Protease activatable cytokines and methods for making and using the same

Abstract

Provided herein are, inter alia, protease-activatable cytokines, as well as uses and methods of treatment. In particular, protease-activatable IL-12 fusion proteins, where the two subunits p40 and p35 are produced as fusion proteins with at least one masking moiety (Mask) linked to the subunit by a release segment (RS), namely, a cleavable linker, especially (i) not cleavable by legumain in human plasma, (ii) cleaved less than 25% compared to EAGRSANHTPAGLTGP (RSR-2295; SEQ ID NO:7048, legumain target) or, (iii) where the release segment comprises EAGRSAXHTPAGLTGP, X is any amino acid other than N (SEQ ID NO:7627). In preferred embodiments, each of the IL-12p35 and IL-12p40 subunits are linked at the N-and C-terminal ends by release segments (cleavable linker) to different masking moieties (Masks). In some embodiments, the fusion proteins further comprise barcodes.

Description

PROTEASE ACTIVATABLE CYTOKINES AND METHODS FOR MAKING AND USING THE SAMECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the United States Provisional Application Serial No. 63 / 607,898. filed December 8, 2023, the content of which is hereby incorporated by reference in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (95KG-385485-WO.xml; Size: 2.427.153 bytes; and Date of Creation: December 5. 2024) is herein incorporated by reference in its entirety.BACKGROUND

[0003] Cytokines have the potential to treat a variety of diseases or conditions. However, the therapeutic utility of cytokines can be limited due to the cellular toxicity, short half-life, need for repetitive or frequent dosing, and the potential to elicit rmdesired immune response in the patients.

[0004] Most cytokine products in the clinical setting are extremely potent. Interleukins, such as IL-2 and IL- 12, and IFN-a are cytokines, produced primarily by cells of the immune system to signal and organize the immune response. In cancer, cytokines facilitate the ability of the immune system to recognize tumor cells as abnormal and harmful to the host. Cytokines further increase the proliferation of, enhance the survival of. and direct a variety of immune cell types to infiltrate the tumor microenvironment and promote potent anti-tumor immune responses resulting in tumor cell killing and tumor clearance. This limits the practical applications of cytokines in a therapeutic setting, particularly in anti-cancer indications.

[0005] Interleukin- 12 (IL- 12) in particular, has been recognized as having potential to be an ideal pay load for tumor immunotherapy. It can activate both the iimate and the adaptive components of the immune system. IL-12 stimulates the production of IFN-y and activates NK cells, as well as CD8+ and CD4+ T cells. In addition, this cytokine also induces antiangiogenic chemokines. remodeling of the tumor extracellular matrix and stimulation of MHC class I molecules expression, making it an extremely attractive anticancer candidate. However, while researchers have shown encouraging preclinical data, the severe toxicity profile of this cytokine has prevented dose escalation and significantly curbed clinical potential as an anticancer agent. Although multiple clinical trials have been on-going since the first human clinical trial of IL-12 in 1996, an FDA-approved IL-12 product remains elusive.

[0006] This presents a significant unmet need for new strategies that can overcome therapeutic index challenges for use of cytokines as anticancer agents. If the potency of cytokines like IL- 12 could be safely harnessed and the toxicity challenges could be controlled, these agents could serve as powerful therapeutics for potential use against a broad spectrum of cancers.BRIEF DESCRIPTION

[0007] The present disclosure provides, among other things, a fusion protein comprising a cytokine, a linker comprising a protease-cleavable release segment, and a mask polypeptide, wherein the linker comprising the release segment is positioned between the cytokine and the mask polypeptide, and wherein the release segment is capable of being cleaved by at least one protease that is present in a tumor. Aspects disclosed herein address a long-felt unmet need for activatable cytokines for cancer therapeutics, that have an increased therapeutic index.

[0008] Various features of this disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings.SUMMARY

[0009] In one aspect, the disclosure provides a fusion protein comprising: a cytokine, a linker (Linkerl) comprising a protease-cleavable release segment (RSI), and a mask polypeptide (Maskl), wherein the linker (Linkerl) comprising the release segment (RSI) is positioned between the cytokine and the mask polypeptide (Maskl); wherein the release segment is capable of being cleaved by at least one protease that is present in a tumor; and wherein the release segment (RSI) (i) is not capable of being cleaved by legumain in human plasma, (ii) is cleaved by legumain in human plasma at a rate that is less than 25% of the rate that EAGRSANHTPAGLTGP (RSR-2295; SEQ ID NO:7048) is cleaved by legumain in human plasma, or (iii) comprises an amino acid sequence comprising the sequence EAGRSAXHTPAGLTGP, wherein X is any amino acid other than N (SEQ ID NO:7627).

[0010] In some embodiments, the cytokine is an interleukin, a transforming growth factor, an interferon, a tumor necrosis factor, a chemokine, or granulocyte macrophage -colony stimulating factor.

[0011] In some embodiments, the cytokine is an interleukin. In some embodiments, the interleukin is selected from IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, and IL-25. In some embodiments, the interleukin is IL-12. In some embodiments, the IL-12 is a disulfide-linked heterodimer comprising a p35 polypeptide and a p40 polypeptide, and wherein the linker and mask are covalently joined to (i) the N-terminus or C- terminus of the p35 polypeptide, or (ii) the N-terminus or C-tenninus of the p40 polypeptide.

[0012] In one aspect, the disclosure provides a fusion protein comprising: (a) a first polypeptide subunit comprising the following elements in an N-to-C or C-to-N terminal orientation: i. a first mask polypeptide (Maskl), ii. a first linker (Linkerl) comprising a first protease-cleavable release segment (RSI), iii. an IL-12 p35 polypeptide, iv. a second linker (Linker2) comprising a second protease- cleavable release segment (RS2), and v. a second mask polypeptide (Mask2); and (b) a second polypeptide submit comprising the following elements in an N-to-C or C-to-N terminal orientation: i. a third mask polypeptide (Mask3), ii. a third linker (Linker3) comprising a third protease-cleavable release segment (RS3), iii. an IL-12 p40 polypeptide, iv. a fourth linker (Linker4) comprising a fourth protease-cleavable release segment (RS4), and v. a fourth mask polypeptide (Mask4); wherein the IL- 12 p35 polypeptide of the first polypeptide subunit and the IL- 12 p40 polypeptide of the second polypeptide subunit are disulfide-linked thereby forming a disulfide -linked heterodimer: wherein each release segment (RSL RS2, RS3. and RS4) is capable of being cleaved by at least one protease that is present in a tumor; and wherein each release segment (RSI, RS2, RS3. and RS4) (i) is not capable of being cleaved by legumain in human plasma, (ii) is cleaved by legumain in human plasma at a rate that is less than 25% of the rate that RSR-2295 (EAGRSANHTPAGLTGP; SEQ ID NO:7048) is cleaved by legumain in human plasma, or (iii) comprises an amino acid sequence comprising the sequence EAGRSAXHTPAGLTGP, wherein X is any amino acid other than N (SEQ ID NO:7627).

[0013] In some embodiments, the first mask polypeptide (Maskl) comprises a first a first barcode fragment (Barcode 1), the second mask polypeptide (Mask2) comprises a second barcode fragment (Barcode2). the third mask polypeptide (Mask3) comprises a third barcode fragment (Barcode3). and the fourth mask polypeptide (Mask4) comprises a fourth barcode fragment (Barcode4); and wherein each barcode fragment (Barcode 1, Barcode2, Barcode3, and Barcode4) is releasable from the fusion protein upon digestion with a non-mammalian protease, and each barcode fragment differs in both sequence and molecular weight from all other barcode fragments or peptide fragments that are releasable from the fusion protein upon complete digestion of the fusion protein by the nonmammalian protease.

[0014] In one aspect, the disclosure provides a fusion protein comprising: (a) a first polypeptide subunit comprising the following elements in an N-to-C or C-to-N terminal orientation: i. a first mask polypeptide (Maskl) comprising a first barcode fragment (Barcodel), ii. a first linker (Linkerl) comprising a first protease-cleavable release segment (RSI), iii. an IL-12 p35 polypeptide, iv. a second linker (Linker2) comprising a second protease-cleavable release segment (RS2), and v. a second mask polypeptide (Mask2) comprising a second barcode fragment (Barcode2); and (b) a second polypeptide subunit comprising the following elements in an N-to-C or C-to-N terminal orientation: i. a third mask polypeptide (Mask3) comprising a third barcode fragment (Barcode3). ii. a third linker (Linker3) comprising a third protease-cleavable release segment (RS3), iii. an IL- 12 p40 polypeptide, iv. a fourth linker (Linker4) comprising a fourth protease-cleavable release segment(RS4), and v. a fourth mask polypeptide (Mask4) comprising a fourth barcode fragment (Barcode4); wherein the IL-12 p35 polypeptide of the first subunit and the IL-12 p40 polypeptide of the second unit are disulfide -linked thereby forming a disulfide -linked heterodimer; wherein each release segment (RSI, RS2, RS3, and RS4) is capable of being cleaved by at least one protease that is present in a tumor; and wherein each barcode fragment (Barcode 1. Barcode2, Barcode3, and Barcode4) is releasable from the fusion protein upon digestion with a non-mammalian protease, and each barcode fragment differs in both sequence and molecular weight from all other barcode fragments or peptide fragments that are releasable from the fusion protein upon complete digestion of the fusion protein by the non-mammalian protease.

[0015] In some embodiments, each release segment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence of Table 6a.

[0016] In some embodiments, each release segment (RSI, RS2, RS3, and RS4) (i) is not capable of being cleaved by legumain in human plasma, (ii) is cleaved by legumain in human plasma at a rate that is less than 25% of the rate that RSR-2295 (EAGRSANHTPAGLTGP; SEQ ID NO:7048) is cleaved by legumain in human plasma, or (iii) comprises an amino acid sequence comprising the sequence EAGRSAXHTPAGLTGP, wherein X is any amino acid other than N (SEQ ID NO:7627).

[0017] In some embodiments, each release segment is a substrate for at least one protease of Table 5.

[0018] In some embodiments, each release segment is capable of being cleaved by uPA, STH, MMP2. MMP7, MMP9, and MMP14.

[0019] In some embodiments, each release segment is not capable of being cleaved by legumain in human plasma.

[0020] In some embodiments, each release segment is not capable of being cleaved by legumain in human blood, plasma, or serum.

[0021] In some embodiments, each release segment is not capable of being cleaved upon incubation with about InM or less legumain for about 20 hours.

[0022] In some embodiments, each release segment is cleaved by legumain in human plasma at a rate that is less than 25%, less than 15%, less than 10%, less than 5%. or less than 2.5% of the rate that RSR-2295 (EAGRSANHTPAGLTGP; SEQ ID NO: 7048) is cleaved by legumain in human plasma.

[0023] In some embodiments, each release segment comprises the amino acid sequence of RSR-3213 (EAGRSASHTPAGLTGP; SEQ ID NO:7628)

[0024] In some embodiments, each mask polypeptide is an extended length non-natural polypeptide (ELNN).

[0025] In some embodiments, each ELNN is, independently, characterized in that: at least 90% of the amino acid residues of each ELNN are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), proline (P), or any combination thereof; and each ELNN comprises at least 3 types of amino acid residues selected from the group consisting of G, A, S, T, E, and P.

[0026] In some embodiments, each ELNN, independently, comprises a plurality of non-overlapping sequence motifs that are each from 9 to 14 amino acid residues in length, wherein the plurality' of nonoverlapping sequence motifs comprises a set of non-overlapping sequence motifs, wherein each nonoverlapping sequence motif of the set of non-overlapping sequence motifs is repeated at least two times in the ELNN.

[0027] In some embodiments, the plurality of non-overlapping sequence motifs comprises at least one non-overlapping sequence motif that occurs only once within the ELNN.

[0028] In some embodiments, the non-overlapping sequence motifs comprise one of or any combination of the sequence motifs listed in Table lb.

[0029] In some embodiments, the non-overlapping sequence motifs comprise at least 2. 3, or 4 of the sequence motifs listed in Table lb.

[0030] In some embodiments, the non-overlapping sequence motifs comprise any one of or any combmation of GTSTEPSEGSAP (SEQ ID NO: 189), GTSESATPESGP (SEQ ID NO: 188), GSGPGTSESATP (SEQ ID NO:201), GSEPATSGSETP (SEQ ID NO: 187), GSPAGSPTSTEE (SEQ ID NO: 186), and GTSPSATPESGP (SEQ ID NO:202).

[0031] In some embodiments, each ELNN comprises at least 4 types of amino acid residues selected from the group consisting of G, A, S, T, E, and P.

[0032] In some embodiments, the amino acid residues of each ELNN consists of A, E, G. S, P, and / or T.

[0033] In some embodiments, each ELNN, independently, has a length of least 100 amino acid residues, at least 200 amino acid residues, or at least 250 amino acid residues.

[0034] In some embodiments, each ELNN, independently, has a length of 100 to 1000 amino acid residues, 100 to 500 amino acid residues, 200 to 100 amino acid residues, 200 to 500 amino acid residues, 200 to 300 amino acid residues, 250 to 1000 amino acid residues, 250 to 500 amino acid residues, or 250 to 300 amino acid residues.

[0035] In some embodiments, each ELNN has a length of between 250 and 300 amino acid residues.

[0036] In some embodiments, each ELNN has a length of 287 or 288 amino acid residues.

[0037] In some embodiments, each ELNN comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence listed in Table 3a or 3b.

[0038] In some embodiments, the first polypeptide mask is a first ELNN (ELNN1), and wherein ELNN1 comprises an amino sequence that has at least 85%, 90%, 91%. 92%, 93%, 94%, 95%. 96%, 97%, 98%, or 99% identity, or 100% identity to:ASGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSESATPESGPGSEPATSGSETPGT SESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGT SESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE GSAPGSEPATSGSETPGTSESAT (SEQ ID NO: 8026).

[0039] In some embodiments, the second polypeptide mask is a second ELNN (ELNN2), and wherein ELNN2 comprises an amino sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, or 99% identity, or 100% identity to:ATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET PGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESG PGTSASATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE PSEGSAPGSEPATSGSETPGEPEA (SEQ ID NO: 8028).

[0040] In some embodiments, the third polypeptide mask is a third ELNN (ELNN3), and wherein ELNN3 comprises an amino sequence that has at least 85%, 90%, 91%. 92%, 93%, 94%, 95%. 96%, 97%, 98%, or 99% identity, or 100% identity to:ASGPGTSTEPSEGSAPGTSESATPESGPGSGPATSESATPGTSESATPESGPGSEPATSGSETPGT SESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGT SESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE GSAPGSEPATSGSETPGTSESAT (SEQ ID NO: 8023).

[0041] In some embodiments, the fourth polypeptide mask is a fourth ELNN (ELNN4), and wherein ELNN4 comprises an amino sequence that has at least 85%, 90%, 91%. 92%, 93%, 94%, 95%. 96%, 97%, 98%, or 99% identity', or 100% identity to:ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSA PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTE PSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTE EGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSPSATPESGPGSEPA TSGSETPGSEPATSGSETPGSPAG (SEQ ID NO: 8025).

[0042] In some embodiments, the IL-12 p35 polypeptide is a human IL-12 p35 polypeptide and the IL12 p40 polypeptide is a human IL-12 p40 polypeptide.

[0043] In some embodiments, the human IL-12 p35 polypeptide comprises an amino acid sequence that has at least 85%. 90%, 91%, 92%, 93%. 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity7to:RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEAC LPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLM DPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDR VMSYLNAS (SEQ ID NO: 8050).

[0044] In some embodiments, the human IL-12 p35 polypeptide comprises the amino acid sequence of:RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEAC LPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLM DPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDR VMSYLNAS (SEQ ID NO: 8050).

[0045] In some embodiments, the human IL-12 p35 polypeptide is an N195 aglycosylated variant.

[0046] In some embodiments, the human IL- 12 p35 polypeptide comprises the amino acid sequence of:RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKT STVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVE FKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIK LCILLHAFR1RAVT1DRVMSYLXAS (SEQ ID NO: 8055), wherein X is any amino acid residue other than asparagine (N).

[0047] In some embodiments, the human IL-12 p35 polypeptide comprises an amino acid sequence selected from:RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEAC LPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLM DPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDR VMSYLSSA (SEQ ID NO: 8052);RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEAC LPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLM DPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDR VMSYLQAS (SEQ ID NO: 8053); andRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLM DPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDR VMSYLDAS (SEQ ID NO: 8054).

[0048] In some embodiments, the human IL-12 p40 polypeptide comprises an amino acid sequence that has at least 85%. 90%, 91%, 92%, 93%. 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity7to:IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFG DAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWW LTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPI EVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSL TFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 8051).

[0049] In some embodiments, the human IL-12 p40 polypeptide comprises the amino acid sequence of:IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFG DAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWW LTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPI EVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSL TFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 8051).

[0050] In some embodiments, the IL-12 p35 polypeptide is a mouse IL-12 p35 polypeptide and the IL-12 p40 polypeptide is a mouse IL-12 p40 polypeptide.

[0051] In some embodiments, each linker further comprises a spacer.

[0052] In some embodiments, each spacer is positioned between a release segment and a cytokine, an IL- 12 p35 polypeptide, or an IL- 12 p40 polypeptide.

[0053] In some embodiments, each spacer is, independently, characterized in that: at least 90% of the amino acid residues of each spacer are selected from gly cine (G), alanine (A), serine (S), threonine (T), glutamate (E), proline (P), or any combination thereof; and each spacer comprises at least 3 ty pes of amino acid residues selected from the group consisting of G, A, S, T, E, and P.

[0054] In some embodiments, each spacer is, independently. 5 to 14 amino acid residues in length.10055| In some embodiments, each spacer comprises at least 4 types of amino acid residues selected from the group consisting of G. A, S. T, E, and P.

[0056] In some embodiments, the amino acid residues of each spacer comprise only amino acid residues selected from G, A, S, T, E, and / or P.

[0057] In some embodiments, each spacer is cleavable by a non-mammalian protease.

[0058] In some embodiments, the non-mammalian protease is Glu-C.

[0059] In some embodiments, each spacer, independently, comprises an amino acid sequence having at least 85%. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table C.

[0060] In some embodiments, each spacer, independently, comprises an amino acid sequence that has at least 85%. 90%, 91%, 92%, 93%. 94%, 95%, 96%, 97%. 98%, or 99% identity, or 100% identity to TSESATPES (SEQ ID NO:98). GTATPESGPG (SEQ ID NO:97), GTPESATPES (SEQ ID NO:99), or GTATPESAP (SEQ ID NO: 100).

[0061] In some embodiments, no spacer shares 100% sequence identity with any other spacer.

[0062] In some embodiments, the fusion protein does not comprise a His-tag, wherein a His-tag is a polypeptide sequence comprising an uninterrupted chain of between 5 and 12 histidine residues.

[0063] In some embodiments, each barcode fragment is releasable from the fusion protein upon digestion with a non-mammalian protease and the non-mammalian protease is Glu-C.

[0064] In some embodiments, none of the barcode fragments include any of the following: the N- terminal amino acid of the first polypeptide subunit, the C-terminal acid of the first polypeptide subunit, the N-terminal amino acid of the second polypeptide subunit, and the C-terminal amino acid of the polypeptide subunit.

