers to a polymer containing a majority, i.e., greater than 50%, of -OCH2CH2- repeating subunits. With regard to specific configurations, PEG can be of any number of different molecular weights, as described in more detail below, and can further be of "branched," "linear," "forked," "multifunctional," and other structures or geometries. PEG is not limited to a particular structure and can be linear (e.g., end-capped, e.g., alkoxyPEG or bifunctional PEG), branched or multi-armed (e.g., forked PEG or PEG attached to a polyol core), or dendritic (or star) architecture, each with or without one or more degradable linkages. Furthermore, the internal structure of PEG can be organized in any number of different repeating patterns and can be selected from the group consisting of homopolymers, alternating copolymers, random copolymers, block copolymers, alternating tripolymers, random tripolymers, and block tripolymers. PEG can be conjugated to polypeptides and peptides by any suitable method. Typically, a reactive PEG derivative (e.g., N-hydroxysuccinamidyl ester PEG) is reacted with a peptide or polypeptide containing an amino acid (e.g., cysteine, lysine, asparagine, glutamine, threonine, tyrosine, serine, aspartic acid, and glutamic acid) having a side chain containing an amine, sulfhydryl, carboxylic acid, or hydroxyl functional group.

[0194] c. Targeting and Retention Domains In certain applications, it may be desirable to maximize the amount of time the construct resides in a desired location in the body. This can be achieved by including an additional domain in the chimeric polypeptide (fusion protein) to affect its movement within the body. For example, the chimeric nucleic acid can encode a domain that directs the polypeptide to a location in the body (e.g., tumor cells or sites of inflammation) (this domain is referred to as a "targeting domain") and / or a domain that retains the polypeptide at a location in the body (e.g., tumor cells or sites of inflammation) (this domain is referred to as a "retention domain"). In some embodiments, a domain can function as both a targeting domain and a retention domain. In some embodiments, the targeting domain and / or retention domain is specific for a protease-rich environment. In some embodiments, the encoded targeting domain and / or retention domain is specific for regulatory T cells (Tregs), for example, targeting the CCR4 or CD39 receptor. Other suitable targeting and / or retention domains include those with cognate ligands (e.g., IL-1 receptor or IL-6 receptor) that are overexpressed in inflamed tissues. In other embodiments, suitable targeting and / or retention domains include those with cognate ligands (e.g., Epcam, CEA, or mesothelin) that are overexpressed in tumor tissue. In some embodiments, the targeting domain is attached to the interleukin via a linker that is cleaved at the site of action (e.g., by an inflammatory or cancer-specific protease), releasing the fully active interleukin at the desired site. In some embodiments, the targeting and / or retention domain is attached to the interleukin via a linker that is not cleaved at the site of action (e.g., by an inflammatory or cancer-specific protease), allowing the cytokine to remain at the desired site.

[0195] The selected antigen is optionally expressed on the surface of diseased cells or tissues (e.g., tumor or cancer cells). Antigens useful for tumor targeting and retention include, but are not limited to, EpCAM, EGFR, HER-2, HER-3, c-Met, FoIR, and CEA. The pharmaceutical compositions disclosed herein also include proteins containing two targeting and / or retention domains that bind to two different target antigens known to be expressed on diseased cells or tissues. Exemplary pairs of antigen-binding domains include, but are not limited to, EGFR / CEA, EpCAM / CEA, and HER-2 / HER-3.

[0196] Suitable targeting and / or retention domains include antigen-binding domains, e.g., antibodies and fragments thereof, including polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-chain variable fragments (scFv), single-domain antibodies, e.g., heavy chain variable domains (VH), light chain variable domains (VL), and variable domains of camelid-type nanobodies (VHH), cytokine-binding sdAbs, etc. Other suitable antigen-binding domains include antibody binding and / or structurally mimicking non-immunoglobulin proteins, e.g., anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, finomers, Kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds, e.g., SpA, GroEL, fibronectin, lipocalin, and CTLA4 scaffolds. Further examples of antigen-binding polypeptides include ligands of a desired receptor, ligand-binding portions of a receptor, lectins, and peptides that bind to or associate with one or more target antigens.

[0197] In some embodiments, the targeting and / or retention domain specifically binds to a cell surface molecule. In some embodiments, the targeting and / or retention domain specifically binds to a tumor antigen. In some embodiments, the targeting polypeptide specifically and independently binds to a tumor antigen selected from at least one of EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FoIR. In some embodiments, the targeting polypeptide specifically and independently binds to two different antigens, and at least one of the antigens is a tumor antigen selected from EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FoIR.

[0198] The targeting antigen and / or retention antigen may be a tumor antigen expressed on tumor cells. Tumor antigens are well known in the art and include, for example, EpCAM, EGFR, HER-2, HER-3, c-Met, FolR, PSMA, CD38, BCMA, and CEA, 5T4, AFP, B7-H3, cadherin-6, CAIX, CD117, CD123, CD138, CD166, CD19, CD20, CD205, CD22, CD30, CD33, CD352, CD37, CD44, CD52, CD56, CD70, CD71, CD74, CD79b, DLL3, EphA2, FAP, FGFR2, FGFR3, GPC3, gpA33, FLT-3, gpNMB, HPV-16 E6, HPV-16 These include E7, ITGA2, ITGA3, SLC39A6, MAGE, mesothelin, Muc1, Muc16, NaPi2b, nectin-4, P-cadherin, NY-ESO-1, PRLR, PSCA, PTK7, ROR1, SLC44A4, SLTRK5, SLTRK6, STEAP1, TIM1, Trop2, and WT1.

[0199] The targeting antigen and / or retention antigen can be an immune checkpoint protein, including, but not limited to, CD27, CD137, 2B4, TIGIT, CD155, ICOS, HVEM, CD40L, LIGHT, TIM-1, OX40, DNAM-1, PD-L1, PD1, PD-L2, CTLA-4, CD8, CD40, CEACAM1, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, IDO1, IDO2, TDO, KIR, LAG-3, TIM-3, or VISTA.

[0200] The targeting antigen and / or retention antigen may be a cell surface molecule, such as a protein, lipid, or polysaccharide. In some embodiments, the targeting antigen and / or retention antigen is present on tumor cells, virus-infected cells, bacteria-infected cells, damaged red blood cells, arterial plaque cells, or inflamed or fibrotic tissue cells. The targeting antigen and / or retention antigen may comprise an immune response modifier. Examples of immune response modifiers include, but are not limited to, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-12 (IL-12), interleukin-15 (IL-15), B7-1 (CD80), B7-2 (CD86), GITRL, CD3, or GITR.

[0201] The targeting antigen and / or retention antigen may be a cytokine receptor. Examples of cytokine receptors include, but are not limited to, type I cytokine receptors, such as GM-CSF receptor, G-CSF receptor, type I IL receptor, Epo receptor, LIF receptor, CNTF receptor, and TPO receptor; type II cytokine receptors, such as IFN-alpha receptor (IFNAR1, IFNAR2), IFB-beta receptor, IFN-gamma receptor (IFNGR1, IFNGR2), and type II IL receptor; CC chemokine receptors, such as CXC chemokine receptor and CX3C chemokine. Receptors, XC chemokine receptors; tumor necrosis receptor superfamily receptors, such as TNFRSF5 / CD40, TNFRSF8 / CD30, TNFRSF7 / CD27, TNFRSF1A / TNFR1 / CD120a, TNFRSF1B / TNFR2 / CD120b; TGF-beta receptors, such as TGF-beta receptor 1 and TGF-beta receptor 2; Ig superfamily receptors, such as IL-1 receptor, CSF-1R, PDGFR (PDGFRA, PDGFRB), and SCFR.

[0202] d. linker As described above, the pharmaceutical composition includes one or more linker sequences. The linker sequence functions to provide flexibility between the polypeptides, for example, so that a blocking moiety can inhibit the activity of the cytokine polypeptide. The linker sequence can be disposed between any or all of the cytokine polypeptide, the serum half-life extending element, and / or the blocking moiety. As described herein, at least one of the linkers is protease-cleavable and includes one or more cleavage sites for one or more desired proteases. Preferably, the desired proteases are enriched or selectively expressed at the desired cytokine activity site (e.g., tumor microenvironment). Thus, the fusion protein is preferentially or selectively cleaved at the desired cytokine activity site.

[0203] The orientation of the components of the pharmaceutical composition is largely a matter of design choice, and it is recognized that multiple orientations are possible, all of which are intended to be encompassed by the present disclosure. For example, the blocking moiety can be placed C-terminally or N-terminally to the cytokine polypeptide.

