Separation part and method of use thereof

Abstract

To provide separation moieties, and methods of use thereof.SOLUTION: Provided herein are separation moieties that are suitable for use in conjunction with a variety of therapeutic payloads. The separation moieties serve to generate conditionally active macromolecules whereby the macromolecules have reduced or minimal biological activity until the separation moieties are modified under specific conditions. Provided herein are compositions and methods to generate and use high efficiency separation moieties and / or linkers. The linkers can confer site-selectivity with regards to biological activity of the attached payload or payloads. In some embodiments, the separation moieties and / or linkers are used in conjugation with therapeutic proteins to treat a disease or disorder, such as proliferative disease, a tumorous disease, an inflammatory disease, an immunological disease, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, and a graft-versus-host disease.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] Sequence List This application includes a sequence listing submitted electronically in ASCII format, the entirety of which is incorporated herein by reference. The aforementioned ASCII copy was created on 14 May 2020, named 761146_000140_SL.txt, and has a size of 896,815 bytes. [Background technology]

[0003] Recombinant fusion proteins containing two or more functional polypeptides are utilized 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, thereby extending the plasma half-life and / or achieving therapeutic effects. 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 misfolding of the fusion protein (Zhao et al, (2008), Protein Expr. Purif., 61:73-77) and low yield of protein production (Amet). This can lead to many undesirable outcomes, including those beyond biological activity (Bai et al., (2006) Proc. Natl Acad. Sci. USA, 102: 7292-7296). One approach to overcome these problems is to use linker sequences between the component polypeptides or domains of the fusion protein. However, selecting a linker suitable 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. The characteristics of the linker sequence (e.g., length, hydrophobicity, amino acid composition, secondary structure, and overall folding) can affect linker compatibility and should be considered when designing and selecting an appropriate linker. In addition, linkers capable of cleaving active therapeutic agents (e.g., therapeutic polypeptides) under selected conditions or at selected biological sites (e.g., tumor microenvironment) can deliver targeted pharmacological activity of therapeutic agents and reduce undesirable systemic effects. This further complicates the design of suitable linkers. There is a need for improved liner sequences that can be used for the preparation of stable fusion proteins, including linkers that can be cleaved under selected conditions. Therefore, novel isolation segments or linkers are disclosed herein. Using the isolation segments or linkers disclosed herein, for example, prodrugs (e.g., conditionally active and / or targeted cytokines) can be specifically delivered to target sites where the linkers are treated 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 [Overview of the Initiative] [Means for solving the problem]

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

[0007] This specification discloses recombinant polypeptides comprising an isolated portion, wherein the isolated portion comprises 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-activating 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 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 moiety may include amino acid sequences that are substrates of at least two proteases. In another embodiment, the separation moiety may include two or more cleavable moieties, each of which is a substrate of a protease. The separation moiety may include a first cleavable moiety containing a first amino acid sequence that is a substrate of a first protease, and a second cleavable moiety containing a second amino acid sequence that is a substrate of a second protease. In embodiments, the disclosure relates to a recombinant polypeptide comprising a separation moiety containing a protease cleavage motif disclosed herein. The recombinant polypeptide may include a separation moiety containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 195-220, or an amino acid sequence having 90% or more identity with SEQ ID NOs: 195-220. Preferred separation moieties include the sequences GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198). The disclosure also relates to functional variants of the separation moiety containing SEQ ID NOs: 195-220. A functional variant of Sequence ID No. 195 may include any of Sequence ID Nos. 258-331. A functional variant of Sequence ID No. 198 may include any of Sequence ID No. 199 or Sequence ID Nos. 332-408. The separable portion disclosed herein may include formula I:[D1]-[L1]-[D2], where D1 is the first target domain.L1 is a segregation that connects or binds D1 to D2, and this segregation contains an amino acid sequence selected from SEQ ID NOs. 195-220, or an amino acid sequence having at least approximately 90% identity to SEQ ID NOs. 195-220. D2 is the second target domain.

[0008] In one embodiment, the recombinant polypeptide may further include a non-cleavable linker sequence. The recombinant polypeptide may include therapeutic proteins, such as cytokines, chemokines, growth factors, soluble receptors, or antigen-binding moieties of antibodies (e.g., scFV, dAb). In another embodiment, the recombinant polypeptide includes cytokines, chemokines, growth factors, soluble receptors, or any combination thereof. In yet another embodiment, the recombinant polypeptide includes at least one of an extracellular domain, a transmembrane domain, and an intracellular domain. In one embodiment, the recombinant polypeptide includes a cell surface receptor, a chimeric antigen receptor (CAR), or a T cell receptor (TCR) subunit. In one embodiment, the recombinant polypeptide includes an antigen-binding polypeptide, an antibody, or its antigen-binding moiety.

[0009] In one embodiment, the cleavable portion 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, 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). One or more proteases may also include proteases selected from cathepsins (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 native polypeptide substrate of FAPα, CTSL1, ADAM (selected from ADAM8, ADAM9, ADAM10, ADAM12, ADAM17, and ADAMTS1) and MMP (selected from MMP1, MMP2, MMP9, and MMP14), 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 than 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 hepatic 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 isolation portions. In one embodiment, the recombinant polypeptide is responsively bound to a portion selected from the group consisting of a polypeptide portion, a lipid portion, a nucleic acid portion, a detectable portion, and a small molecule.

[0011] This specification provides a recombinant proprotein comprising a recombinant polypeptide containing a cleavable moiety that is a substrate for a protease, wherein the cleavable moiety contains 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 the protease produces a polypeptide with unattenuated biological activity. In one embodiment, the biologically active polypeptide includes cytokines, chemokines, growth factors, soluble receptors, or combinations thereof. In some preferred embodiments, the linker is a component of a therapeutically useful fusion protein, which is not cleaved or is cleaved with low efficiency in the peripheral circulation, but is cleaved with high efficiency at desired locations in the body, e.g., tumor microenvironment or inflammatory sites. In another embodiment, the biologically active polypeptide includes at least one of an extracellular domain, a transmembrane domain, and an intracellular domain. In another embodiment, the biologically active polypeptide includes 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 its antigen-binding moiety.

[0012] The 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 specific blocking moiety, and a combination thereof; and c. a peptide linker comprising a cleavage moiety having at least one protease-cleaving sequence (e.g., a cleavage moiety disclosed herein). In one embodiment, the fusion protein has (a) and (b) functionally linked by (c). In another embodiment, the fusion protein has a peptide linker comprising two or more copies of the same cleavage moiety. In another embodiment, the fusion protein has a steric blocking moiety comprising human serum albumin (HSA) or an anti-HSA antibody. In another embodiment, the fusion protein has a signaling protein that 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 has a signaling protein that is an interleukin 2 amino acid sequence. In another embodiment, the fusion protein further comprises one or more half-life extension domains, the domains not being specific blockers. In another embodiment, the fusion protein is generated by rearranging the non-native N-terminus and / or C-terminus in a circular fashion.

[0013] The fusion polypeptides provided herein may comprise a first polypeptide fusion partner bound to a ligand by a protease-cleaving linker, wherein the catalytic efficiency of the cleaving linker is optimized, the ligand is optionally modified, and the first polypeptide fusion partner is a blocking moiety that prevents the modified ligand from binding to the target receptor or a subunit of the target receptor until cleavage by the protease-cleaving linker.

[0014] Furthermore, the fusion polypeptide provided herein may also be a fusion polypeptide comprising a first polypeptide fusion partner bound to a ligand by a protease-cleaving linker, wherein the catalytic efficiency of the cleaving linker is optimized, the ligand is optionally modified, including by cyclic rearrangement, to create a non-native N-terminus and a novel C-terminus compared to the native ligand, at least one of the novel N-terminus or novel C-terminus of the modified ligand is responsively bound to the first polypeptide fusion partner to form a fusion polypeptide, the first polypeptide fusion partner being a blocking portion that prevents binding of the modified ligand to the target receptor or a subunit of the target receptor until cleavage by the protease-cleaving 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 including a second blocking moiety. In another embodiment, the second polypeptide fusion partner is a different type of blocking moiety than that of the first polypeptide fusion partner. In another embodiment, the first polypeptide fusion partner is albumin, and the second polypeptide fusion partner is a domain containing 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 part of a cytokine receptor, a novel affinity peptide specific to cytokines, 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 that of the first polypeptide fusion partner.

[0016] In one embodiment, the fusion protein further comprises a tumor antigen-binding component. In another embodiment, the fusion protein further comprises a serum half-life extension domain. In another embodiment, the ligands include helix bundle proteins and cytokines (but are 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 (IL-27p28), 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), and β-trefoil proteins (but are not limited to IL-1α, IL-1β, IL-1Ra, IL-18, IL-33, IL-36Ra). (including 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 protein (including, but not limited to, triose phosphate isomerase), beta sandwich protein (including, but not limited to, galectin-1, galectin-3), TNF-beta, 7 β-propeller proteins, Class 1 The ligand is selected from the group consisting of MHC α1α2 domains, integrin I domains, GYF domains, C1 domains, C2 domains (e.g., cPLA2, PKC, synaptotagmin), PDZ domains, C3d, and C5a. In one embodiment, the ligand comprises one or more fragments of the IL-2 polypeptide or its equivalent.In another embodiment, the protease-cleavable linker polypeptide comprises a sequence that can be cleaved 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 proteases. In another embodiment, the cytokine or its fragment or mutein is substantially dissociated from the cytokine-blocking moiety after the protease-cleavable polypeptide linker has been cleaved by the protease.

[0017] This specification discloses a fusion polypeptide comprising at least one of the following: a cytokine polypeptide or a functional fragment or mutain [A]; a cytokine blocking moiety [B]; and an optimized protease-cleaving polypeptide linker [L], wherein the blocking moiety is selected from the group consisting of antibodies, antibody fragments, and albumins, and the cytokine comprises a cyclically rearranged cytokine. In some embodiments, the fusion protein further comprises a tumor antigen-binding component and / or a serum half-life extension domain.In some embodiments, the fusion protein comprises a cytokine peptide or its functional fragment or mutain, a helix bundle protein and cytokines (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 (IL-27p28), 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), FAP (e.g., Fapα), ADAM (selected from ADAM8, ADAM9, ADAM10, ADAM12, ADAM17, and ADAMTS1), β-Trefoil Proteins (not limited to, but including 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, The cytokine peptide 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, and FGF-23), α / β (TIM) barrel proteins (including, but not limited to, triose phosphate isomerase), beta sandwich proteins (including, but not limited to, galectin-1 and galectin-3), TNF-β, seven β-propeller proteins, class 1 MHC α1α2 domains, integrin I domains, GYF domains, C1 domains, C2 domains (e.g., cPLA2, PKC, synaptotagmin), PDZ domains, C3d, and C5a. In one embodiment, the cytokine peptide or its functional fragment or mutein contains IL-2.In another embodiment, the cytokine blocking moiety includes a ligand-binding domain or fragment or mutein of a cytokine's homologous receptor, a single-domain antibody or scFv that binds to a cytokine polypeptide or its functional fragment or mutein, 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 its fragment.

[0018] This specification discloses fusion polypeptides comprising a protease-cleavable moiety, wherein the sequence is catalytically optimized for cleavage by a particular protease, and the protease cleavage enables the composition to be induceable within the tumor microenvironment. In one embodiment, the fusion protein further comprises a biologically inactive polypeptide, and cleavage of the protease-cleavable moiety 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 its antigen-binding moiety.

[0019] This disclosure further relates to nucleic acids encoding any polypeptide disclosed herein, vectors comprising any nucleic acid encoding any polypeptide disclosed herein, and host cells comprising such vector.

[0020] This specification provides a method for preparing a pharmaceutical composition, comprising culturing host cells containing a vector comprising nucleic acid encoding any polypeptide disclosed herein under conditions suitable for the expression and harvesting of a desired polypeptide. This specification also describes a method for using any polypeptide disclosed herein, comprising administering an effective amount of such polypeptide-containing pharmaceutical composition to a subject in need. For example, a use for treating a subject with a disease or disorder disclosed herein is described.

[0021] This specification provides pharmaceutical compositions comprising any recombinant polypeptide, any proprotein, any fusion protein, any fusion polypeptide, any nucleic acid, any vector, or any host cell containing such vector as disclosed herein. For example, pharmaceutical compositions for treating subjects having a disease or disorder as disclosed herein are provided.

[0022] Furthermore, the use of recombinant polypeptides, proproteins, fusion proteins, fusion polypeptides, nucleic acids, vectors, or host cells containing such vectors disclosed herein for the manufacture of pharmaceuticals for the treatment of diseases or disorders 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. 195-220, or an amino acid sequence having at least approximately 90% identity with SEQ ID NOs. 195-220. (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) The polypeptide according to item 2, wherein the functional variant of sequence number 195 contains any of sequence numbers 258 to 331. (Item 4) The polypeptide according to item 2, wherein the functional variant of sequence number 198 includes any of sequence numbers 199 or 332-408. (Item 5) The polypeptide described in item 2, wherein the polypeptide includes sequence number 195. (Item 6) The polypeptide described in item 2, wherein the polypeptide includes sequence number 198. (Item 7) A polypeptide comprising formula I, [D1]-[L1]-[D2] In the formula, D1 is the first target domain, L1 is a separation portion that connects or binds D1 to D2, and the separation portion includes an amino acid sequence selected from SEQ ID NOs. 195-220, or an amino acid sequence having at least about 90% identity with SEQ ID NOs. 195-220. The polypeptide wherein D2 is the second target domain. (Item 8) The polypeptide according to item 1 or 7, wherein the separated portion contains 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) The polypeptide according to any one of items 1 to 8, wherein the separated portion includes a cleavable portion that is a substrate for two or more proteases. (Item 10) The polypeptide described in 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) The polypeptide described in 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) The polypeptide according to any one of items 1 to 7, wherein the separated portion comprises an amino acid sequence that is a substrate of at least one protease present in the tumor microenvironment of a human tumor. (Item 13) The polypeptide according to any one of items 1 to 8, wherein the separation portion comprises two or more cleavable portions, and each of the cleavable portions is a substrate of a protease. (Item 14) A polypeptide according to any one of items 1 to 8, comprising a first cleavable moiety containing a first amino acid sequence that is a substrate for a first protease, and a second cleavable moiety containing a second amino acid sequence that is a substrate for a second protease. (Item 15) A polypeptide according to any one of items 1 to 8, further comprising a non-cleavable linker sequence. (Item 16) The polypeptide described in any 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) 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) The polypeptide according to any one of items 1 to 8, wherein the polypeptide comprises a cell surface receptor, a chimeric antigen receptor (CAR), or a T cell receptor (TCR) subunit. (Item 19) The polypeptide described above is a 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) The polypeptide according to any one of items 1 to 14, wherein the cleavable portion 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. (Item 21) The polypeptide described in item 20, wherein 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) The polypeptide described in 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) The polypeptide described in item 21, wherein the aforementioned 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) The polypeptide according to any one of items 1 to 23, wherein the cleavable portion is cleaved by one or more proteases with a catalytic efficiency lower than that of the reference polypeptide sequence. (Item 25) The polypeptide according to item 24, wherein the cleavable portion is cleaved with reduced catalytic efficiency by one or more serum proteases. (Item 26) The polypeptide according to item 25, wherein the cleavable portion is cleaved with reduced catalytic efficiency by one or more hepatic proteases. (Item 27) The cleavable portion is reduced by one or more factor Xa, hepsin, or thrombin. The polypeptide described in item 25 is cleaved with the catalytic efficiency described above. (Item 28) The polypeptide according to any one of items 1 to 25, wherein the polypeptide is responsively bound to a portion selected from the group consisting of a polypeptide portion, a lipid portion, a nucleic acid portion, a detectable portion, and a small molecule. (Item 29) Recombinant proprotein, 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. A polypeptide having biological activity is included, The recombinant proprotein wherein the biological activity of the proprotein is attenuated, and cleavage of the cleavable portion by the protease produces a polypeptide in which the biological activity is not attenuated. (Item 30) The recombinant proprotein according to item 29, wherein the polypeptide having biological activity comprises a cytokine, a chemokine, a growth factor, a soluble receptor, or a combination thereof. (Item 31) The recombinant proprotein according to item 29, wherein the biologically active polypeptide comprises at least one of an extracellular domain, a transmembrane domain, and an intracellular domain. (Item 32) The recombinant proprotein according to 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) The recombinant proprotein according to item 29, wherein the biologically active polypeptide comprises an antigen-binding polypeptide, an antibody, or an antigen-binding moiety thereof. (Item 34) It is a fusion protein, a. Signaling proteins or molecules, b. A blocking portion selected from three-dimensional blocking portions, specific blocking portions, and combinations thereof, c. The fusion protein comprising a peptide linker having a cleavable moiety as defined in item 29. (Item 35) The fusion protein described in item 34, wherein (a) and (b) are functionally bound by (c). (Item 36) The fusion protein described in item 34, wherein the peptide linker contains two or more copies of the same cleavable region. (Item 37) The fusion protein according to item 34, wherein the three-dimensional blocking portion contains human serum albumin (HSA) or an anti-HSA antibody. (Item 38) The fusion protein according to item 34, wherein the signal transduction protein is an interleukin 2-amino acid sequence including (i) a non-native N-terminus and / or (ii) a non-native C-terminus. (Item 39) The fusion protein described in item 34, wherein the aforementioned signal transduction protein is an interleukin 2-amino acid sequence. (Item 40) The fusion protein described in item 34 further comprises one or more half-life extension domains, wherein the domains are not specific blockers. (Item 41) A fusion protein as described in item 40, produced by rearranging the non-native N-terminus and / or C-terminus in a circular fashion. (Item 42) A fusion polypeptide comprising a first polypeptide fusion partner bound to a ligand by a protease-cleaving linker, wherein the catalytic efficiency of the cleaving linker is optimized, the ligand is optionally modified, and the first polypeptide fusion partner is a blocking portion that prevents binding of the modified ligand to a target receptor or a subunit of the target receptor until cleavage by the protease-cleaving linker. (Item 43) A fusion polypeptide comprising a first polypeptide fusion partner bound to a ligand by a protease-cleaving linker, wherein the catalytic efficiency of the cleaving linker is optimized, the ligand is optionally modified, including by cyclic rearrangement, to produce a non-native N-terminus and a novel C-terminus compared to a native ligand, at least one of the novel N-terminus or the novel C-terminus of the modified ligand is responsively bound to the first polypeptide fusion partner to form a fusion polypeptide, the first polypeptide fusion partner being a blocking portion that prevents binding of the modified ligand to a target receptor or a subunit of a target receptor until cleavage by the protease-cleaving linker. (Item 44) The fusion polypeptide according to item 42 or 43, wherein the first polypeptide fusion partner is selected from the group consisting of antibodies, antibody fragments, and albumin molecules. (Item 45) The fusion polypeptide according to item 42 or 43, further comprising a second polypeptide fusion partner including a second blocking portion. (Item 46) The fusion polypeptide according to item 42 or 43, wherein the second polypeptide fusion partner is a blocking portion of a different type than the first polypeptide fusion partner. (Item 47) The fusion polypeptide according to item 42 or 43, wherein the first polypeptide fusion partner is albumin, and the second polypeptide fusion partner is a domain containing a complementary amino acid sequence that blocks cytokine activity. (Item 48) The fusion polypeptide according to 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 to a cytokine, or an antibody or antibody fragment that specifically binds to the cytokine of the fusion polypeptide. (Item 49) The fusion polypeptide according to item 47, wherein the second polypeptide fusion partner is the same type of blocking portion as the first polypeptide fusion partner. (Item 50) A fusion polypeptide according to any one of items 34, 42, or 43, further comprising a tumor antigen-binding component. (Item 51) A fusion polypeptide as described in any one of items 34, 42, or 43, further comprising a serum half-life extension domain. (Item 52) The ligands include helix bundle proteins and cytokines (but are 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 (IL-27p28), 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), and β-trefoil proteins (but are not limited to IL-1α, IL-1β, IL-1Ra, IL-18, 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 A fusion polypeptide as described in item 42 or 43, selected from the group consisting of FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23, α / β(TIM) barrel protein (including, but not limited to, triose phosphate isomerase), beta sandwich protein (including, but not limited to, galectin-1, galectin-3), TNF-beta, 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, C5a. (Item 53) The fusion polypeptide according to item 42 or 43, wherein the ligand comprises one or more fragments thereof of an IL-2 polypeptide. (Item 54) A fusion polypeptide according to any one of items 34, 42, or 43, wherein the protease-cleavable linker polypeptide comprises a sequence that can be 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 proteases. (Item 55) A fusion polypeptide according to any one of items 34, 42, or 43, wherein, after a protease-cleavable polypeptide linker is cleaved by a protease, the cytokine or its fragment or mutein substantially dissociates from the cytokine-blocking portion. (Item 56) It is a fusion polypeptide, a. Cytokine polypeptide or its functional fragment or mutein [A], b. Cytokine blocking portion [B], and c. Optimized protease-cleaving polypeptide linker [L] The fusion polypeptide comprises at least one of each of the following, wherein the blocking portion is selected from the group consisting of antibodies, antibody fragments, and albumin, and the cytokine comprises a cyclically rearranged cytokine. (Item 57) A fusion polypeptide as described in item 56, further comprising a tumor antigen-binding component. (Item 58) A fusion polypeptide as described in item 56, further comprising a serum half-life extension domain. (Item 59) Cytokine peptides or their functional fragments or mutains are helix bundle proteins and cytokines (but are 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, I L-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), FAP, ADAM (selected from ADAM8, ADAM9, ADAM10, ADAM12, ADAM17, and ADAMTS1), β-Trefoil protein (limited) It is not something that can be done, but 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 A fusion polypeptide as described in item 56, selected from the group consisting of FGF-14, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23, α / β(TIM) barrel protein (including, but not limited to, triose phosphate isomerase), beta sandwich protein (including, but not limited to, galectin-1, galectin-3), TNF-beta, 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, C5a. (Item 60) A fusion polypeptide as described in item 56, wherein the cytokine peptide or its functional fragment or mutain contains IL-2. (Item 61) The fusion polypeptide according to item 56, wherein the cytokine blocking portion comprises a ligand-binding domain or fragment or mutein of a homologous receptor of the cytokine, a single-domain antibody or scFv that binds to the cytokine polypeptide or its functional fragment or mutein, or an antibody or antibody fragment that binds to the receptor of the cytokine. (Item 62) The fusion polypeptide described in item 56, wherein the antibody is a single-domain antibody or scFv. (Item 63) The fusion polypeptide according to item 56, wherein the blocking portion extends the serum half-life of the cytokine or its fragment. (Item 64) A fusion polypeptide comprising a protease-cleavable moiety, wherein the sequence is catalytically optimized for cleavage by a particular protease, and the protease cleavage enables the composition to be induced within the tumor microenvironment. (Item 65) The present invention further comprises a biologically inactive polypeptide, wherein cleavage of the cleavable portion by the protease converts the biologically inactive polypeptide into a biologically active polypeptide. Replace with the fusion polypeptide described in item 64. (Item 66) The fusion polypeptide according to item 65, wherein the biologically inactive polypeptide comprises a cytokine, chemokine, growth factor, or soluble receptor. (Item 67) The fusion polypeptide according to item 65, wherein the biologically inactive polypeptide comprises at least one of an extracellular domain, a transmembrane domain, and an intracellular domain. (Item 68) The fusion polypeptide according to 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) The fusion polypeptide according to item 65, wherein the biologically inactive polypeptide comprises an antigen-binding polypeptide, an antibody, or an antigen-binding portion thereof. (Item 70) A nucleic acid encoding a polypeptide as described in any one of items 7 through 69. (Item 71) A vector containing nucleic acids as described in item 70. (Item 72) Host cells containing the vector described in item 71. (Item 73) A method for preparing a pharmaceutical composition, comprising culturing host cells described in item 72 under conditions suitable for the expression and harvesting of a desired polypeptide. (Item 74) A method using a polypeptide described in 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 drawing]