[0065] In some embodiments, the entirety of each barcode fragment is 5 to 100, 5 to 50, 10 to 100, or 10 to 50 amino acid residues from any of the following: the N-terminal amino acid of the first polypeptide subunit, the C-terminal acid of the first polypeptide subunit, the N-terminal amino acid of the second polypeptide subunit, and the C-terminal amino acid of the polypeptide subunit.

[0066] In some embodiments, each barcode fragment is, independently, at least 4 amino acid residues in length.

[0067] In some embodiments, each barcode fragment is, independently, 4 to 20, 5 to 15, 6 to 12. 7 to 10, 8 to 13, 10 to 13, or 11 to 12 amino acid residues in length.

[0068] In some embodiments, each barcode fragment comprises a glutamate at the C-terminus thereof.

[0069] In some embodiments, each barcode fragment does not include a second glutamate at a position other than the C-terminus of the barcode fragment unless the second glutamate is immediately followed by a proline.

[0070] In some embodiments, the amino acid residue that is directly N-terminal to each barcode fragment in the primary amino acid sequence of the fusion protein is a glutamate.

[0071] In some embodiments, the glutamate that is directly N-terminal to each barcode fragment is not immediately adjacent to another glutamate.

[0072] In some embodiments, none of the barcode fragments includes a glutamate that is immediately adjacent to another glutamate, if present, in the mask polypeptide that contains the barcode fragment.

[0073] In some embodiments, each barcode fragment is, independently, selected from Table 2 or Table 3 a.

[0074] In some embodiments, each barcode fragment comprises only amino acid residues selected from A, E, G, S, P, and T.

[0075] In some embodiments, each barcode fragment comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%. 94%, 95%, 96%, 97%. 98%, or 99% identity, or 100% identity, to SGPGSGPGTSE (SEQ ID NO:78), SGPGTSASATPE (SEQ ID NO:1029), SGPGSGPATSE (SEQ ID NO: 1028), or SGPGTSPSATPE (SEQ ID NO:79).

[0076] In some embodiments, each barcode fragment is selected from the following four amino acid sequences: SGPGSGPGTSE (SEQ ID NO:78), SGPGTSASATPE (SEQ ID NO: 1029), SGPGSGPATSE (SEQ ID NO: 1028). or SGPGTSPSATPE (SEQ ID NO:79).

[0077] In some embodiments, the first barcode fragment (Barcodel) comprises the amino acid sequence of SGPGSGPGTSE (SEQ ID NO:78).

[0078] In some embodiments, the second barcode fragment (Barcodc2) comprises the amino acid sequence of SGPGTSASATPE (SEQ ID NO: 1029).

[0079] In some embodiments, the third barcode fragment (Barcode3) comprises the amino acid sequence of SGPGSGPATSE (SEQ ID NO: 1028).

[0080] In some embodiments, the fourth barcode fragment (Barcode4) comprises the amino acid sequence of SGPGTSPSATPE (SEQ ID NO:79).

[0081] In some embodiments, the fusion protein comprises a Glu-C cleavage site comprising one of the following amino acid sequences: ATPESGPG (SEQ ID NO:2223), SGSETPGT (SEQ ID NO:2242), and GTSESATP (SEQ ID NO:204).

[0082] In some embodiments, the fusion protein comprises at least one of the following amino acid sequences: SGPE.SGPGXnSGPE.SGPG (SEQ ID N0:2001), SGPE.SGPGXnATPE.SGPG (SEQ ID N0:2002), SGPE.SGPGXnGTSE.SATP (SEQ ID N0:2003), SGPE.SGPGXnTTPE.SGPG (SEQ ID N0:2004), SGPE.SGPGXnSTPE.SGPG (SEQ ID N0:2005), SGPE.SGPGXnGTPE.SGPG (SEQ IDN0:2006), SGPE.SGPGXnGTPE.TPGS (SEQ ID N0:2007), SGPE.SGPGXnGTPE.TPGS (SEQ ID N0:2007), SGPE.SGPGXnSGSE.TGTP (SEQ ID NO:2008), SGPE.SGPGXnGTPE.GSAP (SEQ ID N0:2009), SGPE.SGPGXnEPSE.SATP (SEQ ID N0:2010), ATPE.SGPGXnSGPE.SGPG (SEQ ID NO:2011), ATPE.SGPGXiiATPE.SGPG (SEQ ID NO:2012), ATPE.SGPGXnGTSE.SATP (SEQ ID NO:2013), ATPE.SGPGXnATSE.SATP (SEQ ID NO:2014), ATPE.SGPGXnTTPE.SGPG (SEQ ID NO:2015), ATPE.SGPGXnSTPE.SGPG (SEQ ID NO:2016), ATPE.SGPGXnGTPE.SGPG (SEQ ID NO:2017), ATPE.SGPGXnGTPE.TPGS (SEQ ID NO:2018), ATPE.SGPGXnSGSE.TGTP (SEQ ID NO:2019), ATPE.SGPGXnGTPE.GSAP (SEQ ID N0:2020), ATPE.SGPGXnEPSE.SATP (SEQ ID NO:2021), GTSE.SATPXnSGPE.SGPG (SEQ ID NO:2022), GTSE.SATPXnATPE.SGPG (SEQ ID NO:2023), GTSE.SATPXnGTSE.SATP (SEQ ID NO:2024), GTSE.SATPXnTTPE.SGPG (SEQ ID NO:2025), GTSE.SATPXnSTPE.SGPG (SEQ ID NO:2026), GTSE.SATPXnGTPE.SGPG (SEQ ID NO:2027), GTSE.SATPXnGTPE.TPGS (SEQ ID NO:2028), GTSE.SATPXnSGSE.TGTP (SEQ ID NO:2029), GTSE.SATPXnGTPE.GSAP (SEQ ID N0:2030), GTSE.SATPXnEPSE.SATP (SEQ ID NO:2031), TTPE.SGPGXnSGPE.SGPG (SEQ ID NO:2032). TTPE.SGPGXnATPE.SGPG (SEQ ID NO:2033). TTPE.SGPGXnGTSE.SATP (SEQ ID NO:2034). TTPE.SGPGXnTTPE.SGPG (SEQ ID NO:2035). TTPE.SGPGXnSTPE.SGPG (SEQ ID NO:2036);TTPE.SGPGXnGTPE.SGPG (SEQ ID NO:2037). TTPE.SGPGXnGTPE.TPGS (SEQ ID NO:2038). TTPE.SGPGXnSGSE.TGTP (SEQ ID NO:2039). TTPE.SGPGXnGTPE.GSAP (SEQ ID N0:2040). TTPE.SGPGXnEPSE.SATP (SEQ ID NO:2041). STPE.SGPGXnSGPE.SGPG (SEQ ID NO:2042). STPE.SGPGXnATPE.SGPG (SEQ ID NO:2043), STPE.SGPGXnGTSE.SATP (SEQ ID NO:2044), STPE.SGPGXnTTPE.SGPG (SEQ ID NO:2045), STPE.SGPGXnSTPE.SGPG (SEQ ID NO:2046), STPE.SGPGXnGTPE.SGPG (SEQ ID NO:2047), STPE.SGPGXnGTPE.TPGS (SEQ ID NO:2048), STPE.SGPGXnSGSE.TGTP (SEQ ID NO:2049), STPE.SGPGXnGTPE.GSAP (SEQ ID NO:2050), STPE.SGPGXnEPSE.SATP (SEQ ID NO:2051), GTPE.SGPGXnSGPE.SGPG (SEQ ID NO:2052), GTPE.SGPGXnATPE.SGPG (SEQ ID NO:2053), GTPE.SGPGXnGTSE.SATP (SEQ ID NO:2054), GTPE.SGPGXnTTPE.SGPG (SEQ ID NO:2055), GTPE.SGPGXnSTPE.SGPG (SEQ ID NO:2056), GTPE.SGPGXnGTPE.SGPG (SEQ ID NO:2057), GTPE.SGPGXnGTPE.TPGS (SEQ ID NO:2058), GTPE.SGPGXnSGSE.TGTP (SEQ ID NO:2059), GTPE.SGPGXnGTPE.GSAP (SEQ ID N0:2060), GTPE.SGPGXnEPSE.SATP (SEQ ID NO:2061), GTPE.TPGSXnSGPE.SGPG (SEQ ID NO:2062), GTPE.TPGSXnATPE.SGPG (SEQ ID NO:2063), GTPE.TPGSXnGTSE.SATP (SEQ ID NO:2064), GTPE.TPGSXnTTPE.SGPG (SEQ ID NO:2065), GTPE.TPGSXnSTPE.SGPG (SEQ ID NO:2066), GTPE.TPGSXnGTPE.SGPG (SEQ ID NO:2067), GTPE.TPGSXnGTPE.TPGS (SEQ ID NO:2068), GTPE.TPGSXnSGSE.TGTP (SEQ ID NO:2069), GTPE.TPGSXnGTPE.GSAP (SEQ ID N0:2070), GTPE.TPGSXnEPSE.SATP (SEQ ID NO:2071), SGSE.TGTPXnSGPE.SGPG (SEQ ID NO:2072), SGSE.TGTPXnATPE.SGPG (SEQ ID NO:2073), SGSE.TGTPXnGTSE.SATP (SEQ ID NO:2074), SGSE.TGTPXnTTPE.SGPG (SEQ ID NO:2075), SGSE.TGTPXnSTPE.SGPG (SEQ ID NO:2076), SGSE.TGTPXnGTPE.SGPG (SEQ ID NQ:2077), SGSE.TGTPXnGTPE.TPGS (SEQ ID NQ:2078), SGSE.TGTPXnSGSE.TGTP (SEQ IDNO:2079), SGSE.TGTPXnGTPE.GSAP (SEQ ID NO:2080), SGSE.TGTPXnEPSE.SATP (SEQ ID NO:2081), GTPE.GSAPXnSGPE.SGPG (SEQ ID NO:2082), GTPE.GSAPXnATPE.SGPG (SEQ ID NO:2083), GTPE.GSAPXnGTSE.SATP (SEQ ID NO:2084), GTPE.GSAPXnTTPE.SGPG (SEQ ID NO:2085), GTPE.GSAPXnSTPE.SGPG (SEQ ID NO:2086), GTPE.GSAPXnGTPE.SGPG (SEQ ID NO:2087), GTPE.GSAPXnGTPE.TPGS (SEQ ID NO:2088), GTPE.GSAPXnSGSE.TGTP (SEQ ID NO:2089), GTPE.GSAPXnGTPE.GSAP (SEQ ID N0:2090), GTPE.GSAPXnEPSE.SATP (SEQ ID NO:2091), EPSE.SATPXnSGPE.SGPG (SEQ ID NO:2092), EPSE.SATPXnATPE.SGPG (SEQ ID NO:2093), EPSE.SATPXnGTSE.SATP (SEQ ID NO:2094). EPSE.SATPXnTTPE.SGPG (SEQ ID NO:2095), EPSE.SATPXnSTPE.SGPG (SEQ ID NO:2096). EPSE.SATPXnGTPE.SGPG (SEQ ID NO:2097), EPSE.SATPXnGTPE.TPGS (SEQ ID NO:2098). EPSE.SATPXnSGSE.TGTP (SEQ ID NO:2099), EPSE.SATPXnGTPE.GSAP (SEQ ID N0:2100), or EPSE.SATPXnEPSE.SATP (SEQ ID NO:21()1), wherein each is a Glu-C cleavage site and n is any integer from 0 to 50.

[0083] In some embodiments, the fusion protein comprises at least one of the following amino acid sequences: ATPE.SGPGXnATPE.SGPG (SEQ ID NO:2102), ATPE.SGPGXnGTSE.SATP (SEQ ID NO:2103), ATPE.SGPGXnATSE.SATP (SEQ ID NO:2104), GPE.SGPGXnATPE.SGPG (SEQ ID NO:2116), ATPE.SGPGXnGTSE.SATP (SEQ ID NO:2103), ATPE.SGPGXnTTPE.SGPG (SEQ ID NO:2106), ATPE.SGPGXnSTPE.SGPG (SEQ ID NO:2107), ATPE.SGPGXnATPE.SGPG (SEQ ID NO:2102), ATPE.SGPGXnGTPE.SGPG (SEQ ID NO:2108), ATPE.SGPGXnGTPE.SGPG (SEQ ID NO:2108), ATPE.SGPGXnATPE.SGPG (SEQ ID NO:2102), GTPE.SGPGXnGTPE.SGPG (SEQ ID NO:2109), GTPE.SGPGXnSTPE.SGPG (SEQ ID NO:2110), GTPE.SGPGXnTTPE.SGPG (SEQ ID NO:2111), GTPE.SGPGXnSTPE.SGPG (SEQ ID NO:2110), GTPE.TPGSXnSGSE.TGTP (SEQ ID NO:2112), GTPE.GSAPXnEPSE.SATP (SEQ ID NO:2113), ATPE.SGPGXnGTPE.SGPG (SEQ ID NO:2108). ATPE.SGPGXnGTPE.SGPG (SEQ ID NO:2108). ATPE.SGPGXnATPE.SGPG (SEQ ID NO:2102). ATPE.SGPGXnGTPE.SGPG (SEQ ID NO:2108). TTPE.SGPGXnTTPE.SGPG (SEQ ID NO: 2114). or STPE.SGPGXnSTPE.SGPG (SEQ ID NO: 2115). wherein each is a Glu-C cleavage site and n is any integer from 0 to 30.

[0084] In some embodiments, the fusion protein comprises at least one of the following amino acid sequence ATPE.SGPGXnATSE.SATP. wherein is a Glu-C cleavage site and n is any integer from O to 30 (SEQ ID NO:2104).

[0085] In some embodiments, n is any integer from 1 to 20, 5 to 15, 3 to 7, 2 to 5, or 5 to 10. In some embodiments, n is 9. In some embodiments, n is 4. In some embodiments, n is 3.

[0086] In some embodiments, Xn is PGTGTSAT (SEQ ID NO:2233), PGSGPGT (SEQ ID NO:2221), PGTTPGTT (SEQ ID NO:2241), PGTPPTST (SEQ ID NO:2235). PGTSPSAT (SEQ ID NO:2238), PGTGSAGT (SEQ ID NO:2230), PGTGGAGT (SEQ ID NO:2228). PGTSPGAT (SEQ ID NO:2237), PGTSGSGT (SEQ ID NO:2236), PGTSSAST (SEQ ID NO:2239), PGTGAGTT (SEQID NO:2227), PGTGSTST (SEQ ID NO:2232), GSEPATSG (SEQ ID NO:2224), APGTSTEP (SEQ ID NO:2222), PGTAGSGT (SEQ ID NO:2226), PGTSSGGT (SEQ ID NO:2240), PGTAGPAT (SEQ ID NO:2225), PGTPGTGT (SEQ ID NO:2234), PGTGGPTT (SEQ ID NO:2229), or PGTGSGST (SEQ ID NO:2231).

[0087] In some embodiments, Xn is TSAS (SEQ ID NO:2214), TGTS (SEQ ID NO:2211), SGP, TTPG (SEQ ID NO:2220), TPPT (SEQ ID NO:2213), TSPS (SEQ ID NO:2217), TGSA (SEQ ID NO:2208), TGGA (SEQ ID NO:2206), TSPG (SEQ ID NO:2216), TSGS (SEQ ID NO:2215), TSSA (SEQ ID NO:2218), TGAG (SEQ ID NO:2205), TGST (SEQ ID NO:2210), EPAT (SEQ ID NO:2201), GTST (SEQ ID NO:2202), TAGS (SEQ ID NO:2204), TSSG (SEQ ID NO:2219), TAGP (SEQ ID NO:2203), TPGT (SEQ ID NO:2212), TGGP (SEQ ID NO:2207), or TGSG (SEQ ID NO:2209). In some embodiments, Xn is SGP.

[0088] In some embodiments, the first polypeptide subunit comprises an amino acid sequence that has at least 85%. 90%, 91%, 92%, 93%. 94%, 95%, 96%, 97%. 98%, or 99% identity, or 100% identity , to a sequence listed in Table Al (SEQ ID NOs: 1000-1005).

[0089] In some embodiments, the first polypeptide subunit comprises an amino acid sequence that has at least 85%. 90%, 91%, 92%. 93%. 94%, 95%, 96%. 97%. 98%, or 99% identity, or 100% identity, to: ASGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSESATPESGPGSEPATSGSETPGT SESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGT SESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE GSAPGSEPATSGSETPGTSESATEAGRSASHTPAGLTGPGTPESATPESRNLPVATPDPGMFP CLHHSONLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNS RETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYOVEFKTMNAKLLMDPKROIFLDQNMLA VIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGTATPE SAPEAGRSASHTPAGLTGPATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGT SESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGT SESATPESGPGTSESATPESGPGTSASATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT STEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGEPEA (SEQ ID NO: 1000).

[0090] In some embodiments, the second polypeptide subunit comprises an amino acid sequence that has at least 85%. 90%, 91%, 92%, 93%. 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity’, to a sequence listed in Table A2 (SEQ ID NOs: 1033-1036).

[0091] In some embodiments, the second polypeptide subunit comprises an amino acid sequence that has at least 85%. 90%, 91%, 92%. 93%. 94%, 95%, 96%, 97%. 98%, or 99% identity, or 100%identity, to:ASGPGTSTEPSEGSAPGTSESATPESGPGSGPATSESATPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATEAGRSASHTPAGLTGPTSESATPESIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDOSSEVLGSGKTLTIQVKEFGDAGOYTCHKGGEVL SHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSR GSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSROVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKRE KKDRVFTDKTSATV1CRKNAS1SVRAQDRYYSSSWSEWASVPCSGTATPESGPGEAGRSASH TPAGLTGPATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSPSATP ESGPGSEPATSGSETPGSEPATSGSETPGSPAG (SEQ ID NO: 1033).

[0092] In some embodiments, the first polypeptide subunit comprises the amino acid sequence of: ASGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATEAGRSASHTPAGLTGPGTPESATPESRNLPVATPDPGMFPCLHHSONLLRAVSNMLOKAROTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNS RETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYOVEFKTMNAKLLMDPKROIFLDONMLA VIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGTATPESAPEAGRSASHTPAGLTGPATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSASATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT STEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGEPEA (SEQ ID NO: 1000). and wherein the second polypeptide subunit comprises the amino acid sequence of:ASGPGTSTEPSEGSAPGTSESATPESGPGSGPATSESATPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGT SESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE GSAPGSEPATSGSETPGTSESATEAGRSASHTPAGLTGPTSESATPESIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDOSSEVLGSGKTLTIQVKEFGDAGOYTCHKGGEVL SHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSR GSSDPOGVTCGAATLSAERVRGDNKEYEYSVECOEDSACPAAEESLPIEVMVDAVHKLKYE NYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVOVQGKSKRE KKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGTATPESGPGEAGRSASH TPAGLTGPATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGT STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSPSATP ESGPGSEPATSGSETPGSEPATSGSETPGSPAG (SEQ ID NO: 1033).