[0204] Pharmaceutical compositions comprising the polypeptide sequences are provided herein. As with all peptides, polypeptides, and proteins (including fragments thereof), it is understood that the amino acid sequence of the chimeric polypeptide may undergo additional modifications (amino acid sequence variants) that do not alter the properties or function of the peptide, polypeptide, or protein. Such modifications include conservative amino acid substitutions, which are described in more detail below.

[0205] The compositions provided herein have a desired function. The compositions are composed of at least a cytokine polypeptide (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, or IFNγ) or a chemokine (e.g., CXCL10, CCL19, CCL20, CCL21), a blocking moiety (e.g., a steric-blocking polypeptide), an optional serum half-life extending element, and an optional targeting polypeptide, with one or more linkers connecting each polypeptide in the composition. A first polypeptide, e.g., an IL-2 mutein, is provided as an active agent. A blocking moiety is provided for blocking the activity of an interleukin. A linker polypeptide, e.g., a protease-cleavable polypeptide, is provided to be cleaved by a protease specifically expressed in the intended target of the active agent. Optionally, the blocking moiety blocks the activity of the first polypeptide by binding the interleukin polypeptide. In some embodiments, the blocking moiety (e.g., a steric blocking peptide) is attached to the interleukin via a protease-cleavable linker that is cleaved at the site of action (e.g., by an inflammation-specific protease) to release the fully active cytokine at the desired site.

[0206] In some embodiments, the linker is a glycine-glycine, a sortase recognition motif, or a sortase recognition motif and a peptide sequence (Gly4Ser). n (SEQ ID NO: 238), or (Gly3Ser) n (SEQ ID NO:239) where n is 1, 2, 3, 4, or 5. In one embodiment, the sortase recognition motif comprises the peptide sequence LPXTG, where X is any amino acid (SEQ ID NO:237). In one embodiment, the covalent bond is between a reactive lysine residue attached to the C-terminus of the cytokine polypeptide and a reactive aspartic acid attached to the N-terminus of the blocking moiety or other moiety. In one embodiment, the covalent bond is between a reactive aspartic acid residue attached to the N-terminus of the cytokine polypeptide and a reactive lysine residue attached to the C-terminus of the blocking moiety or other moiety.

[0207] e. Cleavage and inducibility As described herein, the activity of the cytokine polypeptide in the context of the fusion protein is attenuated, and protease cleavage at the desired active site (e.g., tumor microenvironment) releases a form of cytokine from the fusion protein that is significantly more active as a cytokine receptor agonist than the fusion protein. For example, the cytokine receptor activating (agonist) activity of the fusion polypeptide can be at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, or at least about 1000-fold less than the cytokine receptor activating activity of the cytokine polypeptide as a separate molecular entity. A cytokine polypeptide that is part of a fusion protein exists as a separate molecular entity when it contains substantially identical amino acids to the cytokine polypeptide, is substantially free of additional amino acids, and is not associated (covalently or noncovalently) with other molecules. If necessary, the cytokine polypeptide as a separate molecular entity may contain some additional amino acid sequence (e.g., a tag or short sequence to aid in expression and / or purification).

[0208] In other examples, the cytokine receptor activating (agonist) activity of the fusion polypeptide is at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, or at least about 1000-fold lower than the cytokine receptor activating activity of a polypeptide comprising a cytokine polypeptide produced by cleavage of a protease-cleavable linker in the fusion protein. In other words, the cytokine receptor activating (agonist) activity of a polypeptide comprising a cytokine polypeptide produced by cleavage of a protease-cleavable linker in the fusion protein is at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, or at least about 1000-fold higher than the cytokine receptor activating activity of the fusion protein. In other examples, the recombinant polypeptide is conjugated to a cleavable moiety, wherein the cleavable moiety is cleaved with reduced catalytic efficiency by one or more proteases compared to the reference polypeptide sequence.

[0209] In some embodiments, the cleavable moiety is resistant to proteolytic cleavage by one or more proteases. A cleavable moiety is resistant to a protease if the sequence contains a binding site that is altered from the canonical cleavage motif sequence for a particular protease. In some embodiments, the binding site is altered compared to a reference sequence by making one or more substitutions within the protease cleavage motif sequence. For example, the protease cathepsin S cleaves the sequence GAVVRGA (SEQ ID NO: 240). The sequence can be substituted by substituting an arginine (R) with a glutamine (Q), changing a charged residue to a shorter, more polar residue and reducing the ability of cathepsin S to bind and cleave the sequence. Such semi-conservative amino acid substitutions in the protease target sequence motif result in reduced binding ability, and therefore, such an altered sequence motif is protease-resistant. Uncleavable portions can be created by inserting disruptive amino acids, such as proline (which causes a curve in the secondary structure of the peptide) or histidine (which causes steric interference with other amino acid side chains), into the target sequence motif of the protease. As used herein, a "protease-resistant" peptide linker is a peptide linker that exhibits reduced or undetectable cleavage by one or more specified proteases. Exemplary protease-resistant peptide linkers can be tested, for example, in vitro, by incubating with a specific protease and then analyzing the digestion products by Western blot.

[0210] f. Polypeptide substitution The polypeptides described herein can contain components (e.g., cytokines, blocking moieties) that have the same amino acid sequence as the corresponding native protein (e.g., IL-2, IL-15, HSA) or that have a different amino acid sequence from the native protein, so long as the desired function is maintained. It is understood that one way of defining any known or possible modifications and derivatives of the proteins and nucleic acids encoding them of the present disclosure is by defining sequence variants in terms of identity to a particular known reference sequence. Specifically, disclosed are polypeptides and nucleic acids that have at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the chimeric polypeptides provided herein. For example, provided are polypeptides having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent identity to any nucleic acid or polypeptide sequence described herein, including polypeptides or nucleic acids having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent identity to the sequence of a cleavable linker provided herein. This also includes the variant of linker or derivative polypeptide that comprises 1, 2, 3, 4, 5 or 6 variants from the cleavage domain sequence.Those skilled in the art can easily understand how to determine the identity of two polypeptides or two nucleic acids.For example, identity can be calculated by aligning two sequences and then making identity its highest level.

[0211] Alternative methods for calculating identity can be performed using published algorithms. Optimal sequence alignment for comparison can be performed using the local identity algorithm of Smith and Waterman, Adv. Appl. Math 2:482 (1981), the identity alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection.

[0212] The same type of identity can also be obtained for nucleic acids by the algorithms disclosed in, for example, Zuker, Science 244:48-52 (1989); Jaeger et al., Proc. Natl. Acad. Sci. USA 86:7706-7710 (1989); Jaeger et al., Methods Enzymol. 183:281-306 (1989), which are incorporated herein by reference at least for the material related to nucleic acid alignment. Any method can typically be used, and although the results of these various methods may vary in certain cases, those skilled in the art will understand that if identity is found by at least one of these methods, the sequence has the described identity and is considered to be disclosed herein.

[0213] Protein modifications include modifications of amino acid sequences. Amino acid sequence modifications can occur naturally as allelic variations (e.g., due to genetic polymorphisms), can arise due to environmental influences (e.g., exposure to ultraviolet light), or can be caused by human intervention (e.g., mutagenesis of cloned DNA sequences), resulting in, for example, induced point mutations, deletion mutations, insertion mutations, and substitution mutations. These modifications can result in changes in amino acid sequence, silent mutations, restriction site modifications, or other specific mutations. Amino acid sequence modifications typically fall into one or more of three classes: substitution, insertion, or deletion. Insertions include amino- and / or carboxyl-terminal fusions and intrasequence insertions of single or multiple amino acid residues. Insertions are usually smaller than amino- or carboxyl-terminal fusion insertions, e.g., insertions of about 1 to 4 residues. Deletions are characterized by the removal of one or more amino acid residues from a protein sequence. Typically, no more than about 2 to 6 residues are deleted at any one site within a protein molecule. Amino acid substitutions are typically single residue substitutions, but may occur at multiple different positions at once. Insertions typically involve 1 to 10 amino acid residues, while deletions can range from 1 to 30 residues. Deletions or insertions are preferably made in adjacent pairs, i.e., a deletion of two residues or an insertion of two residues. Substitutions, deletions, insertions, or any combination thereof can be combined to arrive at a final construct. Mutations should not place the sequence out of reading frame and preferably do not create complementary regions that could produce secondary mRNA structure. Substitutional modifications involve the removal of at least one residue and the insertion of a different residue in its place. Such substitutions are generally made according to Table 2 below and are referred to as conservative substitutions. [Table 2]

[0214] Modifications, including specific amino acid substitutions, are made by known methods. For example, modifications can be made by site-specific mutagenesis of nucleotides in the DNA encoding the polypeptide, thereby producing DNA encoding the modification, followed by expression of the DNA in recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, such as M13 primer mutagenesis and PCR mutagenesis.