[0023] [Figure 1A] This is a schematic diagram showing a protease-activated cytokine or chemokine containing a blocking region. The blocking region may optionally function as a serum half-life extension domain. The diagram to the left of the arrow shows that the cytokine is connected to the blocking region via a protease-cleaving 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, the protease cleaves at the protease-cleaving site on the linker, releasing the blocking region and allowing the cytokine to bind to its receptor. [Figure 1B]This is a schematic diagram showing a protease-activated cytokine or chemokine, where the HSA (blocking portion) is directly bound to the target cytokine or chemokine, and there is a protease cleavage site between the HSA and the target cytokine or chemokine. The diagram to the left of the arrow shows that the cytokine is connected to the blocking portion via a protease-cleaving 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, the protease cleaves at the protease cleavage site on the linker, releasing the blocking portion and allowing the cytokine to bind to its receptor. [Figure 1C] This is a schematic diagram showing a protease-activated cytokine or chemokine, with multiple HSAs (blocking moieties) directly bound to the target molecule. Optionally, one or more HSAs may be bound 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-cleaving linker, thus blocking the cytokine's ability to bind to the receptor. The diagram to the right of the arrow shows that in an inflammatory or tumor environment, the protease cleaves at the protease cleavage site on the linker, releasing the blocking moiety and allowing the cytokine to bind to the receptor. The cytokine then has similar pK properties (e.g., a short half-life) compared to the native cytokine. [Figure 1D] This schematic diagram shows a protease-activated cytokine or chemokine containing multiple cytokines of the same or different types, each bound to a binding domain via a protease-cleaving linker. The diagram to the left of the arrow shows that the cytokine is connected to a blocking region via a protease-cleaving 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, the protease cleaves at the protease-cleaving site on the linker, releasing the blocking region and allowing the cytokine to bind to its receptor. [Figure 2] This is a schematic diagram showing a protease-activated cytokine or chemokine comprising a cytokine or chemokine polypeptide linked by at least one protease-cleaving linker, a blocking moiety, and a serum half-life extension domain. The diagram to the left of the arrow shows that the cytokine is linked to the blocking moiety via the protease-cleaving linker, thus blocking the cytokine's ability to bind to its receptor. The cytokine is also bound to a separate half-life extension element, which extends its half-life in serum. The diagram to the right of the arrow shows that in an inflammatory or tumor environment, the protease cleaves at the protease-cleaving site on the linker, releasing the serum half-life extension element and blocking moiety, allowing the cytokine to bind to its receptor. The cytokine then has similar pK properties (e.g., a short half-life) compared to the native cytokine. [Figure 3] This is a schematic diagram showing a protease-activated cytokine or chemokine, comprising a cytokine or chemokine polypeptide linked by at least one protease-cleaving linker, a blocking moiety, and a targeting domain. The diagram to the left of the arrow shows that the cytokine is linked to the blocking moiety and targeting domain via the protease-cleaving linker, thus blocking the cytokine's ability to bind to its receptor. The diagram to the right of the arrow shows that, in the inflammatory or tumor microenvironment, the protease cleaves at the protease-cleaving site within the linker, releasing the targeting domain and blocking moiety, allowing the cytokine to bind to its receptor. [Figure 4A]This is a schematic diagram showing a protease-activated cytokine or chemokine comprising a cytokine or chemokine polypeptide linked by at least one protease-cleaving linker, a blocking moiety, a targeting domain, and a serum half-life extension domain, where the cytokine polypeptide and targeting domain are linked by the protease-cleaving linker. The diagram to the left of the arrow shows that the cytokine or chemokine is linked to the targeting domain, blocking moiety, and half-life extension element via the protease-cleaving linker(s), thus blocking the cytokine or chemokine's ability to bind to its receptor. The diagram to the right of the arrow shows that in the inflammatory or tumor microenvironment, the protease cleaves at the protease-cleaving site on the linker(s), releasing the serum half-life extension element, targeting domain, and blocking moiety, allowing the cytokine to bind to its receptor. The cytokine then has similar pK properties (e.g., short half-life) compared to the native cytokine. [Figure 4B] This is a schematic diagram showing a protease-activated cytokine or chemokine comprising a cytokine or chemokine polypeptide linked by at least one protease-cleaving linker, a blocking moiety, a targeting domain, and a serum half-life extension domain. The diagram to the left of the arrow shows that the cytokine is linked to the targeting domain, blocking moiety, and half-life extension element via the protease-cleaving linker(s), thus blocking the cytokine's ability to bind to the receptor. The diagram to the right of the arrow shows that in the inflammatory or tumor microenvironment, the protease cleaves at the protease-cleaving site on the linker(s), releasing the serum half-life extension element and blocking moiety, allowing the cytokine to bind to the receptor. The targeting moiety maintains the binding, retaining the cytokine within the tumor microenvironment. The cytokine now has similar pK properties (e.g., short half-life) compared to the native cytokine. [Figure 5]The graph shows that linker 2 (GPAGLYAQ, SEQ ID NO: 195) and linker 3 (ALFKSSFP, SEQ ID NO: 198) are minimally cleaved in lung cells, kidney cells, and hepatocytes. [Figure 6] Graphs A and B show that polypeptides containing recombinant human IL-2 and 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] This graph shows 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 a luminescence-based cell viability assay using CellTiter-Glo® (Promega). The proliferation assay was performed without HSA. Each fusion protein contained an anti-HSA conjugate, and both uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in the assay. [Figure 7B] This graph shows 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 a luminescence-based cell viability assay using CellTiter-Glo® (Promega). The proliferation assay was performed with 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 7C] This graph shows 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 a luminescence-based cell viability assay using CellTiter-Glo® (Promega). The proliferation assay was performed without HSA. Each fusion protein contained an anti-HSA conjugate, and both uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in the assay. [Figure 7D] This graph shows 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 a luminescence-based cell viability assay using CellTiter-Glo® (Promega). The proliferation assay was performed with 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 7E] This graph shows 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 a luminescence-based cell viability assay using CellTiter-Glo® (Promega). The proliferation assay was performed without HSA. Each fusion protein contained an anti-HSA conjugate, and both uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in the assay. [Figure 7F] This graph shows 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 a luminescence-based cell viability assay using CellTiter-Glo® (Promega). The proliferation assay was performed with 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 7G]This graph shows 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 a luminescence-based cell viability assay using CellTiter-Glo® (Promega). The proliferation assay was performed without HSA. Each fusion protein contained an anti-HSA conjugate, and both uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in the assay. [Figure 7H] This graph shows 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 a luminescence-based cell viability assay using CellTiter-Glo® (Promega). The proliferation assay was performed with 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 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 a luminescence-based cell viability assay using CellTiter-Glo(Promega). Both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 9A] This 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 a luminescence-based cell viability assay using CellTiter-Glo(Promega). Both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 9B]This 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 a luminescence-based cell viability assay using CellTiter-Glo(Promega). Both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 9C] This 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 a luminescence-based cell viability assay using CellTiter-Glo(Promega). Both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 9D] This 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 a luminescence-based cell viability assay using CellTiter-Glo(Promega). Both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 9E] This 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 a luminescence-based cell viability assay using CellTiter-Glo(Promega). Both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 9F]This 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 a luminescence-based cell viability assay using CellTiter-Glo(Promega). Both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 9G] This 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 a luminescence-based cell viability assay using CellTiter-Glo(Promega). Both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 9H] This 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 a luminescence-based cell viability assay using CellTiter-Glo(Promega). Both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 9I] This 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 a luminescence-based cell viability assay using CellTiter-Glo(Promega). Both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 9J]This 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 a luminescence-based cell viability assay using CellTiter-Glo(Promega). Both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 9K] This 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 a luminescence-based cell viability assay using CellTiter-Glo(Promega). Both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 9L] This 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 a luminescence-based cell viability assay using CellTiter-Glo(Promega). Both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 9M] This 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 a luminescence-based cell viability assay using CellTiter-Glo(Promega). Both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 9N]This 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 a luminescence-based cell viability assay using CellTiter-Glo(Promega). Both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 9O] This 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 a luminescence-based cell viability assay using CellTiter-Glo(Promega). Both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 9P] This 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 a luminescence-based cell viability assay using CellTiter-Glo(Promega). Both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 9Q] This 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 a luminescence-based cell viability assay using CellTiter-Glo(Promega). Both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 9R]This 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 a luminescence-based cell viability assay using CellTiter-Glo(Promega). Both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 9S] This 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 a luminescence-based cell viability assay using CellTiter-Glo(Promega). Both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 9T] This 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 a luminescence-based cell viability assay using CellTiter-Glo(Promega). Both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 9U] This 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 a luminescence-based cell viability assay using CellTiter-Glo(Promega). Both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 9V]This 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 a luminescence-based cell viability assay using CellTiter-Glo(Promega). Both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 9W] This 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 a luminescence-based cell viability assay using CellTiter-Glo(Promega). Both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 9X] This 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 a luminescence-based cell viability assay using CellTiter-Glo(Promega). Both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 9Y] This 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 a luminescence-based cell viability assay using CellTiter-Glo(Promega). Both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 9Z]This 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 a luminescence-based cell viability assay using CellTiter-Glo(Promega). Both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 10] The results of the protein cleavage assay are shown. The fusion protein ACP16 was analyzed on an SDS-PAGE gel in both cleaved and uncleaved forms. As can be seen on the gel, the cleavage was complete. [Figure 11] A-B are a series of graphs showing the results of the HEK-Blue IL-12 reporter assay performed with human p40 / mouse p35 IL12 fusion protein and recombinant human IL12 (Rec hIL-12). The analysis was performed based on the quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the activity of the IL12 protein fusion protein. [Figure 12A] This graph shows the HEK-blue assay results for four IL-12 fusion proteins before and after cleavage by MMP9. The analysis was performed using QUANTI-Blue (InvivoGen) reagents, based on the quantification of secreted alkaline phosphatase (SEAP) activity. This data indicates that cleaved IL-12 proteins exhibit greater activity than the complete fusion protein. The construct tested was ACP06. [Figure 12B] This graph shows the HEK-blue assay results for four IL-12 fusion proteins before and after cleavage by MMP9. The analysis was performed using QUANTI-Blue (InvivoGen) reagents, based on the quantification of secreted alkaline phosphatase (SEAP) activity. This data indicates that cleaved IL-12 proteins exhibit greater activity than the complete fusion protein. The construct tested was ACP08. [Figure 12C]This graph shows the HEK-blue assay results for four IL-12 fusion proteins before and after cleavage by MMP9. The analysis was performed using QUANTI-Blue (InvivoGen) reagents, based on the quantification of secreted alkaline phosphatase (SEAP) activity. This data indicates that cleaved IL-12 proteins exhibit greater activity than the complete fusion protein. The construct tested was ACP07. [Figure 12D] This graph shows the HEK-blue assay results for four IL-12 fusion proteins before and after cleavage by MMP9. The analysis was performed using QUANTI-Blue (InvivoGen) reagents, based on the quantification of secreted alkaline phosphatase (SEAP) activity. This data indicates that cleaved IL-12 proteins exhibit greater activity than the complete fusion protein. The construct tested was ACP09. [Figure 12E] This graph shows the HEK-blue assay results for four IL-12 fusion proteins before and after cleavage by MMP9. The analysis was performed using QUANTI-Blue (InvivoGen) reagents, based on the quantification of secreted alkaline phosphatase (SEAP) activity. This data indicates that cleaved IL-12 proteins exhibit greater activity than the complete fusion protein. The construct tested was ACP10. [Figure 12F] This graph shows the HEK-blue assay results for four IL-12 fusion proteins before and after cleavage by MMP9. The analysis was performed using QUANTI-Blue (InvivoGen) reagents, based on the quantification of secreted alkaline phosphatase (SEAP) activity. This data indicates that cleaved IL-12 proteins exhibit greater activity than the complete fusion protein. The construct tested was ACP11. [Figure 13] The results of the protein cleavage assay are shown. The fusion protein ACP11 was analyzed on an SDS-PAGE gel in both cleaved and uncleaved forms. As can be seen on the gel, the cleavage was complete. [Figure 14]This schematic diagram illustrates a non-limiting example of an inducible cytokine protein, where the construct is activated when a linker attached between two subunits of the cytokine is cleaved by a protease. [Figure 15A] This graph shows the results of HEK-Blue assays performed with human p40 / mouse p35 IL12 fusion protein and recombinant human IL12 (Rec hIL-12). The results support the activity of the IL12 protein fusion protein. Each proliferation assay was performed with and without HSA. [Figure 15B] This graph shows the results of HEK-Blue assays performed with human p40 / mouse p35 IL12 fusion protein and recombinant human IL12 (Rec hIL-12). The results support the activity of the IL12 protein fusion protein. Each proliferation assay was performed with and without HSA. [Figure 15C] This graph shows the results of HEK-Blue assays performed with human p40 / mouse p35 IL12 fusion protein and recombinant human IL12 (Rec hIL-12). The results support the activity of the IL12 protein fusion protein. Each proliferation assay was performed with and without HSA. [Figure 15D] This graph shows the results of HEK-Blue assays performed with human p40 / mouse p35 IL12 fusion protein and recombinant human IL12 (Rec hIL-12). The results support the activity of the IL12 protein fusion protein. Each proliferation assay was performed with and without HSA. [Figure 16] Figures 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 the WEHI 279 cell survival assay. Each assay was performed in either HSA-containing medium (+HSA) or HSA-free medium (-HSA). Each fusion protein contained an anti-HSA conjugate, and both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in the assays. [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 the B16 reporter assay. Each assay was performed in medium containing HSA (+HSA) or medium without HSA (-HSA). Each fusion protein contained an anti-HSA conjugate, and both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in the assays. [Figure 18] Figures A and B show the results of protein cleavage assays. Two constructs, ACP31 (IFN-α fusion protein; A) and ACP55 (IFN-γ fusion protein; B), were performed on SDS-PAGE gels in both cleaved and uncleaved forms. As can be seen in the gel, the cleavage was complete. [Figure 19] A–B are a series of graphs showing the activity of exemplary IFNγ fusion proteins, compared to the activity of a mouse IFNγ control using the B16 reporter assay. Each assay was performed in a culture medium containing HSA. Each fusion protein contained an anti-HSA conjugate. Both the uncleaved and MMP9 protease-cleaved versions of the fusion protein were used in each assay. [Figure 20] A–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 the B16 reporter assay. Each assay was performed in a medium containing HSA. Each fusion protein contained an anti-HSA conjugate. Both the 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 effects of IFNγ and IFNγ fusion protein on tumor growth when injected intraperitoneally (IP) using different dose levels and schedules (μg = micrograms, BID = twice daily, BIW = twice weekly, QW = once weekly). D shows the effects 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 protein using the B16 reporter assay. Each fusion protein contains an anti-HSA binding agent 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 uncleaved fusion proteins using the B16 reporter assay. Each fusion protein contains IFNγ directly fused to albumin. [Figure 24] Figures A and B are 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 patients and cancer patients. Figure A shows the 24-hour 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). Figure 24B shows the 72-hour 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). [Figure 25A] This graph shows the analysis of ACP16 in the HEKBlue IL-2 reporter assay in the presence of HSA. [Figure 25B] This graph shows the analysis of ACP124 in the HEKBlue IL-2 reporter assay in the presence of HSA. Circles represent the activity of the uncut polypeptide, squares represent the activity of the cut polypeptide, and triangles represent IL-2 alone as a control. [Figure 25C]This graph shows the results of the CTLL-2 proliferation assay. CTLL2 cells (ATCC) were suspended at a concentration of 500,000 cells / well in culture medium containing or without 40 mg / ml human serum albumin (HSA), cultured on plates, and stimulated with a dilution series of recombinant hIL2 or activatable hIL2 at 37°C and 5% CO2 for 72 hours. The activity of uncleaved and cleavage-activatable ACP16 was tested. Cleavage-activatable hIL2 was generated by incubation with active MMP9. Cell activity was evaluated using the CellTiter-Glo (Promega) luminescence-based cell viability assay. Triangles represent wile-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 ACP11 (human p40 / mouse p35 IL12 fusion protein) with and without cuts. 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 cut 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 cut polypeptide (ACP136 + MMP9). The EC50 values ​​for each are shown in the table insert. [Figure 27A] This graph shows the activity of cleaved IL-12 polypeptides in the HEKBlue IL2 reporter assay. Fusion proteins are evaluated both with and without cleavage (circles), and in Figure 27A, wild-type IL2 is used as a control + HSA. Data for ACP31 + HSA are shown. The EC50 values ​​for each are shown in the table below each graph. [Figure 27B]This graph shows the activity of cleaved IL-12 polypeptides in the HEKBlue IL2 reporter assay. Fusion proteins are evaluated both with and without cleavage (circles), and in Figure 27B, wild-type IL2 is used as a control + HSA. Data for ACP125 + HSA are shown. The EC50 values ​​for each are shown in the table below each graph. [Figure 27C] This graph shows the activity of cleaved IL-12 polypeptides in the HEKBlue IL2 reporter assay. Fusion proteins are evaluated both with and without cleavage (circles), and in Figure 27C, wild-type IL2 is used as a control + HSA. Data for ACP126 + HSA are shown. The EC50 values ​​for each are shown in the table below each graph. [Figure 27D] This graph shows the activity of cleaved IL-12 polypeptides in the HEKBlue IL2 reporter assay. Fusion proteins are evaluated both with and without cleavage (circles), and in Figure 27D, ACP131 is used as a control (triangle). Data for ACP127 is shown. The EC50 values ​​for each are shown in the table below each graph. [Figure 27E] This graph shows the activity of cleaved IL-12 polypeptides in the HEKBlue IL2 reporter assay. Fusion proteins are evaluated both with and without cleavage (circles), and in Figure 27E, ACP131 is used as a control (triangle). Data for ACP128 is shown. The EC50 values ​​for each are shown in the table below each graph. [Figure 27F] This graph shows the activity of cleaved IL-12 polypeptides in the HEKBlue IL2 reporter assay. Fusion proteins are evaluated both with and without cleavage (circles), and ACP131 is used as a control (triangle) in Figure 27F. Data for ACP129 is also shown. The EC50 values ​​for each are shown in the table below each graph. [Figure 28A]This graph shows the activity of APC56 in the HEKBlue IFNα reporter assay. For each fusion, activity was tested with and without cuts (squares). Mouse IFNγ analysis is included in each graph for comparison. [Figure 28B] This graph shows the activity of APC57 in the HEKBlue IFNα reporter assay. For each fusion, activity was tested with and without cuts (squares). Mouse IFNγ analysis is included in each graph for comparison. [Figure 28C] This graph shows the activity of APC58 in the HEKBlue IFNα reporter assay. For each fusion, activity was tested with and without cuts (squares). Mouse IFNγ analysis is included in each graph for comparison. [Figure 28D] This graph shows the activity of APC59 in the HEKBlue IFNα reporter assay. For each fusion, activity was tested with and without cuts (squares). Mouse IFNγ analysis is included in each graph for comparison. [Figure 28E] This graph shows the activity of APC60 in the HEKBlue IFNα reporter assay. For each fusion, activity was tested with and without cuts (squares). Mouse IFNγ analysis is included in each graph for comparison. [Figure 28F] This graph shows the activity of APC61+HSA in the HEKBlue IFNα reporter assay. For each fusion, activity was tested with and without cuts (squares). Mouse IFNγ analysis is included in each graph for comparison. [Figure 28G] This graph shows the activity of ACP30+HSA in the HEKBlue IFNα reporter assay. For each fusion, activity was tested with and without cuts (squares). Mouse IFNγ analysis is included in each graph for comparison. [Figure 28H]This graph shows the activity of ACP73 in the HEKBlue IFNα reporter assay. For each fusion, activity was tested with and without cuts (squares). Mouse IFNγ analysis is included in each graph for comparison. [Figure 28I] This graph shows the activity of ACP70+HSA in the HEKBlue IFNα reporter assay. For each fusion, activity was tested with and without cuts (squares). Mouse IFNγ analysis is included in each graph for comparison. [Figure 28J] This graph shows the activity of ACP71 in the HEKBlue IFNα reporter assay. For each fusion, activity was tested with and without cuts (squares). Mouse IFNγ analysis is included in each graph for comparison. [Figure 28K] This graph shows the activity of ACP72 in the HEKBlue IFNα reporter assay. For each fusion, activity was tested with and without cuts (squares). Mouse IFNγ analysis is included in each graph for comparison. [Figure 28L] This graph shows the activity of ACP73 in the HEKBlue IFNα reporter assay. For each fusion, activity was tested with and without cuts (squares). Mouse IFNγ analysis is included in each graph for comparison. [Figure 28M] This graph shows the activity of ACP74 in the HEKBlue IFNα reporter assay. For each fusion, activity was tested with and without cuts (squares). Mouse IFNγ analysis is included in each graph for comparison. [Figure 28N] This graph shows the activity of ACP75 in the HEKBlue IFNα reporter assay. For each fusion, activity was tested with and without cuts (squares). Analysis of mouse IFNγ is included in each graph for comparison. [Figure 29]Graphs A and B show the results of the analysis of ACP31 (mouse IFNα1 fusion protein) and ACP11 (human p40 / mouse p35 IL12 fusion protein) in a tumor xenograft model. Graph A shows the tumor volume over time in mice treated with 33 μg ACP31 (circles), 110 μg ACP31 (triangles), 330 μg ACP31 (diamonds), and controls of 1 μg mouse wild-type IFNα1 (dashed line, squares) and 10 μg mIFNα1 (dashed line, small circles). The vehicle alone is indicated by a large white circle. The data show that tumor volume decreases over time in a dose-dependent manner in mice treated with ACP31. B shows the tumor volume over time in mice treated with 17.5 μg ACP11 (square), 175 μg ACP31 (triangle), 525 μg ACP31 (circle), and as controls, 2 μg ACP04 (dashed line, triangle) and 10 μg ACP04 (dashed line, diamond). Vehicle alone is indicated by a large white circle. This data shows a dose-dependent decrease in tumor volume over time in mice treated with both ACP11 and ACP04 (human p40 / mouse p35 IL12 fusion protein). [Figure 30] A-F are a series of spaghetti plots showing the time course of tumor volume in mouse xenograft tumor models 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), respectively. Each line represents a different mouse. [Figure 31]A-C show three graphs illustrating the analysis results of ACP16 and ACP124 in tumor xenograft models. A shows the tumor volume over time in mice treated with 4.4 μg ACP16 (square), 17 μg ACP16 (triangle), 70 μg ACP16 (inverted triangle), 232 μg ACP16 (dark circle), and, as a comparison, 12 μg wild-type IL-2 (dashed line, triangle) and 36 μg wild-type IL-2 (dashed line, diamond). The vehicle alone is indicated by a large white circle. The data show that the tumor volume in mice treated with high concentrations of ACP16 decreases over time in a dose-dependent manner. B shows the tumor volume over time in mice treated with 17 μg ACP124 (square), 70 μg ACP124 (triangle), 230 μg ACP124 (downward triangle), and 700 μg ACP124. Vehicle alone is indicated by a large white circle. C shows the tumor volume over time in mice treated with 17 μg ACP16 (triangle), 70 μg ACP16 (circle), 232 μg ACP16 (dark circle), and, as a comparison, 17 μg ACP124 (dashed line, triangle), 70 μg ACP124 (dashed line, diamond), and 230 μg ACP124 (dashed line, diamond). Vehicle alone is indicated by a dark inverted triangle. The data show that in mice treated with ACP16 instead of ACP124, tumor volume decreases over time in a dose-dependent manner. [Figure 32A] These are a series of spaghetti plots showing the activity of fusion proteins in the MC38 mouse xenograft model. Each line in the plot represents a different mouse. [Figure 32B-1] These are a series of spaghetti plots showing the activity of fusion proteins in the MC38 mouse xenograft model. Each line in the plot represents a different mouse. [Figure 32B-2] Same as above. [Figure 32C-1] These are a series of spaghetti plots showing the activity of fusion proteins in the MC38 mouse xenograft model. Each line in the plot represents a different mouse. [Figure 32C-2] Same as above. [Figure 33]This graph shows the tumor volume over time in mouse xenograft models illustrating tumor growth in control mice (white circles) and AP16-treated mice (squares). [Figure 34] This is a series of survival plots showing the time course of survival in mice treated 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] These are a series of spaghetti plots showing the activity of fusion proteins in an MC38 mouse xenograft model. All mouse groups received a total of four doses, except for up to three doses of ACP132, which showed lethal toxicity after 1 week / 2 doses. Vehicle alone (top), 17, 55, 70, and 230 μg ACP16 (all upper rows), 9, 28, 36, and 119 μg ACP132 (all middle rows), and 13, 42, 54, and 177 μg ACP21 (all lower rows) are shown. Each line in the plot represents an individual animal. [Figure 35-2] Same as above. [Figure 36] This is a schematic diagram showing the substrate cleavage activity in conditionally complete (+FBS) medium by the FRET endpoint assay in four cell lines. The tumor vs. control activity ratio was estimated by averaging three tumor cell lines and comparing them to a control myofibroblast cell line in which the signal was detectable. SEQ ID NOs. 201, 198, 197, 196, and 195 are disclosed in order of appearance in Figure 36. [Figure 37] This is a schematic diagram showing the dynamics of ADAM17_2 substrate in cell cultures. Figure 37 discloses Sequence ID No. 235. [Figure 38]This is a schematic diagram showing the dynamics of the FAPα_1 substrate in a controlled medium. Figure 38 discloses Sequence ID No. 197. [Figure 39] This is a schematic diagram showing the dynamics of FAPα_1 substrates in cell lysates. Figure 39 discloses Sequence ID No. 197. [Figure 40] This is a schematic diagram illustrating the dynamics of the MMP9_1 substrate in cell lysates. Figure 40 discloses Sequence ID No. 196. [Figure 41] This is a schematic diagram showing the substrate cleavage activity in cell lysates as determined by the FRET endpoint assay. Figure 41 discloses sequence numbers 198 and 197 in order of appearance. [Figure 42] This is a schematic diagram illustrating the dynamics of the CTSL1_1 substrate in cell lysates. Figure 42 discloses sequence number 198. [Figure 43] This is a schematic diagram showing the dynamics of the MMP14_1 substrate in cell lysates. Figure 43 discloses Sequence ID No. 195. [Figure 44] This is a schematic diagram showing the calculated enzyme equivalent concentration per cell culture-derived sample. In Figure 44, sequence numbers 201, 198, 197, 196, and 195 are disclosed in the order of their appearance. [Figure 45] This is a schematic diagram showing the enzyme progression curve of CTSL1 cleavage between CSTL1_2 and CTSL1_1. In Figure 45, sequence numbers 198, 199, and 236 are disclosed in order of their appearance. [Figure 46] This is a schematic diagram showing a 30-mer cut between CTSL1_1 (ALFKSSFP, SEQ ID NO: 198) and CTSL1_2 (ALFFSSPP, SEQ ID NO: 199). [Figure 47] This is a schematic diagram showing the sensitivity of CTSL1 FRET substrates to cleavage of CTSK. The rate of product formation was measured as specific activity per unit pmol / min-1 μg-1. The threshold for the reference substrate Z-LR-AMC is shown by the dashed line. In Figure 47, sequence numbers 198 and 199 are disclosed in order of appearance. [Figure 48]This is a schematic diagram illustrating the degradation of 30-mer substrates by MMP9. A "+" indicates the relative degradation rate of the substrates, while a "-" indicates non-cleaved substrates. Figure 48 displays sequence numbers 204, 205, 214, 216, 202, 217, 203, 211, 219, 207, 215, 212, 213, 206, 208, 209, 210, 218, and 220 in order of appearance. [Figure 49] This is a schematic diagram illustrating the tandem MMP14_1 motif decomposition by MMP9. Top: Trace of substrate decomposition, modeled by primary kinetics. Bottom: Trace of product formation showing complex kinetics. In Figure 49, sequence numbers 202-205 are disclosed in order of appearance. [Figure 50] This is a schematic diagram illustrating the degradation of 30-mer substrates by FAPα. Substrates are ranked by their relative degradation rate, indicated by "+", and non-cleaved substrates are indicated by "-". Figure 50 displays sequence numbers 205, 204, 206, 217, 203, 218, 219, 213, 216, 207, 214, 210, 202, 211, 208, 209, 212, 215, and 220 in order of appearance. [Figure 51] This is a schematic diagram illustrating the 30-mer substrate degradation by CTSL1. Substrates are ranked by their relative degradation rate ("+"), and non-cleaved substrates are indicated by "-". Figure 51 displays sequence numbers 207, 208, 202, 218, 219, 212, 215, 217, 211, 209, 214, 206, 213, 210, 216, 203, 204, 205, and 220 in order of appearance. [Figure 52] This is a schematic diagram illustrating the degradation of 30-mer substrates by ADAM17. A "+" indicates the relative degradation rate of the substrates, while a "-" indicates non-cleaved substrates. Figure 52 displays sequence numbers 208, 209, 211, 214, 217, 219, 213, 218, 215, 210, 212, 216, 207, 206, 202, 203, 204, 205, and 220 in order of appearance. [Figure 53]This is a schematic diagram illustrating the 30-mer substrate degradation by factor Xa. A "+" indicates the relative degradation rate of the substrates, while a "-" indicates non-cleaved substrates. Figure 53 displays sequence numbers 220, 206, 202, 214, 208, 209, 215, 210, 218, 217, 207, 213, 216, 211, 212, 219, 203, 204, and 205 in order of appearance. [Figure 54] This is a schematic diagram illustrating the thrombin-mediated degradation of 30-mer substrates. Substrates are ranked by their relative degradation rate ("+"), while non-cleaved substrates are indicated by "-". Figure 54 displays sequence numbers 220, 204, 202, 207, 205, 211, 212, 215, 209, 218, 219, 217, 210, 213, 216, 214, 208, 206, and 203 in order of appearance. [Figure 55] This is a schematic diagram illustrating the degradation of 30-mer substrates by hepsin. Substrates are ranked by their relative degradation rate ("+"), while non-cleaved substrates are indicated by "-". Figure 55 displays sequence numbers 220, 209, 216, 215, 210, 213, 206, 214, 212, 207, 217, 208, 211, 218, 219, 202, 203, 204, and 205 in order of appearance. [Figure 56A] The image shows Western blots probed with IL-2 antibody, demonstrating the stability of ACP16 in 90% serum. Serum was pooled from three human donors. The target construct was incubated with PBS, serum, or MMP9 protease, and cleavage was evaluated at T=0 and T=24 hours. [Figure 56B] The images show Western blots probed with IL-2 antibody, demonstrating the stability of ACP153 in 90% serum. Serum was pooled from three human donors. The target construct was incubated with PBS, serum, or MMP9 protease, and cleavage was evaluated at T=0 and T=24 hours. [Figure 56C]The image shows Western blots probed with IL-2 antibody, demonstrating the stability of ACP157 in 90% serum. Serum was pooled from three human donors. The target construct was incubated with PBS, serum, or MMP9 protease, and cleavage was evaluated at T=0 and T=24 hours. [Figure 57A] Western blots using IL-2 antibody are shown, indicating that ACP153, ACP155, ACP156, ACP16, and ACP372 are stable in 90% serum. Serum was pooled from three human donors. The target construct was incubated with PBS, serum, or MMP9 protease, and cleavage was evaluated at T=24 hours and T=72 hours. Figure 57A shows the results when human serum was used. [Figure 57B] Western blots using IL-2 antibody are shown, indicating that ACP153, ACP155, ACP156, ACP16, and ACP372 are stable in 90% serum. Serum was pooled from three human donors. The target construct was incubated with PBS, serum, or MMP9 protease, and cleavage was evaluated at T=24 and T=72 hours. Figure 57B shows the results when mouse serum was used. [Figure 58A] This shows a series of spaghetti plots illustrating the activity of fusion proteins in the MC38 mouse xenograft model. Vehicle alone (Figure 58A, top), and 17, 55, and 230 μg ACP16 (Figure 58A) are shown. Each line in the plot represents an individual animal. [Figure 58B] This shows a series of spaghetti plots illustrating 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] This shows a series of spaghetti plots illustrating 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]This shows a series of spaghetti plots illustrating 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] The graph shows the results of a STAT activation reporter assay performed on IL-2 fusion protein and recombinant human IL-2 (Rec hIL-2). The analysis was performed using QUANTI-Blue (InvivoGen) reagents, based on the quantification of secreted alkaline phosphatase (SEAP) activity. [Figure 60] The graph shows the results of a STAT activation reporter assay performed on IL-2 fusion protein and recombinant human IL-2 (Rec hIL-2). The analysis was performed using QUANTI-Blue (InvivoGen) reagents, based on the quantification of secreted alkaline phosphatase (SEAP) activity. [Figure 61] The graph shows the results of a STAT activation reporter assay performed on IL-2 fusion protein and recombinant human IL-2 (Rec hIL-2). The analysis was performed using QUANTI-Blue (InvivoGen) reagents, based on the quantification of secreted alkaline phosphatase (SEAP) activity. [Figure 62-1] The graph shows the results of a STAT activation reporter assay performed on IL-2 fusion protein and recombinant human IL-2 (Rec hIL-2). The analysis was performed using QUANTI-Blue (InvivoGen) reagents, based on the quantification of secreted alkaline phosphatase (SEAP) activity. [Figure 62-2] Same as above. [Figure 63-1]This specification shows a table reporting the degree of cleavage observed using the manipulated cleavage substrates and associated proteases described herein. Flanking sequences are shown in lowercase, the first cleavage sequence in underline, the second cleavage sequence in bold, and the third cleavage sequence in italics. In some cases, there is overlap between cleavage sequences, which is indicated accordingly. Figure 63 discloses sequence numbers 202-220 in order of appearance. [Figure 63-2] Same as above. [Figure 64] This is a schematic diagram of the form of an inducible tetravalent antibody. [Figure 65] Figures A and B demonstrate that polyvalent 4-1BB antibodies can induce stimulation of 4-1BB. [Modes for carrying out the invention]