[0093] In one aspect, the disclosure provides a fusion protein comprising a first polypeptide subunit and a second polypeptide subunit, wherein the first polypeptide subunit and the second polypeptide subunit are disulfide-linked thereby forming a disulfide-linked heterodimer, wherein the first polypeptide subunit comprises an amino acid sequence having 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to:ASGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSESATPESGPGSEPATSGSETPGT SESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGT SESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE GSAPGSEPATSGSETPGTSESATEAGRSASHTPAGLTGPGTPESATPESRNLPVATPDPGMFPC LHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSR ETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAV IDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGTATPES APEAGRSASHTPAGLTGPATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSE SATPESGPGTSESATPESGPGTSASATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTST EEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGEPEA (SEQ ID NO: 1000), and wherein the second polypeptide subunit comprises an amino acid sequence having 85%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, or 99% identity, or 100% identity, to:ASGPGTSTEPSEGSAPGTSESATPESGPGSGPATSESATPGTSESATPESGPGSEPATSGSETPGT SESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGT SESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE GSAPGSEPATSGSETPGTSESATEAGRSASHTPAGLTGPTSESATPESIWELKKDVYVVELD WYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIOVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSR GSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYE NYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVOVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGTATPESGPGEAGRSASH TPAGLTGPATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGT STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSPSATP ESGPGSEPATSGSETPGSEPATSGSETPGSPAG (SEQ ID NO: 1033).

[0094] In some embodiments, the first polypeptide subunit comprises the amino acid sequence of: ASGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSESATPESGPGSEPATSGSETPGT SESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATEAGRSASHTPAGLTGPGTPESATPESRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKAROTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYOVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGTATPESAPEAGRSASHTPAGLTGPATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSASATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT STEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGEPEA (SEQ ID NO: 1000). and wherein the second polypeptide subunit comprises the amino acid sequence of:ASGPGTSTEPSEGSAPGTSESATPESGPGSGPATSESATPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATEAGRSASHTPAGLTGPTSESATPESIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDOSSEVLGSGKTLTIQVKEFGDAGOYTCHKGGEVL SHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSR GSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKRE KKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGTATPESGPGEAGRSASH TPAGLTGPATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSPSATPESGPGSEPATSGSETPGSEPATSGSETPGSPAG (SEQ ID NO: 1033).

[0095] In one aspect, the disclosure provides a pharmaceutical composition comprising the fusion disclosed herein and at least one pharmaceutically acceptable excipient.

[0096] In one aspect, the disclosure provides a polynucleotide sequence encoding the fusion protein disclosed herein.

[0097] In one aspect, the disclosure provides an expression vector comprising the polynucleotide sequence described herein.

[0098] In one aspect, the disclosure provides a host cell comprising the expression vector described herein.

[0099] In one aspect, the disclosure provides a method of producing the fusion protein described herein. In some embodiments, the method further comprises isolating the fusion protein from a host cell.

[0100] In one aspect, the disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering an effective amount of the fusion protein described herein or the pharmaceutical composition described herein to the subject.

[0101] In some embodiments, the cancer comprises a solid tumor.BRIEF DESCRIPTION OF THE DRAWINGS

[0102] FIG. 1 depicts a schematic of a protease-activatable IL- 12 fusion protein.

[0103] FIG. 2 depicts IL-12R activity assay curves of various protease-activatable cytokine fusion proteins based on the ability to bind to and activate IL-12R in HEK-Blue IL- 12 reporter cells.“X.AP####” refers to the masked fusion protein and “AP####” refers to the MMP9 unmasked or partially unmasked fusion protein.

[0104] FIG. 3 depicts IL-12R activity assay curves of various protease-activatable cytokine fusion proteins based on the ability to bind to and activate IL-12R in HEK-Blue IL-12 reporter cells.“X.AP####” refers to the masked fusion protein and “AP####” refers to the MMP9 unmasked or partially unmasked fusion protein.

[0105] FIG. 4 depicts IL-12R activity assay curves of various protease-activatable cytokine fusion proteins based on the ability to bind to and activate IL-12R in HEK-Blue IL- 12 reporter cells.“X.AP####" refers to the masked fusion protein and “AP####" refers to the MMP9 unmasked or partially umnasked fusion protein. Recombinant murine IL- 12 is used as a reference.

[0106] FIG. 5 depicts an IL-12R activity assay curve of a quadruple masked protease-activatable cytokine fusion protein, and its unmasked counterpart, based on the ability to bind to and activate IL- 12R in HEK-Blue IL-12 reporter cells. Recombinant murine IL-12 is used as a reference.

[0107] FIG. 6 depicts mouse splenocyte in vitro assay data comparing protease-activatable cytokine fusion proteins: P26M (having 4 x 210 amino acid ELNNs) to P24M (having 4 x 288 amino acid ELNNs).

[0108] FIG. 7 depicts the mean tumor volume of B16F10 tumor model mice treated with P26M (having 4 x 210 amino acid ELNNs) or P24M (having 4 x 288 amino acid ELNNs) at 3 pg QW, as compared to diluent. A scatter plot of mean tumor volume at Day 21 from the same experiment is also provided.

[0109] FIG. 8 depicts a schematic of a protease -activatable IL-12 fusion protein with barcode fragments (C-35 BC; N-35 BC; C-40 BC, andN-40 BC) incorporated into the ELNNs.

[0110] FIG. 9A depicts an alignment of the RSR-2295 and RSR-3213 amino acid sequences and proteases capable of cleaving them. FIG. 9B depicts in vitro protease digestion of protease activatable T cell engagers (paTCEs) fusion proteins employing RSR-2295 or RSR-3213. The RSR-3213 sequence is modified to substantially reduce cleavage by legumain. paTCE refers to the intact fusion protein having two ELNNs (N-tcrminal and C-tcrminal) flanking abispccific TCE core. Unmasked TCE (uTCE) refers to the unmasked TCE core. Ix-C and Ix-N refer to the C-terminal or N-terminal single-masked metabolites, respectively.

[0111] FIG. 10A and FIG. 10B depict relative plasma stability of paTCEs employing RSR-2295 or RSR-3213, measured at Day 0 and Day 7. In FIG. 10A, RSR-2295 employed the SCy5.5 fluorophore and RSR-3213 employed the Scy7.5 fluorophore. In FIG. 10B, the RSR-2295 employed the Scy7.5 fluorophore and RSR-3213 employed the Scy5.5 fluorophore. FIG. 10C depicts the observed cleavability in vivo from tumor homogenates from 3 different mouse tumor models. For each set of bar graphs (i.e., % Ix-C, % Ix-N. % uTCE), each bar from left to right represents Bl, B2, B3, B4, Al, A2, A3, A4, 43-1, 43-2, 43-3, and 43-4. B1-B4 represent 4 different mice from a first tumor model (NCI-N87). A1-A4 represent 4 different mice from a second tumor model (HT-29). 43-1 - 43-4 represent 4 different mice from a third tumor model (HT-55). FIG. 10D depicts the % of total for the 3 metabolites plus the paTCE (paTCE, Ix-N, Ix-C, and uTCE) when employing RSR-2295 or RSR- 3213.

[0112] FIG. 11 depicts relative tumor uptake of paTCEs employing RSR-2295 or RSR-3213. The plasma:tumor ratio was calculated in 3 different mouse tumor models (4 mice per tumor model).There is a “Mouse T’ for each of the 3 different tumor models, a “Mouse 2” for each of the 3 different tumor models, a “Mouse 3“ for each of the different tumor models, and a “Mouse 4“ for each of the 3 different tumor models.

[0113] FIG. 12 depicts mouse splenocyte in vitro assay data comparing protease-activatable cytokine fusion proteins: P24M (having RSR-3213) to P01M (having RSR-2295).

[0114] FIG. 13 depicts the mean tumor volume of B16F10 tumor model mice treated with P24M (having RSR-3213) or P01M (having RSR-2295) at 3 pg QW. as compared to diluent. A scatter plot of mean tumor volume at Day 21 from the same experiment is also provided.

[0115] FIG. 14 depicts a plot of IFNy induction in a mouse splenocyte assay. Masked and unmasked version of protease-activatable cytokine fusion proteins P71M (mouse surrogate for P72H) and P70M (mouse surrogate from P74H) were tested at various concentrations. Recombinant mouse IL-12 was included as reference.

[0116] FIG. 15 depicts pharmacokinetic data of masked and umnasked versions of protease- activatable cytokine fusion proteins P70M (mouse surrogate for P72H) and P71M (mouse surrogate for P74H) in C57BL / 6 mice following IV administration of each fusion protein.

[0117] FIG. 16 depicts efficacy data (as measured by tumor volume) and toxicity data (as measured by survival) in a MC38 colon carcinoma C57BL / 6 mouse model. Protease-activatable cytokine fusion proteins P70M (mouse surrogate for P72H) and P71M (mouse surrogate for P74H) in masked versions only were tested.

[0118] FIG. 17 depicts IFNy induction from PBMCs incubated with increasing concentrations of protease-activatable cytokine fusion proteins P72H or P74H, in masked and unmasked versions.DETAILED DESCRIPTION

[0119] There is a significant unmet need in cancer therapeutics for an activatable cytokine that is efficacious against cancer. The fusion proteins of the disclosure comprise a cytokine to which one or more (e.g., two, three, or four) unstructured polypeptide masks are attached. In some embodiments, these unstructured polypeptide masks sterically reduce target engagement of the cytokine to said cytokine target receptor (e.g., IL-12 being masked to reduce binding to IL-12 receptor), and also extend protein half-life. In some embodiments, the unstructured polypeptide masks are extended length non-natural polypeptides (ELNNs).

[0120] In some embodiments, the properties of ELNNs also minimize the potential for immunogenicity, as their lack of stable tertiary structures disfavors cytokine binding, and the absence of hydrophobic, aromatic, and positively charged residues that sen e as anchor residues for peptide MHC II binding reduces the potential for T cell epitopes.

[0121] In some embodiments, protease cleavage sites at the base of the ELNN or ELNNs enable proteolytic activation of fusion proteins in the tumor microenvironment, unleashing smaller, highly potent cytokines that are capable of binding to said cytokine's target receptor. In some embodiments, in healthy tissues, where protease activity is tightly regulated, fusion proteins remain predominantly inactive, thus expanding the therapeutic index compared to unmasked cytokines.

[0122] In some embodiments, in addition to localized activation, the short half-life of tire unmasked cytokines form further widens the therapeutic index while providing the potency of T-cell immunity to improve the eradication of solid tumors. In some embodiments, the release sites used in tire fusion proteins can be cleaved across a broad array of tumors by proteases that arc collectively involved in every cancer hallmark (growth; survival and death; angiogenesis; invasion and metastasis; inflammation; and immune evasion). Thus, cytokine activity of the fusion proteins is localized to tumors by exploiting the enhanced protease activity that is upregulated in all stages of cancer and tumor development but is tightly regulated in healthy tissues.TERMINOLOGY

[0123] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0124] As used in the specification and claims, the singular forms “a.” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof, unless the context clearly dictates otherw ise.

[0125] Furthermore, "and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: “A, B. and C”; “A, B. or C”; “A or C”; “A or B”; “B or C”; “A and C”; “A and B”; “B and C”; “A” (alone); “B” (alone); and “C” (alone).

[0126] It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and / or “consisting essentially of’ are also provided.

[0127] Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy' orientation. The headings provided herein are not limitations of the various aspects of the disclosure. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0128] The term “about” is used herein to mean approximately, roughly, around, or in the regions of. When the term “about” is used in conjunction yvith a numerical range, it modifies that range byextending the boundaries above and below the numerical values set forth. In general, the term “about” can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower). In some embodiments, tire term indicates deviation from the indicated numerical value by ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%. or ±0.01%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±10%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±5%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±4%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±3%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±2%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±1%. In some embodiments, "about” indicates deviation from the indicated numerical value by ±0.9%. In some embodiments, "about” indicates deviation from the indicated numerical value by ±0.8%. In some embodiments, "about” indicates deviation from the indicated numerical value by ±0.7%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.6%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.5%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.4%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.3%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.1%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.05%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.01%.

[0129] With respect to naturally occurring compounds, the term “isolated” refers to a compound (i.e. , a polypeptide or polynucleotide) that is not in its native state (e.g.. free to varying degrees from components that naturally accompany the compound in nature). No particular level of purification is required. For example, an isolated polypeptide can simply be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the disclosure, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique. “Isolate” and “isolated” may also denote a degree of separation from an original source or surrounding, depending on context.

[0130] The term “polypeptide” refers to any polymer of two or more amino acids. Thus, the terms peptide, dipeptide, tripeptide, oligopeptide, protein, amino acid chain, or any other term used to refer to a chain of two or more amino acids, is included within the definition of “polypeptide.” The term “polypeptide” also encompasses an amino acid polymer that has been modified (e.g.. by post- translational modification), for example, by disulfide bond formation, glycosylation, lipidation. acetylation, phosphorylation, or any other manipulation, such as conjugation with a labelingcomponent. Depending on context, the term “polypeptide” may also be used to refer to a protein comprising two or more polymers of two or more amino acids.

[0131] A “host cell” includes an individual cell (e.g., in culture) which that comprises an exogenous polynucleotide. Host cells may include progeny of a single host cell. The progeny may not necessarily be completely identical (in morphology or in genomic of total DNA complement) to the original parent cell due to naturally occurring or genetically engineered variation.

[0132] A “fusion” or “chimeric” polypeptide or protein comprises a first polypeptide portion linked to a second polypeptide portion with which it is not naturally linked in nature. In some embodiments, the portions may normally exist in separate proteins and are brought together in the fusion polypeptide; they may normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide; or the portions may be brought together from different sources. In some embodiments, a fusion or chimeric protein comprises tw o or more moieties that do not occur in nature e.g., are created, designed, or otherwise generated by humans, such as binding domains, masks, linkers, barcodes, and other polypeptides provided herein). A chimeric protein may be created, for example, by chemical synthesis, or by recombinant expression (e.g., comprising creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship).

[0133] “Conjugated”, “linked,” “fused,” and “fusion” may be used interchangeably herein, depending on context. These terms may refer to the covalent joining together of two more chemical (e.g., polypeptide) elements or components, by whatever means including chemical conjugation or recombinant means.

[0134] The term “cytokine” as used herein refers to any of a class of immunoregulatory proteins that are secreted by cells especially of the immune system and are immunomodulators. Cytokine polypeptides that can be used in the fusion proteins disclosed herein include, but are not limited to interleukins, such as IL-1. IL-1 alpha, IL-2, IL-3, IL-4. IL-5, IL-6, IL-7. IL-8, IL-9, IL-10, IL-11. IL- 12 (e.g., IL-12p35 and IL-12p40). IL-13, IL-14, IL-15, IL-16, IL-17. IL-18. IL-21 and IL-25, transforming growth factors, such as TGF-alpha. and TGF-beta (e.g., TGF-betal. TGF-beta2. TGF- beta3); interferons, such as interferon-alpha. interferon-beta, interferon-gamma, interferon-kappa and interferon-omega; tumor necrosis factors, such as tumor necrosis factor alpha and lymphotoxin; chemokines (e.g., C-X-C motif chemokine 10 (CXCL10), CCL19, CCL20, CCL21). and granulocyte macrophage-colony stimulating factor (GM-CS), as w ell as functional fragments thereof that retain receptor agonist activity.

[0135] The term “chemokine” refers to any of a family of small cytokines with the ability to induce directed chemotaxis in nearby responsive cells.

[0136] As used herein, the terms “activatable,” “activate,” “induce,” and “inducible” refer to the ability of a protein, i.e., a cytokine, that is part of a fusion protein, to bind its receptor and effectuate activity upon cleavage of a mask polypeptide from tire fusion protein.

[0137] As known in the art, “sequence identity” betw een two polypeptides is determined by comparing the amino acid sequence of one polypeptide to the sequence of a second polypeptide. Similarly, “sequence identity” between two polynucleotides is determined by comparing the nucleotide sequence of one polynucleotide to the sequence of a second polynucleotide. The terms “% identical”, “% identity " or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids (as applicable) which arc identical in an optimal aligmnent between the sequences to be compared. Said percentage may be purely statistical, and the differences between the tw o sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of tw o sequences are usually carried out by comparing tire sequences, after optimal alignment, with respect to a segment or “w indow of comparison”, in order to identify local regions of corresponding sequences. For example, the optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm by Neddleman and Wunsch, 1970. J. Mol. Biol. 48, 443, with the aid of the similarity' search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using the algorithms (GAP. BESTFIT, FASTA, BLAST P. BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group. 575 Science Drive, Madison. Wis.). In some embodiments, percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the United States National Center for Biotechnology Information (NCBI) website (e.g. at blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK _LOC=align2seq). In some embodiments, the algorithm parameters used for BLASTN algorithm on the NCBI w'ebsite include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match / Mismatch Scores set to 1, -2; (v) Gap Costs set to Linear; and (vi) the filter for low' complexity regions being used. In some embodiments, the algorithm parameters used for BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment. When discussed herein, whether any particular polypeptide is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to another polypeptide can be determined using methods and computer programs / software known in the art such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). BESTFIT uses the localhomology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology betw een tw o sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present disclosure, the parameters are set, of course, such that the percentage of identity is calculated over tire full-length of the reference polypeptide sequence and that gaps in homology of up to 5% of the total number of amino acids in the reference sequence are allow ed.

[0138] As used herein, the terms “mask polypeptide”, “mask”, and “masking moiety” refer to a polypeptide that is capable of reducing the binding of a cytokine to the target cytokine receptor in the context of a fusion protein (such as a chimeric polypeptide) provided herein. Exemplary mask polypeptides include, but are not limited to, the ELNN polypeptides described herein. Additional mask polypeptides include albumin, polypeptides consisting of proline, serine and alanine, coiled-coil domains, albumin binding domains, Fc domains, and binding domains with specificity to conserved regions of an antibody variable domain. Mask polypeptides are described in further detail in Lucchi et al. (ACS Cent Sci. 2021 May 26; 7(5): 724-738).

[0139] As used herein, the terms “ELNN polypeptides” and “ELNNs” are synonymous and refer to extended length polypeptides comprising non-naturally occurring, substantially non-repetitive sequences (e.g., polypeptide motifs) that arc composed mainly of small hydrophilic amino acids, with the sequence having a low degree or no secondary or tertiary structure under physiologic conditions. ELNN polypeptides include unstructured hydrophilic polypeptides comprising repeating motifs of 6 natural amino acids (G, A, P, E, S, and / or T). In some embodiments, an ELNN polypeptide comprises multiple motifs of 6 natural amino acids (G. A, P, E. S, T), wherein the motifs are the same or comprise a combination of different motifs. In some embodiments, ELNN polypeptides can confer certain desirable pharmacokinetic, physicochemical, and pharmaceutical properties when linked to proteins, including T-cell engagers as disclosed herein. Such desirable properties may include but are not limited to enhanced pharmacokinetic parameters and solubility characteristics, as well as improved therapeutic index. ELNN polypeptides are known in the art, and non-limiting descriptions relating to and examples of ELNN polypeptides known as XTEN® polypeptides are available in Schellenberger et al., (2009) Nat Biotechno 27(12): 1186-90; Brandl et al., (2020) Journal of Controlled Release 327: 186-197; and Radon et al., (2021) Advanced Functional Materials 31, 2101633 (pages 1-33), the entire contents of each of which are incorporated herein by reference.