[0215] Modifications can be selected to optimize binding. For example, affinity maturation techniques can be used to alter the binding of scFvs by introducing random mutations within the complementarity-determining regions (CDRs). Such random mutations can be introduced using a variety of techniques, including radiation, chemical mutagens, or error-prone PCR. Multiple rounds of mutation and selection can be performed, for example, using phage display.

[0216] The present disclosure also relates to nucleic acids encoding the chimeric polypeptides described herein and the use of such nucleic acids to produce the chimeric polypeptides and for therapeutic purposes. For example, the present invention includes DNA and RNA molecules (e.g., mRNA, self-replicating RNA) that encode the chimeric polypeptides, and to the therapeutic uses of such DNA and RNA molecules.

[0217] g. Exemplary Compositions Exemplary fusion proteins of the invention combine the elements described above in various orientations. The orientations described in this section are intended as examples and should not be considered limiting.

[0218] In some embodiments, the fusion protein comprises a cytokine, a blocking moiety, and a half-life extending element. In some embodiments, the cytokine is located between the half-life extending element and the blocking moiety. In some embodiments, the cytokine is N-terminal to the blocking moiety and the half-life extending element. In some such embodiments, the cytokine is proximal to the blocking moiety, and in some such embodiments, the cytokine is proximal to the half-life extending element. All embodiments must include at least one protease-cleavable linker so that the cytokine becomes active upon cleavage. In some embodiments, the cytokine is C-terminal to the blocking moiety and the half-life extending element. Additional elements can be attached to each other by a cleavable linker, a non-cleavable linker, or by direct fusion.

[0219] In some embodiments, the blocking domain used is capable of extending half-life and the cytokine is located between two such blocking domains, hi some embodiments, the cytokine is located between two blocking domains, one of which is capable of extending half-life.

[0220] In some embodiments, two cytokines are contained in the same construct. In some embodiments, each cytokine is connected to two blocking domains (for a total of three in one molecule), with one blocking domain between the two cytokine domains. In some embodiments, one or more additional half-life extending domains can be included to optimize pharmacokinetic properties.

[0221] In some embodiments, three cytokines are included in the same construct, and in some embodiments, the third cytokine may function to block the other two cytokines in place of the blocking domain between the two cytokines. Preferred half-life extending elements for use in the fusion protein are human serum albumin (HSA), an antibody or antibody fragment (e.g., scFV, dAb) that binds to serum albumin, human or humanized IgG, or a fragment of any of the foregoing. In some preferred embodiments, the blocking moiety is human serum albumin (HSA), or an antibody or antibody fragment that binds to serum albumin, an antibody that binds to a cytokine and prevents cytokine receptor binding or activation, another cytokine, or a fragment of any of the foregoing. In preferred embodiments that include an additional targeting domain, the targeting domain is an antibody that binds to a cell surface protein that is enriched on the surface of cancer cells, such as EpCAM, FOLR1, and fibronectin.

[0222] vii. Other Uses The detachment moieties disclosed herein can be used in antibody-antibiotic conjugates. The detachment moieties disclosed herein connect or bind an antimicrobial antibiotic to an antibody specific to a bacterial strain (e.g., Staphylococcus aureus Ab). The antibody-antibiotic conjugate does not exhibit antibacterial activity when the antibody binds to the antibiotic. However, upon internalization within a host cell, the detachment moiety is cleaved by a protease, releasing the free antibiotic. The free antibiotic kills the bacteria within the cell.

[0223] The separation moieties disclosed herein can be used in chemical probes used for protein detection and isolation. Chemical probes are designed based on the interaction of small molecule compounds with proteins. The probes typically contain a covalent binding motif for interacting with the target protein, a detection / purification tag for visualization / purification of the target protein, and a linker group. The separation moieties described herein can be incorporated to enable detection and isolation of the target protein.

[0224] C. Methods of Treatment and Pharmaceutical Compositions Additionally provided are methods for treating a subject having or at risk of developing a disease or disorder, such as a proliferative disease, a neoplastic disease, an inflammatory disease, an immune disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, or graft-versus-host disease. The methods involve administering to a subject in need thereof an effective amount of a fusion protein disclosed herein, typically administered as a pharmaceutical composition. In some embodiments, the methods further include selecting a subject having or at risk of developing such a disease or disorder. The pharmaceutical composition preferably comprises a blocked cytokine activated at a site of inflammation, a fragment or mutein thereof. In one embodiment, the chimeric polypeptide comprises a cytokine polypeptide, a fragment or mutein thereof, and a serum half-life extending element. In another embodiment, the chimeric polypeptide comprises a cytokine polypeptide, a fragment or mutein thereof, and a blocking moiety (e.g., a steric-blocking polypeptide), wherein the steric-blocking polypeptide is capable of sterically blocking the activity of the cytokine polypeptide, the fragment or mutein thereof. In another embodiment, the chimeric polypeptide comprises a cytokine polypeptide, a fragment or mutein thereof, a blocking moiety, and a serum half-life extending element.

[0225] Inflammation is part of the complex biological response of body tissues to harmful stimuli (e.g., pathogens, damaged cells, or irritants) and is a defensive response involving immune cells, blood vessels, and molecular mediators. The function of inflammation is to eliminate the initial cause of cellular injury, remove necrotic cells and tissue damaged by the initial injury and inflammatory process, and initiate tissue repair. Inflammation can arise from infection, as a symptom, or as a disease (e.g., cancer, atherosclerosis, allergies, myopathy, HIV, obesity, or autoimmune disease). Autoimmune diseases are chronic conditions resulting from an abnormal immune response to self-antigens. Autoimmune diseases that can be treated using the polypeptides disclosed herein include, but are not limited to, lupus, celiac disease, type 1 diabetes, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.

[0226] The pharmaceutical composition may include one or more protease-cleavable linker sequences. The linker sequence functions to provide flexibility between polypeptides so that each polypeptide can inhibit the activity of the first polypeptide. The linker sequence can be positioned between any or all of the cytokine polypeptide, its fragment or mutein, the blocking moiety, and the serum half-life extending element. Optionally, the composition includes two, three, four, or five linker sequences. The linker sequence, the two, three, or four linker sequences, may be the same or different. In one embodiment, the linker sequence includes GGGGS (SEQ ID NO: 232), GSGSGS (SEQ ID NO: 233), or G(SGGG)2SGGT (SEQ ID NO: 234). In another embodiment, the linker comprises a protease-cleavable sequence selected from the group consisting of HSSKLQ (SEQ ID NO: 25), GPLGVRG (SEQ ID NO: 221), IPVSLRSG (SEQ ID NO: 222), VPLSLYSG (SEQ ID NO: 223), or SGESPAYYTA (SEQ ID NO: 224). In some embodiments, the linker is cleaved by a protease selected from the group consisting of kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin G, elastase, PR-3, granzyme M, calpain, matrix metalloproteinase (MMP), plasminogen activator, cathepsin, caspase, tryptase, or tumor cell surface protease.

[0227] Further provided is a method for treating a subject having or at risk of developing cancer. The method comprises administering to a subject in need thereof an effective amount of a chimeric polypeptide (fusion protein) disclosed herein, typically administered as a pharmaceutical composition. In some embodiments, the method further comprises selecting a subject having or at risk of developing cancer. The pharmaceutical composition preferably comprises a blocked cytokine, fragment, or mutein thereof that is activated at the tumor site. Preferably, the tumor is a solid tumor. The cancer may be colon cancer, lung cancer, melanoma, sarcoma, renal cell carcinoma, or breast cancer.

[0228] The method may further involve the administration of one or more additional agents for treating cancer, such as a chemotherapeutic agent (e.g., adriamycin, cervidin, bleomycin, alkeran, velban, oncovin, fluorouracil, thiotepa, methotrexate, bisantrene, noanthrone, tiguanine, citalibine, procarabidine), an immuno-oncology agent (e.g., anti-PD-L1, anti-CTLA4, anti-PD-1, anti-CD47, anti-GD2), a cell therapy (e.g., CAR-T, T-cell therapy), an oncolytic virus, or the like.