[0024] This disclosure relates to a novel isolation portion or linker and a polypeptide (e.g., a fusion protein) containing the linker. The linker is preferably protease-cleavable and links a first target amino acid sequence (e.g., a first target domain) to a second target amino acid sequence (e.g., a second target domain).

[0025] The isolation portions of this disclosure confer site selectivity to the action of the attached payload(s). The payload(s) may be therapeutic agents, half-life extenders, blocking agents, or any combination thereof. The isolation portions can be used to attach to any target payload(es) including cytokines, antibodies, cell-based therapeutics, etc. The isolation portions may be used individually, or in tandem, triple, quadruple, etc., as long as the isolation portion is less than about 100 amino acids. The individual isolation portions may be directly linked to each other, or may have non-cleavable linkers scattered throughout, either of which promotes high efficiency and site specificity.

[0026] Various embodiments of this 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 meanings generally understood by those skilled in the art in which the invention pertains. In some cases, terms with generally 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 indicating a difference from the generally understood meanings in the art. The techniques and procedures described or referenced herein are generally well understood by those skilled in the art and are commonly employed using conventional methodologies (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Procedures involving the use of commercially available kits and reagents, where appropriate, are generally carried out according to the protocols and conditions defined by the manufacturer, unless otherwise stated.

[0028] "Cytokines" is a well-known technical term referring to any class of immunomodulatory proteins (e.g., interleukins or interferons) secreted by cells, particularly immune system cells, and acting as immune system modulators. Cytokine polypeptides that can be used in the fusion proteins disclosed herein include, but are not limited to, transforming growth factors, e.g., TGF-α and TGF-β (e.g., TGF-beta 1, TGF-beta 2, TGF-beta 3); interferons, e.g., interferon-α, interferon-β, interferon-γ, interferon-kappa, and interferon-omega; interleukins, e.g., IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 Examples include IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, and IL-25; tumor necrosis factors, e.g., tumor necrosis factor alpha and lymphotoxin; transforming growth factor beta (TGF beta) family proteins, chemokines (e.g., CXC motif chemokine 10 (CXCL10), CCL19, CCL20, CCL21), and granulocyte-macrophage colony-stimulating factor (GM-CS), as well as fragments of such polypeptides that activate homologous receptors of cytokines (i.e., the aforementioned functional fragments). "Chemokine" is a technical term referring to any family of small cytokines that have the ability to induce directed chemotaxis in nearby responsive cells.

[0029] Cytokines are known to have short serum half-lives, often only a few minutes. Even cytokine forms with amino acid sequence modifications intended to extend serum half-life while retaining receptor agonist activity typically have similarly short serum half-lives. As used herein, “short half-life cytokines” refer to cytokines with substantially short half-lives circulating in the serum of the subject, e.g., cytokines with serum half-lives 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 sequences have 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 extend serum half-life while retaining receptor agonist activity. The latter example is not intended to include the addition of heterologous protein domains, e.g., genuine half-life-extending elements, e.g., serum albumin.

[0030] As used herein, “conservative” amino acid substitution generally refers to the substitution of an amino acid residue with another amino acid within a recognized group, which may alter the structure of the peptide but substantially maintain the peptide’s biological activity. 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, those skilled in the art will reasonably expect that replacing leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similarly replacing an amino acid with a structurally related amino acid will not significantly affect the biological activity of the resulting molecule.

[0031] "Saltase" is a transpeptidase that modifies proteins by recognizing and cleaving carboxyl end selection signals embedded in or attached to the terminals of target proteins or peptides. Saltase A catalyzes the cleavage of the LPXTG motif (where X is any standard amino acid) (SEQ ID NO: 237) located between Thr and Gly residues on the target protein, forming an enzyme-thioacyl intermediate through transient attachment of the Thr residue to the active site Cys residue on the enzyme. To complete the peptide transfer and produce a peptide-monomer conjugate, a biomolecule with a nucleophilic group at its N-terminus (typically an oligoglycine motif) attacks the intermediate, substituting saltase A and linking the two molecules.

[0032] As used herein, the term “steric blocker” refers to a polypeptide or polypeptide moiety that can covalently bind to a cytokine polypeptide directly or indirectly, for example, in the form of a chimeric polypeptide (fusion protein), via other moieties such as linkers, but otherwise does not covalently bind to the cytokine polypeptide. Steric blockers can non-covalently bind to cytokine polypeptides, for example, by electrostatic, hydrophobic, ionic, or hydrogen bonds. Due to their proximal position to the cytokine site and their relative size, steric blockers typically inhibit or block the activity of the cytokine moiety.

[0033] In this specification, “half-life extension element” refers to a portion of a chimeric polypeptide that increases the serum half-life and improves pK by, for example, altering its size (e.g., to exceed the renal filtration cutoff), shape, hydrodynamic radius, charge, or parameters of absorption, biodistribution, metabolism, and elimination.

[0034] As used herein, the terms “separation” or “linker” refer to an amino acid sequence, typically less than approximately 100 amino acids, that connects or links a first target amino acid sequence (e.g., an amino acid sequence that folds to form a first protein domain) to a second target amino acid sequence (e.g., an amino acid sequence that folds to form a second protein domain) within a continuous polypeptide chain. Separation or linkers are protease-cleavable because they typically contain one or more protease cleavage sites. A “tandem linker” refers to a linker containing two or more protease cleavage sites that can be cleaved by the same or different proteases. Tandem linkers can be positioned in any desired direction, for example, one cleavage site may be adjacent to the other, one cleavage site may overlap the other, or one cleavage site may be followed by the other by an amino acid interposed between the two cleavage sites.

[0035] As used herein, the terms “activatable,” “activate,” “inducible,” 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 activity when an additional element is cleaved from the conjugate.

[0036] As used herein, “plasmid” or “viral vector” is an active agent that transports the nucleic acids of this disclosure into a cell without degradation, and includes a promoter that causes the nucleic acid molecule and / or polypeptide to be expressed in the delivered cell.

[0037] As used herein, the terms “peptide,” “polypeptide,” or “protein” are used broadly to mean 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 suggest a specific size or number of amino acids constituting the molecule, and that the peptides of the present invention may contain up to several amino acid residues or more.

[0038] When used in general, “subjects” can refer to vertebrates, more specifically mammals (e.g., humans, horses, cats, dogs, cattle, pigs, sheep, goats, mice, rabbits, rats, and guinea pigs), birds, reptiles, amphibians, fish, and any other animals. The term does not indicate a specific age or sex. Therefore, it is intended to encompass adult and neonatal subjects, regardless of whether they are 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 terms patient or subject include human and veterinary subjects.

[0040] As used herein, the terms “treat,” “treat,” “treat,” or grammatically related terms refer to a disease or condition, or a method of reducing the effects of the symptoms of a disease or condition. Therefore, in the methods of this disclosure, treatment can mean reducing 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 a treatment if one or more symptoms of the disease in question are reduced by 10% compared to a control. Therefore, this reduction may 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. In this field, it is well understood that treatment does not necessarily mean the cure or complete disappearance of a disease, condition, or symptoms of a disease or condition. Desired therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, symptom relief, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, recovery or temporary relief of the disease state, and remission or improved prognosis.

[0041] As used herein, the terms “prevent,” “prevent,” and “prevent” a disease or disorder mean an action that inhibits or delays the onset or exacerbation of one or more symptoms of a disease or disorder, occurring before or approximately simultaneously with the onset of one or more symptoms of the disease or disorder in the subject, such as the administration of a chimeric polypeptide or a nucleic acid sequence encoding a chimeric polypeptide.

[0042] As used herein, references to “reduce,” “mitigate,” or “inhibit” include changes 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%, or at least about 90% or more compared to a preferred control level. Such terms may, but are not necessarily, include the complete disappearance of a function or property (e.g., agonist activity).

[0043] A “attenuated cytokine receptor agonist” is a cytokine receptor agonist whose receptor agonist activity is reduced compared to a naturally occurring agonist of the cytokine receptor. Attenuated cytokine agonists may have agonist activity of at least about 10×, at least about 50×, at least about 100×, at least about 250×, at least about 500×, at least about 1000×, or less compared to a naturally occurring agonist of the receptor. When a fusion protein containing a cytokine polypeptide described herein is described as “attenuated” or having “attenuated activity”, it means that the fusion protein is an attenuated cytokine receptor agonist.

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

[0045] As used herein, “part” refers to a portion of a molecule that has a characteristic function within the molecule, and that function may be performed by the part in the context of another molecule. The part may be a chemical entity having a specific function, or a part of a biological molecule having a specific function. For example, a “blocking portion” in a fusion protein is a portion of a fusion protein that is capable of blocking the activity of part or all of the fusion polypeptide. This may be a protein domain (e.g., serum albumin).

[0046] A. Separation part or linker This disclosure relates to a novel protease-cleavable segregation. As described herein, the protease-cleavable segregation is designed to be efficiently cleaved by a protease at a desired site (e.g., a protease selectively expressed or highly expressed in the tumor microenvironment), but to be stable and not cleaved or cleaved with low efficiency at other sites (e.g., peripheral areas, e.g., healthy tissue or serum).

[0047] The protease-cleavable isolates were designed using a process that prioritized proteases suitable for cleaving the isolate based on its expression in the target indication (e.g., expression in specific types of tumors (e.g., colorectal cancer, lung cancer, breast cancer, melanoma)). Multiple data sources (including mRNA, proteomics, and histological staining data) regarding increased or specific protease expression in the target indication were used. Proteases were also prioritized based on their specific activity and intrinsic specificity, with high specific activity and high intrinsic activity being preferred. Stability in serum was an important design consideration, and proteases independent of arginine in the substrate were selected to avoid the potential for off-target cleavage of the isolate by serum proteases. This is because many off-target enzymes are active against arginine residues.

[0048] In the design process, initiation sequences were selected using diverse peptide libraries as substrates for proteases. The protease cleavage products were detected by mass spectrometry to identify sequence motifs preferred for each candidate protease. For the selected initial motifs, novel peptide libraries were designed, produced, and analyzed, each adjusted to the preferred sequence motif for the candidate proteases. Furthermore, the peptide motifs were counter-screened for cleavage by serum proteases (thrombin and factor Xa) and by hepsin of hepatic / renal proteases. This process yielded peptides containing sequence motifs that are highly efficient cleaved by certain tumor-associated proteases (e.g., matrix metalloproteinase 9 (MMP9), MMP14, and / or cathepsin L), but stable in serum or normal healthy tissue (not cleaved or cleaved with low efficiency) (e.g., by thrombin, factor Xa, and hepsin). The isolated portions disclosed herein represent efficient cleavage by human tumors and minimal cleavage by normal tissue or serum.

[0049] This disclosure relates to a separator or linker that connects a first target amino acid sequence (e.g., a first target domain) to a second target amino acid sequence (e.g., a second target domain). Typically, the first and second target amino acid sequences are not found together in native proteins. For example, the separator can connect or join the first and second target domains of a fusion protein. The separator is an amino acid sequence that can be of any suitable length and is preferably cleaved by a protease.

[0050] The isolation components disclosed herein can impart functionality, including flexibility and the ability to cleave. Flexible linkers are typically applied when linked domains require some degree of movement or interaction. Scleavable linkers are introduced to release released functional domains to a target site in vivo. The isolation components disclosed herein serve to connect at least two target domains. The isolation components can maintain cooperative interdomain interactions or the retention of biological activity. The isolation components can link functional domains (e.g., payloads and half-life extension elements) released from the isolation component at a target site (e.g., a tumor microenvironment).

[0051] In a preferred embodiment, the separated portion is cleavable by a cleaving agent, such as an enzyme. Preferably, the separated portion includes protease cleavage sites. In some cases, the separated portion includes one or more cleavage sites. The separated portion may include a single protease cleavage site. Alternatively, the separated portion may include two or more protease cleavage sites. For example, it may include two, three, four, five, or more cleavage sites. If the separated portion includes two or more protease cleavage sites, the cleavage sites may be cleaved by the same protease or by different proteases. A separated portion containing two or more cleavage sites is referred to as a "tandem linker". The two or more cleavage sites can be arranged in any desired direction (including, but not limited to, cases where one cleavage site is adjacent to the other, where one cleavage site overlaps the other, or where one cleavage site follows the other by an intervening amino acid).