[0140] In some embodiments, the repetitiveness of an ELNN sequence refers to the 3-mer repetitiveness and can be measured by computer programs or algorithms or by other means known in the art. In some embodiments, the 3-mer repetitiveness of an ELNN may be assessed by determining the number of occurrences of the overlapping 3-mer sequences within the polypeptide. For example, a polypeptide of 200 amino acid residues has 198 overlapping 3-amino acid sequences (3-mers), but thenumber of unique 3-mer sequences will depend on the amount of repetitiveness within the sequence. In some embodiments, the score can be generated (hereinafter “subsequence score”) that is reflective of the degree of repetitiveness of the 3-mers in the overall polypeptide sequence. In this context, “subsequence score” means the sum of occurrences of each unique 3-mer frame across a 200 consecutive amino acid sequence of the polypeptide divided by the absolute number of unique 3-mer subsequences within the 200 amino acid sequence. Examples of such subsequence scores derived from the first 200 amino acids of repetitive and non-repetitive polypeptides are presented in Example 73 of International Patent Application Publication No. WO 2010 / 091122 Al, which is incorporated by reference in its entirety.

[0141] In some embodiments, and in the context of ELNNs, a “substantially non-repetitive sequence,” refers to an ELNN sequence, wherein (1) there are few or no instances of four identical amino acids in a row in the ELNN sequence and wherein (2) the ELNN has a subsequence score (defined in the preceding paragraph herein) of 12, or 10 or less or that there is not a pattern in the order, from N- to C-tenninus, of the sequence motifs that constitute the polypeptide sequence.

[0142] A “vector” is a nucleic acid molecule that transfers an inserted nucleic acid molecule into and / or between host cells. In some embodiments, a vector self-replicates in an appropriate host. The term includes vectors that function primarily for insertion of DNA or RNA into a cell, replication of vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for transcription and / or translation of the DNA or RNA. Also included are vectors that provide more than one of the above functions. An “expression vector” is a polynucleotide which, when introduced into an appropriate host cell, can be used for the transcription of mRNA that is translated into a polypeptide(s). In some embodiments, an “expression system” is a suitable host cell comprising an expression vector that can function to yield a desired expression product.

[0143] The terms “treatment” or “treating,” and “ameliorating” may be used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit. By “therapeutic benefit” is meant eradication or amelioration of the underly ing disorder being treated. In some embodiments, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disease condition such that an improvement is observed in the subject, notw ithstanding that the subject may still be afflicted with the underlying disorder. In some embodiments, a therapeutic benefit comprises slowing or halting the growth of one or more tumors. In some embodiments, a therapeutic benefit comprises reducing the size of one or more tumors. In some embodiments, a therapeutic benefit comprises eradicating one or more tumors from a subject. In some embodiments, a therapeutic benefit comprises effecting the death of cancer cells.

[0144] As used herein, the term “therapeutically effective amount" refers to an amount of a biologically active agent (such as a fusion protein provided herein, e.g., as part of a pharmaceutical composition), that is capable of having any detectable, beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition when administered in one or repeated doses to a subject. Such effect need not be absolute to be beneficial. The disease condition can refer to a disorder or a disease, e.g.. cancer or a symptom of cancer.CYTOKINES, CLEAVAGE SEQUENCES, BARCODE FRAGMENTS, AND FUSION POLYPEPTIDES

[0145] The present disclosure provides, inter alia, new and useful fusion proteins comprising (i) one or more mask polypeptides (such as ELNNs), (ii) a cytokine (e.g., IL-12) linked to the mask polypeptide(s), and (iii) one or more protease-cleavable release segments (RS), wherein an RS is positioned between the mask polypeptide(s) and the cytokine.

[0146] In some embodiments, the fusion polypeptide comprises a first ELNN (such as an ELNN described herein). In some embodiments, the polypeptide further comprises a second ELNN (such as an ELNN described herein). In some embodiments, the polypeptide comprises an ELNN at or near its N-tenninus (an “N-terminal ELNN"). In some embodiments, the polypeptide comprises an ELNN at or near its C-tenninus (a “C-terminal ELNN”). In some embodiments, the polypeptide comprises both an N-terminal ELNN and a C-terminal ELNN.

[0147] In some embodiments, a fusion polypeptide comprises a cytokine and a first ELNN is attached to the N-terminus of the cytokine by a first RS and a second ELNN is attached to the C- terminus of the cytokine by a second RS. In some embodiments, each RS is cleavable by a protease mentioned herein. In some embodiments, each RS comprises an RS sequence disclosed herein. In some embodiments, the fusion polypeptide is a cytokine fusion.

[0148] Included herein are polypeptide sequences that may be used. e.g.. to link one polypeptide moiety to another within a fusion protein. For example, useful linkers are provided that are cleaved by multiple proteases but not legumain. In some embodiments, such linkers may be used outside the context of cytokines such as those described herein.

[0149] In some embodiments, a fusion can comprise one or more barcode fragments (e.g., as described herein) releasable (e.g.. configured to be released) the fusion polypeptide upon cleavage or digestion of the fusion polypeptide by a protease. In some embodiments, the protease is a nonmammalian protease. In some embodiments, each barcode fragment differs in sequence and molecular weight from all other peptide fragments (including all other barcode fragments if present) that are releasable from the polypeptide upon complete digestion of the polypeptide by the protease, thereby making it unique and making its presence detectable through techniques such as mass spectrometry'.CYTOKINES

[0150] In general, the therapeutic use of cytokines is strongly limited by their systemic toxicity. TNF, for example, was originally discovered for its capacity of inducing the hemorrhagic necrosis of some tumors, and for its in vitro cytotoxic effect on different tumoral lines, but it subsequently proved to have strong pro -inflammatory activity, which can, in case of overproduction conditions, dangerously affect the human body. As the systemic toxicity is a fundamental problem with the use of pharmacologically active amounts of cytokines in humans, novel derivatives and therapeutic strategies are now under evaluation, aimed at reducing the toxic effects of this class of biological effectors while keeping their therapeutic efficacy.

[0151] One cytokine for use in production of the fusion proteins of the disclosure is Interleukin- 12 (IL-12). IL-12 is a disulfide-linked heterodimer of two separately encoded subunits (p35 and p40), which are linked covalently to give rise to the so-called bioactive heterodimeric (p70) molecule. Apart from forming heterodimers (IL- 12 and IL-23), the p40 subunit is also secreted as a monomer (p40) and a homodimer (p402). It is known in the art that synthesis of the heterodimer as a single chain with a linker connecting the p35 to the p40 subunit preserves the full biological activity of the heterodimer. IL-12 plays a critical role in the early inflammatory response to infection and in the generation of Thl cells, which favor cell-mediated immunity'. It has been found that overproduction of IL-12 can be dangerous to the host because it is involved in the pathogenesis of a number of autoimmune inflammatory diseases (e.g., MS. arthritis, type 1 diabetes).

[0152] IL- 12 is a pleiotropic cytokine, that creates an interconnection between the iimate and adaptive immunity. IL-12 was first described as a factor secreted from PMA-induced EBV- transfonned B-cell lines. Based on its actions, IL-12 has been designated as cytotoxic lymphocyte maturation factor and natural killer cell stimulatory factor. Due to bridging the iimate and adaptive immunity and potently stimulating the production of IFNy. a cytokine coordinating natural mechanisms of anticancer defense, IL- 12 seemed ideal candidate for tumor immunotherapy in humans. However, severe side effects associated with systemic administration of IL-12 in clinical investigations and the very narrow therapeutic index of this cytokine markedly hampered the use of this cytokine in cancer patients. Approaches to IL-12 therapy in which delivery of the cytokine is tumor-targeted, which may diminish some of the previous issues with IL- 12 therapy, are currently in clinical trials for cancers.|0153| The IL-12 receptor (IL-12R) is a heterodimeric complex consisting of 1L-12RP1 and 1L- 12RP2 chains expressed on the surface of activated T-cells and natural killer cells. The IL-12RP1 chain binds to the IL-12p40 subunit, whereas IL-12p35 in association with IL-12RP2 confers an intracellular signaling ability. Signal transduction through IL-12R induces phosphorylation of lanus kinase (Iak2) and tyrosine kinase (Tyk2), that phosphorylate and activate signal transducer andactivator of transcription (STAT)l, STAT3, STAT4, and STATS. The specific cellular effects of IL- 12 are due mainly to activation of STAT4. IL-12 induces natural killer and T-cells to produce cytokines, in particular interferon (IFN)y, that mediate many of the proinflammatory activities of IL- 12, including CD4+ T-cell differentiation toward the Thl phenotype.

[0154] Disclosed herein are compositions and methods comprising conditionally active IL-12 and other cytokines designed to address the risks associated with conventional cytokine therapy and provide much needed immunomodulatory therapeutics.

[0155] The amino acid sequence for exemplary IL-12 p35 and p40 subunits are provided in Table l .Table la. IL-12 amino acid sequences

[0156] In some embodiments, the fusion protein disclosed herein comprises a first polypeptide subunit comprising a wild-type p35 amino acid sequence, such as SEQ ID NO: 8050 and a second polypeptide subunit comprising a wild-type p40 amino acid sequence, such as SEQ ID NO: 8051.|0157| In some embodiments, the fusion protein disclosed herein comprises a first polypeptide subunit comprising a p35 amino acid sequence that has at least 85%, 90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%, 98%. or 99% identity, or 100% identity to SEQ ID NO: 8050. and a second polypeptide subunit comprising a p40 amino acid sequence that has at least 85%. 90%, 91%, 92%, 93%, 94%. 95%. 96%, 97%, 98%. or 99% identity, or 100% identity to SEQ ID NO: 8051.

[0158] In some embodiments, the fusion protein disclosed herein comprises a first polypeptide subunit comprising a wild-type p35 amino acid sequence, such as SEQ ID NO: 8056 and a second polypeptide subunit comprising a wild-type p40 amino acid sequence, such as SEQ ID NO: 8057.

[0159] In some embodiments, the fusion protein disclosed herein comprises a first polypeptide subunit comprising a p35 amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to SEQ ID NO: 8056, and a second polypeptide subunit comprising a p40 amino acid sequence that has at least 85%, 90%, 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to SEQ ID NO: 8057.

[0160] In some embodiments, the fusion protein disclosed herein comprises a first polypeptide subunit comprising an aglycosylation mutant variant p35 amino acid sequence. The aglycosylation mutant variant p35 amino acid sequence comprises at least one amino acid substitution of the NAS amino acid sequence located at the C-tenninus of the wild-ty pe p35 amino acid sequence. In some embodiments, the fusion protein disclosed herein comprises a first polypeptide subunit comprising an aglycosylation mutant variant p35 amino acid sequence that has at least 85%, 90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity', or 100% identity to any one of SEQ ID NOs: 8052- 8055. In some embodiments, the fusion protein disclosed herein comprises a first polypeptide subunit comprising an aglycosylation mutant variant p35 amino acid sequence of any one of SEQ ID NOs: 8052-8055, and a second polypeptide subunit comprising a wild-type p40 amino acid sequence, such as SEQ ID NO: 8051.

[0161] In some embodiments, the cytokine polypeptide in the fusion protein is Intcrlcukin-2 (IL-2). IL-2 exerts both stimulatory' and regulatory functions in the immune system and is, along with other members of the common ' -chain cytokine family, central to immune homeostasis. IL-2 mediates its action by binding to IL-2 receptors (IL-2R). consisting of either trimeric receptors made of IL- 2Roc(CD25), IL-2RP (CD122). and IL-2R-y (y-c. CD132) chains or dimeric p y IL-2Rs.

[0162] In some embodiments, the cytokine polypeptide in the fusion protein is Interleukin- 15 (IL- 15). IL-15, a member of the 4-alpha-helix bundle family of cytokines, is an immunomodulator for the treatment of cancer. IL-15 mediates its action by binding to IL-15Ra, which is expressed on antigen- presenting dendritic cells, monocytes and macrophages.

[0163] In some embodiments, the cy tokine polypeptide in the fusion protein is Interleukin-7 (IL-7). IL-7, also of the IL-2 / IL-15 family, is expressed by stromal cells, epithelial cells, endothelial cells, fibroblasts, smooth muscle cells and keratinocytes, and following activation, by dendritic cells (Alpdogan et al., 2005).

[0164] While the present application is exemplified using IL- 12 as the exemplary cytokine, those of skill in the art will understand that the teachings provided herein may readily be adapted for and describe and enable the use of fusion proteins formed from other cytokines, fragments and muteins, such as IL-2, IL-7, IL-12, IL-15, IL-18, IL-21 IL-23. IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha, lymphotoxin, TGF-betal, TGFbeta2, TGFbeta3, GM-CSF, CXCL10, CCL19, CCL20, CCL21 and functional fragments or mutcins of any of the foregoing. A description of cytokines that may be incorporated into the fusion proteins disclosed herein is provided in WO2021262985A1, incorporated herein by reference.

[0165] Various elements ensure the delivery and activity of the cytokine in the fusion proteins of the invention preferentially at the site of desired cytokine activity and to severely limit systemic exposure to the cytokine via linkage to an ELNN described herein, which allows serum half-life extension for the cytokine of interest. In this serum half-life extension strategy, the fusion protein may circulate for extended times (preferentially 1-2 or more weeks) but the activated version from which the ELNN sequence has been cleaved has the ty pical serum half-life of the cytokine.

[0166] By comparison to a fusion protein, the serum half-life of the underlying cytokine administered intravenously is only about 10 minutes due to distribution into the total body extracellular space. Subsequently, the cytokine is metabolized by the kidneys with a half-life of 2.5 hours.

[0167] In some embodiments of this invention, the fusion protein comprises a release segment which is cleaved at the site of action (e.g.. by inflammation-specific or tumor-specific proteases) thereby releasing the cytokine’s full activity' at the desired site and also separating it from the half-life extension of the uncleaved version. In such embodiments, the fully active and free cytokine would have very different pharmacokinetic (pK) properties— a half-life of hours instead of weeks. In addition, exposure to active cytokine is limited to the site of desired cytokine activity (e.g.. an inflammatory site or the tumor microenvironment) and systemic exposure to active cytokine, and associated toxicity’ and side effects, are reduced.

[0168] Creating a fusion protein from cytokines is an elegant mechanism by which to improve the use of cytokines, as immuno stimulatory agents, for example for treating cancer. For example, in this aspect, the pharmacokinetics and / or pharmacodynamics of the cytokine (e.g., IL -2, IL-7, IL-12, IL-15, IL-18, IL-21 IL-23, IFN-alpha, IFN-beta and IFN-gamma, TNF-alpha, lymphotoxin, TGF-betal. TGF-beta2, TGF-beta3 GM-CSF, CXCL10. CCL19, CCL20, and CCL21 can be tailored to maximally activate effector cells (e.g., effect T cells, NK cells) and / or cytotoxic immune response promoting cells (e g., induce dendritic cell maturation) at a site of desired activity, such as in a tumor or tumor microenvironment, but preferably not systemically.

[0169] Thus, provided herein arc pharmaceutical compositions comprising fusion proteins that arc comprised of at least one cytokine polypeptide, such as interleukins (e.g., IL-2, IL-7, IL-12, IL-15, IL- 18, IL-21, IL-23), interferons (IFNs, including IFN-alpha, IFN-beta and IFN-gamma), tumor necrosis factors (e.g.. TNF-alpha, lymphotoxin), transforming growth factors (e.g., TGF-betal, TGF-beta2, TGF-beta3). chemokines (e.g. CXCL10, CCL19, CCL20, CCL21) and granulocyte macrophagecolony stimulating factor (GM-CS) or a functional fragment or mutein of any of the foregoing.

[0170] In some embodiments, the cytokine polypeptides (including functional fragments) that are included in the fusion proteins disclosed herein are not mutated or engineered to alter the properties of die naturally occurring cytokine, including receptor binding affinity and specificity or serum half-life.EXTENDED RECOMBINANT POLYPEPTIDES (ELNNs)Chain Length and Amino Acid Composition

[0171] In some embodiments, an ELNN comprises at least 100, or at least 150 amino acids. In some embodiments, an ELNN is from 100 to 3,000, or from 150 to 3,000 amino acids in length. In some embodiments, an ELNN is from 100 to 1,000, or from 150 to 1,000 amino acids in length. In some embodiments, an ELNN is at least (about) 100, at least (about) 150. at least (about) 200, at least (about) 250, at least (about) 300, at least (about) 350, at least (about) 400, at least (about) 450, at least(about) 500, at least (about) 550, at least (about) 600, at least (about) 650, at least (about) 700, at least(about) 750, at least (about) 800, at least (about) 850, at least (about) 900, at least (about) 950, at least(about) 1,000, at least (about) 1,100, at least (about) 1,200, at least (about) 1,300, at least (about)1,400, at least (about) 1,500, at least (about) 1,600, at least (about) 1,700, at least (about) 1,800, at least (about) 1,900, or at least (about) 2,000 amino acids in length. In some embodiments, an ELNN is at most (about) 100, at most (about) 150, at most (about) 200, at most (about) 250, at most (about) 300, at most (about) 350, at most (about) 400, at most (about) 450, at most (about) 500, at most (about) 550, at most (about) 600, at most (about) 650, at most (about) 700, at most (about) 750, at most (about) 800, at most (about) 850, at most (about) 900, at most (about) 950. at most (about) 1.000, at most (about) 1,100, at most (about) 1,200, at most (about) 1,300, at most (about) 1.400. at most (about) 1,500, at most (about) 1.600, at most (about) 1,700. at most (about) 1,800, at most (about) 1,900. or at most (about) 2,000 amino acids in length. In some embodiments, an ELNN has(about) 100, (about) 150, (about) 200, (about) 250, (about) 300, (about) 350, (about) 400, (about) 450, (about) 500, (about) 550, (about) 600, (about) 650, (about) 700, (about) 750, (about) 800, (about) 850, (about) 900, (about) 950, (about) 1,000, (about) 1,100, (about) 1,200, (about) 1,300, (about) 1,400, (about) 1,500, (about) 1,600, (about) 1,700, (about) 1,800, (about) 1,900, or (about) 2,000 amino acids in length, or of a range between any two of the foregoing. In some embodiments, at least 90% of the amino acid residues of the ELNN are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) or proline (P). In some embodiments, at least 91%, 92%. 93%, 94%, 95%, 96%. 97%, 98%, 99% or 100% of the amino acid residues of the ELNN are glycine (G), alanine (A), serine (S). threonine (T), glutamate (E) or proline (P). In some embodiments, an ELNN comprises at least 3 different types of amino acids selected from the group consisting of G, A, S. T, E, and P. In some embodiments, an ELNN comprises at least 4 different types of amino acids selected from the group consisting of G, A, S, T, E. and P. In some embodiments, an ELNN comprises at least 5 different types of amino acids selected from the group consisting of G, A. S. T, E, and P. In some embodiments, an ELNN consists of amino acids selected from the group consisting of G. A, S. T. E, and P. In some embodiments, an ELNN comprises G, A. S, T, E. or P amino acids. In some embodiments, an ELNN (e.g.. ELNN1, ELNN2. etc.) is characterized in that: (i) it comprises at least 100. or at least 150 amino acids; (ii) at least 90% of the amino acid residues of the ELNN are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) or proline (P); and (iii) it comprises at least 4 different ty pes of the amino acids from G, A, S, T, E. or P. As used herein, the term “glutamate” is a synonym for “glutamic acid,” and refers to the glutamic acid residue whether or not the side-chain carboxyl is deprotonated. In some embodiments, the ELNN-containing fusion polypeptide comprises a first ELNN and a second ELNN. In some embodiments, the sum of the total number of amino acids in the first ELNN and the total number of amino acids in the second ELNN is at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, or at least 800 amino acids.Non-Overlapping Sequence Motif