[0229] Provided herein is a pharmaceutical formulation or composition comprising a chimeric polypeptide and a pharmaceutically acceptable carrier. The compositions provided herein are suitable for in vitro or in vivo administration. A pharmaceutically acceptable carrier refers to a material that is not biologically or otherwise undesirable, i.e., the material is administered to a subject without causing undesirable biological effects or interacting in a harmful manner with other components of the pharmaceutical formulation or composition in which it is contained. The carrier is selected to minimize degradation of the active ingredient and minimize side effects in the subject.

[0230] For suitable carriers and their formulations, see Remington: The Science and Practice of Pharmacy, 21 stEdition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic; however, the formulation may be made hypertonic or hypotonic as desired. Examples of pharmaceutically acceptable carriers include, but are not limited to, sterile water, saline, buffered solutions such as Ringer's solution, and dextrose solution. The pH of the solution is generally about 5 to about 8 or about 7 to 7.5. Other carriers include sustained-release preparations, such as semipermeable matrices of solid hydrophobic polymers containing the immunogenic polypeptide. The matrices are in the form of shaped articles (e.g., films, liposomes, or microparticles). Certain carriers may be more preferable depending, for example, on the route of administration and the concentration of the composition being administered. The carrier is one suitable for administering the chimeric polypeptide or a nucleic acid sequence encoding the chimeric polypeptide to humans or other subjects.

[0231] Pharmaceutical formulations or compositions can be administered in several ways, depending on whether local or systemic treatment is desired and the area to be treated. The compositions can be administered via any of several routes, including topical, oral, parenteral, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intracavity, transdermal, intrahepatic, intracranial, nebulized / inhaled, or via bronchoscopy. In some embodiments, the compositions are administered locally (non-systemically), including intratumorally, intraarticularly, intrathecally, etc.

[0232] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (e.g., those based on Ringer's dextrose), and the like. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents, and inert gases, may optionally be present.

[0233] Formulations for topical administration include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like may optionally be necessary or desirable.

[0234] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders are optionally desirable.

[0235] Optionally, the chimeric polypeptide or a nucleic acid sequence encoding the chimeric polypeptide is administered via a vector. There are multiple compositions and methods that can be used to deliver nucleic acid molecules and / or polypeptides to cells, either in vitro or in vivo, for example, via expression vectors. These methods and compositions can be broadly categorized into two classes: viral-based delivery systems and non-viral-based delivery systems. Such methods are well known in the art and can be readily adapted for use with the compositions and methods described herein. Such compositions and methods can be used to transfect or transduce cells in vitro or in vivo, for example, to produce cell lines that express, and preferably secrete, the encoded chimeric polypeptide, or to therapeutically deliver nucleic acids to a subject. The components of the chimeric nucleic acids disclosed herein are typically operably linked in-frame to encode a fusion protein.

[0236] As used herein, a plasmid or viral vector is an agent that transports the nucleic acids of the present disclosure into cells without degradation and includes a promoter that drives expression of the nucleic acid molecule and / or polypeptide in the delivered cell. Examples of viral vectors include adenoviruses, adeno-associated viruses, herpes viruses, vaccinia viruses, polioviruses, Sindbis viruses, and other RNA viruses, including viruses with an HIV backbone. Also preferred are any virus families that share the properties of these viruses, making them suitable for use as vectors. Retroviral vectors are generally described in Coffin et al., Retroviruses, Cold Spring Harbor Laboratory Press (1997), which is incorporated herein by reference for information regarding vectors and methods for their construction. The construction of replication-deficient adenoviruses has also been described (Berkner et al., J. Virol. 61:1213-20 (1987); Massie et al., Mol. Cell. Biol. 6:2872-83 (1986); Haj-Ahmad et al., J. Virol. 57:267-74 (1986); Davidson et al., J. Virol. 61:1226-39 (1987); Zhang et al., BioTechniques 15:868-72 (1993)). The advantage and use of these viruses as vectors is that although they can replicate within the initially infected cell, they are unable to form new infectious viral particles, thereby limiting the extent to which they can spread to other cell types. Recombinant adenoviruses have been shown to achieve high efficiency following direct in vivo delivery to respiratory epithelia, hepatocytes, vascular endothelium, central nervous system parenchyma, and several other tissue sites. Other useful systems include, for example, replicating vaccinia virus vectors and host-restricted non-replicating vaccinia virus vectors.

[0237] The provided polypeptides and / or nucleic acid molecules can be delivered via virus-like particles. Virus-like particles (VLPs) consist of viral protein(s) derived from viral structural proteins. Methods for producing and using virus-like particles are described, for example, in Garcea and Gissmann, Current Opinion in Biotechnology 15:513-7 (2004).

[0238] The provided polypeptides can be delivered by viral components called dense bodies (DBs). DBs transport proteins to target cells via membrane fusion. Methods for producing and using DBs are described, for example, in Pepperl-Klindworth et al., Gene Therapy 10:278-84 (2003).

[0239] The provided polypeptides can be delivered by tegument aggregates. Methods for making and using tegument aggregates are described in International Publication No. WO2006 / 110728.

[0240] Non-viral delivery methods can include an expression vector containing a nucleic acid molecule and a nucleic acid sequence encoding a polypeptide, where the nucleic acid is operably linked to an expression control sequence. Suitable vector backbones include those commonly used in the art, such as plasmids, artificial chromosomes, BACs, YACs, or PACs. Numerous vectors and expression systems are commercially available from companies such as Novagen (Madison, Wis.), Clonetech (Pal Alto, Calif.), Stratagene (La Jolla, Calif.), and Invitrogen / Life Technologies (Carlsbad, Calif.). Vectors typically contain one or more regulatory regions. Regulatory regions include, but are not limited to, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcription initiation sites, termination sequences, polyadenylation sequences, and introns. Such vectors can also be used to produce chimeric polypeptides by expression in suitable host cells, such as CHO cells.

[0241] Preferred promoters for controlling transcription from vectors in mammalian host cells can be obtained from a variety of sources, including viral genomes such as polyoma, simian virus 40 (SV40), adenovirus, retrovirus, hepatitis B virus, and most preferably cytomegalovirus (CMV), or heterologous mammalian promoters such as the β-actin promoter or EF1α promoter, or hybrid or chimeric promoters (e.g., the CMV promoter fused with the β-actin promoter). Of course, promoters derived from the host cell or related species are also useful herein.

[0242] An enhancer generally refers to a DNA sequence that functions at no fixed distance from the transcription start site and may be located 5' or 3' to the transcription unit. Furthermore, enhancers can be present within introns or within the coding sequence itself. They are typically 10 to 300 base pairs (bp) in length and function in cis. Enhancers typically function to increase transcription from nearby promoters. Enhancers may also contain response elements that mediate the regulation of transcription. While many enhancer sequences are known from mammalian genes (globin, elastase, albumin, fetoprotein, insulin), enhancers from eukaryotic viruses are typically used for general expression. Preferred examples include the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0243] The promoter and / or enhancer may be inducible (e.g., chemically or physically regulated). Chemically regulated promoters and / or enhancers may be regulated, for example, by the presence of alcohol, tetracycline, steroids, or metals. Physically regulated promoters and / or enhancers may be regulated by environmental factors such as temperature and light. Optionally, the promoter and / or enhancer region can act as a constitutive promoter and / or enhancer to maximize expression of the region of the transcription unit to be transcribed. In certain vectors, the promoter and / or enhancer region may be active in a cell-type-specific manner. Optionally, in certain vectors, the promoter and / or enhancer region may be active in all eukaryotic cells, independent of cell type. Preferred promoters of this type are the CMV promoter, the SV40 promoter, the β-actin promoter, the EF1α promoter, and retroviral long terminal repeats (LTRs).

[0244] Vectors may also contain, for example, an origin of replication and / or a marker. Marker genes can confer a selectable phenotype (e.g., antibiotic resistance) to cells. Marker products are used to determine whether a vector has been delivered to a cell and, once delivered, whether it is being expressed. Examples of selectable markers for mammalian cells include dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, puromycin, and blastidine. When such selectable markers are successfully transferred into mammalian host cells, the transformed mammalian host cells can survive even when placed under selection pressure. Other examples of markers include, for example, the E. coli lacZ gene, green fluorescent protein (GFP), and luciferase. In addition, expression vectors may contain tag sequences designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as GFP, glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, and FLAG™ tags (Kodak, New Haven, Conn.), are typically expressed as fusions with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including at either the carboxyl or amino terminus.