[0052] The present invention particularly focuses on disease-specific protease-cleaving linkers. Furthermore, it is preferable that protease-cleaving linkers are preferentially cleaved at desired locations within the body, such as the tumor microenvironment, compared to peripheral circulation. For example, the rate at which protease-cleaving linkers are 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 at a desired location within the body (e.g., the tumor microenvironment) compared to 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, aspartate proteases, threonine proteases, glutamate proteases, metalloproteinases, asparagine peptide lyases, serum proteases, cathepsins, cathepsins B, C, D, E, G, K, 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. Examples include actinidine, bromelain, calpain, caspase, caspase-3, Mirl-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matryptase, regmaine, plasmmepsin, 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). Proteases capable of cleaving linker amino acid sequences (which may be encoded by chimeric nucleic acid sequences provided herein) can be selected from, for example, the group consisting of prostate-specific antigen (PSA), matrix metalloproteinases (MMPs), disintegrin metalloproteinases (ADAMs), plasminogen activators, cathepsins, caspases, tumor cell surface proteases, and elastases. MMPs may be, for example, matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), and matrix metalloproteinase 14 (MMP14).In addition, or alternatively, the linker may be cleaved by cathepsins (e.g., cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin K, and / or cathepsin L). Preferably, the linker may be cleaved by MMP14 or cathepsin L.

[0054] Table 1 shows proteases useful for linker cleavage and for use in the methods disclosed herein, and Table 1a shows exemplary proteases and their cleavage sites. [Table 1-1] [Table 1-2] [Table 1A-1] [Table 1A-2]

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

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

[0057] Preferably, the linker length varies between 2 and 30 amino acids and is optimized for each condition so as not to impose any constraints on the three-dimensional structure or interactions of the domain to which the linker is bound.

[0058] In some embodiments, the separated portion is the sequence GPAGLYAQ (sequence number 195);GPAGMKGL (sequence number 196);PGGPAGIG (sequence number 197);ALFKSSFP (sequence number 198);ALFFSSPP (sequence number 199);LAQRLRSS (sequence number 200);LAQKLKSS (sequence number 201);GALFKSSFPSGGGPAGLYAQGGSGKGGSGK (sequence number 202);RGSGGGPAGLYAQGSGGGPAGLYAQGGSGK (sequence number 203); KGGGPAGLYAQGPAGLYAQGPAGLYAQGSR (Sequence No. 204);RGGPAGLYAQGGPAGLYAQGGGPAGLYAQK (Sequence No. 205);KGGALFKSSFPGGPAGIGPLAQKLKSSGGS (Sequence No. 206);SGGPGGPAGIGALFKSSFPLAQKLKSSGGG (Sequence No. 207);RGPLAQKLKSSALFKSSFPGGPAGIGGGGK (Sequence 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 Includes SSGPAGLYAQGGK (sequence number 215); RGGGPAGLYAQPLAQKLKSSALFKSSFPGG (sequence number 216); SGPLAQKLKSSGPAGLYAQALFKSSFPGSK (sequence number 217); KGGPGGPAGIGPLAQRLRSSALFKSSFPGR (sequence number 218); KSGPGGPAGIGALFFSSPPLAQKLKSSGGR (sequence number 219); or SGGFPRSGGSFNPRTFGSKRKRRGSRGGGG (sequence number 220).

[0059] Certain preferred isolates include the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198). Isolates disclosed herein may include one or more identical or different cleavage motifs or functional variants. Isolates may include one, two, three, four, five, or more cleavage motifs or functional variants. Isolates containing 30 amino acids may include two cleavage motifs or functional variants, three cleavage motifs or functional variants, or more. A “functional variant” of an isolate retains the ability to be cleaved efficiently at a target site (e.g., a tumor microenvironment expressing high levels of protease) and not cleaved or cleaved efficiently in peripheral areas (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 an isolate containing any one of SEQ ID NOs. 195-220.

[0060] The isolated portion containing multiple cleavage motifs can be selected from SEQ ID NOs: 195-201 and combinations thereof. A preferred isolated portion containing multiple cleavage motifs contains amino acids selected from SEQ ID NOs: 202-220.

[0061] The isolation portion may contain both ALFKSSFP (sequence number 198) and GPAGLYAQ (sequence number 195). The isolation portion may contain two cleavage motifs, each having the sequence GPAGLYAQ (sequence number 195). Alternatively or additionally, the isolation portion may contain two cleavage motifs, each having the sequence ALFKSSFP (sequence number 198). The isolation portion may contain the same or a different third cleavage motif.

[0062] In some embodiments, the isolated portion includes 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 sequence numbers 195–220 over the entire length of sequence numbers 195–220.

[0063] Furthermore, this disclosure also relates to functional variants of the isolated portion including SEQ ID NOs. 195-220. Functional variants of the isolated portion including 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 proteases.

[0064] A functional variant may include at least one amino acid substitution, deletion, or insertion compared to the isolate containing 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 the isolate containing SEQ ID NOs. 195-220. In some preferred embodiments, a functional variant differs from the isolate containing SEQ ID NOs. 195-220 by fewer than 10, 8, 5, 4, 3, 2, or 1 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 compared to SEQ ID NOs. 195-220. The amino acid substitutions may be conserved or non-conservative, but are preferably conserved.

[0065] In other embodiments, the functional variant of the isolated portion may contain one, two, three, four, or five or more non-conservative amino acid substitutions compared to the isolated portion containing SEQ ID NOs. 195-220. Non-conservative amino acid substitutions will be recognizable to those skilled in the art. The functional variant of the isolated portion preferably contains one, two, three, four, or five or fewer amino acid deletions.

[0066] The amino acid sequences disclosed in the isolated region can be described by their relative linear positions within the isolated region with respect to the sissile bond. As will be readily apparent to those skilled in the art, the isolated region containing eight amino acid protease substrates (e.g., SEQ ID NOs. 195-201) contains amino acids at positions P4, P3, P2, P1, P1', P2', P3', and P4', with the sissile bond located between P1 and P1'. For example, the amino acid positions in the isolated region containing the sequence GPAGLYAQ (SEQ ID NOs. 195) can be described as follows: [Table 1B]

[0067] The amino acid positions of the isolated region containing the sequence ALFKSSFP (sequence number 198) can be described as follows: [Table 1C]

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

[0069] In embodiments, the isolated 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, the functional variants of GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) contain one or more amino acid substitutions and can substantially retain the ability to be cleaved by proteases. Specifically, the functional variant of GPAGLYAQ (SEQ ID NO: 195) is cleaved by MMP14, and the functional variant of ALFKSSFP (SEQ ID NO: 198) is cleaved by cathepsin L (CTSL1). The functional variants also retain the ability to be cleaved efficiently at target sites (e.g., tumor microenvironments expressing high levels of proteases). For example, functional variants of GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) retain at least approximately 50%, 55%, 60%, 70%, 80%, 85%, 95%, or higher cleavage efficiency of the isolation portion containing the amino acid sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198), respectively.

[0070] Preferably, the functional variant of GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) contains 1, 2, 3, 4, or 5 or fewer 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 unsubstituted. 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] A functional variant 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 undesirable: 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 contain amino acid substitutions at position P1 and / or P1'.

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

[0073] In some embodiments, amino acid substitutions at position P2 or P2' of GPAGLYAQ (SEQ ID NO: 195) are undesirable.

[0074] In some embodiments, functional variants of GPAGLYAQ (SEQ ID NO: 195) include amino acid sequences selected from SEQ ID NOs: 258-331. Specific functional variants of GPAGLYAQ (SEQ ID NO: 195) include GPAGLLYAQ (SEQ ID NO: 295) and GPAGLKGA (SEQ ID NO: 285).

[0075] Functional variants of LFKSSFP (SEQ ID NO: 198) preferably include hydrophobic amino acid substitutions. Functional variants of LFKSSFP (SEQ ID NO: 198) may preferably include 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) position P1 (f) Phenylalanine, 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, serine, valine, histidine, alanine, or asparagine at position P3.

[0076] The inclusion of aspartic acid and / or glutamic acid in functional variants of SEQ ID NO: 198 is generally undesirable and should be avoided. In functional variants of LFKSSFP (SEQ ID NO: 198), the following amino acid substitutions are also undesirable: (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] The amino acid substitutions of the functional variant of LFKSSFP (SEQ ID NO: 198) preferably include amino acid substitutions at position P4 and / or P1. In some embodiments, amino acid substitutions at position P4' of the functional variant of LFKSSFP (SEQ ID NO: 198) are undesirable.

[0078] In some embodiments, functional variants of LFKSSFP (SEQ ID NO: 198) include amino acid sequences selected from SEQ ID NOs: 332-408. 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 isolated portion disclosed herein can form a stable complex under physiological conditions with the amino acid sequence (e.g., domain) to which it binds, while simultaneously being cleavable by proteases. For example, the isolated portion is stable in circulation (e.g., not cleaved or cleaved with low efficiency) and cleaved with high efficiency at the target site (i.e., the tumor microenvironment). Thus, the fusion polypeptide containing the linker disclosed herein has, if desired, 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 at the desired location (e.g., the tumor microenvironment), the linker can be efficiently cleaved to release the linker-linked component, restoring or nearly restoring the half-life and biological activity of the component as separate molecular entities.

[0080] The separated portion preferably remains stable in 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 longer.

[0081] In some embodiments, the separated portion is cleaved by 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 20%, 5%, or less than 1% in circulation compared to the target site. Furthermore, the separated portion remains stable even in the absence of an enzyme capable of cleaving the linker. However, upon exposure to a suitable enzyme (i.e., a protease), the separated portion is cleaved, resulting in the separation of the bound domain.

[0082] B. Polypeptides and compositions containing separated portions The isolation portions disclosed herein can be used in a wide range of applications. While not limited to these applications, the isolation portions are also suitable for fusion proteins. As further described herein, the isolation portions are particularly useful for preparing therapeutic fusion proteins in which the therapeutic biological activity of the fusion protein is attenuated, and this attenuation is removed by cleavage of the isolation portion. The isolation portions 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. Thermanson (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 isolation portions suitable for use with payloads that target or are targeted to the tumor microenvironment.

[0083] This disclosure relates to recombinant polypeptides in which an isolated portion, as disclosed herein, links a first target amino acid sequence (e.g., a first target domain) to a second target amino acid sequence (e.g., a second target domain). Typically, the first and second target amino acid sequences are not found together in natural proteins. Preferred linkers are SEQ ID NOs: 195-220. In embodiments, at least one of the first and second target amino acid sequences is an amino acid sequence of a therapeutic polypeptide. In some embodiments in which at least one of the first and second target amino acid sequences is an amino acid sequence of a therapeutic polypeptide, the other target amino acid sequence may be an amino acid sequence of a targeted polypeptide, a half-life extension polypeptide, and / or a blocking polypeptide.

[0084] A polypeptide containing a segregated portion can be represented by formula I:[D1]-[L1]-[D2], where D1 is a first target amino acid sequence (e.g., a target domain), L1 is a segregated portion that connects or binds D1 to D2, and D2 is a second target amino acid sequence (e.g., a second target domain). Preferably, L1 is a protease-cleavable segregated portion, and more preferably, L1 includes or consists of any of sequence numbers 195-220.

[0085] The polypeptide can also be represented by formula II:[D1]-[L1]-[D2]-[L2]-[D3], where D1 is the first target amino acid (e.g., the target domain), L1 and L2 are each independently linkers, D2 is the second target amino acid sequence (e.g., the target domain), D3 is the third target amino acid (e.g., the target domain), and at least one of L1 and L2 is a protease-cleavable segregation portion, preferably at least one of L1 and L2 contains or consists of any of sequence numbers 195 to 220.

[0086] Furthermore, the polypeptide can also be represented by formula III:[D1]-[L1]-[D2]-[L2]-[D3]-[L3]-[D4], where D1 is a first target amino acid (e.g., a target domain), L1, L2, and L3 are each independently linkers, D2 is a second target amino acid sequence (e.g., a target domain), D3 is a third target amino acid (e.g., a target domain), D4 is a fourth target amino acid (e.g., a target domain), and at least one of L1, L2, and L3 is a protease-cleavable segregation portion, preferably at least one of L1, L2, and L3 includes or consists of any of sequence numbers 195-220.

[0087] Further specific applications of the separated portion will be described in more detail in this specification.

[0088] i. Delivery of the payload The isolation portions described herein can be used to attach therapeutic drug portions. In this approach, therapeutic drug portions are attached to the isolation portions to create therapeutic drug subcomplexes. The individual drug subcomplexes may be inactive prodrugs until a target protease cleaves the prodrug and releases the drug.

[0089] ii. Antibody-drug conjugates Another application of the isolated portion is in the field of antibody-drug conjugates (ADCs), primarily targeting cancer treatment. ADCs are typically antibodies that bind to a cytotoxic portion (e.g., a cytotoxic drug). ADCs distinguish between healthy and diseased cells, resulting in targeted delivery of the drug (e.g., a cytotoxic drug) to the diseased cells. ADCs typically contain antibodies that target tumor markers specific to tumor cells; the antibody attaches itself to the tumor cells, and the ADC is absorbed into the cells, thereby releasing the cytotoxic component and killing the tumor cells. An important aspect of ADCs is the provision of a stable linker between the antibody component and the cytotoxic drug. In such applications, the linker may be cleavable or non-cleavable. In the case of a non-cleavable linker, the antibody, linker, and cytotoxic unit are taken up into the tumor cells. The properties of the linker typically determine the release profile of the cytotoxic drug. For example, the cleavage linker between an antibody and a cytotoxic drug is typically catalyzed by enzymes within tumor cells or the tumor microenvironment, at which point the antibody and cytotoxic drug are cleaved, releasing the cytotoxic drug.

[0090] In certain embodiments, the separation portion disclosed herein binds or connects the drug portion to the antibody portion.

[0091] iii. Peptide-drug conjugates The isolation portions disclosed herein are suitable for use in peptide-drug conjugates. Such compounds typically contain a cytotoxic payload and a linker, but the peptide-drug conjugate, instead of an antibody, is equipped with a peptide capable of penetrating tumors, thus allowing the cargo to be delivered into the tumor. In some embodiments, the isolation portion conjugates or links the cytotoxic payload to the peptide. The peptide-drug conjugate remains stable and biologically inactive until the isolation portion is cleaved by a target protease.

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

[0093] In one embodiment, the isolated portion is used to tether the targeting portion to the CAR construct. The CAR replaces the endogenous TCR complex with a novel receptor that binds to an external target of cancer cells using a fragment of a human or mouse antibody. The antibody fragment is bound to various intracellular signaling proteins 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, the isolated portion is used to tether the targeting portion to the TCR construct. The TCR is based on a gene for a protein receptor already naturally present in T cells. The gene for the desired TCR may be discovered within a single patient, for example, in a patient capable of initiating an effective immune response against a certain type of cancer. This gene can then be introduced into other patients by incorporating it into a TCR T cell construct, or it can be reengineered to improve its binding interaction with MHC targets. 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] Such types of engineered T cells, whether autologous or allogeneic, include engineered T cell receptor components that include targeting agents such as isolated human antibodies or humanized antibodies. In CAR-T cells and TCR-T cells, the binding affinity of the targeting moiety can be affected by the steric, chemical, or flexibility properties of the spacer moiety that tethers the targeting moiety to the construct and the rest of the T cell. The spacer moieties disclosed herein are suitable for use with engineered constructs for making CAR T cells and TCR T cells.

[0096] v. antigen-binding protein The isolated moieties disclosed herein are suitable for use in antigen-binding proteins. An "antigen-binding protein" (ABP) is a protein that includes 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 specificity and affinity similar to that of a natural antibody. Typically, the isolated moiety binds a polypeptide that blocks the antigen-binding site of the ABP from binding its cognate antigen. However, when the isolated moiety is cleaved, the blocking polypeptide diffuses away from the ABP antigen-binding site, and the ABP can bind 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 of the complementarity determining regions (CDRs) within the antigen-binding site of the ABP. Such blocking peptides can be obtained by screening a library or by screening peptide fragments of the cognate antigen of the ABP of interest. Typically, when the ABP includes an antigen-binding site of an antibody, the isolated moiety and the blocker bind to the amino terminus of the antibody light chain or the amino terminus of the antibody heavy chain, and the blocker is tethered near the antigen-binding site to readily block the antigen-binding site. When the ABP includes an alternative scaffold for the binding site, suitable variations of this approach are used. Similarly, when a single-chain antibody binding site (e.g., scFV of a dAb) is used, the blocker isolated moiety typically binds to the amino terminus near the antigen-binding site. In certain embodiments, the ABP includes an antibody binding site comprising VH and VL, and the blocker isolated moiety is bound to the amino terminus of VL.

[0097] The ABP may be an antibody (for example, 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 (for example, 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, ABP consists of an antibody. In some embodiments, ABP is essentially composed of an antibody. In some embodiments, ABP includes an alternative scaffold. In some embodiments, ABP consists of an alternative scaffold. In some embodiments, ABP is essentially composed of an alternative scaffold. In some embodiments, ABP includes an antibody fragment. In some embodiments, ABP consists of an antibody fragment. In some embodiments, ABP is essentially composed of an antibody fragment.

[0099] In some embodiments, the isolation portions disclosed herein are suitable for use with antibodies. The term “antibody” is used herein in its broadest sense and includes certain types of immunoglobulin molecules that contain 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 polyspecific antibodies. An antibody is one type of ABP.

[0100] In some embodiments, the isolation portions disclosed herein are suitable for use with antigen-binding proteins containing alternative scaffolds. An "alternative scaffold" refers to a molecule in which one or more regions are 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 the antigen or epitope with similar specificity and affinity to an antibody. Exemplary alternative scaffolds include fibronectin (e.g., Adnectin®), β-sandwich (e.g., iMab), lipocalin (e.g., Anticalin®), EETI-II / AGRP, BPTI / LACI-D1 / ITI-D2 (e.g., Knitz 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 on alternative scaffolds can be found 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 referenced in whole. Alternative scaffolds are a type of ABP.

[0102] In some embodiments, the isolation portions disclosed herein are suitable for use with antibody fragments. “Antibody fragment” includes a portion of an intact antibody, for example, the antigen-binding region or variable region of an intact antibody. Examples of antibody fragments include Fv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv(sFv) fragments, and scFv-Fc fragments.

[0103] In some embodiments, the separation portions disclosed herein are suitable for use with one or more Fv, Fab, or F(ab')2 fragments. An "Fv" fragment comprises a non-covalent dimer of one heavy-chain variable domain and one light-chain variable domain. A "Fab" fragment comprises a constant domain of the light chain and a first constant domain (CH1) of the heavy chain, in addition to the heavy-chain and light-chain variable domains. 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') fragments can be dissociated, for example, by treatment with 1-mercaptoethanol.

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

[0105] In some embodiments, the linker is (GGGGS)n (Sequence ID 231). In some embodiments, n = 1, 2, 3, 4, 5, or 6. Antibodies from Escherichia coli. In Rosenberg M. & Moore GP (Eds.), The Pharmacology See Monoclonal Antibodies vol.113 (pp.269-315). Springer-Verlag, New York (the entire text is referenced by reference). The “scFv-Fc” fragment contains an scFv with an attached 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 domain of the scFv (i.e., VH-VL or VL-VH). Any suitable Fc domain known in the art or described herein may be used. In some cases, the Fc domain contains an IgG4 Fc domain.

[0106] In some embodiments, the isolation portions 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 the antibody specifically binds to an antigen without the presence of another variable domain. Single-domain antibodies and their fragments 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 whole). Single-domain antibodies are also known as sdAbs or nanobodies.

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

[0108] In some embodiments, the isolation portions 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 includes antibodies that are substantially similar and bind to the same epitope(s), except for variants that may normally occur during the production of monoclonal antibodies. Such variants are generally present in small amounts. Monoclonal antibodies are typically obtained by a process that involves selecting a single antibody from multiple antibodies. For example, the selection process may involve selecting a unique clone from multiple clones (e.g., a pool of hybridoma clones, phage clones, or recombinant DNA clones). The selected antibody may be further modified, for example, to improve its affinity for TNFR superfamily member proteins ("affinity maturation"), to humanize the antibody, to improve its yield in cell culture, and / or to reduce its immunogenicity in the target.

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

[0110] In some embodiments, the isolation portions disclosed herein are suitable for use with humanized antibodies. The “humanized” form of a non-human antibody is a chimeric antibody containing minimal sequences derived from the non-human antibody. A humanized antibody is generally a human antibody (recipient antibody) in which residues from one or more CDRs are replaced by 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) having the desired specificity, affinity, or biological effect. In some cases, selected framework region residues of the recipient antibody are replaced by corresponding framework region residues from the donor antibody. The humanized antibody may also contain residues not found in either the recipient antibody or the donor antibody. Such modifications may be made to further refine the antibody function. Further details are provided, 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 these is incorporated herein by reference in whole).

[0111] In some embodiments, the isolation portions disclosed herein are suitable for use with human antibodies. “Human antibody” refers to an antibody having an amino acid sequence corresponding to an antibody produced by a human or human cell, or one derived from a non-human source utilizing the human antibody repertoire or a human antibody coding sequence (e.g., obtained from a human source or newly designed). Specifically, human antibodies exclude humanized antibodies.

[0112] In some embodiments, the ABP provided herein specifically binds to the extracellular domain of TNFR superfamily proteins. In some embodiments, the TNFR superfamily proteins are CD27, CD137, CD40, GITR, LT-beta-R, CD30, HVEM, TNFR1, TNFR2, or OX-40. The TNFR superfamily proteins 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 myeloid-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 ABP provided herein is an antibody. In some embodiments, the ABP provided herein is an antibody fragment. In some embodiments, the ABP provided herein is an alternative scaffold.

[0114] In some embodiments, the ABP provided herein comprises an immunoglobulin molecule. In some embodiments, the ABP provided herein consists of an immunoglobulin molecule. In some embodiments, the ABP provided herein is essentially composed of an immunoglobulin molecule. In some embodiments, the immunoglobulin molecule comprises an antibody. In some embodiments, the immunoglobulin molecule consists of an antibody. In some embodiments, the immunoglobulin molecule is essentially composed of an antibody.

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

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

[0117] In some embodiments, the separating portion is connected to a further blocking portion.

[0118] In some embodiments, the separating portion is arranged so that the movement of the antigen-binding protein subunit is restricted.

[0119] a. Multivalent antigen-binding proteins with dual specificity and monospecificity In some embodiments, the isolation portion disclosed herein is suitable for use with a multispecific antigen-binding protein (ABP). The multispecific ABP provided herein binds to multiple antigens. For example, a multispecific antibody can bind to two, three, four, five, or more antigens. Alternatively, a 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 one 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, a multispecific ABP binds to two different epitopes (i.e., a "bispecific ABP"). In some embodiments, a multispecific ABP binds to three different epitopes (i.e., a "trispecific ABP"). In some embodiments, multispecific ABPs bind to four different epitopes (i.e., "tetraspecific ABPs"). In some embodiments, multispecific ABPs bind to five different epitopes (i.e., "pentaspecific ABPs"). In some embodiments, multispecific ABPs bind to six, seven, eight, or more different epitopes. Each binding specificity can exist in any preferred valency.