[0172] In some embodiments, the ELNN comprises, or is formed from, a plurality of nonoverlapping sequence motifs. In some embodiments, at least one of the non-overlapping sequence motifs is recurring (or repeated at least two times in the ELNN). In some embodiments, the ELNN comprises at least one other non-overlapping sequence motif that is non-recurring (or found only once within the ELNN). In some embodiments, the plurality' of non-overlapping sequence motifs comprises(a) a set of (recurring) non-overlapping sequence motifs, wherein each non-overlapping sequence motif of the set of non-overlapping sequence motifs is repeated at least tw o times in the ELNN; and(b) a non-overlapping (non-rccurring) sequence motif that occurs (or is found) only once within the ELNN. In some embodiments, each non-overlapping sequence motif is from 9 to 14 (or 10 to 14, or 11 to 13) amino acids in length. In some embodiments, each non-overlapping sequence motif is 12 amino acids in length. In some embodiments, the plurality of non-overlapping sequence motifscomprises a set of non-overlapping (recurring) sequence motifs, wherein each non-overlapping sequence motif of the set of non-overlapping sequence motifs is (1) repeated at least two times in the ELNN; and (2) is between 9 and 14 amino acids in length. In some embodiments, the set of (recurring) non-overlapping sequence motifs comprises 12-mer sequence motifs identified herein by SEQ ID NOs: 179-200 and 1715-1722 in Table lb. In some embodiments, the set of (recurring) nonoverlapping sequence motifs comprise 12-mer sequence motifs identified herein by SEQ ID NOs: 186-189 in Table lb. In some embodiments, the set of (recurring) non-overlapping sequence motifs comprise at least two, at least three, or all four of 12-mer sequence motifs of SEQ ID NOs: 186-189 in Table lb. In some embodiments, an ELNN further comprises a sequence other than a 12-mer sequence motif shown in Table 1. In some embodiments, an ELNN comprises a sequence that is not in Table lb such as ASSATPESGP (SEQ ID NO:203), GSGPGTSESATP (SEQ ID NO:201), or GTSESATP (SEQ ID NO:204). In some embodiments, an ELNN comprises a sequence that is not in Table lb such as ATPESGP (SEQ ID NO:205), GTSPSATPESGP (SEQ ID NQ:202). or GTSESAGEPEA (SEQ ID NO:206). In some embodiments, an ELNN comprises a barcode sequence.Table lb. Exemplary 12-Mer Sequence Motifs for Construction of ELNNs*Dcnotcs individual motif sequences that, when used together in various permutations, results in a “family sequence”Unstructured Polypeptide Confirmation

[0173] In various embodiments, an ELNN component (or the ELNN components) of a fusion protein has an unstructured conformation under physiological conditions, regardless of the length (e g., extended length) of the polymer. For example, the ELNN is characterized by a large conformational freedom of the peptide backbone. In some embodiments, the ELNN is characterized by a lack of long-range interactions as determined by NMR. In some embodiments, the present disclosure provides ELNNs that, under physiologic conditions, resemble the structure of denatured sequences largely devoid in secondary structure. In some embodiments, the ELNNs can be substantially devoid of secondary structure under physiologic conditions. “Largely devoid,” as used in this context, means that less than 50% of the ELNN amino acid residues of the ELNN contribute to secondary structure as measured or determined by the means described herein. “Substantially devoid,” as used in this context, means that at least about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or at least about 99% of the ELNN amino acid residues of the ELNN sequence do not contribute to secondary structure, as measured or determined by the means described herein.|0174| A variety of methods have been established in the art to discern the presence or absence of secondary and tertiary structures in a given polypeptide. In some embodiments. ELNN secondary structure can be measured spectrophotometrically, e.g., by circular dichroism spectroscopy in the “far- UV” spectral region (190-250 nm). Secondary structure elements, such as alpha-helix and beta-sheet, each give rise to a characteristic shape and magnitude of CD spectra. Secondary structure can also be predicted for a polypeptide sequence via certain computer programs or algorithms, such as the well- known Chou-Fasman algorithm (Chou, P. Y., et al. (1974) Biochemistry, 13: 222-45) and the Gamier- Osguthorpe-Robson (“GOR”) algorithm (Garnier I, Gibrat JF, Robson B. (1996), GOR method for predicting protein secondary structure from amino acid sequence. Methods Enzymol 266:540-553), as described in US Patent Application Publication No. 20030228309A1 (the entire contents of which are incorporated herein by reference). For a given sequence, the algorithms can predict whether there exists some or no secondary structure at all, expressed as the total and / or percentage of residues of the sequence that form, for example, alpha-helices or beta-sheets or the percentage of residues of the sequence predicted to result in random coil formation (which lacks secondary structure).

[0175] In some embodiments, the ELNNs used in a fusion protein composition can have an alphahelix percentage ranging from 0% to less than about 5% as determined by a Chou-Fasman algorithm.In some embodiments, the ELNNs of the fusion protein compositions can have a beta-sheet percentage ranging from 0% to less than about 5% as determined by a Chou-Fasman algorithm. In some embodiments, the ELNNs of the fusion protein compositions can have an alpha-helix percentage ranging from 0% to less than about 5% and a beta-sheet percentage ranging from 0% to less than about 5% as determined by a Chou-Fasman algorithm. In some embodiments, the ELNNs of the fusion protein compositions will have an alpha-helix percentage less than about 2% and a betasheet percentage less than about 2%. In some embodiments, the ELNNs of the fusion protein compositions can have a high degree of random coil percentage, as determined by a GOR algorithm. In some embodiments, an ELNN can have at least about 80%, more preferably at least about 90%, more preferably at least about 91%. more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%. more preferably at least about 95%. more preferably at least about 96%, more preferably at least about 97%. more preferably at least about 98%. and most preferably at least about 99% random coil, as determined by a GOR algorithm.Net Charge

[0176] In some embodiments, the ELNN polypeptides can have an unstructured characteristic imparted by incorporation of amino acid residues with a net charge and / or reducing the proportion of hydrophobic amino acids in the ELNN sequence. The overall net charge and net charge density may be controlled, e.g.. by modifying the content of charged amino acids in the ELNNs. In some embodiments, the net charge density of the ELNN of the compositions may be above +0.1 or below - 0.1 charges / residue. In some embodiments, the net charge of a ELNN can be about 0%. about 1%. about 2%. about 3%. about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10% about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% or more.

[0177] Since most tissues and surfaces in a human or animal have a net negative charge, the ELNNs can optionally be designed to have a net negative charge to minimize non-specific interactions between the ELNN containing compositions and various surfaces such as blood vessels, healthy tissues, or various receptors. Not to be bound by a particular theory, an ELNN may adopt open conformations due to electrostatic repulsion between individual amino acids of the ELNN polypeptide that individually carry a high net negative charge and that are distributed across the sequence of the ELNN polypeptide. Such a distribution of net negative charge in the extended sequence lengths of ELNN can lead to an unstructured conformation that, in turn, can result in an effective increase in hydrodynamic radius. Accordingly, in some embodiments the ELNNs contain glutamic acid such that the glutamic acid is at about 8, 10, 15. 20, 25, or even about 30% of the amino acids in the sequences. The ELNN of the compositions of the present disclosure generally have no or a low content of positively charged amino acids. In some embodiments the ELNN may have less than about 10% amino acid residues with a positive charge, or less than about 7%, or less than about 5%. or less thanabout 2% amino acid residues with a positive charge. However, the present disclosure contemplates polypeptides where a limited number of amino acids with a positive charge, such as lysine, may be incorporated into an ELNN, e.g., to pennit conjugation between the epsilon amine of tire lysine and a reactive group on a peptide, a linker bridge, or a reactive group on a dmg or small molecule to be conjugated to the ELNN backbone.

[0178] In some embodiments, an ELNN may comprise charged residues separated by other residues such as serine or glycine, which may lead to better expression or purification behavior. Based on the net charge, ELNNs of the subject compositions may have an isoelectric point (pl) of 1.0. 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or even 6.5. In some embodiments, the ELNN will have an isoelectric point betw een 1.5 and 4.5. In some embodiments, an ELNN incorporated into a fusion protein carries a net negative charge under physiologic conditions contributes to the unstructured conformation and reduced binding of the ELNN component to mammalian proteins and tissues.

[0179] As hydrophobic ammo acids can impart structure to a polypeptide, in some embodiments the content of hydrophobic amino acids in the ELNN is less than 5%, or less than 2%, or less than 1% hydrophobic amino acid content. In some embodiments, an ELNN has no hydrophobic amino acids. In some embodiments, the amino acid content of methionine and try ptophan in the ELNN component of a fusion protein is less than 5%, or less than 2%, and most preferably less than 1%. In some embodiments, the ELNN has a sequence that has less than 10% amino acid residues with a positive charge, or less than about 7%, or less that about 5%, or less than about 2% amino acid residues with a positive charge, the sum of methionine and try ptophan residues will be less than 2%, and the sum of asparagine and glutamine residues will be less than 10% of the total ELNN sequence. In some embodiments, the ELNN has no methionine or tryptophan residues.Increased Hydrodynamic Radius

[0180] In some embodiments, the ELNN can have a high hydrodynamic radius, conferring a corresponding increased Apparent Molecular Weight to the fusion protein which incorporates the ELNN. The linking of ELNNs to cytokine sequences can result in the fusion protein compositions diat can have increased hydrodynamic radii, increased Apparent Molecular Weight, and increased Apparent Molecular Weight Factor compared to cytokines not linked to an ELNN. For example, in some therapeutic applications in which prolonged half-life is desired, one or more ELNNs with a high hydrodynamic radius are incorporated into a fusion protein comprising a cytokine to effectively enlarge the hydrodynamic radius of the fusion protein beyond the glomerular pore size of approximately 3-5 mn (corresponding to an apparent molecular weight of about 70 kDa) (Caliceti. 2003. Pharmacokinetic and biodistribution properties of polyethylene glycolj-protein conjugates. Adv. Drug Deliv. Rev. 55: 1261-1277), resulting in reduced renal clearance of circulating proteins. In some embodiments, the hydrodynamic radius of a protein is determined by its molecular weight as well as by its structure, including shape and compactness. Not to be bound by a particular theory, theELNN may adopt open conformations due to electrostatic repulsion between individual charges of the peptide or the inherent flexibility imparted by the particular amino acids in the sequence that lack potential to confer secondary structure. In some embodiments, the open, extended and unstructured conformation of the ELNN polypeptide has a greater proportional hydrodynamic radius compared to polypeptides of a comparable sequence length and / or molecular weight that have secondary and / or tertiary structure, such as typical globular proteins. Methods for determining the hydrodynamic radius are well known in the art, such as by the use of size exclusion chromatography (SEC), as described in U.S. Patent Nos. 6,406,632 and 7,294,513. In some embodiments, the addition of increasing lengths of ELNN results in proportional increases in the parameters of hydrodynamic radius, Apparent Molecular Weight, and Apparent Molecular Weight Factor, permitting the tailoring of the fusion to desired characteristic cut-off Apparent Molecular Weights or hydrodynamic radii. Accordingly, in some embodiments, the fusion protein can be configured with an ELNN such that the fusion protein can have a hydrodynamic radius of at least about 5 nm, or at least about 8 nm, or at least about 10 nm, or 12 nm, or at least about 15 nm. In some embodiments, the large hydrodynamic radius conferred by the ELNN in a fusion protein can lead to reduced renal clearance of the resulting fusion protein, leading to a corresponding increase in terminal half-life, an increase in mean residence time, and / or a decrease in renal clearance rate.

[0181] In some embodiments, an ELNN (or multiple ELNNs, such as two ELNNs) of a chosen length and sequence can be selectively incorporated into a cytokine to create a fusion protein that will have, under physiologic conditions, an Apparent Molecular Weight of at least about 150 kDa, or at least about 300 kDa. or at least about 400 kDa, or at least about 500 kDa. or at least about 600 kDa, or at least about 700 kDa. or at least about 800 kDa, or at least about 900 kDa, or at least about 1000 kDa. or at least about 1200 kDa. or at least about 1500 kDa, or at least about 1800 kDa, or at least about 2000 kDa, or at least about 2300 kDa or more. In some embodiments, an ELNN (or multiple ELNNs, such as two ELNNs) of a chosen length and sequence can be selectively linked to a cytokine to result in a fusion protein that has. under physiologic conditions, an Apparent Molecular Weight Factor of at least 3. alternatively of at least 4, alternatively of at least 5, alternatively of at least 6. alternatively of at least 7. alternatively of at least 8, alternatively of at least 9, alternatively of at least 10, alternatively of at least 1 , or an Apparent Molecular Weight Factor of at least 20 or greater. In some embodiments, the fusion protein has, under physiologic conditions, an Apparent Molecular Weight Factor that is about 4 to about 20, or is about 6 to about 15, or is about 8 to about 12, or is about 9 to about 10 relative to the actual molecular weight of the fusion protein. In some embodiments, the fusion polypeptide exhibits an apparent molecular weight factor under physiological conditions that is greater than about 6.Increased Terminal Half-Life

[0182] In some embodiments, a fusion polypeptide comprising an ELNN lias a terminal half-life that is at least two-fold longer, or at least three-fold longer, or at least four -fold longer, or at least five-fold longer, compared to a corresponding biologically active polypeptide that is not linked to the ELNN. In some embodiments, the (fusion) polypeptide has a terminal half-life that is at least two-fold longer compared to the biologically active polypeptide not linked to the ELNN.

[0183] In some embodiments, administration of a therapeutically effective amount of a fusion protein to a subject in need thereof results in a gain in time of at least two-fold, or at least three-fold, or at least four-fold, or at least five-fold or more spent within a therapeutic window for the fusion protein compared to tire corresponding cytokine not linked to the ELNN(s) when administered at a comparable dose to a subject.

[0184] In some embodiments, a cytokine released from a fusion protein upon protease cleavage comprises one or more short polypeptides (e.g., about 30, 25. 20, 15, 14, 13. 12, 11, 10, or less amino acids in length) that has no amino acids other than G, A, P, E, S, and / or T. For example, a short polypeptide that has no amino acids other than G, A. P, E, S, and / or T might be incorporated into one or more spacer or linker sequences of the cytokine, and / or a portion of one or more spacers or linkers that remain part of the cy tokine after cleavage. In some embodiments, a cytokine that is released from a fusion protein comprises a GTSESATPES (SEQ ID NO:96) on the N-tcrminal side (e.g., the closest amino acid of the sequence is within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid positions of the N- terminal amino acid or the sequence includes the N-tenninus) of the cytokine. In some embodiments, a cytokine that is released from a fusion protein comprises a GTATPESGPG on the C-terminal side (e.g., the closest amino acid of the sequence is within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid positions of the N-terminal amino acid or the sequence includes the N-tenninus) of the cytokine.Low Immunogenicity

[0185] In some embodiments, the present disclosure provides compositions in which the ELNNs have a low degree of immunogenicity or arc substantially non-immunogcnic. Several factors can contribute to the low immunogenicity of an ELNN, e.g., the substantially non-repetitive sequence, the unstructured confonnation, tire high degree of solubility, the low degree or lack of self-aggregation, the low degree or lack of proteolytic sites within the sequence, and the low degree or lack of epitopes in the ELNN.

[0186] One of ordinary skill in the art will understand that, in general, polypeptides having highly repetitive short amino acid sequences (e.g., wherein a 200 amino acid-long sequence contain on average 20 repeats or more of a limited set of 3- or 4-mers) and / or having contiguous repetitive amino acid residues (e.g., wherein 5- or 6-mer sequences have identical amino acid residues) have atendency to aggregate or form higher order structures or form contacts resulting in cry stalline or pseudo-crystalline structures.

[0187] In some embodiments, a ELNN sequence is substantially non-repetitive. wherein (1) the ELNN sequence has no three contiguous amino acids that are identical amino acid types, unless the amino acid is serine, in which case no more than three contiguous amino acids can be serine residues; and wherein (2) the ELNN contains no 3-amino acid sequences (3-mers) that occur more than 16, more than 14, more than 12, or more than 10 times within an at least 200 amino acid-long sequence of tire ELNN (e.g., the entire span of an ELNN that is at least amino acids long). Without being bound by any scientific theory', such substantially non-rcpctitivc sequences have less tendency to aggregate and, thus, enable the design of long-sequence ELNNs with a relatively low frequency of charged amino acids that would be likely to aggregate if die sequences or amino acid residues were otherwise more repetitive.

[0188] Conformational epitopes can be formed by regions of protein surfaces that are composed of multiple discontinuous amino acid sequences of a protein antigen. Without being bound by any scientific theory', the precise folding of the protein may bring these sequences into well-defined, stable spatial configurations or epitopes that can be recognized as “foreign” by the host humoral immune system, resulting in the production of antibodies to the protein and / or triggering a cell-mediated immune response. In the latter case, the immimc response to a protein in an individual is heavily influenced by T-cell epitope recognition that is a function of the peptide binding specificity' of that individual’s HLA-DR allotype. Engagement of an MHC Class II peptide complex by a cognate T-cell receptor on die surface of the T-cell, together with the cross-binding of certain other co-receptors such as the CD4 molecule, can induce an activated state within the T-cell. Activation may lead to the release of cytokines further activating other lymphocytes such as B cells to produce antibodies or activating T killer cells as a full cellular immune response.

[0189] Without being bound by any scientific theory', the ability of a peptide to bind a given MHC Class II molecule for presentation on the surface of an APC (antigen presenting cell) may depend on a number of factors; most notably its primary sequence. In some embodiments, a lower degree of immunogenicity may be achieved by designing ELNNs that resist antigen processing in antigen presenting cells, and / or choosing sequences that do not bind MHC receptors well. In some embodiments, ELNN-containing fusion proteins have substantially non-repetitive ELNN polypeptides designed to reduce binding with MHC II receptors, as well as to avoid formation of epitopes for T-cell receptor or antibody binding, resulting in a low degree of immunogenicity. Without being bound by any scientific theory, avoidance of immunogenicity is. in part, a direct result of the conformational flexibility of ELNNs; i.e., the lack of secondary structure due to the selection and order of amino acid residues. For example, of particular interest are sequences having a low tendency to adapt compactly folded conformations in aqueous solution or under physiologic conditions that could result inconformational epitopes. The administration of fusion proteins comprising ELNNs, using conventional therapeutic practices and dosing, would generally not result in the formation of neutralizing antibodies to the ELNNs, and may also reduce the immunogenicity of cytokine fusion partners in the fusion protein compositions.