[0245] As used herein, the terms peptide, polypeptide, or protein are used broadly to refer to two or more amino acids linked by a peptide bond. Protein, peptide, and polypeptide are also used interchangeably herein to refer to an amino acid sequence. It should be recognized that the term polypeptide is not used herein to imply a specific size or number of amino acids comprising the molecule, and that the peptides of the present invention may contain up to a few amino acid residues or more. As used throughout, a subject can be a vertebrate, more specifically a mammal (e.g., human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig), bird, reptile, amphibian, fish, or any other animal. The term does not denote a particular age or sex. Thus, it is intended to encompass adult and newborn subjects, regardless of male or female. As used herein, patient or subject can be used interchangeably and can refer to a subject with a disease or disorder (e.g., cancer). The term patient or subject includes human and veterinary subjects.

[0246] A subject at risk of developing a disease or disorder may have a genetic predisposition to the disease or disorder, e.g., have a family history, or have a mutation in a gene that causes the disease or disorder, or exhibit early signs or symptoms of the disease or disorder. A subject who currently has a disease or disorder may have one or more symptoms of the disease or disorder and may have been diagnosed with the disease or disorder.

[0247] The methods and medicaments described herein are useful for both prophylactic and therapeutic treatments. For prophylactic use, a therapeutically effective amount of a chimeric polypeptide or a chimeric nucleic acid sequence encoding a chimeric polypeptide described herein is administered to a subject pre-onset (e.g., before overt signs of cancer or inflammation) or during the early stages of disease (e.g., at the first signs and symptoms of cancer or inflammation). Prophylactic administration can occur from days to years before the onset of symptoms of cancer or inflammation. Prophylactic administration can be used, for example, in the prophylactic treatment of a subject diagnosed with a genetic predisposition to cancer. Therapeutic treatment involves administering a therapeutically effective amount of a chimeric polypeptide or a nucleic acid sequence encoding a chimeric polypeptide described herein to a subject after the diagnosis or onset of cancer or inflammation (e.g., an autoimmune disease). Prophylactic use can also be applied when a patient is undergoing treatment (e.g., chemotherapy) in which inflammation is expected.

[0248] According to the methods taught herein, a subject is administered an effective amount of an agent (e.g., a chimeric polypeptide). The terms effective amount and effective dosage are used interchangeably. The term effective amount is defined as the amount necessary to produce a desired physiological response. The effective amount and schedule for administering an agent can be determined empirically, and making such a determination is within the skill of one of ordinary skill in the art. The dosage range for administration is large enough to produce the desired effect of affecting (e.g., reducing or delaying) one or more symptoms of a disease or disorder. The dosage should not be so large as to cause substantial adverse side effects (e.g., unwanted cross-reactions, anaphylactic reactions, etc.). Generally, dosage will vary depending on age, condition, sex, type of disease, extent of disease or disorder, route of administration, or whether other drugs are included in the regimen, and can be determined by one of ordinary skill in the art. Dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and one or more doses can be administered daily, for one day, or for several days. Guidance as to appropriate dosages for a given class of pharmaceutical product can be found in the literature.

[0249] As used herein, the terms treatment, treating, or treating refer to a method of reducing the effects of a disease or condition, or the symptoms of a disease or condition. Thus, in the methods of the present disclosure, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or condition, or the symptoms of the disease or condition. For example, a method for treating a disease is considered therapeutic if one or more symptoms of the disease in a subject are reduced by 10% compared to a control. Thus, this reduction can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage reduction between 10% and 100% compared to native or control levels. It should be understood that treatment does not necessarily refer to a cure or complete elimination of a disease, condition, or the symptoms of a disease or condition.

[0250] As used herein, the terms prevent, preventing, and prevention of a disease or disorder refer to an action that inhibits or delays the onset or progression of one or more symptoms of a disease or disorder, occurring before or at about the same time that a subject begins to exhibit one or more symptoms of the disease or disorder, e.g., the administration of a chimeric polypeptide or a nucleic acid sequence encoding a chimeric polypeptide. As used herein, reference to decrease, reduction, or inhibition includes a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more change compared to control levels. Such terms can, but do not necessarily, include complete elimination.

[0251] IL-2 variants that are selective for IL2Rαβγ over IL2Rβγ have been developed (Shanafelt, AB, et al., 2000, Nat Biotechnol. 18:1197-202; Cassell, DJ, et al., 2002, Curr Pharm Des., 8:2171-83). These variants have amino acid substitutions that reduce affinity for IL2RB. Because IL-2 has no detectable affinity for IL2RG, these variants consequently have reduced affinity for the IL2Rβγ receptor complex and reduced ability to activate IL2Rβγ-expressing cells, but retain the ability to bind to IL2RA and to bind and activate the IL2Rαβγ receptor complex.

[0252] One of these variants, IL2 / N88R (Bay 50-4798), has been investigated in clinical trials as a less toxic version of IL-2 as an immune system stimulator, based on the hypothesis that IL2Rβγ-expressing NK cells are a major contributor to toxicity. Bay 50-4798 has been shown to selectively stimulate the proliferation of activated T cells over NK cells and has been evaluated in phase I / II clinical trials in cancer patients (Margolin, K., et al., 2007, Clin Cancer Res., 13:3312-9) and HIV patients (Davey, R.T., et al., 2008, J Interferon Cytokine Res., 28:89-100). These clinical trials demonstrated that Bay 50-4798 was significantly safer and better tolerated than aldesleukin and also increased levels of CD4+CD25+ T cells, a Treg-enriched cell population. Following these clinical trials, research in the field has more fully validated the Treg cell identity and demonstrated that Treg cells selectively express IL2Rαβγ (reviewed in Malek, TR, et al, 2010, Immunity, 33:153-65). Based on this new research, it can now be understood that IL2Rαβγ-selective agonists should be selective for Treg cells.

[0253] In addition, variants can be made that selectively alter affinity for the CD25 chain compared to native Il-2.

[0254] IL-2 can be engineered to produce mutants that bind generally to the IL-2R complex or that bind specifically to the IL-2R α subunit with affinities different from those of the corresponding wild-type IL-2 or a currently available mutant (designated C125S because the cysteine ​​residue at position 125 is replaced with a serine residue).