[0120] In various embodiments, the antigen-binding protein includes a blocking domain. The isolation portion disclosed herein allows the blocking domain to be attached to the first antigen-binding domain, thereby inhibiting (a) the binding affinity or avidity of the antigen-binding protein on the epitope, and / or (b) the agonist or antagonist activity of the antigen-binding protein on the epitope. Preferably, the isolation portion includes a cleavage site. When the isolation portion is cleaved by a disease-specific enzyme (i.e., a protease), (a) the binding affinity or avidity of the antigen-binding protein to the epitope, and / or (b) the agonist or antagonist activity of the antigen-binding protein to the epitope increases. In some embodiments, the antigen-binding protein includes an additional isolation portion that binds or affixes an additional blocking domain to the antigen-binding domain. The additional isolation portion includes a cleavage site.

[0121] In various embodiments, the antigen-binding protein includes additional blocking domains (e.g., second, third, or fourth blocking domains) that are responsively bound to the antigen-binding domain (e.g., second, third, or fourth antigen-binding domain) by a cleavable separation moiety as disclosed herein. The blocking domains may be steric blockers or specific blockers. In some embodiments, the steric blockers are independently selected from the group consisting of extracellular fragments of antibody-binding proteins, serum albumin, fragments of serum albumin, and antibodies or antigen-binding fragments that bind to serum albumin. In some embodiments, the specific blockers are independently selected from antibodies or antigen-binding fragments that bind to the first, second, third, or fourth antigen-binding domain of the antigen-binding protein described herein. Preferably, the blocking domains are serum albumin or fragments thereof, or antibodies or antigen-binding fragments that bind to serum albumin.

[0122] The antigen-binding domain may further include a third antigen-binding domain and a fourth antigen-binding domain, each having binding specificity to the target antigen. TNFR superfamily target antigens are well known in the art and are included in this 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-beta-R, GITR, nerve growth factor receptor, or OX-40 (CD34). Preferred TNFR superfamily target antigens are CD27, CD137, and OX40.

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

[0124] In some embodiments, the antigen-binding polypeptide includes an Fc region. In one form, two antigen-binding domains are located at opposite ends of the Fc region. In another form, two antibody arms are attached to the N-terminus of the Fc region, each arm containing two antigen-binding domains. The antigen-binding domain may also include a half-life extension domain. The half-life extension domain may be albumin, an antigen-binding domain that recruits albumin, or immunoglobulin Fc or a fragment thereof. In some embodiments, the half-life extension domain is responsively bound to the antigen-binding polypeptide by a cleavable linker.

[0125] The disclosure also relates to an antigen-binding protein comprising at least a first polypeptide and a second polypeptide. The first polypeptide comprises at least a portion of the antibody heavy chain constant region and the antibody heavy chain variable region (VH). The second polypeptide comprises at least a portion of the antibody light chain constant region and the antibody light chain variable region (VL). At least one of the first and second polypeptides further comprises a blocking domain that is responsively bound to the VH or VL via a protease-cleaving linker. The first polypeptide associates with the second polypeptide, and the VH and VL form an antigen-binding site that has binding specificity to 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 specific to 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, ABP is monospecific polyvalent ABP. Such forms can have various structures and can be prepared using suitable antibody manipulation techniques. For example, a bivalent Fab antibody can be prepared containing a heavy chain with the structure VH-CH1-uncleavable liner-VH-CH1-CH2-CH3. Such a heavy chain can be expressed and paired with two light chains. The heavy chain can be dimerized via conventional interchain disulfide bonds to form an antibody form containing four Fab antigen-binding sites. In such a form, the separation portion described herein can be bound to the amino terminus of the light chain polypeptide, and a blocker can be bound to each of the Fab antigen-binding sites. Other suitable forms of monospecific polyvalent ABP can be readily conceived by those skilled in the art. In one such example, a heavy chain having 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 form. In this form, the isolation portion described herein can be attached to the amino terminus of a light chain polypeptide, allowing the blocker to be attached to each of the Fab antigen-binding sites. The binding activity of such monospecific tetravalent ABP is shielded by a blocking domain, which is removed when the isolation domain is cleaved (e.g., in the tumor microenvironment), allowing the ABP to bind to its homologous antigen.

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

[0128] In embodiments, the polyvalent antigen-binding protein may include a first antigen-binding site specific to a target antigen (e.g., CD27 or TNFR1), a second antigen-binding site specific to a target antigen, a blocking polypeptide, at least one protease-cleaving linker, and an optional half-life-extending element. In such embodiments, the first blocking polypeptide is responsively bound to the first antigen-binding site by a protease-cleaving linker, and optionally, the second blocking polypeptide is responsively bound to the second antigen-binding site by a protease-cleaving linker. Preferably, the blocking polypeptide is responsively bound to the second antigen-binding site by a protease-cleaving linker. The blocking polypeptide inhibits (a) the binding affinity or avidity of the antigen-binding protein to the target antigen, and / or (b) the agonist or antagonist activity of the antigen-binding protein to the target antigen, and when the protease-cleaving linker is cleaved, (a) the binding affinity or avidity of the antigen-binding protein to the target antigen, and / or (b) the agonist activity of the antigen-binding protein to the target antigen increases. An optional half-life extension element may be admissibly bound to the first antigen-binding site and / or the second antigen-binding site via an optional protease-cleaving linker. In such embodiments, the first and second binding sites may be specific to the same epitope or different epitopes.

[0129] In the embodiment, the antigen-binding protein further includes a third antigen-binding site specific to the same target antigen. The antigen-binding protein may further include a fourth antigen-binding site specific to the same target antigen as the first, second, and third antigen-binding sites. The third and fourth antigen-binding sites may each further include a blocking polypeptide that is competently bound to the antigen-binding site via a protease-cleaving linker.

[0130] The disclosure also relates to a tetravalent antigen-binding protein, which may comprise a first polypeptide comprising at least a portion of the constant region and variable region (VH) of the antibody heavy chain, and a second polypeptide comprising at least a portion of the constant region and variable region (VL) of the antibody light chain. The first polypeptide associates with the second polypeptide, and the VH and VL form an antigen-binding site having binding specificity to the target antigen. The tetravalent antigen-binding protein further comprises a blocking polypeptide that is responsively bound to the VH or VL via a protease-cleaving linker. The blocking polypeptide inhibits the antigen-binding site from binding 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 having specificity to the 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 at least a portion of the antibody heavy chain constant region and the antibody heavy chain variable region (VH). The fourth polypeptide may comprise at least a portion of the antibody light chain constant region and the antibody light chain variable region (VL). The fifth polypeptide may comprise at least a portion of the antibody heavy chain constant region and the antibody heavy chain variable region (VH). The sixth polypeptide may comprise at least a portion of the antibody light chain constant region and the antibody light chain variable region (VL). The seventh polypeptide may comprise at least a portion of the antibody heavy chain constant region and the antibody heavy chain variable region (VH). The eighth polypeptide may comprise at least a portion of the antibody light chain constant region and the antibody light chain variable region (VL).

[0132] In some embodiments, at least one of the third and fourth polypeptides further comprises a blocking polypeptide that is competently bound to VH or VL via a protease-cleaving linker. The third polypeptide associates with the fourth polypeptide, VH and VL form an antigen-binding site with binding specificity to the 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 include a blocking polypeptide that is competently bound to VH or VL via a protease-cleaving linker. The fifth polypeptide associates with the sixth polypeptide, and VH and VL form an antigen-binding site that has binding specificity to the target antigen, while the blocking domain inhibits the antigen-binding site from binding to the target antigen.

[0134] In some embodiments, the seventh and eighth polypeptides further include a blocking polypeptide that is competently bound to VH or VL via a protease-cleaving linker. The seventh polypeptide associates with the eighth polypeptide, and VH and VL form an antigen-binding site that has binding specificity to the target antigen, while the blocking domain inhibits the antigen-binding site from binding to the target antigen.

[0135] vi. Inducible cytokines This specification discloses methods and compositions for manipulating 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 thought to have a short serum half-life and high potency. As a result, therapeutic administration of cytokines leads to undesirable systemic effects and toxicity. This effect and toxicity are further exacerbated by the need to administer large amounts of cytokine to achieve the desired level of cytokine at the intended site of 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 the treatment of tumors.

[0136] This specification discloses a fusion protein that overcomes the toxicity and short half-life issues that have severely limited the clinical use of cytokines in oncology. The fusion protein contains a cytokine polypeptide having receptor agonist activity. However, in the context of the fusion protein, the agonist activity of the cytokine receptor 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 active site (e.g., tumor), and is typically concentrated or selectively present at the desired active site. Thus, the fusion protein is preferentially (or selectively) and efficiently cleaved at the desired active site, substantially limiting cytokine activity to the desired active site, such as the tumor microenvironment. Upon protease cleavage at the desired active site (e.g., tumor microenvironment), a form of cytokine with much higher activity as a cytokine receptor agonist than the fusion protein (typically at least about 100 times more active than the fusion protein) is released from the fusion protein. The cytokines released upon cleavage of fusion proteins typically have short half-lives, often substantially similar to those of native cytokines, further limiting cytokine activity against the tumor microenvironment. Even with extended fusion protein half-lives, toxicity is dramatically reduced or eliminated because the circulating fusion protein is attenuated and the active cytokines are targeted to the tumor microenvironment. The fusion proteins described herein enable, for the first time, the administration of effective therapeutic doses of cytokines to treat tumors by substantially limiting cytokine activity to the tumor microenvironment, dramatically reducing or eliminating the undesirable systemic effects and toxicity of cytokines.

[0137] Generally, the therapeutic use of cytokines is severely limited by their systemic toxicity. For example, TNF was initially discovered to have the ability to induce hemorrhagic necrosis in some tumors and to exert in vitro cytotoxic effects on various tumor lines, but it was later found to possess potent pro-inflammatory activity and, under conditions of overproduction, could have dangerous effects on the human body. Systemic toxicity is a fundamental problem when using pharmacologically active amounts of cytokines in humans, and currently, novel derivatives and therapeutic strategies are being evaluated to maintain therapeutic efficacy while reducing the toxic effects of this class of biological effectors.

[0138] IL-2 plays a stimulating and regulatory role in the immune system and, along with other members of the common gamma chain (γc) cytokine family, is central to immune homeostasis. IL-2 mediates its action by binding to the IL-2 receptor (IL-2R). This receptor consists of either a trimer receptor composed of IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (γc, CD132) chains, or a dimer βγIL-2R (1, 3). Both IL-2R variants can transmit signals when IL-2 binds to them. However, the trimer αβγIL-2R has approximately 10 to 100 times higher affinity for IL-2 than the dimer βγIL-2R (3). This suggests that CD25 confers high-affinity binding of IL-2 to the receptor, but is not essential for signal transduction. Trimeric IL-2R is found in activated T cells and CD4+ forkhead box P3 (FoxP3)+ T regulatory cells (Tregs), and these cells are sensitive to IL-2 in vitro and in vivo. Conversely, antigen-experienced (memory) CD8+, CD44 high-memory phenotype (MP) CD8+, and natural killer (NK) cells are conjugated with high levels of dimeric βγIL-2R, and these cells also respond actively to IL-2 in vitro and in vivo.

[0139] The expression of high-affinity IL-2R is essential for T cells to respond to low concentrations of IL-2 that are transiently available in vivo. IL-2Rα expression is not observed in naive T cells or 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 strictly regulated and is constitutively expressed by all lymphoid cells. When high-affinity IL-2R is induced by an antigen, IL-2R signaling upregulates IL-2Rα expression, partly through the regulation of Stat5-dependent IL2ra transcription (Kim et al., 2001). This process corresponds to a mechanism that maintains high-affinity IL-2R expression and preserves IL-2 signaling while a source of IL-2 remains available.

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

[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 to produce a stable quaternary high-affinity IL-2R (Kd 10-11M, i.e., 10pM). The formation of the high-affinity quaternary IL-2-IL-2R complex leads to signal transduction via tyrosine kinases Jak1 and Jak3 (which associate 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, while IL-2Rα is recycled on the cell surface (Hemar et al., 1995; Yu and Malek, 2001). Thus, such functional activity, which requires sustained IL-2R signaling, necessitates a continuous supply of IL-2 to engage with IL-2Rα and form further IL-2-IL-2R signaling complexes.

[0142] Interleukin-15 (IL-15), another member of the 4-alpha-helix bundle family of cytokines, is also emerging as an immunomodulator for cancer treatment. IL-15 is initially captured via IL-15Rα, which is expressed on antigen-presenting dendritic cells, monocytes, and macrophages. IL-15 exhibits broad activity, inducing differentiation and proliferation of T cells, B cells, and natural killer (NK) cells through signaling via IL-15 / IL-2-R-β (CD122) and the common γ chain (CD132). It also interacts with CD8 + CD8 cells also enhance the cytolytic activity of T cells and have long-lasting antigen experience. +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, it is thought that selectively boosting IL-15 activity in the tumor microenvironment enhances innate and specific immunity, enabling the fight against tumors (Waldmann et al., 2012). IL-15 was initially identified as having the ability to stimulate T cell proliferation in a manner similar to IL-2, via a common receptor component (IL-2R / 15Rβ-γc) and signaling mediated by 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 promoting the induction of cytotoxic T lymphocytes and the generation, proliferation, and activation of NK cells (Waldmann et al., 1999). However, unlike IL-2, which is required to maintain forkhead box P3 (FOXP3)-expressing CD4+CD25+ Treg cells and keep these cells in the periphery, IL-15 has little effect on Tregs (Berger et al., 2009). This is important because CD4+CD25+ Tregs expressing FOXP3 inhibit effector T cells, thereby inhibiting immune responses, including those against tumors. IL-2 also plays a crucial role in initiating activation-induced cell death (AICD), a process that leads to the elimination of autoreactive T cells, while 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.). (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 evident in the fact that IL-15Rα - / - and IL-15 - / - This is further emphasized by the mouse phenotype. Knockout mice have been shown to have a reduced total number of CD8+ T cells and to be deficient in several subsets of memory phenotypic CD8+ T cells, NK cells, NK / T cells, and intestinal intraepithelial lymphocytes, indicating that IL-15 imparts essential positive homeostatic functions to these cell subsets (Lodolce et al., 1996; Kennedy et al., 1998). The phenotypic similarities between these two lines of knockout mice suggest that IL-15Rα is important for maintaining physiologically appropriate IL-15 signaling.

[0144] IL-15 is trans-presented on the IL-15Rβγc complex on the surface of T cells and natural killer (NK) cells via the IL-15 receptor alpha chain (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 has been shown to have limited effects on cancer patients because it relies on IL-15Ra, which is frequently downregulated in cancer patients. Therefore, the fusion protein RLI, in which the sushi+ domain of IL15Ra is bound to IL-15 via a linker, has been proposed as an alternative approach to IL-15 therapy (Bessard et al., 2009). Administration of soluble IL-15 / IL-15Rα complex has been shown to significantly enhance 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, modulates phagocytosis, and stimulates the secretion of IL-8 and IL-1R antagonists. IL-15 functions by activating JAK2, p38, ERK1 / 2 MAPK, Syk kinase, and NF-κB transcription factors (Pelletier et al., 2002). In mast cells, IL-15 functions as a growth factor and apoptosis inhibitor. In such cells, IL-15 activates the JAK2 / STAT5 pathway without requiring γc binding (Tagaya et al., 1996). Furthermore, IL-15 induces proliferation and differentiation of B lymphocytes and increases immunoglobulin secretion (Armitage et al., 1995). Furthermore, it prevents Fas-mediated apoptosis and enables the induction of an antibody response partially independently of CD4 help (Demerci et al., 2004; Steel et al., 2010). Monocytes, macrophages, and dendritic cells effectively transcribe and translate IL-15. They also 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 CD83, CD86, CD40, and MHC class II expression, are resistant to apoptosis, and exhibit enhanced interferon-γ secretion (Anguille et al., 2009).

[0146] Furthermore, IL-15 has been shown to act on non-blood cells, including muscle cells, adipocytes, endothelial cells, and nerve cells. IL-15 can exert anabolic effects on muscle and may direct muscle cell differentiation (Quinn et al., 1995). IL-15 can stimulate muscle cells and muscle fibers to accumulate contractile proteins and can also slow muscle wasting in rats with cancer-related cachexia (Figueras et al., 2004). In addition, IL-15 has been shown to stimulate angiogenesis (Angiolillo et al., 1997) and induce microglial growth and survival (Hanisch et al., 1997).

[0147] Interleukin-7 (IL-7), also belonging to 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 after activation, also expressed by dendritic cells (Alpdogan et al., 2005). Initially, IL-7 was described as a growth and differentiation factor for precursor B lymphocytes, but subsequent studies have shown that it is critically involved in T lymphocyte development and differentiation. Interleukin-7 signaling is essential for the establishment of optimal CD8 T cell function, homeostasis, and memory (Schluns et al., 2000), is necessary for the survival of most T cell subsets, and its expression is proposed to be important for regulating T cell number.

[0148] IL-7 is IL-7Rα and γ cIL-7Rα binds to a dimeric receptor containing IL-7Rα to form a ternary complex, which plays a fundamental role in extracellular matrix remodeling, T cell and B cell development, and homeostasis (Mazzucchelli and Durum, 2007). Furthermore, IL-7Rα cross-reacts with thymic stromal lymphocyte necrosis factor (TSLP) and its receptor (TSLPR) to form a ternary complex, activating the TSLP pathway. This results in the proliferation of T cells and dendritic cells in humans, and further B cell development in mice (Leonard, 2002). Therefore, tight regulation of the signaling cascade activated by this complex is essential for normal cellular function. Mutations in the IL-7Rα external domain lead to IL-7 pathway deficiency, inhibiting T cell 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 even limited thymic function by strengthening immune reconstitution and promoting peripheral expansion of even a small number of thymic extruders. Therefore, IL-7 therapy has potential to repair the immune system depleted by cytotoxic chemotherapy (Capitini et al., 2010).

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

[0151] The IL-12 receptor (IL-12R) is a heterodimer complex consisting of 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 capability (Benson et al., 2011). Signaling via IL-12R induces phosphorylation of Janus kinase (Jak2) and tyrosine kinase (Tyk2), which phosphorylate and activate signaling transcription factors (STAT)1, STAT3, STAT4, and STAT5. The specific cellular effects of IL-12 are mainly due to the activation of STAT4. IL-12 induces natural killer cells and T cells to produce cytokines, specifically interferon (IFN)γ, and mediates many of IL-12's pro-inflammatory activities, including the differentiation of CD4+ T cells into the Th1 phenotype (Montepaone et al., 2014).

[0152] Treg cells actively suppress the activation of the immune system, preventing pathological autoreactivity and resulting autoimmune diseases. The development of drugs and methods to selectively activate regulatory T cells for the treatment of autoimmune diseases has been a subject of intense research, and much of it was unsuccessful until the present invention was developed, which can selectively deliver activated interleukins to the site 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 and play a major role in maintaining tolerance to autoantigens 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 associated with deficiencies in Treg cell count and Treg function.

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

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

[0155] Chronic GVHD patients (Koreth, J., et al., 2011, N Engl J Clinical trials of low-dose IL-2 therapy in patients with HCV-associated autoimmune vasculitis (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 being initiated to investigate the efficacy of IL-2 in several other autoimmune and inflammatory diseases. The reason for using so-called low-dose IL-2 is to take advantage of the high IL-2 affinity of the trimer IL-2 receptor constitutively expressed on Tregs, while also inactivating other T cells that do not express the high-affinity receptor. Aldesleukin (marketed as Proleukin® by Prometheus Laboratories, San Diego, CA), a recombinant form of IL-2 used in these trials, is associated with high toxicity. Aldesleukin is approved for the treatment of metastatic melanoma and metastatic renal cell carcinoma, but due to its extremely serious side effects, its use is only recommended in hospital settings with access to intensive care (Web address: www.proleukin.com / assets / pdf / proleukin.pdf).

[0156] In clinical trials of IL-2 in autoimmune diseases, low doses of IL-2 are used to target Treg cells. This is because Treg cells, by expressing IL2R alpha, 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 such low doses, safety and tolerability issues arise, and the treatments using IL-2 involve either daily subcutaneous injections on a chronic basis or intermittent 5-day treatment courses. Therefore, there is a need for autoimmune disease therapies that enhance the number and function of Treg cells, target Treg cells more specifically than IL-2, are safer and more tolerable, and require less frequent administration.

[0157] One proposed approach to improve the therapeutic index of IL-2-based therapy is to use variants of IL-2 that are more selective to Treg cells than other immune cells. The IL-2 receptor is 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 the multifaceted effects on the immune system and high systemic toxicity. Specifically, activated T effector cells express IL2Rαβγ, as do lung epithelial cells. However, activation of T effector cells is directly contrary to the objective of downmodulating and controlling the immune response, and activation of lung epithelial cells leads to known dose-limiting side effects of IL-2, including pulmonary edema. In fact, the main side effect of high-dose IL-2 immunotherapy is vascular leakage syndrome (VLS), in which fluid accumulates in the blood vessels of organs such as the lungs and liver, subsequently causing pulmonary edema and hepatocyte damage. There is no treatment for VLS other than discontinuation of IL-2. Low-dose IL-2 regimens are being tested in patients to avoid VLS, but at the cost of suboptimal treatment outcomes.

[0158] According to the literature, VLS is thought to be caused by the release of inflammatory cytokines from IL-2 activated NK cells. However, there is strong evidence that pulmonary edema arises from IL-2 directly binding to pulmonary endothelial cells expressing functional αβγ IL-2R at low to moderate levels. Furthermore, pulmonary edema associated with the interaction between IL-2 and pulmonary endothelial cells can be treated in CD25-deficient host mice by inhibiting binding to CD25 with an anti-CD25 monoclonal antibody (mAb), or by using CD122-specific IL-2 / anti-IL-2 The use of an mAb (IL-2 / mAb) complex suppressed the condition, thus preventing VLS.

[0159] Therapies using interleukin cytokines other than IL-2 are more limited. IL-15 exhibits similar immune cell stimulating activity to IL-2 but without the same inhibitory effects, making it a promising immunotherapy candidate. Clinical trials using recombinant human IL-15 in the treatment of metastatic melanoma or renal cell carcinoma demonstrated clear changes in the distribution, proliferation, and activation of immune cells, suggesting potential antitumor activity (Conlon et al., 2014). Currently, IL-15 is 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 exacerbation 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 continuation of naive and memory T cell homeostasis in the periphery. Furthermore, IL-7 is important for lymph node (LN) organogenesis and for maintaining 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 an increase in both CD4+ and CD8+ T cells, but the number of regulatory T cells monitored by FoxP3 expression did not significantly increase (Sportes et al., 2008). Clinical trials reported in 2006, 2008, and 2010 involved subcutaneous injection of different doses of IL-7 into patients with different types of cancer, such as metastatic melanoma or sarcoma. Aside from transient fever and mild erythema, toxicity was minimal. Circulating levels of CD4+ and CD8+ T cells increased significantly, and Treg counts decreased. TCR repertoire diversity increased after IL-7 therapy. However, the antitumor activity of IL-7 was not adequately evaluated (Gao et. al., 2015). The results suggest that IL-7 therapy may enhance and broaden the immune response.