[0190] In some embodiments, the ELNNs utilized in the subject fusion proteins can be substantially free of epitopes recognized by human T cells. The elimination of such epitopes for the purpose of generating less immunogenic proteins has been disclosed previously; see for example WO 98 / 52976, WO 02 / 079232, and WO 00 / 3317 which are incorporated by reference herein. Assays for human T cell epitopes have been described (Stickler, M., et al. (2003) J Immunol Methods, 281 : 95-108). Of particular interest are peptide sequences that can be oligomerized without generating T cell epitopes or non-human sequences. This can be achieved by testing direct repeats of these sequences for the presence of T-cell epitopes and for the occurrence of 6 to 15-mer and, in particular, 9-mer sequences that are not human, and then altering the design of the ELNN sequence to eliminate or disrupt the epitope sequence. In some embodiments, the ELNNs are substantially non-immunogenic by the restriction of the numbers of epitopes of the ELNN predicted to bind MHC receptors. With a reduction in the numbers of epitopes capable of binding to MHC receptors, there is a concomitant reduction in the potential for T cell activation as well as T cell helper function, reduced B cell activation or upregulation and reduced antibody production. The low degree of predicted T-cell epitopes can be determined by epitope prediction algorithms such as. e.g., TEPITOPE (Stumiolo, T._ et al. (1999) Nat Biotechnol. 17: 555-61). as shown in Example 74 of International Patent Application Publication No. WO 2010 / 144502 A2, which is incorporated by reference in its entirety. Aspects of the TEPITOPE score of a given peptide frame within a protein are disclosed in Stumiolo, T. et al. (1999) Nature Biotechnology’ 17:555). The score ranges over at least 20 logs, from about 10 to about -10 (corresponding to binding constraints of 10e10KDto 10e10KD). and can be reduced by avoiding hydrophobic amino acids that can serve as anchor residues during peptide display on MHC, such as M, I, L, V. or F. In some embodiments, an ELNN component incorporated into a fusion protein does not have a predicted T-cell epitope at a TEPITOPE score of about -5 or greater, or -6 or greater, or -7 or greater, or -8 or greater, or at a TEPITOPE score of -9 or greater. As used herein, a score of “-9 or greater” would encompass TEPITOPE scores of 10 to -9, inclusive, but would not encompass a score of -10, as -10 is less than -9.

[0191] In some embodiments, the ELNNs, including those incorporated into the subject fusion proteins, can be rendered substantially non-immunogenic by the restriction of known proteolytic sites from the sequence of the ELNN, reducing the processing of ELNN into small peptides that can bind to MHC II receptors. In some embodiments, the ELNN sequence can be rendered substantially non- immunogenic by the use a sequence that is substantially devoid of secondary structure, conferring resistance to many proteases due to the high entropy of the structure. Accordingly, the reducedTEPITOPE score and elimination of known proteolytic sites from the ELNN may render the ELNN compositions, including the ELNN of the fusion protein compositions, substantially unable to be bound by mammalian receptors, including those of the immune system. In some embodiments, an ELNN of a fusion protein can have >100 nM KD binding to a mammalian receptor, or greater than 500 nM KD, or greater than 1 pM KD towards a mammalian cell surface or circulating polypeptide receptor.

[0192] Additionally, the substantially non-repetitive sequence and corresponding lack of epitopes of such embodiments of ELNNs can limit the ability’ of B cells to bind to or be activated by the ELNNs. In some embodiments, while an ELNN can make contacts with many different B cells over its extended sequence, each individual B cell may only make one or a small number of contacts with an individual ELNN. As a result, ELNNs typically may have a much lower tendency to stimulate proliferation of B cells and thus an immune response. In some embodiments, the fusion protein may have reduced immunogenicity as compared to the corresponding cytokine that is not fused to a mask polypeptide such as an ELNN. In some embodiments, the administration of up to three parenteral doses of a fusion protein to a mammal may result in detectable anti- fusion protein IgG at a serum dilution of 1: 100 but not at a dilution of 1 : 1000. In some embodiments, the administration of up to three parenteral doses of a fusion protein to a mammal may result in detectable anti-cytokine IgG at a serum dilution of 1 :100 but not at a dilution of 1: 1000. In some embodiments, the administration of up to three parenteral doses of n fusion protein to a mammal may result in detectable anti-ELNN IgG at a serum dilution of 1 : 100 but not at a dilution of 1 : 1000. In some embodiments, the mammal can be, e.g.. a mouse, a rat. a rabbit, cynomolgus monkey, or human. In some embodiments, tire mammal is a human.

[0193] An additional feature of certain ELNNs with substantially non-repetitive sequences relative to those less non-repetitive sequences (such as one having three contiguous amino acids that are identical) can be that non-repetitive ELNNs form weaker contacts with cytokines (e.g., monovalent interactions), thereby resulting in less likelihood of immune clearance such that the fusion protein compositions can remain in circulation for an increased period of time.

[0194] In some embodiments, a biologically active polypeptide (such as a cytokine) comprising an ELNN is less immunogenic compared to the fusion polypeptide not linked to any ELNN, wherein immunogenicity is ascertained by measuring production of IgG antibodies that selectively bind to the biologically active polypeptide after administration of comparable doses to a subject.BARCODE FRAGMENT

[0195] In some embodiments, a polypeptide (e.g., a fusion polypeptide or a portion thereof such as an ELNN) comprises one or more barcode fragments (e.g., a first, second, or third barcode fragment) releasable from the polypeptide upon digestion by a protease. In some embodiments, the protease is anon-maminalian protease. In some embodiments, the protease is a prokaryotic protease. As used herein, the term ’‘barcode fragment" (or “barcode," or “barcode sequence") can refer to either the portion of the polypeptide cleavably fused within the polypeptide, or the resulting peptide fragment released from the polypeptide.

[0196] In some embodiments, a barcode fragment (1) is a portion of an ELNN that includes at least part of the (non-recurring, non-overlapping) sequence motif that occurs (or is found) only once within tire ELNN; and (2) differs in sequence and molecular weight from all other peptide fragments that are releasable from the polypeptide upon cleavage or complete digestion of the polypeptide by the protease.

[0197] In some embodiments, a barcode fragment does not include the N-terminal amino acid or the C-tenninal amino acid of the fusion polypeptide. As described herein, in some embodiments, a barcode fragment is releasable (e.g., configured to be released) upon Glu-C digestion of the fusion polypeptide. In some embodiments, a barcode fragment is in an ELNN and does not include a glutamic acid that is immediately adjacent to another glutamic acid, if present, in the ELNN. In some embodiments, a barcode fragment has a glutamic acid at its C-terminus. One of ordinary skill in the art will understand that the C-tenninus of a barcode fragment can refer to the “last” (or the most C- tenninal) amino acid residue within the barcode fragment, when cleavably fused within a polypeptide (such as an ELNN), even if other non-barcodc amino acid residues arc positioned C -terminal to the barcode fragment within the polypeptide (e.g., ELNN). In some embodiments, a barcode fragment has an N-terminal amino acid that is immediately preceded by a glutamic acid residue. In some embodiments, the glutamic acid residue that precedes the N-terminal amino acid is not immediately adjacent to another glutamic acid residue. In some embodiments, a barcode fragment does not include a (second) glutamic acid residue at a position other than the C-terminus of the barcode fragment unless the glutamic acid is immediately followed by a proline. In some embodiments, a barcode fragment is positioned a distance from either the N-terminus of the polypeptide or the C-terminus of the polypeptide, wherein the distance is from 10 to 150, or 10 to 125 amino acids. In some embodiments, a barcode fragment is positioned within, or at a location of, 300, 280. 260, 250, 240. 220, 200, 190. 180, 170, 160. 150, 140. 130, 120, 110. 100, 90. 80. 70, 60, 50, 48. 40, 36, 30, 24. 20. 12, or 10 amino acids from the N-terminus of the polypeptide, or at a location in a range between any of the foregoing. In some embodiments, a barcode fragment is positioned within 200, within 150, within 100, or within 50 amino acids of the N-terminus of the polypeptide. In some embodiments, a barcode fragment is positioned at a location that is between 10 and 200. between 30 and 200. between 40 and 150, or between 50 and 100 amino acids from the N-terminus of the polypeptide. In some embodiments, a barcode fragment is positioned within, or at a location of. 300, 280, 260. 250, 240, 220. 200, 190, 180, 170, 160, 150, 140, 130. 120, 110, 100. 90, 80, 70, 60, 50. 48, 40, 36, 30. 24, 20, 12, or 10 amino acids from the C-terminus of the polypeptide, or at a location in a range between anyof the foregoing. In some embodiments, a barcode fragment is positioned within 200, within 150, within 100, or within 50 amino acids of the C-tenninus of the polypeptide. In some embodiments, a barcode fragment is positioned at a location that is betw een 10 and 200, between 30 and 200, between 40 and 150, or between 50 and 100 amino acids from the C-tenninus of the polypeptide. In some embodiments, a barcode fragment (BAR) is characterized in that: (i) it does not include a glutamic acid that is immediately adjacent to another glutamic acid, if present, in the ELNN; (ii) it has a glutamic acid at its C-tenninus; (iii) it has an N-terminal amino acid that is immediately preceded by a glutamic acid residue; and (iv) it is positioned a distance from either the N-terminus of the polypeptide or the C-terminus of the polypeptide, wherein the distance is from 10 to 150 amino acids, or from 10 to 125 amino acids in length. In some embodiments, a barcode fragment is in an ELNN and (i) does not include the N-terminal amino acid or the C-terminal amino acid of the polypeptide; (ii) does not include a glutamic acid that is immediately adjacent to another glutamic acid in the ELNN; (iii) has a glutamic acid at its C-terminus; (iv) has an N-terminal amino acid that is immediately preceded by a glutamic acid residue; and (v) is positioned a distance from either the N- terminus of the polypeptide or the C-terminus of the polypeptide, wherein the distance is from 10 to 150. or 10 to 125 amino acids in length. In some embodiments, the glutamic acid residue that precedes the N-terminal amino acid is not immediately adjacent to another glutamic acid residue. In some embodiments, a barcode fragment does not include a glutamic acid residue at a position other than the C-terminus of the barcode fragment unless the glutamic acid is immediately followed by a proline. Depending on context herein and when referring to placement within a polypeptide sequence, the term “distance-’ can refer to the number of amino acid residues from the N-terminus of the polypeptide to the most N-terminal amino acid residue of the barcode fragment, or from the C- tenninus of the polypeptide to the most C-terminal amino acid residue of the barcode fragment. In some embodiments, for a barcoded ELNN fused to a biologically active polypeptide, at least one barcode fragment (or at least tw o barcode fragments, or three barcode fragments) contained in the barcoded ELNN is positioned at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300 amino acids from the biologically active polypeptide. In some embodiments, a barcode fragment is at least 4, at least 5, at least 6, at least 7, or at least 8 amino acids in length. In some embodiments, a barcode fragment is at least 4 amino acids in length. In some embodiments, a barcode fragment is 4, 5, 6, 7. 8, 9. 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25 amino acids in length, or in a range between any of the foregoing values. In some embodiments, a barcode fragment is between 4 and 20, between 5 and 15, between 6 and 12, or between 7 and 10 amino acids in length. In some embodiments, a barcode fragment comprises an amino acid sequence identified herein by SEQ ID NOs: 68-79 and SEQ ID NOs: 1010-1029 in Table 2.Table 2. Exemplary Barcode Fragments Releasable Upon Glu-C Digest

[0198] In some embodiments, each barcode fragment differs in both sequence and molecular weight from all other peptide fragments that are releasable from the chimeric polypeptides described herein upon complete digestion the chimeric polypeptide by a non-mammalian protease. In some embodiments, the non-mammalian protease is Glu-C.

[0199] In some embodiments, the chimeric polypeptides disclosed herein comprises a Glu-C cleavage site comprising one of the following amino acid sequences: ATPESGPG (SEQ ID NO:2223), SGSETPGT (SEQ ID NO:2242), and GTSESATP (SEQ ID NO:204).

[0200] In some embodiments, the chimeric polypeptides disclosed herein comprises at least one of the following amino acid sequences: PE.GSXnPE.SG (SEQ ID NO:2243), PE.GSXnSE.GG (SEQ ID NO:2244), PE.GSXnSE.TG (SEQ ID NO:2246), PE.GSXnSE.SA (SEQ ID NO:2245), PE.SGXnPE.SG (SEQ ID NO:2247), PE.SGXnSE.GG (SEQ ID NO:2248), PE.SGXnSE.TG (SEQ ID NO:2250), PE.SGXnSE.SA (SEQ ID NO:2249). and PE.TPXnPE.SG (SEQ ID NO:2251), PE.TPXnSE.GG (SEQ ID NO:2252), PE.TPXnSE.TG (SEQ ID NO:2254), PE.TPXnSE.SA (SEQ ID NO:2253), wherein each is a Glu-C cleavage site and n is any integer from 0 to 50. In some embodiments, the chimeric polypeptides disclosed herein comprises at least one of the following amino acid sequences: PE.SGXnPE.SG (SEQ ID NO:2247), PE.GSXnSE.GG (SEQ ID NO:2244), PE.TPXnSE.TG (SEQ ID NO:2254), PE.SGXnSE.SA (SEQ ID NO:2249). In some embodiments, n is any integer from 1 to 20. In some embodiments, n is any integer from 5 to 15. In some embodiments, n is any integer from 5 to 10. In some embodiments, n is 9. In some embodiments, n is any integer from 5 to 15. In some embodiments, Xnis SGPGTGTSATPE (SEQ ID NO: 1010), SGPGSGPGTSE (SEQ ID NO:78), SGPGTTPGTTPE (SEQ ID NO:1011), SGPGTPPTSTPE (SEQ ID NQ: 1012). SGPGTSPSATPE (SEQ ID NO:79), SGPGTGSAGTPE (SEQ ID NO: 1013), SGPGTGGAGTPE (SEQ ID NO: 1014), SGPGTSPGATPE (SEQ ID NO: 1015), SGPGTSGSGTPE (SEQ ID NO: 1016), SGPGTSSASTPE (SEQ ID NO: 1017), SGPGTGAGTTPE (SEQ ID NO: 1018), SGPGTGSTSTPE (SEQ ID NO: 1019), TPGSEPATSGSE (SEQ ID NO: 1020), GSAPGTSTEPSE (SEQ ID NO: 1021), SGPGTAGSGTPE (SEQ ID NO:1022), SGPGTSSGGTPE (SEQ ID NO:1023). SGPGTAGPATPE (SEQ ID NO: 1024), SGPGTPGTGTPE (SEQ ID NO: 1025), SGPGTGGPTTPE (SEQ ID NO: 1026), or SGPGTGSGSTPE (SEQ ID NO: 1027).