[0255] Accordingly, the present invention provides mutant interleukin-2 (IL-2) polypeptides that comprise an amino acid sequence that is at least 80% identical (e.g., 85, 87, 90, 95, 97, 98, or 99% identical) to wild-type IL-2 and that bind to the IL-2 trimeric receptor more highly than the dimeric IL-2 receptor compared to wild-type IL-2. *) polypeptides. Typically, the muteins also bind to the IL-2 receptor alpha subunit (IL-2Rα) with a higher affinity than wild-type IL-2 binds to IL-2Rα. The amino acid sequence within the mutant IL-2 polypeptide can vary from SEQ ID NO: 1 (UniProtKB Accession No. P60568) by including (or including only) one or more amino acid substitutions, which can be considered conservative or non-conservative substitutions. Unnatural amino acids can also be incorporated. Alternatively or additionally, the amino acid sequence can vary from SEQ ID NO: 1 (which can be considered the "reference" sequence) by including, as well as adding and / or deleting, one or more amino acid residues. More specifically, the amino acid sequence may differ from that of SEQ ID NO: 1 by mutation of at least one of the following positions (or a combination thereof): 1, 4, 8, 9, 10, 11, 13, 15, 26, 29, 30, 31, 35, 37, 46, 48, 49, 54, 61, 64, 67, 68, 69, 71, 73, 74, 75, 76, 79, 88, 89, 90, 92, 99, 101, 103, 114, 125, 128, or 133 of SEQ ID NO: 1. As noted above, only one of these positions may be altered, or two, three, four, five, six, seven, eight, nine, ten, or eleven or more (up to and including all) of these positions may be altered. For example, the amino acid sequence may differ from SEQ ID NO: 1 at positions 69 and 74, and additionally at one or more of positions 30, 35, and 128.The amino acid sequence may also differ from SEQ ID NO:2 (as disclosed in US7569215, incorporated herein by reference) at one of the following sets of positions: (a) positions 64, 69, and 74; (b) positions 69, 74, and 101; (c) positions 69, 74, and 128; (d) positions 30, 69, 74, and 103; (e) positions 49, 69, 73, and 76; (f) positions 69, 74, 101, and 133; (g) positions 30, 69, 74, and 128; (h) positions 69, 74, 88, and 99; (i) positions 30, 69, 74, and 128; (j) positions 9, 11, 35, 69, and 74; (k) positions (l) positions 1, 46, 49, 61, 69, and 79; (l) positions 48, 68, 71, 90, 103, and 114; (m) positions 4, 10, 11, 69, 74, 88, and 133; (n) positions 15, 30, 31, 35, 48, 69, 74, and 92; (O) positions 30, 68, 69, 71, 74, 75, 76, and 90 (p) positions 30, 31, 37, 69, 73, 74, 79, and 128; (q) positions 26, 29, 30, 54, 67, 69, 74, and 92; (r) positions 8, 13, 26, 30, 35, 37, 69, 74, and 92, and (s) positions 29, 31, 35, 37, 48, 69, 71, 74, 88, 89. Other than the mutations at these positions, the amino acid sequence of the variant IL-2 polypeptide may otherwise be identical to SEQ ID NO:1. For certain substitutions, the amino acid sequence may differ from SEQ ID NO: 1 by having one or more of the following mutations: A1T, S4P, K8R, K9T, T10A, Q11R, Q13R, E15K, N26D, N29S, N30S, N30D, N30T, Y31H, Y31C, K35R, T37A, T37R, M46L, K48E, K 49R, K49E, K54R, E61D, K64R, E67G, E68D, V69A, N71T, N71A, N71R, A73V, Q74P, S75P, K76E, K76R, H79R, N88D, I89V, N90H, I92T, S99P, T101A, F103S, I114V, I128T, I128A, T133A, or T133N. Our nomenclature is consistent with that used in the scientific literature, i.e., the single-letter code for the amino acid in the wild-type or reference sequence is given, followed by the position of the code in the sequence, and then the single-letter code for the substituted amino acid.Thus, A1T indicates that the alanine residue at position 1 has been substituted with threonine. Other variant polypeptides within the scope of the invention include those comprising variants of SEQ ID NO:2 having substitutions at V69 (e.g., A) and Q74 (e.g., P). For example, the amino acid sequence can comprise one of the following sets of mutations with respect to SEQ ID NO:2: (a) K64R, V69A, and Q74P; (b) V69A, Q74P, and T101A; (c) V69A, Q74P, and I128T; (d) N30D, V69A, Q74P, and F103S; (e) K49E, V69A, A73V, and K76E; (f) V69A, Q74P, T101A, and T133N; (g) N30S, V69A, Q74P, and I128T. 128A; (h) V69A, Q74P, N88D, and S99P; (i) N30S, V69A, Q74P, and I128T; (j) K9T, Q11R, K35R, V69A, and Q74P; (k) A1T, M46L, K49R, E61D, V69A, and H79R; (l) K48E, E68D, N71T, N90H, F103S, and I114V; (m) S4P , T10A, Q11R, V69A, Q74P, N88D, and T133A; (n) E15K, N30S, Y31H, K35R, K48E, V69A, Q74P, and I92T; (o) N30S, E68D, V69A, N71A, Q74P, S75P, K76R, and N90H; (p) N30S, Y31C, T37A, V69A, A73V, Q74P, H79R and I128T; (q) N26D, N29S, N30S, K54R, E67G, V69A, Q74P, and I92T; (r) K8R, Q13R, N26D, N30T, K35R, T37R, V69A, Q74P, and I92T; and (s) N29S, Y31H, K35R, T37A, K48E, V69A, N71R, Q74P, N88D, and I89V. SEQ ID NO: 2 is disclosed in U.S. Patent No. 7,569,215, which is incorporated herein by reference as an exemplary IL-2 polypeptide sequence that may be used in the present invention.

[0256] As noted above, any variant IL-2 polypeptide disclosed herein can include the recited sequence, or the polypeptide can be limited to the recited sequence and otherwise identical to SEQ ID NO: 1. Additionally, any variant IL-2 polypeptide described herein can optionally include a substitution of the cysteine ​​residue at position 125 with another residue (e.g., serine) and / or can optionally include a deletion of the alanine residue at position 1 of SEQ ID NO: 1.

[0257] The variant IL-2 polypeptides disclosed herein have a K of less than about 28 nM (e.g., less than about 25 nM; less than about 5 nM; less than about 1 nM; less than about 500 pM; or less than about 100 pM). d More specifically, the mutant IL-2 polypeptide may have an affinity equilibrium constant of less than 1.0 nM (e.g., about 0.8, 0.6, 0.4, or 0.2 nM). Affinity may also be expressed as the relative rate of dissociation from the IL-2R α subunit or IL-2 receptor complex (e.g., a complex expressed on the cell surface or otherwise membrane-bound). For example, the mutant IL-2 polypeptide may bind to the IL-2R α subunit at a rate lower than the wild-type polypeptide or an IL-2-based therapeutic (e.g., IL-2 * ) dissociates from, for example, IL-2Rα at a reduced rate compared to IL-2 * It can be characterized as the time, or mean time, that a polypeptide remains on the surface of a cell, e.g., expressing IL-2R. * The polypeptide can remain on the receptor for at least about 2, 5, 10, 50, 100, or 250 times (or more).

[0258] Disclosed are materials, compositions, and components that can be used for, used in conjunction with, used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and where combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that specific reference to each of the various individual and collective combinations and permutations of these compounds cannot be explicitly disclosed, but each is specifically contemplated and described herein. For example, where a method is disclosed and discussed, and multiple modifications that can be made to multiple molecules comprising the method are discussed, all combinations and permutations of the method and possible modifications are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of the present disclosure, including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are various additional steps that may be performed, it should be understood that each of these additional steps may be performed with any specific method step or combination of method steps of the methods of the present disclosure, and that each such combination or subset of combinations should be considered to be specifically contemplated and disclosed.

[0259] Publications cited herein and the material for which they are cited are specifically incorporated herein by reference in their entirety. 6. Incorporation by Reference All publications, patents, and patent applications mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. However, the citation of a reference herein should not be construed as an admission that such reference is prior art to the present invention. In the event that definitions or terminology set forth in a reference incorporated by reference differ from the terms or discussion set forth herein, the current terms and definitions shall control. [Example]

[0260] The following are examples of methods and compositions of the present invention. It will be understood that various other embodiments may be practiced given the general description provided herein.

[0261] Example 1. Detection of IL-2, IL-2 muteins, IL-2Rα, and IL-2Rγ in fusion proteins by ELISA IL-2 muteins are detected using commercially available antibodies, such as anti-IL-2 monoclonal antibody (JES6-1A12) (BD Pharmingen; San Jose, Calif.). Positive controls are used to demonstrate whether the monoclonal antibody recognizes the cytokine or mutein. Antibodies against the IL-2Rα and IL-2Rγ chains are also used. Wells of a 96-well plate are coated with the antibody (2.5 μg / ml) in PBS. The wells are blocked with 5% nonfat milk and 0.2% Tween® 20 in PBS (PBS-M-Tw), and the fusion protein is added for 1–2 hours at 37°C. After washing, an anti-IL-2 biotin-labeled antibody (e.g., JES5H4 (BD Pharmingen)) is added, and binding is detected using streptavidin-HRP (Southern Biotechnology Associates; Birmingham, Ala.). The ELISA plate is developed by adding 50 μl of O-phenylenediamine (OPD) (Sigma-Aldrich) in 0.1 M citric acid pH 4.5 and 0.04% H 2 O 2 , stopped by adding 50 μl / well of 2N H 2 SO 4 , and the absorbance is read at 490 nm.

[0262] Example 2: Protease cleavage of fusion proteins by MMP9 protease Those skilled in the art will be familiar with how to set up a protein cleavage assay. 100 μg of protein in 1×PBS pH 7.4 was cleaved with 1 μg of active MMP9 (Sigma catalog no. SAE0078-50 or Enzo catalog no. BML-SE360) and incubated at room temperature for up to 16 hours. The digested protein was then used in functional assays or stored at −80° C. before testing. The extent of cleavage was monitored by SDS-PAGE using methods well known in the art. As shown in Figures 10, 13, 18A, 18b, and 27A, complete cleavage of the fusion protein by MMP9 protease is observed.

[0263] Example 3: CTLL-2 assay CTLL2 cells (ATCC) were plated at 500,000 cells / well in culture medium with or without 40 mg / ml human serum albumin (HSA) and stimulated with serial dilutions of recombinant hIL2 or activatable hIL2 for 72 hours at 37°C and 5% CO2. The activity of uncleaved and cleaved activatable hIL2 was tested. Cleaved activatable hIL2 was generated by incubation with active MMP9. Cell viability was assessed using the CellTiter-Glo® (Promega) luminescent cell viability assay. The results are shown in Figures 8A-8F, 9A-9Z, and 25C.