[0161] IL-12 is a pleiotropic cytokine whose action facilitates the interaction between innate and adaptive immunity. Initially, IL-12 was reported as a factor secreted from PMA-induced EBV-transformed B cell lines. Based on its action, IL-12 is called a cytotoxic lymphocyte maturation factor and a natural killer cell stimulator. Because IL-12 bridges the gap between innate and adaptive immunity and strongly stimulates the production of IFNγ, a cytokine that modulates the natural mechanisms of anti-cancer defense, it seemed like an ideal candidate for tumor immunotherapy in humans. However, serious side effects associated with systemic administration of IL-12 in clinical studies, and the very narrow therapeutic index of this cytokine, significantly reduced interest in using it in cancer patients (Lasek et al., 2014). An approach to IL-12 therapy that directs cytokine delivery to the tumor may overcome the past problems of IL-12 therapy and is currently being pursued in clinical trials for cancer.

[0162] Direct use of IL-2 as an agonist to bind to IL-2R and modulate the immune response is problematic due to well-established therapeutic risks (e.g., its short half-life and high toxicity). These risks also limit the therapeutic development and use of other cytokines. Novel forms of cytokines that mitigate these risks are needed. This specification discloses compositions and methods comprising IL-2 and IL-15, as well as other cytokines, functional fragments and mutaines of cytokines, and conditionally active cytokines, designed to address such risks and provide the necessary immunomodulatory therapeutics.

[0163] The present invention is designed to address the shortcomings of direct IL-2 therapy and other cytokine-based therapies by using, for example, cytokine-blocking moieties, such as steric blocking polypeptides, serum half-life-extending polypeptides, targeted polypeptides, binding polypeptides (including protease-cleaving 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, lymphotoxins), transforming growth factors (e.g., TGFβ1, TGFβ2, TGFβ3), chemokines (CXC-motif chemokines 10 (CXCL10), CCL19, CCL20, CCL21), and granulocyte-macrophage colony-stimulating factor (GM-CS), are highly potent when administered to patients. As used herein, “chemokine” means a family of small molecule cytokines that have the ability to induce directed chemotaxis in nearby responsive cells. While cytokines can provide potent therapies, their clinical use has been limited due to undesirable effects that are difficult to control clinically. This disclosure relates to novel forms of cytokines that can be used in patients with reduced or eliminated undesirable effects. More specifically, this disclosure relates to a pharmaceutical composition comprising a chimeric polypeptide (fusion protein) and the aforementioned pharmaceutical formulation comprising a nucleic acid encoding the fusion protein and a cytokine or cytokine active fragment or mutein having reduced activity in activating cytokine receptors compared to the corresponding cytokine. However, under selected conditions or in selected biological environments, the chimeric polypeptide activates the homologous receptor with the same or greater potency as the corresponding native cytokine. As described herein, this is typically achieved using a cytokine-blocking moiety that, under general conditions, blocks or inhibits the receptor-activating function of the cytokine, its active fragment or mutein, but does not block or inhibit such function under selected conditions, for example, when present at a desired site of cytokine activity (e.g., an inflammatory site or tumor).

[0164] Chimeric polypeptides and nucleic acids encoding chimeric polypeptides can be prepared using any suitable method. For example, nucleic acids encoding chimeric polypeptides can be prepared 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 prepared in a similar manner, for example, by expressing a suitable nucleic acid using synthetic or semi-synthetic chemical techniques. In some embodiments, the blocking moiety can be attached to the cytokine polypeptide by saltase-mediated conjugation. "Saltase" is a transpeptidase that modifies proteins by recognizing and cleaving a carboxyl end sorting signal embedded in or attached to the terminal of a target protein or peptide. Saltase A catalyzes the cleavage of the LPXTG motif (where X is any standard amino acid) (SEQ ID NO: 237) located between the Thr and Gly residues on the target protein, forming an enzyme-thioacyl intermediate by transient attachment of the Thr residue to the active site Cys residue on the enzyme. To complete the peptide transfer and create a peptide-monomer conjugate, a biomolecule with a nucleophilic group at its N-terminus (typically an oligoglycine motif) attacks the intermediate, substituting for saltase A and linking the two molecules.

[0165] To form a cytokine blocking subconjugate, the N-terminus of the cytokine polypeptide is first tagged with a polyglycine sequence, or the C-terminus is tagged with an LPXTG (SEQ ID NO: 237) motif. The blocking subconjugate or other element is attached to the respective peptide that functions as the receptor site of the tagged polypeptide. When conjugating with a domain containing the LPXTG (SEQ ID NO: 237) receptor peptide attached via the N-terminus, the N-terminus of the polypeptide is tagged with a polyglycine stretch. When conjugating with a domain containing a polyglycine peptide attached via the C-terminus, the C-terminus of the polypeptide is tagged with the LPXTG (SEQ ID NO: 237) saltase recognition sequence. Saltase, recognizing polyglycine and the LPXTG (SEQ ID NO: 237) sequence, forms a peptide bond between the polymer peptide and the tagged polypeptide. The saltase reaction cleaves a glycine residue as an intermediate and is performed at room temperature.

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

[0167] Any suitable linker can be used. For example, the linker may be glycine-glycine, a saltase recognition motif, or a saltase recognition motif and a peptide sequence (Gly4Ser). n (Sequence number 238) or (Gly3Ser) nThis may include (SEQ ID NO: 239) (where n is 1, 2, 3, 4, or 5). Typically, the saltase recognition motif includes the peptide sequence LPXTG (SEQ ID NO: 237) (where X is any amino acid). In some embodiments, the covalent bond exists between a reactive lysine residue attached to the C-terminus of the cytokine polypeptide and a reactive aspartate attached to the N-terminus of a blocker or other domain. In other embodiments, the covalent bond exists between a reactive aspartate 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] Therefore, as will be described in detail herein, the cytokine blocking moiety used may be a steric blocker. As used herein, “steric blocker” refers to a polypeptide or polypeptide moiety that can be covalently bound to a cytokine polypeptide directly or indirectly, for example in the form of a chimeric polypeptide (fusion protein), via other moieties such as a linker, but not otherwise covalently bound to the cytokine polypeptide. A steric blocker can be non-covalently bound to the cytokine polypeptide, for example, by electrostatic, hydrophobic, ionic, or hydrogen bonds. Due to its proximal position to the cytokine site and its relative size, a steric blocker typically inhibits or blocks the activity of the cytokine moiety. Steric inhibition of the cytokine moiety can be removed by spatially separating the cytokine moiety from the steric blocker, which can be done, for example, by enzymatically cleaving a fusion protein containing the steric blocker and the cytokine polypeptide at the site between the steric blocker and the cytokine polypeptide.

[0169] As described in detail herein, the blocking function may be combined with, or attributable to, additional functional components in the pharmaceutical composition (e.g., targeting domains, serum half-life extension elements, and protease-cleaving binding polypeptides). For example, serum half-life extension polypeptides may also be steric blockers.

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

[0171] Various elements ensure preferential delivery and activation of IL-2 at the desired IL-2 active site, and severely limit systemic exposure to interleukins through blocking and / or targeting strategies preferentially linked to serum half-life extension strategies. In this serum half-life extension strategy, the blocked version of the interleukin circulates for a long period (preferably 1-2 weeks or longer), while the activated version has the typical serum half-life of the interleukin.

[0172] Compared to the serum half-life extended version, the serum half-life of intravenously administered IL-2 is only about 10 minutes. This is because it is distributed within the large extracellular space of the entire body, approximately 15 L in an average-sized adult. Subsequently, IL-2 is metabolized by the kidneys with a half-life of approximately 2.5 hours (see Smith, K. "Interleukin 2 immunotherapy." Therapeutic Immunology 240 (2001)). Other measurements indicate that 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). In some embodiments of the present invention, the half-life extended element is bound to interleukin via a linker, which 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 non-cleaved half-life extended element. In such embodiments, fully active and free interleukins are thought to have very different pharmacokinetic (pK) properties, with half-lives of several hours rather than several weeks. In addition, exposure to active cytokines is limited to sites with desired cytokine activity (e.g., inflammatory sites or tumors), reducing systemic exposure to active cytokines and associated toxicity and side effects.

[0173] Other cytokines envisioned in this invention have pharmacological properties similar to IL-2 (e.g., IL-15 (reported by Blood 2011 117:4787-4795; doi:doi.org / 10.1182 / blood-2010-10-311456)), and therefore the design of this invention addresses the drawbacks of using these agents directly and provides chimeric polypeptides that have an extended half-life and / or can be targeted to a desired active site (e.g., an inflammatory site or a tumor).

[0174] As desired, IL-2 can be designed to bind generally to the IL-2R complex, or specifically to one of the three IL-2R subunits, with a different affinity than the corresponding wild-type IL-2, to selectively activate, for example, Treg or Teff (effector T cells). For example, an IL-2 polypeptide that is said to have a higher affinity in the trimer form for the dimeric beta / gamma form of the IL-2 receptor compared to wild-type IL-2 may have an amino acid sequence containing one of the following mutant sets with respect to SEQ ID NO: 1 (mature IL-2 protein containing amino acids 21-153 of human IL-2 with UniProt accession number P60568-1): (a) K64R, V69A, and Q74P; (b) V69A, Q 74P, 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 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 mutains of other cytokines (including interleukins (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-23), interferons (IFN 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, mutains with a desired binding affinity to homologous receptors can be prepared.

[0175] As described above, any variant IL-2 polypeptide disclosed herein may include, and may not include, the sequence described herein, and may otherwise be identical to SEQ ID NO: 1. Furthermore, any variant IL-2 polypeptide disclosed herein may optionally include substitution of the cysteine ​​residue at position 125 with another residue (e.g., serine), and / or optionally include 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 action demonstrated that in vitro, human T cell proliferation requires exposure to effective concentrations of IL-2 for at least 5–6 hours (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 for intravenous administration and 3.3 hours for 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 levels above the level required to stimulate T cell proliferation for the required duration necessitates high doses or frequent administrations that result in peak IL-2 levels significantly above the EC50 of Treg cells. Such high IL-2 peak levels can activate the IL2Rβγ receptor, potentially leading to 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 at lower doses and lower peak levels than IL-2. Therefore, such IL-2 analogs require less dose or administration frequency than IL-2 to effectively stimulate Treg cells. Fewer subcutaneous administrations of IL-2 drugs also improve patient tolerability. Therapeutic agents with these characteristics clinically lead to improved pharmacological efficacy, reduced toxicity, and improved patient compliance with therapy. Alternatively, IL-2 or IL-2 mutaine (hereinafter referred to as "IL-2*") may be selectively targeted to the intended site of action (e.g., the site of inflammation). This targeting can be achieved by adding a domain containing a blocker of the cleaved IL-2 (or mutaine) to the administered drug, by targeting the domain, or by one of several strategies including a combination of these two.

[0177] In some embodiments, IL-2 * Partial agonists can be engineered 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 of the receptor subunits and not to the others. Unlike full agonists or full antagonists, this type of partial agonist can tune the signaling properties to an amplitude that elicits the desired functional properties and does not meet the threshold of undesirable properties. Considering the different activities of partial agonists, it is thought that the IL-2 repertoire can be designed to exhibit a more refined degree of characteristic signaling activity, ranging from near full agonism to partial agonism and full antagonism.

[0178] In some embodiments, IL-2 * has a variable affinity for IL-2Rα. In some embodiments, IL-2 * has a higher affinity for IL-2Rα than wild-type IL-2. In other embodiments, IL-2 * has a variable affinity for IL-2Rβ. In one embodiment, IL-2 * has enhanced binding affinity for IL-2Rβ (e.g., the N-terminus of IL-2Rβ), thereby eliminating the functional requirement for IL-2Rα. In another embodiment, IL-2 is generated that has increased binding affinity for IL-2Rβ but shows a decrease in binding to IL-2Rγ, whereby the heterodimerization and signaling of IL-2Rβγ are incomplete. *

[0179] ​Furthermore, blocking portions, which are described in more detail below, may also be used to favorably facilitate binding to or activation of one or more receptors. In one embodiment, a blocking portion is added to block the binding or activation of IL-2Rβγ but not alter the binding or activation of IL-2Rα. In another embodiment, a blocking portion is added to attenuate the binding or activation of IL-2Rα. In yet another embodiment, a blocking portion is added to inhibit the binding and activation of three receptors. This blocking may be mitigated by removing the blocking portion under specific conditions, for example, by proteolytic cleavage of the linker that binds one or more blocking portions to cytokines.

[0180] A similar approach can be applied to the improvement of other cytokines, and in detail, they can be used as immunostimulants, for example, 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, lymphotoxins, TGF-β1, TGFβ2, TGFβ3, GM-CSF, CXCL10, CCL19, CCL20, and CCL21) can be tuned to maximize the activation of effector cells (e.g., T cells, NK cells) and / or cytotoxic immune response-promoting cells (e.g., those that induce dendritic cell maturation) at desired active sites (e.g., within tumors, but preferably not systemically).

[0181] Accordingly, this specification provides pharmaceutical compositions comprising at least one cytokine polypeptide, for example, 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., TNF, lymphotoxins), transforming growth factors (e.g., TGF-β1, TGFβ2, TGFβ3), chemokines (e.g., CXCL10, CCL19, CCL20, CCL21), and granulocyte-macrophage colony-stimulating factor (GM-CS), or any of the aforementioned functional fragments or mutaines. The polypeptide also typically includes at least one linker amino acid sequence, which in certain embodiments can be cleaved by an endogenous protease. In one embodiment, the linker comprises an amino acid sequence including 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 sterically 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, 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 sterically 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-cleaving domain. In some embodiments, blockade and reduction of cytokine activity are achieved simply by attaching an additional domain having a very short linker to the N or C terminus of the interleukin domain.In such embodiments, it is expected that blockade will be mitigated by protease digestion of the blocking portion or by protease digestion of the short linker that tethers the blocker to the interleukin. Once the domain is clipped or released, it will no longer be able to block cytokine activity.

[0182] A pharmaceutical composition, such as a chimeric polypeptide, may contain two or more cytokines, which may be the same cytokine polypeptide or different cytokine polypeptides. For example, two or more different types of cytokines may 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 may have an activity that modulates 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) contained in the fusion proteins disclosed herein are not mutated or manipulated to alter the properties of native cytokines, including receptor binding affinity and specificity or serum half-life. However, changes in the amino acid sequence from native (including wild-type) cytokines are permissible to facilitate cloning and to achieve desired expression levels.

[0184] a. Blocking section The blocking portion may be any portion that inhibits the cytokine's ability to bind to and / or activate its receptor. The blocking portion can inhibit the cytokine's ability 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 portions include the full-length or cytokine-binding fragment or mutain of the cytokine's homologous receptor. Antibodies and their fragments 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 (VHH) of camelid-type nanobodies, and cytokine-binding sdAbs. Other suitable antigen-binding domains that bind to cytokines may also be used, including binding domains based on non-immunoglobulin proteins that bind and / or mimic the structure of antibodies, such as anticarin, affilin, affibody molecules, affimers, affitins, alphabodies, avimers, DARPin, finomers, Knitz domain peptides, monobodies, and other manipulated scaffolds, such as SpA, GroEL, fibronectin, lipocalin, and the CTLA4 scaffold. Further examples of suitable blocking polypeptides include polypeptides that sterically inhibit or block cytokines from binding to their homologous receptors. Advantageously, such portions may also function as half-life extension 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 mutains of such polypeptides, can also be used.Furthermore, antibodies and antigen-binding domains that bind to proteins with long serum half-lives (e.g., HSA, immunoglobulins, or transferrin) or receptors recycled on the plasma membrane (e.g., FcRn or transferrin receptors) can also inhibit cytokines, particularly when bound to these 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 camelid-type nanobody variable domains (VHH), and sdAbs. Other suitable antigen-binding domains that bind to cytokines may also be used, including binding domains based on non-immunoglobulin proteins that mimic antibody binding and / or structure, such as anticarin, affilin, affibody molecules, affimers, affitins, alphabodies, avimers, DARPin, finomers, Knitz domain peptides, monobodies, and other manipulated scaffolds, such as SpA, GroEL, fibronectin, lipocalin, and the CTLA4 scaffold.

[0185] In exemplary cases, if IL-2 is a chimeric polypeptide cytokine, the blocking portion may be the alpha chain of the IL-2 receptor (IL-2Rα), or the full-length or fragment or mutaine of 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 its fragment, and an anti-HSA sdAb or scFv, etc.

[0186] b. In vivo half-life extension element Preferably, the chimeric polypeptide contains an in vivo half-life extension element. Increasing the in vivo half-life of therapeutic molecules with short natural half-lives allows for more tolerable and manageable dosing regimens without sacrificing efficacy. As used herein, “half-life extension element” is a part of a chimeric polypeptide that extends the in vivo half-life and improves pK by, for example, altering size (e.g., 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 an element within the polypeptide chain that binds to receptors that are recycled to the cell plasma membrane rather than being degraded by lysosomes (e.g., FcRn receptors and transferrin receptors on endothelial cells). Three types of proteins, e.g., human IgG, HSA (or its fragments), and transferrin, persist in human serum much longer than would be predicted from their size, due to their ability to bind to receptors that are recycled without being degraded by lysosomes. These proteins or fragments that retain FcRn binding typically bind to other polypeptides to extend their serum half-life. In one embodiment, the half-life extension element is a human serum albumin (HSA) binding domain. HSA (SEQ ID NO: 2) may be bound directly to the pharmaceutical composition or via a short linker. Fragments of HSA can also be used. HSA and its fragments can function as both blocking moieties and half-life extension elements. Human IgG can perform similar functions.

[0187] The serum half-life extension element may be an antigen-binding polypeptide that binds to long-lived serum proteins such as serum albumin and transferrin. Examples of such polypeptides include antibodies and their fragments, 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 camelid-type nanobody variable domains (VHH), cytokine-binding sdAbs, etc. Other suitable antigen-binding domains include binding domains based on non-immunoglobulin proteins that mimic antibody binding and / or structure, e.g., anticarin, affin, affibody molecules, affimers, affitins, alpha bodies, avimers, DARPin, finomers, Knitz domain peptides, monobodies, and other engineered scaffolds, e.g., SpA, GroEL, fibronectin, lipocalin, and CTLA4 scaffolds. Further examples of antigen-binding polypeptides include ligands for desired receptors, ligand-binding moieties for receptors, lectins, and peptides that bind to or associate with one or more target antigens.

[0188] Some preferred serum half-life extension elements are polypeptides containing a complementation-determining region (CDR) and, optionally, a non-CDR loop. Advantageously, such serum half-life extension elements can extend the serum half-life of cytokines 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 extension element is a domain derived from an immunoglobulin molecule (Ig molecule) or an engineered protein scaffold that mimics antibody structure and / or binding activity. Ig may be any class or subclass (IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, etc.). The polypeptide chain of the Ig molecule folds into a series of parallel β-chains linked by loops. Within the variable region, the three loops constitute a "complementation-determining region" (CDR) that determines the antigen-binding specificity of the molecule. An IgG molecule comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding fragment thereof. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are highly sequence-variable and / or involved in antigen recognition and / or usually form structurally defined loops, interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments of this disclosure, at least some or all of the amino acid sequences of FR1, FR2, FR3, and FR4 are part of the “non-CDR loop” of the binding site described herein.The variable domain of an immunoglobulin molecule has several β-strands arranged in two sheets. Both the variable domains of the immunoglobulin light chain and heavy chain contain three hypervariable loops, i.e., complementarity-determining regions (CDRs). The three CDRs of the V domain (CDR1, CDR2, CDR3) are clustered at one end of the β-barrel. The CDRs are loops connecting the β-strands BC, C'-C'', and FG of the immunoglobulin folding, the bottom loop connecting the β-strands AB, CC', C''-D, and EF of the immunoglobulin folding, and the top loop connecting the DE strand of the immunoglobulin folding are non-CDR loops. In some embodiments of this 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 following: AB, CD, EF, and DE loops of the C1 set domain of Ig or an Ig-like molecule; AB, CC', EF, FG, BC, and EC' loops of the C2 set domain of Ig or an Ig-like molecule; and DE, BD, GF, A(A1A2)B, and EF loops of the I(intermediate) set domain of Ig or an Ig-like molecule.

[0189] Within the variable domain, CDRs are thought to be responsible for antigen recognition and binding, while FR residues are considered scaffolds for CDRs. However, in certain cases, some FR residues play a crucial role in antigen recognition and binding. Framework region residues that influence Ag binding can be divided into two categories. First, there are FR residues that come into contact with the antigen and form part of the binding site, and some of these residues are sequence-wise close to CDRs. Other residues are sequence-wise far removed from CDRs, but the 3D structure of the molecule is close to CDRs (e.g., the heavy chain loop).

[0190] The binding site is any type of polypeptide. For example, in certain cases, the binding site is a native 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 site is an engineered scaffold. Engineered scaffolds include, for example, sdAb, scFv, Fab, VHH, fibronectin type III domain, immunoglobulin-like scaffolds (as suggested in Halaby et al., 1999. Prot Eng 12(7):563-571), DARPin, cystine knot peptide, lipocalin, a 3-helix bundle scaffold, a protein G-associated albumin-binding module, or a DNA or RNA aptamer scaffold.

[0191] In some cases, the serum half-life extension element includes a binding site for bulk serum proteins. In some embodiments, the CDR provides a binding site for bulk serum proteins. Bulk serum proteins are, in some examples, globulin, albumin, transferrin, IgG1, IgG2, IgG4, IgG3, IgA monomer, factor XIII, fibrinogen, IgE, or pentameric IgM. In some embodiments, the CDR forms a binding site for immunoglobulin light chains, such as Igκ free light chain or Igλ free light chain.

[0192] The serum half-life extension element may be any type of binding domain, including, but is not limited to, domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, and humanized antibodies. In some embodiments, the binding site is a single-chain variable fragment (scFv), a single-domain antibody, e.g., a heavy-chain variable domain (VH), a light-chain variable domain (VL), and a variable domain (VHH) from a camelid nanobody. In other embodiments, the binding site is a non-Ig binding domain, i.e., an antibody mimetic, e.g., antikalin, affilin, affibody molecule, affimer, afitin, alpha-body, avimer, DARPin, finomer, Knitz domain peptide, or monobody.

[0193] In other embodiments, the serum half-life extension element may be a water-soluble polymer or a peptide conjugated with a water-soluble polymer such as PEG. As used herein, “PEG,” “polyethylene glycol,” and “poly(ethylene glycol)” are interchangeable and encompass any non-peptide water-soluble poly(ethylene oxide). Furthermore, the term “PEG” also refers to polymers containing a majority, i.e., more than 50%, of the –OCH2CH2– repeating subunits. Regarding specific forms, PEG can take on any number of different molecular weights, as will be described in more detail below, and can also take on structures or geometric arrangements such as “branched,” “linear,” “fork-type,” and “multifunctional.” PEG is not limited to any particular structure and can have linear (e.g., end-capped, e.g., alkoxyPEG or bifunctional PEG), branched or multi-armed (e.g., fork-type PEG or PEG attached to a polyol core), or dendritic (or star-type) architectures, each with or without one or more degradable bonds. 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 trippolymers, random trippolymers, and block trippolymers. PEG can be conjugated with polypeptides and peptides in any suitable manner. Typically, a reactive PEG derivative (e.g., N-hydroxysuccinamidyl PEG) is reacted with a peptide or polypeptide containing an amine, sulfhydryl, carboxylic acid, or an amino acid having a side chain containing a hydroxyl functional group (e.g., cysteine, lysine, asparagine, glutamine, threonine, tyrosine, serine, aspartic acid, and glutamic acid).