[0201] In some embodiments, a chimeric polypeptide comprises at least one of the following amino acid sequences:SGPE.SGPGXnSGPE.SGPG (SEQ ID N0:2001), SGPE.SGPGXnATPE.SGPG (SEQ ID N0:2002), SGPE.SGPGXnGTSE.SATP (SEQ ID N0:2003), SGPE.SGPGXnTTPE.SGPG (SEQ ID N0:2004), SGPE.SGPGXnSTPE.SGPG (SEQ ID N0:2005), SGPE.SGPGXnGTPE.SGPG (SEQ ID N0:2006), SGPE.SGPGXnGTPE.TPGS (SEQ ID N0:2007), SGPE.SGPGXnGTPE.TPGS (SEQ ID N0:2007), SGPE.SGPGXnSGSE.TGTP (SEQ ID N0:2008), SGPE.SGPGXnGTPE.GSAP (SEQ ID N0:2009), SGPE.SGPGXnEPSE.SATP (SEQ ID NQ:2010), ATPE.SGPGXnSGPE.SGPG (SEQ ID NO:2011), ATPE.SGPGXnATPE.SGPG (SEQ ID NQ:2012), ATPE.SGPGXnGTSE.SATP (SEQ ID NQ:2013), ATPE.SGPGXnATSE.SATP (SEQ ID NQ:2014), ATPE.SGPGXnTTPE.SGPG (SEQ ID NQ:2015), ATPE.SGPGXnSTPE.SGPG (SEQ ID NQ:2016), ATPE.SGPGXnGTPE.SGPG (SEQ ID NQ:2017), ATPE.SGPGXnGTPE.TPGS (SEQ ID NO:2018), ATPE.SGPGX„SGSE.TGTP (SEQ ID NO:2019), ATPE.SGPGXnGTPE.GSAP (SEQ ID N0:2020), ATPE.SGPGXnEPSE.SATP (SEQ ID NO:2021), GTSE.SATPXnSGPE.SGPG (SEQ ID NO:2022), GTSE.SATPXnATPE.SGPG (SEQ ID NO:2023), GTSE.SATPXnGTSE.SATP (SEQ ID NO:2024), GTSE.SATPXnTTPE.SGPG (SEQ ID NO:2025).GTSE.SATPXnSTPE.SGPG (SEQ ID NO:2026), GTSE.SATPX„GTPE.SGPG (SEQ ID NO:2027), GTSE.SATPXnGTPE.TPGS (SEQ ID NO:2028), GTSE.SATPX„SGSE.TGTP (SEQ ID NO:2029), GTSE.SATPXnGTPE.GSAP (SEQ ID N0:2030), GTSE.SATPXnEPSE.SATP (SEQ ID NO:2031), TTPE.SGPGXnSGPE.SGPG (SEQ ID NO:2032), TTPE.SGPGX„ATPE.SGPG (SEQ ID NO:2033), TTPE.SGPGXnGTSE.SATP (SEQ ID NO:2034), TTPE.SGPGX„TTPE.SGPG (SEQ ID NO:2035), TTPE.SGPGXnSTPE.SGPG (SEQ ID NO:2036), TTPE.SGPGXnGTPE.SGPG (SEQ ID NO:2037), TTPE.SGPGXnGTPE.TPGS (SEQ ID NO:2038), TTPE.SGPGXnSGSE.TGTP (SEQ ID NO:2039), TTPE.SGPGXnGTPE.GSAP (SEQ ID N0:2040), TTPE.SGPGXnEPSE.SATP (SEQ ID NO:2041), STPE.SGPGXnSGPE.SGPG (SEQ ID NO:2042), STPE.SGPGX„ATPE.SGPG (SEQ ID NO:2043), STPE.SGPGXnGTSE.SATP (SEQ ID NO:2044), STPE.SGPGXnTTPE.SGPG (SEQ ID NO:2045), STPE.SGPGXnSTPE.SGPG (SEQ ID NO:2046), STPE.SGPGXnGTPE.SGPG (SEQ ID NO:2047), STPE.SGPGXnGTPE.TPGS (SEQ ID NO:2048), STPE.SGPGX„SGSE.TGTP (SEQ ID NO:2049), STPE.SGPGXnGTPE.GSAP (SEQ ID NQ:2050), STPE.SGPGXnEPSE.SATP (SEQ ID NQ:2051). GTPE.SGPGXnSGPE.SGPG (SEQ ID NQ:2052), GTPE.SGPGXnATPE.SGPG (SEQ ID NQ:2053), GTPE.SGPGXnGTSE.SATP (SEQ ID NQ:2054), GTPE.SGPGXnTTPE.SGPG (SEQ ID NO:2055). GTPE.SGPGXnSTPE.SGPG (SEQ ID NO:2056), GTPE.SGPGXnGTPE.SGPG (SEQ ID NO:2057), GTPE.SGPGXnGTPE.TPGS (SEQ ID NO:2058), GTPE.SGPGXnSGSE.TGTP (SEQ ID NO:2059), GTPE.SGPGXnGTPE.GSAP (SEQ ID N0:2060), GTPE.SGPGXnEPSE.SATP (SEQ ID NO:2061), GTPE.TPGSXnSGPE.SGPG (SEQ ID NO:2062), GTPE.TPGSXnATPE.SGPG (SEQ ID NO:2063), GTPE.TPGSXnGTSE.SATP (SEQ ID NO:2064), GTPE.TPGSXnTTPE.SGPG (SEQ ID NO:2065). GTPE.TPGSXnSTPE.SGPG (SEQ ID NO:2066), GTPE.TPGSX„GTPE.SGPG (SEQ ID NO:2067), GTPE.TPGSXnGTPE.TPGS (SEQ ID NO:2068), GTPE.TPGSX„SGSE.TGTP (SEQ ID NO:2069), GTPE.TPGSXnGTPE.GSAP (SEQ ID N0:2070), GTPE.TPGSX„EPSE.SATP (SEQ ID NO:2071), SGSE.TGTPXnSGPE.SGPG (SEQ ID NO:2072), SGSE.TGTPX„ATPE.SGPG (SEQ ID NO:2073), SGSE.TGTPXnGTSE.SATP (SEQ ID NO:2074), SGSE.TGTPX„TTPE.SGPG (SEQ ID NO:2075), SGSE.TGTPXnSTPE.SGPG (SEQ ID NO:2076), SGSE.TGTPX„GTPE.SGPG (SEQ ID NO:2077), SGSE.TGTPXnGTPE.TPGS (SEQ ID NO:2078), SGSE.TGTPX„SGSE.TGTP (SEQ ID NO:2079), SGSE.TGTPXnGTPE.GSAP (SEQ ID N0:2080), SGSE.TGTPXnEPSE.SATP (SEQ ID NO:2081), GTPE.GSAPXnSGPE.SGPG (SEQ ID NO:2082), GTPE.GSAPXnATPE.SGPG (SEQ ID NO:2083), GTPE.GSAPXnGTSE.SATP (SEQ ID NO:2084), GTPE.GSAPXnTTPE.SGPG (SEQ ID NO:2085), GTPE.GSAPXnSTPE.SGPG (SEQ ID NO:2086), GTPE.GSAPXnGTPE.SGPG (SEQ ID NO:2087), GTPE.GSAPXnGTPE.TPGS (SEQ ID NO:2088), GTPE.GSAPXnSGSE.TGTP (SEQ ID NO:2089), GTPE.GSAPXnGTPE.GSAP (SEQ ID NQ:2090), GTPE.GSAPXnEPSE.SATP (SEQ ID NO:2091), EPSE.SATPXnSGPE.SGPG (SEQ ID NO:2092), EPSE.SATPX„ATPE.SGPG (SEQ ID NO:2093). EPSE.SATPXnGTSE.SATP (SEQ ID NO:2094), EPSE.SATPX„TTPE.SGPG (SEQ ID NO:2095). EPSE.SATPXnSTPE.SGPG (SEQ ID NO:2096), EPSE.SATPX„GTPE.SGPG (SEQ ID NO:2097). EPSE.SATPXnGTPE.TPGS (SEQ ID NQ:2098), EPSE.SATPXnSGSE.TGTP (SEQ ID NQ:2099),EPSE.SATPXnGTPE.GSAP (SEQ ID N0:2100), or EPSE.SATPX„EPSE.SATP (SEQ ID NO:2101), wherein each is a Glu-C cleavage site and n is any integer from 0 to 50. In some embodiments, the chimeric polypeptide comprises at least one of the following amino acid sequences:SGPE.SGPGXnATPE.SGPG (SEQ ID NO:2105), ATPE.SGPGXnGTSE.SATP (SEQ ID NO:2103), ATPE.SGPGXnATSE.SATP (SEQ ID NO:2104), ATPE.SGPGXnTTPE.SGPG (SEQ ID NO:2106), ATPE.SGPGXnSTPE.SGPG (SEQ ID NO:2107), ATPE.SGPGXnATPE.SGPG (SEQ ID NO:2102), ATPE.SGPGXnGTPE.SGPG (SEQ ID NO:2108), ATPE.SGPGXnGTPE.SGPG (SEQ ID NO:2108), ATPE.SGPGXnATPE.SGPG (SEQ ID NO:2102), GTPE.SGPGXnGTPE.SGPG (SEQ ID NO:2109), GTPE.SGPGXnSTPE.SGPG (SEQ ID NO:2HO), GTPE.SGPGXnTTPE.SGPG (SEQ ID NO:2111), GTPE.SGPGXnSTPE.SGPG (SEQ ID NO:2HO), GTPE.TPGSX„SGSE.TGTP (SEQ ID NO:2112), GTPE.GSAPXnEPSE.SATP (SEQ ID NO:2113), ATPE.SGPGXnGTPE.SGPG (SEQ ID NO:2108), ATPE.SGPGXnGTPE.SGPG (SEQ ID NO:2108), ATPE.SGPGXnATPE.SGPG (SEQ ID NO:2102), ATPE.SGPGXnGTPE.SGPG (SEQ ID NO:2108), TTPE.SGPGXnTTPE.SGPG (SEQ ID NO:2114), or STPE.SGPGXnSTPE.SGPG (SEQ ID NO:2115), wherein each is a Glu-C cleavage site and n is any integer from 0 to 30. In some embodiments, n is any integer from 1 to 20. In some embodiments, n is any integer from 5 to 15. In some embodiments, n is any integer from 3 to 7. In some embodiments, n is any integer from 5 to 10. In some embodiments, n is 9. In some embodiments, n is 4. In some embodiments, n is 3. In some embodiments, n is any integer from 5 to 15. In some embodiments, wherein Xnis PGTGTSAT (SEQ ID NO:2233), PGSGPGT (SEQ ID NO:2221), PGTTPGTT (SEQ ID NO:2241). PGTPPTST (SEQ ID NO:2235). PGTSPSAT (SEQ ID NO:2238), PGTGSAGT (SEQ ID NO:2230), PGTGGAGT (SEQ ID NO:2228). PGTSPGAT (SEQ ID NO:2237), PGTSGSGT (SEQ ID NO:2236), PGTSSAST (SEQ ID NO:2239), PGTGAGTT (SEQ ID NO:2227). PGTGSTST (SEQ ID NO:2232). GSEPATSG (SEQ ID NO:2224). APGTSTEP (SEQ ID NO:2222). PGTAGSGT (SEQ ID NO:2226), PGTSSGGT (SEQ ID NQ:2240), PGTAGPAT (SEQ ID NO:2225). PGTPGTGT (SEQ ID NO:2234), PGTGGPTT (SEQ ID NO:2229), or PGTGSGST (SEQ ID NO:2231). In some embodiments. Xnis TSAS (SEQ ID NO:2214), TGTS (SEQ ID NO:2211), SGP, TTPG (SEQ ID NO:2220), TPPT (SEQ ID NO:2213), TSPS (SEQ ID NO:2217), TGSA (SEQ ID NO:2208), TGGA (SEQ ID NO:2206), TSPG (SEQ ID NO:2216), TSGS (SEQ ID NO:2215), TSSA (SEQ ID NO:2218), TGAG (SEQ ID NO:2205), TGST (SEQ ID NO:2210), EPAT (SEQ ID NO:2201), GTST (SEQ ID NO:2202), TAGS (SEQ ID NO:2204), TSSG (SEQ ID NO:2219), TAGP (SEQ ID NO:2203), TPGT (SEQ ID NO:2212), TGGP (SEQ ID NO:2207), or TGSG (SEQ ID NO:2209).

[0202] In some embodiments, barcodes are designed to have improved analytical properties. In some embodiments, such barcodes can be released with relatively modest concentrations of a nonmammalian protease such as Glu-C. This facilitates better detection, e.g., through LC / MS. and alsoallows measurement of peptides that are generated from the cleavable linker thereby allowing a measurement of cleavage products using, e.g., LC / MS.

[0203] In some embodiments of fusion proteins comprising an ELNN, the fusion protein has a single polypeptide chain, and the polypeptide chain comprises a barcode fragment that is at a position within the polypeptide chain that is from 10 to 200 amino acids or from 10 to 125 amino acids from the N- terminus or the C-terminus of the polypeptide chain. In some embodiments, a fusion protein comprises a first ELNN and a second ELNN, the first ELNN is at the N-terminal side of the cytokine, and the first barcode fragment is positioned within 200, 150, 100, or 50 amino acids of the N-tenninus of the fusion protein. In some embodiments, the second ELNN is at the C-tcnninal side of the cytokine, and the second barcode fragment is positioned within 200, 150, 100, or 50 amino acids of the C-terminus of the chimeric polypeptide.

[0204] In some embodiments, an ELNN further comprises one or more additional barcode fragments, wherein the one or more additional barcode fragments each differs in sequence and molecular weight from all other peptides fragments that are releasable from the polypeptide upon complete digestion of the polypeptide by the protease. In some embodiments, a barcoded ELNN comprises only one barcode fragment. In some embodiments, a barcoded ELNN comprises a set of barcode fragments, comprising a first barcode fragment, such as those described herein. In some embodiments, the set of barcode fragments comprises a second barcode fragment (or a further barcode fragment), such as those described herein. In some embodiments, the set of barcode fragments comprises a third barcode fragment, such as those described herein.

[0205] A set of barcode fragments fused within an N-terminal ELNN can be referred to as an N- terminal set of barcodes (an “N-terminal set”). A set of barcode fragments fused within a C-terminal ELNN can be referred to as a C-terminal set of barcodes (a “C-terminal set”). In some embodiments, the N-terminal set comprises a first barcode fragment and a second barcode fragment. In some embodiments, the N-terminal set further comprises a third barcode fragment. In some embodiments, the C -terminal set comprises a first barcode fragment and a second barcode fragment. In some embodiments, the C-terminal set further comprises a third barcode fragment. In some embodiments, the polypeptide comprises a set of barcode fragments that includes a first barcode fragment, a further (second) barcode fragment, and at least one additional barcode fragment, wherein each barcode fragment of the set of barcode fragments (1) is a portion of the second ELNN and (2) differs in sequence and molecular weight from all other peptides fragments that are releasable from the polypeptide upon complete digestion of the polypeptide by the protease.

[0206] Included herein is a mixture comprising a plurality of polypeptides of varying length; the mixture comprising a first set of polypeptides and a second set of polypeptides. In some embodiments, each polypeptide of the first set of polypeptides comprises a barcode fragment that (a) is releasablefrom the polypeptide by digestion with a protease and (b) has a sequence and molecular weight that differs from the sequence and molecular weight of all other fragments that are releasable from the first set of polypeptides. In some embodiments, the second set of polypeptides lack the barcode fragment of the first set of polypeptides (e.g., due to truncation). In some embodiments, both the first set of polypeptides and the second set of polypeptides each comprise a reference fragment that (a) is common to the first set of polypeptides and the second set of polypeptides and (b) releasable by digestion with the protease. In some embodiments, the ratio of the first set of polypeptides to polypeptides comprising the reference fragment is greater than 0.70. In some embodiments, the ratio of the first set of polypeptides to polypeptides comprising the reference fragment is greater than 0.80, 0.90, 0.95, or 0.98. In some embodiments, the reference fragment occurs no more than once in each polypeptide of the first set of polypeptides and the second set of polypeptides. In some embodiments, the protease is a protease that cleaves on the C-terminal side of glutamic acid residues. In some embodiments, the protease is a Glu-C protease. In some embodiments, the protease is not trypsin. In some embodiments, the polypeptides of varying lengths comprise polypeptides comprising at least one ELNN, such as any described herein. In some embodiments, the first set of polypeptides comprises a full-length polypeptide, wherein the barcode fragment is a portion of the full-length polypeptide. In some embodiments, the full-length polypeptide is a (fusion) polypeptide, such as any described hereinabove or described anywhere else herein. In some embodiments, the polypeptides of varying lengths in a mixture differ from one another due to N-terminal trimcation, C-terminal trimcation, or both N- and C-terminal truncation of a full-length polypeptide. In some embodiments, the first set of polypeptides and the second set of polypeptides may differ in one or more pharmacological properties.

[0207] The present disclosure also provides methods for assessing, in a mixture comprising polypeptides of varying length, a relative amount of a first set of polypeptides in the mixture to a second set of polypeptides in the mixture, wherein (1) each polypeptide of the first set of polypeptides shares a barcode fragment that occurs once and only once in the polypeptide and (2) each polypeptide of the second set of polypeptides lacks the barcode fragment that is shared by polypeptides of the first set, wherein individual polypeptides of both the first of polypeptides and the second set of polypeptides each comprises a reference fragment. In some embodiments, the methods comprise contacting the mixture with a protease to produce a plurality of proteolytic fragments that result from cleavage of the first set of polypeptides and the second set of polypeptides, wherein the plurality’ of proteolytic fragments comprise a plurality of reference fragments, and a plurality’ of barcode fragments. In some embodiments, the methods can further comprise determining a ratio of the amount of barcode fragments to the amount of reference fragments, thereby assessing the relative amounts of the first set of poly peptides to the second set of polypeptides. In some embodiments, the barcode fragment occurs no more than once in each polypeptide of the first set of polypeptides. In someembodiments, the reference fragment occurs no more than once in each polypeptide of the first set of polypeptides and the second set of polypeptides. In some embodiments, the plurality of proteolytic fragments comprises a plurality of reference fragments, and a plurality of barcode fragments. In some embodiments, the protease cleaves the first and second sets of polypeptides (or the polypeptides of varying length) on the C-terminal side of glutamic acid residues that are not followed by a proline residue. In some embodiments, the protease is a Glu-C protease. In some embodiments, the protease is not trypsin. In some embodiments, the step of determining a ratio of the amount of barcode fragments to the amount of reference fragments comprises identifying barcode fragments and reference fragments from the mixture after it has been contacted with the protease. In some embodiments, the barcode fragments and the reference fragments are identified based on their respective masses. In some embodiments, the barcode fragments and the reference fragments are identified via mass spectrometry.

[0208] In some embodiments, the barcode fragments and reference fragments are identified via liquid chromatography -mass spectrometry (LC-MS). In some embodiments, the step of detennining a ratio of the barcode fragments to the reference fragments comprises isobaric labeling. In some embodiments, the step of determining a ratio of the barcode fragments to the reference fragments comprises spiking the mixture with one or both of an isotope-labeled reference fragment and an isotope labeled barcode fragment. In some embodiments, the polypeptides of varying lengths comprise polypeptides that comprise at least one ELNN. as described hereinabove or described anywhere else herein. In some embodiments, the ELNN is characterized in that (i) it comprises at least 100. or at least 150 amino acids; (ii) at least 90% of the amino acid residues of the ELNN are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) or proline (P); and (iii) it comprises at least 4 different types of amino acids that are G, A. S, T, E. or P. In some embodiments, the barcode fragment, when present, is a portion of the ELNN. In some embodiments, the mixture of polypeptides of varying lengths comprises a polypeptide as any described hereinabove or described anywhere else herein. In some embodiments, the polypeptides of varying length comprise a full- length polypeptide and truncated fragments thereof. In some embodiments, the polypeptides of varying length consist essentially of the full-length polypeptide and truncated fragments thereof. In some embodiments, the polypeptides of varying lengths in a mixture differ from one another due to N-tenninal truncation, C-terminal truncation, or both N- and C-terminal truncation of a full-length polypeptide. In some embodiments, the full-length polypeptide is a polypeptide as described hereinabove or described anywhere else herein. In some embodiments, the ratio of the amount of barcode fragments to reference fragments is greater than 0.50, 0.60, 0.70, 0.80, 0.90, 0.95, 0.98, or 0.99.Isobaric Labeling-Based Quantification of Peptides

[0209] In some embodiments, isobaric labeling can be used for determining a ratio of the barcode fragments to the reference fragments. Isobaric labeling is a mass spectrometry strategy used in quantitative proteomics, wherein peptides or proteins (or portions thereof) are labeled with various chemical groups that are isobaric (identical in mass) but vary in terms of distribution of heavy isotopes around their structure. In some embodiments, these tags, commonly referred to as tandem mass tags, are designed so that the mass tag is cleaved at a specific linker region upon high-energy collision-induced dissociation (CID) during tandem mass spectrometry, thereby yielding reporter ions of different masses. Some of the most common isobaric tags are amine-reactive tags.Exemplary Barcoded ELNN Polypeptides

[0210] Included herein are ELNNs comprising barcode fragments that are portions of the ELNNs.|02111 Amino acid sequences of exemplary barcoded ELNNs, containing one barcode (e.g., SEQ ID NOs: 8002-8003, 8005-8009, and 8013-8022), or two barcodes (e.g.. SEQ ID NOS: 8001. 8004. and 8012), or three barcodes (e.g., SEQ ID NO: 8011), are illustrated in Table 3a. In some embodiments, among these exemplary barcoded ELNNs. 12 (SEQ ID NOs: 8001-8003, 8008-8009, 8011, 8015- 8019, and 8022) are to be fused to a biologically -active protein (such as a cytokine) at the C-terminal of the biologically -active protein, and 10 (SEQ ID NOS: 8004-8007, 8010, 8012-8014. 8020. and 8021) are to be fused at the N-terminal of the biologically -active protein. In some embodiments, the ELNN has at least 90%, at least 92%, at least 95%. at least 98%, at least 99% or 100% sequence identity to a sequence identified herein by SEQ ID NOs: 8001-8022 in Table 3a.Table 3a. Exemplary Barcoded ELNNs

[0212] In some embodiments, a barcoded ELNN can be obtained by making one or more mutations to existing ELNN, such as any listed in Table 3b, according to one or more of the following criteria: to minimize the sequence change in the ELNN, to minimize the amino acid composition change in the ELNN, to substantially maintain the net charge of the ELNN, to substantially maintain (or improve) low immunogenicity of the ELNN, and to substantially maintain (or improve) the pharmacokineticproperties of the ELNN. In some embodiments, the ELNN sequence has at least 90%, at least 92%, at least 95%. at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 601-659 listed in Table 3b. In some embodiments, the ELNN sequence, having at least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) but less than 100% sequence identity to any of SEQ ID NOs: 601-659 listed in Table 3b, is obtained by one or more mutations (e g., less than 10, less than 8, less than 6, less than 5. less than 4, less than 3, less than 2 mutations) of the corresponding sequence from Table 3b. In some embodiments, the one or more mutations comprise deletion of a glutamic acid residue, insertion of a glutamic acid residue, substitution of a glutamic acid residue, or substitution for a glutamic acid residue, or any combination thereof. In some embodiments, where the ELNN sequence differs from, but has at least 90% (e.g., at least 92%, at least 95%. at least 98%, or at least 99%) sequence identity to. any one of SEQ ID NOs: 601-659 listed in Table 3b. at least 80%, at least 90%, at least 95%. at least 97%, or about 100% of the difference between the ELNN sequence and the corresponding sequence of Table 3b involve deletion of a glutamic acid residue, insertion of a glutamic acid residue, substitution of a glutamic acid residue, or substitution for a glutamic acid residue, or any combination thereof. In some such embodiments, at least 80%. at least 90%, at least 95%, at least 97%, or about 100% of the difference betw een the ELNN sequence and the corresponding sequence of Table 3b involve a substitution of a glutamic acid residue, or a substitution for a glutamic acid residue, or both.