[0264] Example 4: Protease cleavage of IL-2 / IL-2Rα / IL-2Rγ chimeric polypeptides increases accessibility to antibodies and biologically active IL-2 muteins The IL-2 mutein fusion proteins are biochemically characterized before and after cleavage with a protease (e.g., PSA). Immunoblot analysis demonstrates that the fusion proteins can be cleaved by PSA, and that the intensity of the predicted low-molecular-weight cleavage product of approximately 20 kDa, reactive with anti-IL-2 antibodies, increases after treatment of the samples with PSA. The extent of cleavage depends on the amount of PSA and the incubation time. Interestingly, when the fusion proteins are analyzed by ELISA before and after PSA treatment, the apparent amount of IL-2 increases after PSA cleavage. In this experiment, the apparent amount of IL-2 detected using this sandwich ELISA increases approximately two- or four-fold, depending on the construct. This suggests that antibody binding is partially blocked by the intact fusion protein. Aliquots of the same samples are also analyzed after PSA treatment with the CTLL-2 cell line, which requires IL-2 for growth and survival. Cell viability can be confirmed using a colorimetric MTT assay. In this assay, the supernatant contains more biologically active IL-2 at higher dilutions, and the amount of biologically active IL-2 increases after PSA cleavage. The increased amount of IL-2 mutein suggests an increase in the predicted low molecular weight cleavage fragment of approximately 20 kDa that is reactive with anti-IL-2 antibodies after PSA cleavage, increased antibody accessibility, and, most importantly, an increased amount of biologically active IL-2 mutein.

[0265] Example 5. In vivo delivery of protease-activated fusion proteins reduces tumor growth The chimeric polypeptides will be examined to determine whether they can have biological effects in vivo. For these experiments, we use a system in which tumor cells injected intraperitoneally initially rapidly and preferentially attach and grow in milky spots (a series of organized immune aggregates found on the omentum) (Gerber et al., Am. J. Pathol. 169:1739-52 (2006)). This system allows for multiple intraperitoneal delivery of fusion proteins and allows tumor growth to be analyzed by examining dissociated omental cells, providing a convenient method for examining the effect of fusion protein treatment on tumor growth. In these experiments, we can use the Colon38 cell line, a rapidly growing tumor cell line that expresses both MMP2 and MMP9 in vitro. Normally, omental tissue expresses relatively low amounts of MMP2 and MMP9, but the presence of Colon38 tumors in the omentum increases MMP levels. This tumor model will be used to examine the ability of IL-2 mutein fusion proteins to affect tumor growth. Colon38 cells are injected intraperitoneally and allowed to attach and grow for 1 day, after which the fusion protein is administered intraperitoneally daily. On day 7, the animals are sacrificed and the omentum is examined for tumor growth using flow cytometry and colony formation assays.

[0266] Example 6: Quantification of antigen affinity by flow cytometry Human CD20, an activatable interleukin protein + Cells and cynomolgus monkey CD20 + The binding affinity to the cells is tested.

[0267] CD20 +The cells were incubated with 100 μL of serial dilutions of activatable interleukin protein and at least one protease. After washing three times with FACS buffer, the cells were incubated with 0.1 mL of 10 μg / mL mouse monoclonal anti-idiotypic antibody in the same buffer for 45 minutes on ice. After a second washing cycle, the cells were incubated with 0.1 mL of 15 μg / mL FITC-conjugated goat anti-mouse IgG antibody under the same conditions as before. As a control, the cells were incubated with anti-His IgG followed by FITC-conjugated goat anti-mouse IgG antibody without activatable IL2 protein. The cells were then washed again and resuspended in 0.2 mL of FACS buffer containing 2 μg / mL propidium iodide (PI) to exclude dead cells. 1 × 10 4 The fluorescence of live cells is measured using a Beckman-Coulter FC500 MPL flow cytometer (Beckman-Coulter, Krefeld, Germany) with MXP software or a Millipore Guava EasyCyte flow cytometer (Merck Millipore, Schwalbach, Germany) with Incyte software. The mean fluorescence intensity of the cell samples is calculated using CXP software (Beckman-Coulter, Krefeld, Germany) or Incyte software (Merck Millipore, Schwalbach, Germany). After subtracting the fluorescence intensity values ​​of cells stained with secondary and tertiary reagents only, the K value is calculated using the equation for one-site binding (hyperbolic) in GraphPad Prism (version 6.00 for Windows, GraphPad Software, La Jolla, California, USA). D Calculate the value.

[0268] CD20 binding and cross-reactivity were measured using human CD20 + Evaluate on tumor cell lines. K D The ratio was determined in CHO cell lines expressing recombinant human or recombinant cynomolgus antigens. D Calculate using the value.

[0269] Example 7: Cytotoxicity assay activatable interleukin protein CD20 + Mediating an immune response against target cells is assessed in vitro.

[0270] Fluorescently labeled CD20 + REC-1 cells (mantle cell lymphoma cell line, ATCC CRL-3004) were incubated with isolated PBMCs from random donors or CB15 T cells (a standardized T cell line) as effector cells in the presence of activatable IL2 protein and at least one protease. After 4 hours of incubation at 37°C in a humidified incubator, the release of a fluorescent dye from the target cells into the supernatant was quantified using a spectrofluorometer. Target cells incubated without activatable IL2 protein and target cells completely lysed by adding saponin at the end of incubation served as negative and positive controls, respectively.

[0271] Based on the measurement of remaining viable target cells, calculate the percentage of specific cell lysis according to the following formula: [1 - (number of viable targets)] (試料) / number of raw targets (自発) )] × 100%. Sigmoidal dose-response curve and EC 50 Values ​​are calculated by nonlinear regression / four-parameter logistic fit using GraphPad software. Lysis values ​​obtained at a given antibody concentration are used to calculate a sigmoidal dose-response curve by four-parameter logistic fit analysis using Prism® GraphPad® software.

[0272] Example 8: Pharmacokinetics of activatable interleukin proteins The half-life of activatable interleukin proteins is assessed in animal studies.

[0273] The activatable IL2 protein is administered as a 0.5 mg / kg bolus injection into the saphenous vein of cynomolgus monkeys. Another group of cynomolgus monkeys receives an IL2 construct of equivalent size but lacking the serum half-life extending element. Groups 3 and 4 receive an IL2 construct with a serum half-life extending element, and CD20 and a cytokine with a serum half-life extending element, respectively, both of equivalent size to the activatable interleukin protein. Each test group consists of five monkeys. Serum samples are collected at the indicated time points and serially diluted, followed by quantitation of protein concentration using a binding ELISA against CD20.

[0274] Pharmacokinetic analysis is performed using plasma concentrations of the test article. When plotted against time after administration, the group mean plasma data for each test article fit a multi-exponential profile. The data are fit by a standard two-compartment model with a bolus input and first-order rate constants for the distribution and elimination phases. The general equation that best fits intravenous data is: c(t) = Ae -αt +Be -βt (c(t) is the plasma concentration at time t, A and B are the Y-axis intercepts, and α and β are the apparent first-order rate constants for the distribution and elimination phases, respectively. The α phase is the initial phase of clearance and reflects distribution of the protein to all extracellular fluids of the animal, while the second or β portion of the decay curve represents the true plasma clearance. Methods for fitting such equations are well known in the art. For example, A=D / V(α-k21) / (α-β), B=D / V(β-k21) / (α-β), and α and β (if α>β) are fitted to a quadratic equation: r with estimated parameters: V=volume of distribution, k10=excretion rate, k12=rate of transfer from compartment 1 to compartment 2, k21=rate of transfer from compartment 2 to compartment 1, and D=administered dose. 2 +(k12+k21+k10)r+k21k10=0 is a root of

[0275] Data Analysis. Concentration versus time profile graphs are generated using KaleidaGraph (KaleidaGraph™ V.3.09 Copyright 1986-1997. Synergy Software. Reading, Pa.). Values ​​reported as less than reportable (LTR) are not included in the PK analysis and are not displayed on the graphs. Pharmacokinetic parameters are determined by compartmental analysis using WinNonlin software (WinNonlin® Professional V.3.1 WinNonlin™ Copyright 1998-1999. Pharsight Corporation. Mountain View, Calif.). Pharmacokinetic parameters are calculated as described in Ritschel WA and Kearns GL, 1999, IN: Handbook of Basic Pharmacokinetics Including Clinical Applications, 5th edition, American Pharmaceutical Assoc., Washington, DC.