[0194] c. Targeting domains and retention domains For certain applications, it may be desirable to maximize the amount of time a construct is present at a desired location within the body. This can be achieved by including an additional domain in a chimeric polypeptide (fusion protein) to influence its movement within the body. For example, a chimeric nucleic acid may encode a domain that directs the polypeptide to a location within the body (e.g., a tumor cell or an inflammatory site) (this domain is referred to as the “targeting domain”), and / or a domain that retains the polypeptide at that location within the body (e.g., a tumor cell or an inflammatory site) (this domain is referred to as the “retention domain”). In some embodiments, the domain may function as both a targeting domain and a retention domain. In some embodiments, the targeting domain and / or retention domain are specific to protease-rich environments. In some embodiments, the encoded targeting domain and / or retention domain are specific to regulatory T cells (Tregs) and target, for example, the CCR4 or CD39 receptor. Other suitable targeting and / or retention domains include those having a homologous ligand (e.g., the IL-1 receptor or IL-6 receptor) that is overexpressed in inflammatory tissue. In other embodiments, preferred targeting and / or retention domains include those having a congeneral ligand (e.g., Epcam, CEA, or mesothelin) that is overexpressed in tumor tissue. In some embodiments, the targeting domain is bound to the interleukin via a linker, which is cleaved at the site of action (e.g., by an inflammation or cancer-specific protease) to release a fully active interleukin at the desired site. In some embodiments, the targeting and / or retention domain is bound to the interleukin via a linker that is not cleaved at the site of action (e.g., by an inflammation or cancer-specific protease), allowing the cytokine to remain at the desired site.

[0195] The antigens selected may, in some cases, be 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 comprising 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, such as those found in 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 camelid-type nanobody variable domains (VHH), and cytokine-binding sdAbs, as well as antibodies and their fragments. Other suitable antigen-binding domains include non-immunoglobulin proteins that bind to and / or mimic the structure of antibodies, such as anticarin, affin, affibody molecules, affimers, affitins, alpha bodies, avimers, DARPin, finomers, Knitz domain peptides, monobodies, and other engineered scaffold-based binding domains, such as SpA, GroEL, fibronectin, lipocalin, and the CTLA4 scaffold. Further examples of antigen-binding polypeptides include ligands for desired receptors, ligand-binding moieties for receptors, 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 cell surface molecules. In some embodiments, the targeting and / or retention domain specifically binds to tumor antigens. In some embodiments, the targeted polypeptide specifically and independently binds to tumor antigens selected from at least one of EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FoIR. In some embodiments, the targeted polypeptide specifically and independently binds to two different antigens, at least one of which is a tumor antigen selected from EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FoIR.

[0198] Targeted antigens and / or retaining antigens may be tumor antigens 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 This includes 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] Targeted antigens and / or retained antigens may be immune checkpoint proteins. Examples of immune checkpoint proteins include, but are 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] Targeted antigens and / or retained antigens may be cell surface molecules such as proteins, lipids, and polysaccharides. In some embodiments, targeted antigens and / or retained antigens are present on tumor cells, virus-infected cells, bacterial-infected cells, damaged erythrocytes, arterial plaque cells, and inflammatory or fibrotic tissue cells. Targeted antigens and / or retained antigens may include immune response modifiers. Examples of immune response modifiers, but 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] Targeted antigens and / or retained antigens may be cytokine receptors. Examples of cytokine receptors, but are not limited to, type I cytokine receptors, e.g., GM-CSF receptor, G-CSF receptor, type I IL receptor, Epo receptor, LIF receptor, CNTF receptor, TPO receptor; type II cytokine receptors, e.g., IFN-alpha receptors (IFNAR1, IFNAR2), IFB-beta receptor, IFN-gamma receptors (IFNGR1, IFNGR2), type II IL receptor; and CC chemokine receptors, e.g., CXC chemokine receptor, CX3C chemokine receptor. Receptors include XC chemokine receptors; tumor necrosis receptor superfamily receptors, e.g., TNFRSF5 / CD40, TNFRSF8 / CD30, TNFRSF7 / CD27, TNFRSF1A / TNFR1 / CD120a, TNFRSF1B / TNFR2 / CD120b; TGF-beta receptors, e.g., TGF-beta receptor 1, TGF-beta receptor 2; and Ig superfamily receptors, e.g., IL-1 receptor, CSF-1R, PDGFR (PDGFRA, PDGFRB), and SCFR.

[0202] d. Linker As described above, the pharmaceutical composition comprises one or more linker sequences. The linker sequences function to provide flexibility between polypeptides, for example, so that a blocking portion can inhibit the activity of the cytokine polypeptide. The linker sequences can be positioned between any or all of the cytokine polypeptide, serum half-life extension elements, and / or blocking portions. As described herein, at least one of the linkers is protease-cleaving and contains one or more cleavage sites for one or more desired proteases. Preferably, the desired proteases are concentrated or selectively expressed at the desired cytokine active site (e.g., the tumor microenvironment). Thus, the fusion protein is preferentially or selectively cleaved at the desired cytokine active site.

[0203] The orientation of components in a pharmaceutical composition is primarily a matter of design choice, and it is recognized that multiple orientations are possible, all of which are intended to be covered in this disclosure. For example, the blocking portion can be located at the C-terminal or N-terminal end of the cytokine polypeptide.

[0204] This specification provides pharmaceutical compositions comprising polypeptide sequences. It should be understood that, as with all peptides, polypeptides, and proteins (including their fragments), the amino acid sequence of a 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 further detail below.

[0205] The compositions provided herein have desired functions. The compositions consist 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 extension element, and an optional targeted polypeptide, wherein one or more linkers link each polypeptide in the composition. A first polypeptide, e.g., IL-2 mutein, is provided as the active agent. A blocking moiety for blocking the activity of interleukins is provided. A linker polypeptide, e.g., a protease-cleavable polypeptide, is provided to be cleaved by a protease specifically expressed at the intended target of the active agent. Optionally, the blocking moiety blocks the activity of the first polypeptide by binding to an interleukin polypeptide. In some embodiments, the blocking moiety (e.g., a steric blocking peptide) is bound to the interleukin via a protease-cleavable linker, which is cleaved at the site of action (e.g., by an inflammation-specific protease) to release a fully active cytokine at the desired site.

[0206] In some embodiments, the linker is glycine-glycine, a saltase recognition motif, or a saltase recognition motif and a peptide sequence (Gly4Ser) n (Sequence ID 238), or (Gly3Ser) n (SEQ ID NO: 239) (where n is 1, 2, 3, 4, or 5). In one embodiment, the saltase recognition motif comprises the peptide sequence LPXTG (where X is any amino acid (SEQ ID NO: 237)). In one embodiment, the covalent bond exists between a reactive lysine residue attached to the C-terminus of the cytokine polypeptide and a reactive aspartate attached to the N-terminus of the blocking moiety or other moiety. In one embodiment, the covalent bond exists between a reactive aspartate 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. Cutting and inductive properties As described herein, the activity of cytokine polypeptides in the context of fusion proteins is attenuated, and protease cleavage at the desired active site (e.g., tumor microenvironment) releases cytokine forms from the fusion protein that are far more active as cytokine receptor agonists than the fusion protein. For example, the cytokine receptor activating (agonist) activity of a fusion polypeptide may be at least about 10 times, at least about 50 times, at least about 100 times, at least about 250 times, at least about 500 times, or at least about 1000 times lower 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 if it contains substantially the same amino acids as the cytokine polypeptide, substantially no additional amino acids, and is not associated with other molecules (by covalent or non-covalent bonds). If necessary, the cytokine polypeptide as a separate molecular entity may contain some additional amino acid sequences (e.g., tags or short sequences to aid expression and / or purification).

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

[0209] In some embodiments, the cleavage portion is resistant to proteolytic cleavage by one or more proteases. The cleavage portion is resistant to proteases if the sequence contains a binding site that has been modified from a canonical cleavage motif sequence for a particular protease. In some embodiments, the binding site is modified compared to the 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 replacing arginine (R) with glutamine (Q), thereby changing the charged residue to a shorter, more polar residue and reducing the ability of cathepsin S to bind to and cleave the sequence. Such semi-conservative amino acid substitutions in the protease target sequence motif result in reduced binding ability, and therefore, such modified sequence motifs are protease resistant. Uncleavable regions can be created by inserting disruptive amino acids, such as proline (which causes a curve in the peptide's secondary structure) or histidine (which causes steric interference with other amino acid side chains), into the target sequence motif of a protease. As used herein, a “protease-resistant” peptide linker is a peptide linker in which cleavage by one or more specified proteases is reduced or undetectable. Exemplary protease-resistant peptide linkers can be tested, for example, in vitro by incubation with a specific protease and then analysis of the digested product by Western blotting.

[0210] f. polypeptide substitution Polypeptides described herein may include 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, insofar as the desired function is maintained. One way of defining any known or possible modifications and derivatives of the proteins and nucleic acids encoding them in this disclosure is to be understood as defining sequence variants in terms of identity with respect to a particular known reference sequence. Specifically, polypeptides and 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, and 99 percent identity with respect to the chimeric polypeptides provided herein. For example, a polypeptide is provided 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 with any nucleic acid or polypeptide sequence described herein. This includes a polypeptide or nucleic acid 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 with any nucleic acid or polypeptide sequence described herein. This also includes variants of linkers or inducible polypeptides containing one, two, three, four, five, or six variants from the cleavage domain sequence. Those skilled in the art will readily understand how to determine the identity of two polypeptides or two nucleic acids. For example, identity can be calculated such that the identity is at its highest level after aligning the two sequences.

[0211] Another method for calculating identity can be performed by publicly available algorithms. Optimal sequence alignment for comparison can be achieved by 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 similarity search 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 algorithms disclosed, for example, in Zuker, Science 244:48-52 (1989); Jaeger et al., Proc. Natl Acad. Sci. USA 86:7706-7710 (1989); and Jaeger et al., Methods Enzymol. 183:281-306 (1989), which are incorporated herein by reference, at least with respect to materials relating to nucleic acid alignment. While any method may typically be used, and in certain particular cases the results of these various methods may differ, those skilled in the art will understand that if identity is found by at least one of these methods, the sequence is considered to have the described identity and is disclosed herein.

[0213] Protein modifications include modifications of the amino acid sequence. Amino acid sequence modifications can occur spontaneously as allele variations (e.g., due to genetic polymorphism), due to environmental influences (e.g., exposure to ultraviolet light), or through human intervention (e.g., mutagenesis of cloned DAN sequences), resulting in induced point mutants, deletion mutants, insertion mutants, and substitution mutants. These modifications can result in changes in the amino acid sequence, silent mutations, restriction site modifications, or other specific mutations. Amino acid sequence modifications are typically classified into one or more of three classes: substitution, insertion, or deletion. Insertions include amino and / or carboxyl-terminus fusions and intrasequence insertions of one or more amino residues. Insertions are usually smaller than amino or carboxyl-terminus fusion insertions, typically involving 1 to 4 residues. Deletions are characterized by the removal of one or more amino acid residues from a protein sequence. Typically, approximately 2 to 6 residues or fewer are deleted at any single site within the protein molecule. Amino acid substitutions are typically single-residue substitutions, but may occur at multiple different locations simultaneously. Insertions usually involve 1 to 10 amino acid residues, while deletions range from 1 to 30 residues. Deletions or insertions are preferably in adjacent pairs, i.e., deletions of two residues or insertions of two residues. Substitutions, deletions, insertions, or any combination thereof can be combined to obtain the final construct. Mutations should not place the sequence outside the reading frame and, preferably, do not create complementary regions capable of producing secondary mRNA structures. Substitutional modifications are modifications in which at least one residue is removed and a different residue is inserted in its place. Such substitutions generally occur according to Table 2 below and are referred to as conservative substitutions. [Table 2]

[0214] Modifications, including specific amino acid substitutions, are carried out by known methods. For example, modifications are performed by site-directed mutagenesis of nucleotides in the polypeptide-encoding DNA, which produces DNA encoding the modification, and then this DNA is expressed in recombinant cell cultures. Techniques for inducing substitutional mutations at specific sites in DNA with known sequences are well known, such as M13 primer mutagenesis and PCR mutagenesis.

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

[0216] Furthermore, this disclosure relates to nucleic acids encoding chimeric polypeptides as described herein, and to the use of such nucleic acids for producing chimeric polypeptides and for therapeutic purposes. For example, the present invention includes DNA and RNA molecules (e.g., mRNA, self-replicating RNA) encoding chimeric polypeptides, and relates to the therapeutic use of such DNA and RNA molecules.

[0217] Shell. Exemplary Composition The exemplary fusion proteins of the present invention combine the elements described above in various directions. The directions described in this section are intended as examples only and should not be considered limiting.

[0218] In some embodiments, the fusion protein comprises a cytokine, a blocking moiety, and a half-life extension element. In some embodiments, the cytokine is located between the half-life extension element and the blocking moiety. In some embodiments, the cytokine is N-terminal to the blocking moiety and the half-life extension 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 extension element. All embodiments must include at least one protease-cleaving 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 extension element. Additional elements can be attached to each other by cleaving linkers, non-cleaving linkers, or direct fusion.

[0219] In some embodiments, the blocking domains used have an extended half-life, and the cytokine is located between two such blocking domains. In some embodiments, the cytokine is located between two blocking domains, one of which has an extended half-life.

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

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

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

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

[0224] C. Methods of treatment and pharmaceutical compositions Furthermore, the present invention provides a method for treating subjects who have or are at risk of developing a disease or disorder, such as proliferative disorders, neoplastic diseases, inflammatory diseases, immune disorders, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, graft-versus-host diseases, etc. The method typically involves administering an effective amount of the fusion protein disclosed herein, typically administered as a pharmaceutical composition, to a subject in need. In some embodiments, the method further includes selecting subjects who have or are at risk of developing such a disease or disorder. The pharmaceutical composition preferably comprises a blocked cytokine, its fragment, or mutein that is activated at the site of inflammation. In one embodiment, the chimeric polypeptide comprises a cytokine polypeptide, its fragment, or mutein, and a serum half-life extension element. In another embodiment, the chimeric polypeptide comprises a cytokine polypeptide, its fragment, or mutein, and a blocking moiety (e.g., a steric blocking polypeptide), in which case the steric blocking polypeptide is capable of sterically blocking the activity of the cytokine polypeptide, its fragment, or mutein. In another embodiment, the chimeric polypeptide comprises a cytokine polypeptide, its fragment or mutain, a blocking moiety, and a serum half-life extension element.

[0225] Inflammation is part of a complex biological response of body tissues to harmful stimuli (e.g., pathogens, damaged cells, or irritants), and is a defense 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 tissues damaged in the initial injury and inflammatory processes, and initiate tissue repair. Inflammation can arise from infections, symptoms, or diseases (e.g., cancer, atherosclerosis, allergies, myopathy, HIV, obesity, or autoimmune diseases). Autoimmune diseases are chronic diseases resulting from an abnormal immune response to autoantigens. Autoimmune diseases that can be treated with 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 contain one or more protease-cleaving linker sequences. The linker sequences function to provide flexibility between polypeptides so that each polypeptide can inhibit the activity of the first polypeptide. The linker sequences can be positioned between any or all of the cytokine polypeptide, its fragment or mutain, blocking moieties, and serum half-life extension elements. Optionally, the composition may contain two, three, four, or five linker sequences. The linker sequence, two, three, or four linker sequences may be the same linker sequence or different linker sequences. 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), GPLVGRG (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 proteases.

[0227] Furthermore, the present invention provides a method for treating subjects who have cancer or are at risk of developing cancer. The method comprises administering an effective amount of a chimeric polypeptide (fusion protein) disclosed herein, typically administered as a pharmaceutical composition, to a subject in need. In some embodiments, the method further comprises selecting a subject who has cancer or is at risk of developing cancer. The pharmaceutical composition preferably comprises a blocked cytokine, its fragment, or a mutain 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 drugs to treat cancer, such as chemotherapeutic agents (e.g., Adriamycin, Seruvidine, Bleomycin, Alkeran, Verban, Oncovin, Fluorouracil, Thiotepa, Methotrexate, Bisanthren, Noantrone, Tiguanine, Cytaribine, Procalabidine), immuno-oncology agents (e.g., anti-PD-L1, anti-CTLA4, anti-PD-1, anti-CD47, anti-GD2), cell therapies (e.g., CAR-T, T-cell therapy), or oncolytic viruses.

[0229] This specification provides pharmaceutical formulations or compositions 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 means 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 containing it. The carrier is selected to minimize the degradation of the active ingredient and minimize adverse effects on the subject.

[0230] For suitable carriers and their formulations, see Remington: The Science and Practice of Pharmacy, 21 stThis is described in Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). Typically, an appropriate amount of pharmaceutically acceptable salt is used in the formulation to make it isotonic, although the formulation may be hypertonic or hypotonic as desired. Examples of pharmaceutically acceptable carriers include, but are not limited to, sterile water, physiological saline, buffer solutions such as Ringer's solution, and dextrose solutions. The pH of the solutions 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 immunogenic polypeptides. The matrices take the form of molded articles (e.g., films, liposomes, or microparticles). Certain carriers may be more preferred depending, for example, on the route of administration and the concentration of the composition to be administered. The carrier is suitable for administering chimeric polypeptides or nucleic acid sequences encoding chimeric polypeptides to humans or other subjects.

[0231] Pharmaceutical formulations or compositions are administered in multiple ways, depending on whether topical or systemic treatment is preferred and the area to be treated. Compositions are administered via one of several routes of administration, including topical, oral, parenteral, intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, intracavitary, percutaneous, intrahepatic, intracranial, spray / inhalation, or placement via bronchoscopy. In some embodiments, compositions are administered topically (non-systemically), including in tumors, intra-articular, or intrathecal spaces.

[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, alcohol / aqueous solutions, emulsions, or suspensions (including saline and buffer media). Parenteral vehicles include sodium chloride solutions, ringer's dextrose, dextrose and sodium chloride, Ringer's lactate, or fixative oils. Vehicles for intravenous administration include liquids and nutritional supplements, electrolyte supplements (e.g., those based on ringer's dextrose), etc. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may be optionally present.

[0233] Topical formulations include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, and thickeners are optionally required or desirable.

[0234] Compositions for oral administration include powders or granules, suspensions or solutions in water or a non-aqueous medium, capsules, sachets, or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing agents, or binders are optional and desirable.

[0235] Optionally, a chimeric polypeptide or a nucleic acid sequence encoding a chimeric polypeptide is administered by a vector. There are several 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 an expression vector. These methods and compositions can be broadly classified into two classes: virus-based delivery systems and non-virus-based delivery systems. Such methods are well known in the art and are readily adaptable for use with the compositions and methods described herein. Such compositions and methods can be used in vitro or in vivo to translocate or transduce cells to, for example, produce cell lines that express and preferably secrete an encoded chimeric polypeptide, or to therapeutically deliver nucleic acids to a target. The components of the chimeric nucleic acids disclosed herein are typically conjugated in-frame to encode a fusion protein.

[0236] As used herein, a plasmid or viral vector is an active agent that transports the nucleic acids of this disclosure into a cell without degradation, and includes a promoter that expresses the nucleic acid molecule and / or polypeptide in the delivered cell. Viral vectors include, for example, adenoviruses, adeno-associated viruses, herpesviruses, vaccinia viruses, polioviruses, Sindobis viruses, and other RNA viruses, including viruses having the HIV skeleton. Any viral family that shares the properties of these viruses and is therefore suitable for use as a vector is also preferred. Retroviral vectors are generally described in Coffin et al., Retroviruses, Cold Spring Harbor Laboratory Press (1997), and vectors and methods for their preparation are incorporated herein by reference. 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 initial infected cell, they cannot form new infectious viral particles, thus limiting their ability to spread to other cell types. Recombinant adenoviruses have been shown to achieve high efficiency after direct in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, central nervous system parenchyma, and several other tissue sites. Other useful systems include, for example, replicated vaccinia virus vectors and host-restricted non-replicated 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 proteins (or more) derived from the structural proteins of a virus. Methods for constructing and using virus-like particles are described, for example, in Garcea and Gissmann's Current Opinion in This is described in Biotechnology 15:513-7 (2004).

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

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

[0240] Non-viral-based delivery methods may include an expression vector comprising a nucleic acid molecule and a nucleic acid sequence encoding a polypeptide, wherein the nucleic acid is admissibly bound to an expression regulatory sequence. Suitable vector skeletons include, for example, 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 start sites, termination sequences, polyadenylation sequences, and introns. Using such vectors, chimeric polypeptides can also be created by expression in suitable host cells, such as CHO cells.

[0241] Preferred promoters for controlling transcription from a vector within mammalian host cells can be obtained from a variety of sources, for example, from viral genomes, such as polyomas, Simian virus 40 (SV40), adenoviruses, retroviruses, hepatitis B virus, most preferably cytomegalovirus (CMV), or from heterologous mammalian promoters, such as the β-actin promoter or EF1α promoter, or from hybrid or chimeric promoters (e.g., a CMV promoter fused with the β-actin promoter). Naturally, promoters derived from host cells or related species are also useful herein.

[0242] An enhancer is generally a DNA sequence that functions without a fixed distance from the transcription start site and can be either 5' or 3' relative to the transcription unit. Furthermore, enhancers can be found within introns or within the coding sequence itself. They are typically 10–300 base pairs (bp) long and function in cis. Enhancers usually function to increase transcription from nearby promoters. Enhancers may also include response elements that mediate transcriptional regulation. Many enhancer sequences are known from mammalian genes (globin, elastase, albumin, fetoprotein, insulin), but typically enhancers from eukaryotic viruses are used for general expression. Preferred examples include the SV40 enhancer located posterior to the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer located posterior to the origin of replication, and the adenovirus enhancer.

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

[0244] Furthermore, vectors may also include, for example, origins of replication and / or markers. Marker genes can confer selectable phenotypes (e.g., antibiotic resistance) to cells. Marker products are used to determine whether the vector has been delivered to cells and, if delivered, whether it is expressed. Examples of selective markers for mammalian cells include dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, puromycin, and blastidine. If such selective markers successfully translocate to mammalian host cells, the transformed mammalian host cells can survive even under selective pressure. Other examples of markers include, for example, the E. coli lacZ gene, green fluorescent protein (GFP), and luciferase. In addition, expression vectors may include tag sequences designed to facilitate the 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® tag (Kodak, New Haven, Conn.), are typically expressed as fusions with the encoded polypeptide. Such tags can be inserted at any position within the polypeptide, including either a carboxyl or amino terminus.