[0276] Activatable interleukin proteins are expected to have improved pharmacokinetic parameters, such as increased elimination half-life, compared to proteins lacking serum half-life extending elements.

[0277] Example 9: Xenograft tumor model The activatable IL2 protein is evaluated in a xenograft model.

[0278] Female immunodeficient NOD / scid mice were irradiated with sublethal doses of radiation (2 Gy) and 4 × 10 6 Ramos RA1 cells were subcutaneously inoculated into the right dorsal region. 3 Once the number reaches 10, animals are assigned to three treatment groups: Groups 2 and 3 (8 animals each) receive 1.5 x 10 7Activated human T cells are injected intraperitoneally. Three days later, animals in Group 3 are subsequently treated with a total of nine intravenous doses of 50 μg of activatable interleukin protein. Groups 1 and 2 are treated with vehicle only. Body weights and tumor volumes are determined for 30 days.

[0279] Animals treated with activatable interleukin proteins are expected to have a statistically significant delay in tumor growth compared to the respective vehicle-treated control groups.

[0280] Example 10: Mouse IFNγ WEHI cell viability assay WEHI279 cells (ATCC) were plated at 25,000 cells / well in culture medium with or without 1.5% human serum albumin (HSA) and stimulated with serial dilutions of recombinant mIFNγ or inducible mIFNγ for 72 hours at 37°C and 5% CO2. The activity of uncleaved and cleaved inducible mIFNγ was tested. Cleaved inducible mIFNγ was generated by incubation with active MMP9. Cell viability was assessed using a CellTiter-Glo (Promega) luminescence-based cell viability assay. The EC50 values ​​of the cleaved inducible mIFNγ molecules were at least 100-fold more potent than the uncleaved inducible mIFNγ molecules. As shown in Figures 16A-16B, greater induction was observed in assays in which the culture medium contained human serum albumin.

[0281] Example 11: Reserved Example 12: Mouse IFNγ B16 reporter cell assay B16-Blue IFNγ cells (InvivoGen) were plated at 75,000 cells / well in culture medium with or without 1.5% human serum albumin (HSA) and stimulated with serial dilutions of recombinant mIFNγ or inducible mIFNγ for 24 hours at 37°C and 5% CO2. The activity of uncleaved and cleaved inducible mIFNγ was tested. Cleaved inducible mIFNγ was generated by incubation with activated MMP-9. Supernatants were collected, and QUANTI-Blue reagent (InvivoGen) was added. The cells were incubated at 37°C for 2 hours. SEAP activation was assessed by measuring absorbance at 620 nm. The EC50 values ​​of the cleaved inducible mIFNγ molecules were at least 100-fold more potent than those of the uncleaved inducible mIFNγ molecules. The results are shown, for example, in Figures 19A-19B, 22A-22B, and 23A-23B. This experiment was repeated for the IFNα conjugate using B16-Blue IFNα / β cells. The EC50 values ​​for the cleaved mIFNα molecules were at least 100-fold more potent than the uncleaved mIFNα molecules. See Figures 20A-20B. Example 13. In vivo delivery of protease-activated fusion proteins reduces tumor growth

[0282] The chimeric polypeptides will be examined to determine whether they can have biological effects in vivo. For these experiments, we use a system in which intraperitoneally injected tumor cells initially rapidly and preferentially attach and grow in milky spots (a series of organized immune aggregates found on the omentum) (Gerber et al., Am. J. Pathol. 169:1739-52 (2...

Claims

1. A polypeptide consisting of the protease-cleavable amino acid sequence of SEQ ID NO:

195.

2. A polypeptide comprising Formula I, [D1]-[L1]-[D2] wherein D1 is a first domain of interest; L1 is a separate moiety that connects or joins D1 to D2, said separate moiety comprising the amino acid sequence of SEQ ID NO: 195; The polypeptide, wherein D2 is a second domain of interest.

3. The polypeptide of claim 2, wherein the detached portion comprises an amino acid sequence that is a substrate for at least one protease present in the tumor microenvironment of a human tumor.

4. 3. The polypeptide of claim 2, wherein the separating portion comprises two or more cleavable moieties, each of the cleavable moieties being a substrate for a protease.

5. 3. The polypeptide of claim 2, comprising a first cleavable portion comprising a first amino acid sequence that is a substrate for a first protease, and a second cleavable portion comprising a second amino acid sequence that is a substrate for a second protease.

6. The polypeptide of claim 2, further comprising a non-cleavable linker sequence.

7. The polypeptide of claim 2 , wherein the polypeptide comprises a cytokine, a chemokine, a growth factor, a soluble receptor, or any combination thereof.

8. The polypeptide of claim 2 , wherein the polypeptide comprises at least one of an extracellular domain, a transmembrane domain, and an intracellular domain.

9. The polypeptide of claim 2, wherein the polypeptide comprises a subunit of a cell surface receptor, a chimeric antigen receptor (CAR), or a T cell receptor (TCR).

10. The polypeptide of claim 2 , wherein the polypeptide comprises an antigen-binding polypeptide, an antibody, or an antigen-binding portion thereof.

11. 3. The polypeptide of claim 2, wherein the polypeptide is operably linked to a moiety selected from the group consisting of a polypeptide moiety, a lipid moiety, a nucleic acid moiety, a detectable moiety, and a small molecule.

12. A recombinant proprotein comprising: a. a recombinant polypeptide comprising a cleavable moiety that is a substrate for a protease, wherein the cleavable moiety comprises the amino acid sequence of SEQ ID NO: 195; b. A polypeptide having biological activity The recombinant proprotein comprising:

13. 13. The recombinant proprotein of claim 12, wherein the polypeptide having biological activity comprises a cytokine, a chemokine, a growth factor, a soluble receptor, or a combination thereof.

14. 13. The recombinant proprotein of claim 12, wherein the biologically active polypeptide comprises at least one of an extracellular domain, a transmembrane domain, and an intracellular domain.

15. 13. The recombinant proprotein of claim 12, wherein the biologically active polypeptide comprises a cell surface receptor, a chimeric antigen receptor (CAR), or a T cell receptor (TCR) subunit.

16. 13. The recombinant proprotein of claim 12, wherein the polypeptide having biological activity comprises an antigen-binding polypeptide, an antibody, or an antigen-binding portion thereof.

17. 13. The recombinant proprotein of claim 12, wherein the cleavable moiety links the biologically active polypeptide to another amino acid sequence.

18. 13. The recombinant proprotein of claim 12, wherein the cleavable moiety that is a substrate for a protease links the biologically active polypeptide to another amino acid sequence.

19. 13. The recombinant proprotein of claim 12, wherein the biological activity of the recombinant proprotein is attenuated, and cleavage of the cleavable moiety by the protease produces a polypeptide having non-attenuated biological activity.

20. 13. The recombinant proprotein of claim 12, further comprising a blocking moiety capable of blocking the biological activity of the recombinant proprotein, selected from a steric blocking moiety, a specific blocking moiety, and combinations thereof.

21. 21. The recombinant proprotein of claim 20, wherein the blocking moiety comprises a steric blocking moiety comprising human serum albumin (HSA), an anti-HSA antibody, immunoglobulin Fc, or a fragment of any of the foregoing.

22. 21. The recombinant proprotein of claim 20, wherein the blocking moiety comprises a specific blocking moiety comprising an antibody or antigen-binding fragment thereof having binding specificity for the biologically active polypeptide, or a ligand-binding portion or ligand-binding fragment thereof of a receptor having binding specificity for the biologically active polypeptide.

23. 13. The recombinant proprotein of claim 12, further comprising a half-life prolonging domain.

24. 13. The recombinant proprotein of claim 12, wherein the biologically active polypeptide comprises at least one of an extracellular domain, a transmembrane domain, and an intracellular domain.

25. A nucleic acid encoding a polypeptide according to any one of claims 1 to 11 or a recombinant proprotein according to any one of claims 12 to 24.

26. A vector comprising the nucleic acid of claim 25.

27. A host cell comprising the vector of claim 26.

28. 28. A method for producing a pharmaceutical composition, comprising culturing the host cell of claim 27 under conditions suitable for the expression and harvesting of a desired polypeptide.

29. A pharmaceutical composition for treating a subject in need thereof, comprising a polypeptide according to any one of claims 1 to 11 or a recombinant proprotein according to any one of claims 12 to 24.

Images