[0245] As used herein, the terms peptide, polypeptide, or protein are used broadly to mean 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 suggest a specific size or number of amino acids constituting the molecule, and that the peptides of the present invention may contain up to a few amino acid residues or more. As used in whole, subjects may be vertebrates, more specifically mammals (e.g., humans, horses, cats, dogs, cattle, pigs, sheep, goats, mice, rabbits, rats, and guinea pigs), birds, reptiles, amphibians, fish, and any other animals. The terms do not indicate a specific age or sex; therefore, adult and neonatal subjects, male or female, are intended to be covered. As used herein, patient or subject may be used interchangeably to refer to a subject with a disease or disorder (e.g., cancer). The term patient or subject includes human and veterinary subjects.

[0246] Individuals at risk of developing a disease or disability may have a genetic predisposition to the disease or disability, for example, having a family history, having a mutation in their genes that causes the disease or disability, or showing early signs or symptoms of the disease or disability. Individuals currently having a disease or disability may have one or more symptoms of the disease or disability and may have been diagnosed with the disease or disability.

[0247] The methods and agents described herein are useful for both prophylactic and therapeutic treatment. For prophylactic use, a therapeutically effective dose of the chimeric polypeptide or chimeric nucleic acid sequence encoding the chimeric polypeptide described herein is administered to the subject before the onset of disease (e.g., before the obvious 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 be carried out for several days to several 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 dose of the chimeric polypeptide or chimeric nucleic acid sequence encoding the chimeric polypeptide described herein to the subject after the diagnosis or onset of cancer or inflammation (e.g., autoimmune disease). Prophylactic use can also be applied when the patient is receiving treatment in which inflammation is expected (e.g., chemotherapy).

[0248] According to the methods taught herein, subjects are administered an effective dose of the drug (e.g., a chimeric polypeptide). The terms effective dose and effective drug dose are used interchangeably. The term effective dose is defined as the amount required to produce a desired physiological response. The effective dose and schedule for administering the drug can be determined empirically, and making such determinations is within the scope of the skill of those skilled in the art. The drug dose range for administration is a range large enough to produce the desired effect, affecting (e.g., reducing or delaying) one or more symptoms of the disease or disorder. The drug dose should not be so large as to cause substantially harmful side effects (e.g., undesirable cross-reactions, anaphylactic reactions, etc.). Generally, drug doses vary depending on age, condition, sex, type of disease, severity of disease or disorder, route of administration, or whether other drugs are included in the regimen, and can be determined by those skilled in the art. Drug doses can be adjusted by individual physicians in the event of any contraindications. Drug doses may vary and may be administered as one or more doses daily, daily, or for several days. Guidelines for appropriate dosages in a given class of pharmaceutical products can be found in the literature.

[0249] As used herein, the terms treatment, cure, or cure mean a disease or condition, or a method of reducing the effects of the symptoms of a disease or condition. Therefore, in the methods of this disclosure, treatment may mean a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or condition, or in the symptoms of such disease or condition. For example, a method for treating a disease is considered a treatment if one or more symptoms of the disease in question are reduced by 10% compared to a control. Therefore, this reduction may 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 mean a cure or complete disappearance of a disease, condition, or the symptoms of a disease or condition.

[0250] As used herein, the terms preventing, inhibiting, and sole prevention of disease or disorder refer to an action that inhibits or delays the onset or exacerbation of one or more symptoms of a disease or disorder, occurring before or approximately simultaneously with the onset of one or more symptoms of the disease or disorder in a subject, for example, the administration of a chimeric polypeptide or a nucleic acid sequence encoding a chimeric polypeptide. As used herein, references to reduction, reduction, or inhibition include changes of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to a control level. Such terms may, but may not, include complete disappearance.

[0251] IL-2 variants that are more selective to IL2Rαβγ than 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. Since IL-2's affinity for IL2RG is undetectable, these variants consequently have reduced affinity for the IL2Rβγ receptor complex and a reduced ability to activate IL2Rβγ-expressing cells, but they retain their ability to bind to IL2RA and to bind to and activate the IL2Rαβγ receptor complex.

[0252] One of these variants, IL2 / N88R (Bay 50-4798), was clinically tested as a less toxic version of IL-2 as an immune system stimulant, based on the hypothesis that IL2Rβγ-expressing NK cells are the primary contributor to toxicity. Bay 50-4798 has been shown to selectively stimulate the proliferation of activated T cells compared to NK cells and was 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, RT, et.al., 2008, J Interferon Cytokine Res., 28:89-100). These clinical trials showed that Bay 50-4798 is considerably safer and more tolerable than aldezleukin, and also showed to increase levels of CD4+CD25+ T cells, a cell population rich in Treg cells. Following these clinical trials, research in this field has more fully confirmed the contents of Treg cells, demonstrating 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, it is possible to create mutants that selectively alter the affinity for the CD25 chain compared to native Il-2.

[0254] IL-2 can be engineered to produce mutants that bind to the IL-2R complex in general, or mutants that bind specifically to the IL-2Rα subunit, with different affinities than the corresponding wild-type IL-2 or currently available mutants (referred to as C125S because the cysteine ​​residue at position 125 is replaced with a serine residue).

[0255] Therefore, the present invention relates to a mutant interleukin-2 (IL-2) comprising an amino acid sequence that is at least 80% identical to wild-type IL-2 (e.g., 85, 87, 90, 95, 97, 98, or 99% identical), and which binds more highly to the IL-2 trimer receptor than to the dimeric IL-2 receptor compared to wild-type IL-2. *The mutant IL-2 polypeptide is characterized by a polypeptide. Typically, muteins also bind to the IL-2 receptor α 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 be altered from SEQ ID NO: 1 (UniProtKB accession number P60568) by including (or including only) one or more amino acid substitutions that may be considered conserved or non-conserved substitutions. Non-native amino acids may also be incorporated. Alternatively or additionally, the amino acid sequence may be altered from SEQ ID NO: 1 (which may be considered a “reference” sequence) by including, and by adding and / or deleting, one or more amino acid residues. More specifically, the amino acid sequence may differ from the sequence of SEQ ID NO: 1 by mutations at at least one of the following positions (or combinations thereof) of SEQ ID NO: 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. As mentioned above, only one of these positions may be changed, or two, three, four, five, six, seven, eight, nine, ten, or eleven, or more (up to all of them) of these positions may be changed. For example, the amino acid sequence may differ from SEQ ID NO: 1 at positions 69 and 74, and further at one or more of positions 30, 35, and 128.Furthermore, the amino acid sequence may 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) position 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. Apart from these positional mutations, the amino acid sequence of the mutant IL-2 polypeptide may otherwise be identical to SEQ ID NO: 1. For specific 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. The inventors' nomenclature is consistent with scientific literature nomenclature, namely, the single-letter code of an amino acid in the wild-type sequence or reference sequence, followed by the position of the code in the sequence, and then the single-letter code of the substituted amino acid.Therefore, A1T indicates that the alanine residue at position 1 is substituted with threonine. Other variant polypeptides within the scope of the present invention include variants of SEQ ID NO: 2 having substitutions at V69 (e.g., A) and Q74 (e.g., P). For example, the amino acid sequence may include 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 I 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. Sequence 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 described above, any mutant IL-2 polypeptide disclosed herein may include the described sequence, and may be limited to the described sequence and otherwise identical to SEQ ID NO: 1. Furthermore, any mutant IL-2 polypeptide described herein may optionally include substitution of the cysteine ​​residue at position 125 with another residue (e.g., serine), and / or optionally include deletion of the alanine residue at position 1 of SEQ ID NO: 1.

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

[0258] Materials, compositions, and components that may be used for, in conjunction with, in preparation of, or products thereof, are disclosed herein. Where these and other materials are disclosed herein, and combinations, subsets, interactions, groups, etc., of these materials are disclosed, specific references to various individual and collective combinations and rearrangements of each of these compounds cannot be expressly disclosed, but each is intended and described herein. For example, where a method is disclosed and discussed, and multiple modifications that may be made to multiple molecules including the method are discussed, all combinations and rearrangements of the method, as well as possible modifications, are intended unless otherwise specifically indicated. Similarly, any subsets or combinations thereof are also intended and disclosed. This concept applies to all aspects of the disclosure, including steps in methods using the compositions of the disclosure, but is not limited to these. Therefore, where various additional steps may be implemented, each of these additional steps may be implemented in any specific method step or combination of method steps of the Method Disclosure, and each such combination or subset of combinations should be considered as specifically contemplated and disclosed.

[0259] Publications cited herein and materials from which they are cited are incorporated herein by reference in their entirety. 6. Reference All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent as each individual publication or patent application is specifically and individually incorporated herein by reference. However, the reference of any reference herein should not be construed as an affirmation that such reference is prior art of the present invention. If any definition or term in any reference is found to differ from any term or discussion herein, the current term and definition shall prevail. [Examples]

[0260] The following are examples of the methods and compositions of the present invention. Considering the general description set forth herein, it will be understood that various other embodiments may be implemented.

[0261] Example 1. Detection of IL-2, IL-2 mutein, IL-2Rα, and IL-2Rγ in fusion proteins by ELISA. IL-2 mutein is detected using a commercially available antibody, such as anti-IL-2 monoclonal antibody (JES6-1A12) (BD Pharmingen; San Jose, Calif.). A positive control is used to indicate whether the monoclonal antibody recognizes the cytokine or mutein. Antibodies against IL-2Rα and IL-2Rγ chains are also used. Wells of a 96-well plate are coated with antibody in PBS (2.5 μg / ml). Wells are blocked with 5% nonfat milk and 0.2% Tween® 20 (PBS-M-Tw) in PBS, and the fusion protein is added at 37°C for 1-2 hours. After washing, anti-IL-2 biotin-labeled antibody (e.g., JES5H4 (BD Pharmingen)) is added, followed by streptavidin HRP (Southern Biotechnology). Binding is detected using Associates (Birmingham, Ala.). The ELISA plate is developed by adding 50 μl of O-phenylenediamine (OPD) (Sigma-Aldrich) to 0.1 M citrate pH 4.5 and 0.04% H2O2, stopping the reaction by adding 50 μl / well of 2N H2SO4, and reading the absorbance at 490 nm.

[0262] Example 2: Protease cleavage of fusion protein by MMP9 protease Those skilled in the art are expected to be familiar with the setup of the protein cleavage assay. 100 μg of protein in 1×PBS pH 7.4 was cleaved with 1 μg of active MMP9 (Sigma catalog number SAE0078-50 or Enzo catalog number BML-SE360) and incubated at room temperature for up to 16 hours. The digested protein was then used for functional assays or stored at -80°C before testing. The degree of cleavage was monitored by SDS-PAGE using methods well known in the art. Complete cleavage of the fusion protein by the MMP9 protease is observed, as shown in Figures 10, 13, 18A, 18b, and 27A.

[0263] Example 3: CTLL-2 assay CTLL2 cells (ATCC) were suspended at a concentration of 500,000 cells / well in culture media containing or without 40 mg / ml human serum albumin (HSA), cultured on plates, and stimulated with a dilution series of recombinant hIL2 or activatable hIL2 at 37°C and 5% CO2 for 72 hours. The activity of non-cleaved and cleavage-activatable hIL2 was tested. Cleavage-activatable hIL2 was generated by incubation with active MMP9. Cell activity was evaluated using the CellTiter-Glo® (Promega) luminescence-based 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 polypeptide improves access to antibodies and biologically active IL-2 mutein. The IL-2 mutein fusion protein is biochemically characterized before and after cleavage with a protease (e.g., PSA). Immunoblot analysis will show that the fusion protein can be cleaved by PSA, and that after treatment with PSA, the intensity of the predicted low molecular weight cleavage product of approximately 20 kDa, which is reactive to the anti-IL-2 antibody, will increase. The degree of cleavage depends on the amount of PSA and the incubation time. Interestingly, when the fusion protein was analyzed by ELISA before and after PSA treatment, an apparent increase in the amount of IL-2 was found after PSA cleavage. In this experiment, the apparent amount of IL-2 detected using this sandwich ELISA increased by approximately 2 or 4 times, depending on the construct. This suggests that antibody binding is partially inhibited in the intact fusion protein. Aliquots of the same sample are also analyzed after PSA treatment using 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 with increasing dilution, and the amount of biologically active IL-2 increases after PSA cleavage. The increase in IL-2 mutein levels suggests an increase in the predicted low molecular weight cleavage fragments of approximately 20 kDa that are reactive to anti-IL-2 antibodies after PSA cleavage, increased antibody accessibility, and most importantly, an increase in the amount of biologically active IL-2 mutein.

[0265] Example 5. In vivo delivery of protease-activating fusion protein reduces tumor growth. We will investigate chimeric polypeptides to determine if they may have biological effects in vivo. For such experiments, we will use a system in which tumor cells injected into the peritoneal cavity rapidly and preferentially attach to and grow in milky spots (a series of organized immunoaggregates found on the omentum) (Gerber et al., Am.J. Pathol. 169:1739-52 (2006)). This system allows for multiple deliveries of the fusion protein into the peritoneal cavity, and tumor growth can be analyzed by examining the dissociated omental cells, thus providing a convenient method for investigating the effects of fusion protein treatment on tumor growth. In these experiments, we can use the rapidly growing Colon38 tumor cell line, which expresses both MMP2 and MMP9 in vitro. Normally, omental tissue expresses relatively small amounts of MMP2 and MMP9, but MMP levels increase when Colon38 tumors are present in the omentum. Using this tumor model, we will investigate the ability of IL-2 mutein fusion proteins to influence tumor growth. Colon38 cells were injected into the peritoneal cavity and allowed to adhere and grow for one day, after which the fusion protein was administered intraperitoneally daily. On day 7, the animals were sacrificed, and tumor growth in the omentum was examined 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 + Test the binding affinity to cells.

[0267] CD20 +Cells are incubated with 100 μL of serially diluted activatable interleukin protein and at least one protease. After washing three times with FACS buffer, cells are incubated on ice for 45 minutes with 0.1 mL of 10 μg / mL mouse monoclonal anti-idiotype antibody in the same buffer. After a second washing cycle, cells are 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, cells are incubated with anti-His IgG and then with FITC-conjugated goat anti-mouse IgG antibody without activatable IL2 protein. Cells are then washed again and resuspended in 0.2 mL of FACS buffer containing 2 μg / mL propidium iodide (PI) to remove dead cells. 1 × 10 4 The fluorescence of living 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 average fluorescence intensity of the cell sample 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 only with secondary and tertiary reagents, the result is calculated using GraphPad Prism (version 6.00 for Windows, GraphPad Software, La Using the equation (hyperbola) of the one-site bond from Jolla, California, USA, K D Calculate the value.

[0268] CD20 binding and cross-reactivity with human CD20 + Evaluate on tumor cell lines. Cross-reactivity K D The ratio was quantified using CHO cell lines expressing recombinant human antigen or recombinant cynomolgus monkey antigen. DCalculate using the values.

[0269] Example 7: Cytotoxic Assay CD20 of Activatable Interleukin Proteins + The mediation of the immune response against target cells will be evaluated 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 (standardized T cell line) as effector cells in the presence of activatable IL2 protein and at least one protease. After incubation at 37°C for 4 hours in a humidified incubator, the release of 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 were used as negative and positive controls, respectively.

[0271] Based on the measured number of remaining target cells, the percentage of specific cell lysis is calculated according to the following formula: [1 - (number of living targets)] (試料) / number of raw targets (自発) )] × 100%. Sigmoid dose-response curve and EC 50 The values ​​are calculated using nonlinear regression / 4-parameter logistic fitting with GraphPad software. Using the lysis values ​​obtained at a given antibody concentration, the sigmoid dose-response curve is calculated using 4-parameter logistic fitting analysis with Prism® GraphPad® software.

[0272] Example 8: Pharmacokinetics of Activatable Interleukin Proteins The half-life elimination of activatable interleukin proteins will be evaluated in animal experiments.

[0273] The activatable IL2 protein is administered as a 0.5 mg / kg bolus via saphenous vein to cynomolgus monkeys. Another group of cynomolgus monkeys receives an IL2 construct of comparable size, but lacking the serum half-life extension element. Groups 3 and 4 receive an IL2 construct with the serum half-life extension element, as well as CD20 and cytokines with the serum half-life extension element, respectively. All of these are of comparable size to the activatable interleukin protein. Each test group consists of 5 monkeys. Serum samples are collected at the indicated time, serially diluted, and the protein concentration is quantified using ELISA bound to CD20.

[0274] Pharmacokinetic analysis is performed using the plasma concentrations of the test items. When plotted against time after administration, the group-mean plasma data for each test item fits a multi-order exponential profile. The data is fitted using a standard two-compartment model with a bolus input and first-order rate constants for the distribution and efflux phases. The general equation most appropriate for intravenous administration data is as follows: c(t)=Ae -αt +Be -βt (c(t) is the plasma concentration at time t, A and B are the Y-intercepts, and α and β are the apparent first-order rate constants of the distribution and efflux phases, respectively). The α phase is the initial phase of clearance, reflecting the distribution of protein into all extracellular fluid of the animal, and the second or β phase 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 given by a quadratic equation using estimation parameters: V = volume of distribution, k10 = efflux rate, k12 = transfer rate from compartment 1 to compartment 2, k21 = transfer rate from compartment 2 to compartment 1, and D = dose administered: r 2 It is a root of +(k12+k21+k10)r+k21k10=0

[0275] Data analysis. The graph of concentration versus time profile is from KaleidaGraph (KaleidaGraph (trademark) V.3.09 Copyright 1986-1997). The analysis is performed using Synergy Software (Reading, Pa.). Values ​​reported as less than reportable (LTR) are not included in the PK analysis and are not displayed in the graphs. Pharmacokinetic parameters are determined by compartment 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 exhibit improved pharmacokinetic parameters, such as increased efflux half-life, compared to proteins lacking serum half-life extension elements.

[0277] Example 9: Xenograft tumor model We will evaluate the activatable IL2 protein using a xenograft model.

[0278] Female immunodeficient NOD / scid mice were irradiated with a sublethal dose of radiation (2 Gy), resulting in 4 × 10⁶ mice. 6 Individual Ramos RA1 cells are subcutaneously inoculated into the right dorsal region. The tumor is 100-200 mm in size. 3 Once the target is reached, the animals are assigned to three treatment groups. Groups 2 and 3 (8 animals each) receive 1.5 × 10 7One activated human T cell was injected intraperitoneally. Three days later, the third group of animals was subsequently treated with a total of nine intravenous doses of 50 μg of activatable interleukin protein. The first and second groups were treated with vehicles only. Body weight and tumor volume were measured for 30 days.

[0279] Animals treated with activatable interleukin proteins are expected to exhibit a statistically significant delay in tumor growth compared to the control group treated with each vehicle.

[0280] Example 10: Mouse IFNγ WEHI cell viability assay WEHI279 cells (ATCC) were suspended at a concentration of 25,000 cells / well in culture media containing or without 1.5% human serum albumin (HSA), cultured on plates, and stimulated with a dilution series of recombinant mIFNγ or inducible mIFNγ at 37°C and 5% CO2 for 72 hours. The activity of non-cleaved and cleavage-inducible mIFNγ was tested. Cleavage-inducible mIFNγ was generated by incubation with active MMP9. Cell viability was assessed using the CellTiter-Glo (Promega) luminescence-based cell viability assay. The EC50 values ​​of cleavage-inducible mIFNγ molecules were at least 100-fold stronger than those of non-cleavage-inducible mIFNγ molecules. Greater inducibility was observed in assays using culture media containing human serum albumin, as shown in Figures 16A-16.

[0281] Example 11: Reserved Example 12: Mouse IFNγ B16 Reporter Cell Assay B16-Blue IFNγ cells (InvivoGen) were cultured at a concentration of 75,000 cells / well in culture media containing or without 1.5% human serum albumin (HSA), and stimulated with a dilution series of recombinant mIFNγ or inducible mIFNγ at 37°C and 5% CO2 for 24 hours. The activity of non-cleaved and cleavage-inducible mIFNγ was tested. Cleavage-inducible mIFNγ was generated by incubation with active MMP9. SEAP activation was evaluated by collecting the supernatant, adding QUANTI-Blue reagent (InvivoGen), incubating at 37°C for 2 hours, and measuring the absorbance at 620 nm. The EC50 value of the cleavage-inducible mIFNγ molecule was at least 100 times stronger than that of the non-cleavage-inducible mIFNγ molecule. The results are shown, for example, in Figures...

Claims

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

198.

2. Formula I: [D1]-[L1]-[D2] A polypeptide comprising, In the formula, D1 is the first target domain, L1 is a separation portion that connects or binds D1 to D2, and the separation portion contains the amino acid sequence of SEQ ID NO:

198. The polypeptide wherein D2 is the second target domain.

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

4. The polypeptide according to claim 2, wherein the separation portion comprises two or more cleavable portions, and each of the cleavable portions is a substrate for a protease.

5. The polypeptide according to claim 2, comprising a first cleavable moiety containing a first amino acid sequence that is a substrate for a first protease, and a second cleavable moiety containing a second amino acid sequence that is a substrate for a second protease.

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

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

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

9. The polypeptide according to 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 according to claim 2, wherein the polypeptide comprises an antigen-binding polypeptide, an antibody or its antigen-binding portion.

11. The polypeptide according to claim 2, wherein the polypeptide is responsively bound to a portion selected from the group consisting of a polypeptide portion, a lipid portion, a nucleic acid portion, a detectable portion, and a small molecule.

12. Recombinant proprotein, 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: 198, b. Polypeptides with biological activity The recombinant proprotein, including the above.

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

14. The recombinant proprotein according to claim 12, wherein the polypeptide having the biological activity comprises at least one of an extracellular domain, a transmembrane domain, and an intracellular domain.

15. The recombinant proprotein according to claim 12, wherein the polypeptide having the biological activity comprises a cell surface receptor, a chimeric antigen receptor (CAR), or a T cell receptor (TCR) subunit.

16. The recombinant proprotein according to claim 12, wherein the polypeptide having the biological activity comprises an antigen-binding polypeptide, an antibody or its antigen-binding portion.

17. The recombinant proprotein according to claim 12, wherein the cleavable portion binds the biologically active polypeptide to another amino acid sequence.

18. The recombinant proprotein according to claim 12, wherein the cleavable portion, which is a substrate of the protease, attaches the biologically active polypeptide to another amino acid sequence.

19. The recombinant proprotein according to claim 12, wherein the biological activity of the recombinant proprotein is attenuated, and cleavage of the cleavable portion by the protease produces a polypeptide in which the biological activity is not attenuated.

20. The recombinant proprotein according to claim 12, further comprising a steric blocking portion, a specific blocking portion, and a combination thereof, which can block the biological activity of the recombinant proprotein.

21. The recombinant proprotein according to claim 20, wherein the blocking portion comprises a steric blocking portion containing human serum albumin (HSA), an anti-HSA antibody, immunoglobulin Fc, or any of the aforementioned fragments.

22. The recombinant proprotein according to claim 20, wherein the blocking portion comprises a specific blocking portion including an antibody or antigen-binding fragment having binding specificity to the biologically active polypeptide, or a ligand-binding portion of a receptor or ligand-binding fragment having binding specificity to the biologically active polypeptide.

23. The recombinant proprotein according to claim 12, further comprising a half-life extension domain.

24. The recombinant proprotein according to claim 12, wherein the polypeptide having the biological activity 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 described in claim 25.

27. A host cell comprising the vector according to claim 26.

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

29. A pharmaceutical composition for treating a subject requiring treatment, 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