[0213] The "a substitution of a first amino acid,” as used herein, refers to replacement of the first amino acid residue with a second amino acid residue, resulting in the second amino acid residue taking its place at the substitution position in the obtained sequence. For example, “a substitution of glutamic acid” refers to replacement of the glutamic acid (E) residue for a non-glutamic acid residue (e.g., serine (S)).Table 3b. Exemplary Existing ELNNs for Engineering into Barcoded ELNN(s)

[0214] In some embodiments, for constructing the sequence of a barcoded ELNN, amino-acid mutations are performed on ELNN of intermediate lengths to those of Table 3b, as well as ELNN of longer lengths than those of Table 3b, such as those in which one or more 12-mer motifs of Table lb are added to the N- or C- terminus of a general-purpose ELNN of Table 3b.

[0215] Additional examples of existing ELNNs that can be used according to the present disclosure are disclosed in U.S. Patent Publication Nos. 2010 / 0239554, 2010 / 0323956, 2011 / 0046060, 2011 / 0046061. 2011 / 0077199, or 2011 / 0172146. or International Patent Publication Nos. WO 2010091122. WO 2010144502. WO 2010144508, WO 2011028228, WO 2011028229, WO2011028344, WO 2014 / 011819, WO 2015 / 023891, WO 2017 / 040344, WO 2019 / 126576, WO 2020 / 264200; WO 2020 / 264208; WO 2021 / 097186, WO 2021 / 262985, or WO 2021 / 263058.

[0216] In some embodiments, a barcoded ELNN fused within a polypeptide chain adjacent to the N- terminus of the polypeptide chain (“N-terminal ELNN”) can be attached to a His tag of HHHHHH (SEQ ID NO: 48) or HHHHHHHH (SEQ ID NO: 49) at the N-terminus to facilitate the purification of the fusion polypeptide. In some embodiments, a barcoded ELNN fused within a polypeptide chain at the C -terminus of the polypeptide chain (“C-tenninal ELNN”) can be comprise or be attached to the sequence EPEA at the C-terminus to facilitate the purification of the fusion polypeptide. In some embodiments, the fusion polypeptide comprises both an N-tcnninal barcoded ELNN and a C-tcnninal barcoded ELNN, wherein the N-terminal barcoded ELNN is attached to a His tag of HHHHHH (SEQ ID NO: 48) or HHHHHHHH (SEQ ID NO: 49) at the N-terminus; and wherein the C-terminal barcoded ELNN is attached to the sequence EPEA at the C-terminus, thereby facilitating purification of the fusion polypeptide, for example, to at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% purity by chromatography methods known in the art, including but not limited to IMAC chromatography, C-tagXL affinity matrix, and other such methods.

[0217] A barcode fragment, as described herein, can be cleavably fused within the ELNN and releasable (i.e., configured to be released) from the ELNN upon digestion of the polypeptide by a protease. In some embodiments, the protease is a Glu-C protease. In some embodiments, the protease cleaves on the C-terminal side of glutamic acid residues diat are not followed by proline. In some embodiments, a barcoded ELNN (an ELNN that contains barcode fragment(s) therewithal) is designed to achieve high efficiency, precision and accuracy of the protease digestion. For example, in some embodiments, adjacent Glu-Glu (EE) residues in an ELNN sequence can result in varying cleavage patterns upon Glu-C digestion. Accordingly, when Glu-C protease is used for barcode release, the barcoded ELNN or the barcode fragment(s) may not contain any Glu-Glu (EE) sequence. Additionally, a di-peptide Glu-Pro (EP) sequence, if present in the fusion polypeptide, may not be cleaved by Glu-C protease during the barcode release process.ACTIVATABLE CYTOKINE FUSION PROTEINS

[0218] In one aspect, the disclosure provides a fusion protein comprising: a cytokine (e g., an interleukin, a transforming growth factor, an interferon, a tumor necrosis factor, a chemokine, or granulocyte macrophage-colony stimulating factor), a linker (Linkerl) comprising a protease- cleavable release segment (RSI), and a mask polypeptide (Maskl). wherein the linker (Linkerl) comprising the release segment (RSI) is positioned between the cytokine and the mask polypeptide (Maskl); wherein the release segment is capable of being cleaved by at least one protease that is present in a tumor; and wherein the release segment (RSI) is not capable of being cleaved by legumain in human plasma,.

[0219] In another aspect, the disclosure provides a fusion protein comprising: a cytokine (e.g., an interleukin, a transforming growth factor, an interferon, a tumor necrosis factor, a chemokine, or granulocyte macrophage-colony stimulating factor), a linker (Linkerl) comprising a protease- cleavable release segment (RSI), and a mask polypeptide (Maskl), wherein the linker (Linkerl) comprising the release segment (RSI) is positioned between the cytokine and the mask polypeptide (Maskl); wherein the release segment is capable of being cleaved by at least one protease that is present in a tumor; and wherein the release segment (RSI) is cleaved by legumain in human plasma at a rate that is less than 25% of the rate that EAGRSANHTPAGLTGP (RSR-2295; SEQ ID NO: 7048) is cleaved by legumain in human plasma.

[0220] In yet another aspect, the disclosure provides a fusion protein comprising: a cytokine (e.g., an interleukin, a transforming growth factor, an interferon, a tumor necrosis factor, a chemokine, or granulocyte macrophage-colony stimulating factor), a linker (Linkerl) comprising a protease- cleavable release segment (RSI), and a mask polypeptide (Maskl), wherein the linker (Linkerl) comprising the release segment (RSI) is positioned between the cytokine and the mask polypeptide (Maskl); wherein the release segment is capable of being cleaved by at least one protease that is present in a tumor; and wherein the release segment (RSI) comprises an amino acid sequence comprising the sequence EAGRSAXHTPAGLTGP. wherein X is any amino acid other than N (SEQ ID NO:7627).

[0221] In some embodiments, the linker is attached to the N-tenninus of the cytokine. In some embodiments, the linker is attached to the C -terminus of the cytokine.

[0222] In another aspect, the disclosure provides a fusion protein comprising the following elements in an N-to-C or C-to-N terminal orientation: a first mask polypeptide (Maskl), a first linker (Linkerl) comprising a first protease-cleavable release segment (RSI), a cytokine (e.g., an interleukin, a transforming growth factor, an interferon, a tumor necrosis factor, a chemokine, or granulocyte macrophage-colony stimulating factor), a second linker (Linker2) comprising a second protease- clcavablc release segment (RS2), and a second mask polypeptide (Mask2), wherein each release segment (RSI and RS2) (i) is not capable of being cleaved by legumain in human plasma, (ii) is cleaved by legumain in human plasma at a rate that is less than 25% of the rate that RSR-2295 (EAGRSANHTPAGLTGP; SEQ ID NO: 7048) is cleaved by legumain in human plasma, or (iii) comprises an amino acid sequence comprising the sequence EAGRSAXHTPAGLTGP, wherein X is any amino acid other than N (SEQ ID NO:7627).

[0223] Cytokines that may be incorporated in the fusion proteins of the disclosure may be composed of more than one subunit polypeptide. For example, IL-12 is composed of an IL-12 p35 polypeptide subunit and an IL-12 p40 polypeptide subunit that come together to form IL-12. Similarly, IL-23 is composed of an IL-23 pl9 polypeptide subunit and an IL- 12 p40 polypeptide subunit that cometogether to form IL -23. Thus, in some embodiments, the fusion protein of the disclosure comprises a first subunit polypeptide comprising a first subunit of a cytokine, and a second subunit polypeptide comprising a second subunit of the cytokine.

[0224] In one aspect, the disclosure provides a fusion protein comprising: a. a first polypeptide submit comprising the following elements in an N-to-C or C-to-N terminal orientation: i. a first mask polypeptide (Maskl, ii. a first linker (Linkerl) comprising a first protease-cleavable release segment (RSI), iii. a first cytokine subunit polypeptide, iv. a second linker (Linker2) comprising a second protease-cleavable release segment (RS2), and v. a second mask polypeptide (Mask2); and b. a second polypeptide submit comprising the following elements in an N-to-C or C-to-N terminal orientation: i. a third mask polypeptide (Mask3), ii. a third linker (Linker3) comprising a third protease-cleavable release segment (RS3), iii. a second cytokine subunit polypeptide, iv. a fourth linker (Linker4) comprising a fourth protease-cleavable release segment (RS4), and v. a fourth mask polypeptide (Mask4); wherein the first cytokine submit polypeptide of the first polypeptide subunit and the second cytokine subunit polypeptide of the second polypeptide submit are linked thereby forming a linked heterodimer; wherein each release segment (RSI, RS2, RS3, and RS4) is capable of being cleaved by at least one protease that is present in a tumor; and wherein each release segment (RSI, RS2, RS3, and RS4) (i) is not capable of being cleaved by legumain in human plasma, (ii) is cleaved by legumain in human plasma at a rate that is less than 25% of the rate that RSR-2295 (EAGRSANHTPAGLTGP; SEQ ID NO:7048) is cleaved by legumain in human plasma, or (iii) comprises an amino acid sequence comprising the sequence EAGRSAXHTPAGLTGP, wherein X is any amino acid other than N (SEQ ID NO:7627).

[0225] In another aspect, the disclosure provides a fusion protein comprising: a. a first polypeptide subunit comprising the following elements in an N-to-C or C-to-N terminal orientation: i. a first mask polypeptide (Maskl), ii. a first linker (Linkerl) comprising a first protcasc-clcavablc release segment (RSI), iii. an IL-12 p35 polypeptide, iv. a second linker (Linker2) comprising a second protease-cleavable release segment (RS2), and v. a second mask polypeptide (Mask2); and b. a second polypeptide subunit comprising the following elements in an N-to-C or C-to-N tenninal orientation: i. a third mask polypeptide (Mask3). ii. a third linker (Linker3) comprising a third protease-cleavable release segment (RS3).iii. an IL- 12 p40 polypeptide, iv. a fourth linker (Linker4) comprising a fourth protease-cleavable release segment (RS4), and v. a fourth mask polypeptide (Mask4); wherein the IL-12 p35 polypeptide of the first polypeptide subunit and the IL-12 p40 polypeptide of the second polypeptide subunit are disulfide-linked thereby forming a disulfide-linked heterodimer; wherein each release segment (RSI, RS2, RS3, and RS4) is capable of being cleaved by at least one protease that is present in a tumor; and wherein each release segment (RSI, RS2, RS3, and RS4) (i) is not capable of being cleaved by legumain in human plasma, (ii) is cleaved by legumain in human plasma at a rate that is less than 25% of the rate that RSR-2295 (EAGRSANHTPAGLTGP; SEQ ID NO: 7048) is cleaved by legumain in human plasma, or (iii) comprises an amino acid sequence comprising the sequence EAGRSAXHTPAGLTGP, wherein X is any amino acid other than N (SEQ ID NO:7627).

[0226] In some embodiments, the first mask polypeptide (Maskl) comprises a first a first barcode fragment (Barcode 1), the second mask polypeptide (Mask2) comprises a second barcode fragment (Barcode2), the third mask polypeptide (Mask3) comprises a third barcode fragment (Barcode3), and the fourth mask polypeptide (Mask4) comprises a fourth barcode fragment (Barcodc4); and wherein each barcode fragment (Barcode 1, Barcode2, Barcode3, and Barcode4) is releasable from the fusion protein upon digestion with a non-mammalian protease, and each barcode fragment differs in both sequence and molecular weight from all other barcode fragments or peptide fragments that are releasable from the fusion protein upon complete digestion of the fusion protein by the nonmammalian protease.

[0227] In another aspect, the disclosure provides a fusion protein comprising: a. a first polypeptide subunit comprising the following elements in an N-to-C or C-to-N terminal orientation: i. a first mask polypeptide (Maskl) comprising a first barcode fragment (Barcode 1). ii. a first linker (Linkerl) comprising a first protease-cleavable release segment (RSI), iii. an IL-12 p35 polypeptide, iv. a second linker (Linker2) comprising a second protease-cleavable release segment (RS2), and v. a second mask poly peptide (Mask2) comprising a second barcode fragment (Barcode2); andb. a second polypeptide subunit comprising the following elements in an N-to-C or C-to-N terminal orientation: i. a third mask polypeptide (Mask3) comprising a third barcode fragment (Barcode3), ii. a third linker (Linker3) comprising a third protease-cleavable release segment (RS3). iii. an IL- 12 p40 polypeptide. iv. a fourth linker (Linker4) comprising a fourth protease-cleavable release segment (RS4), and v. a fourth mask polypeptide (Mask4) comprising a fourth barcode fragment (Barcode4); wherein the IL-12 p35 polypeptide of the first subunit and the IL-12 p40 polypeptide of the second unit are disulfide-linked thereby forming a disulfide-linked heterodimer; wherein each release segment (RSI. RS2, RS3. and RS4) is capable of being cleaved by at least one protease that is present in a tumor; and wherein each barcode fragment (Barcode 1, Barcode2, Barcode3, and Barcode4) is releasable from the fusion protein upon digestion with a non-mammalian protease, and each barcode fragment differs in both sequence and molecular weight from all other barcode fragments or peptide fragments that are releasable from the fusion protein upon complete digestion of the fusion protein by the non-mammalian protease.

[0228] In some embodiments, each release segment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%. 94%, 95%, 96%, 97%. 98%, or 99% identity, or 100% identity, to a sequence of Table 6a.

[0229] In some embodiments, each release segment (RSI, RS2, RS3. and RS4) (i) is not capable of being cleaved by legumain in human plasma, (ii) is cleaved by legumain in human plasma at a rate that is less than 25% of the rate that RSR-2295 (EAGRSANHTPAGLTGP; SEQ ID NO:7048) is cleaved by legumain in human plasma, or (iii) comprises an amino acid sequence comprising the sequence EAGRSAXHTPAGLTGP, wherein X is any amino acid other than N (SEQ ID NO:7627).

[0230] It will be appreciated that various amino acid substitutions (especially conservative amino acid substitutions) can be made in a fusion protein sequence to create variants without departing from the spirit of the present disclosure with respect to the biological activity or pharmacologic properties of. e.g., a protease-activable cytokine fusion protein. Examples of conservative substitutions for amino acids in polypeptide sequences are shown in Table 4a. In addition, variants can also include, for instance, polypeptides wherein one or more amino acid residues are added or deleted at the N- or C-tenninus of the full-length native amino acid sequence of a fusion protein that retains at least a portion of the biological activity of the native peptide.Table 4a: Exemplary conservative amino acid substitutionsSPACERS & C YTOKINE RELEASE SEGMENTS

[0231] Included herein arc fusion proteins comprising a cytokine that either becomes biologically active or has an increase in biological activity upon release from an ELNN by cleavage of an optional cleavage sequence incorporated within optional spacer sequences into the fusion protein, e.g., as described herein.

[0232] In some embodiments, the spacer may be provided to enhance expression of the fusion protein from a host cell and / or to decrease steric hindrance such that the cytokine may assume its desired tertiary structure and / or interact appropriately with its target molecule. For spacers and methods of identifying desirable spacers, see, for example, George, et al. (2003) Protein Engineering 15:871-879, specifically incorporated by reference herein. In some embodiments, the spacer comprises one or more peptide sequences that arc between 1 to 50 amino acid residues in length, or about 1 to 25 residues, or about 1 to 10 residues in length. Spacer sequences, exclusive of cleavage sites, can comprise any of the 20 natural L amino acids, and will preferably comprise hydrophilic amino acids that are sterically unhindered that can include, but not be limited to, glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) or proline (P). In some embodiments, the spacer can be a poly glycine or polyalanine, or predominately a mixture of combinations of glycine and alanine residues. In some embodiments, the spacer polypeptide exclusive of a cleavage sequence is substantially devoid of secondary structure. In some embodiments, one or both spacer sequences in a fusion protein composition may each further contain a cleavage sequence, which may be identical or may be different, wherein the cleavage sequence may be acted on by a protease to release the cytokine from the fusion protein.Table 4b: Exemplary Spacers between a Release Segment and a Cytokine

[0233] In some embodiments of the polypeptides of this disclosure, a release segment (RS) (e.g., a first release segment (RSI), a second release segment (RS2), a third release segment (RS3), a fourth release segment (RS4), etc.) can be fused to a cytokine by a spacer. In some embodiments, a spacer can (each independently) comprise at least 4 types of amino acids that are glycine (G), alanine (A), serine (S). threonine (T), glutamate (E) or proline (P). In some embodiments, the fusion protein of this disclosure can comprise a first release segment fused to the cytokine via a first spacer and a second release segment fused to the cytokine via a second spacer. In some embodiments, a spacer (e.g., a first spacer, a second spacer, etc.) can (each independently) comprise an amino acid sequence having at least (about) 80%. at least (about) 90%. or 100% sequence identity to a sequence set forth in Table C. In some embodiments, the spacer (e.g., the first spacer, the second spacer, etc.) can (each independently) comprise an amino acid sequence identical to a sequence set forth in Table C.

[0234] In some embodiments, the incorporation of the cleavage sequence into a fusion protein is designed to permit release of a cytokine that becomes active or more active upon its release from one or more ELNNs. In some embodiments, the cleavage sequences are located sufficiently close to the cytokine sequences, generally within 18, or within 12, or within 6, or within 2 amino acids of the cytokine sequence terminus, such that any remaining residues attached to the cytokine after cleavage do not appreciably interfere with the activity (e.g.. such as binding to a receptor) of the cytokine yet provide sufficient access to the protease to be able to effect cleavage of the cleavage sequence. In some embodiments, the cleavage site is a sequence that can be cleaved by a protease endogenous to the mammalian subject such that a fusion protein can be cleaved after administration to a subject. In such cases, the fusion protein can serve as a circulating depot for the cytokine. Examples of cleavage sites contemplated herein include, but are not limited to, a polypeptide sequence cleavable by a mammalian endogenous protease listed in Table 5.

[0235] In some embodiments, a fusion protein comprises spacer sequences that comprise one or more cleavage sequences configured to release the cytokine from the fusion protein when acted on by a protease. In some embodiments, a spacer sequence does not comprise a cleavage sequence. In some embodiments, the one or more cleavage sequences can be a sequence having at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 96%. at least about 97%, at least about 98%. at least about 99%, or 100%) sequence identify to a sequence from Table 6a or 6b.

[0236] In some embodiments, the disclosure provides cytokine release segment polypeptides (or release segments (RSs)) that are substrates for one or more mammalian proteases associated with or produced by disease tissues or cells found in proximity to disease tissues. The one or more mammalian proteases are present in a tumor. The one or more mammalian proteases may be expressed by cancer cells within the tumor or by non-cancerous cells within the tumor. Such proteases can include, but not be limited to the classes of proteases such as metalloproteinases, cysteine proteases, aspartate proteases, and serine proteases, including, but not limited to, the proteases of Table 5. The RSs are useful for, amongst other things, incorporation into the subject recombinant polypeptides, conferring an inactive fonnat that can be activated by the cleavage of the RSs by mammalian proteases. As described herein, the RSs are incorporated into the subject recombinant polypeptide compositions, linking the incorporated cytokines to the ELNN (exemplar,' configurations of which are described herein) such that upon cleavage of the RSs by action of the one or more proteases for which the RSs are substrates, the cytokines and ELNN are released from the composition and the cytokines, no longer shielded by the ELNN. regain their full potential to bind their ligands.Table 5: Proteases of Target Tissues

[0237] In some embodiments, the disclosure provides activatable recombinant polypeptides (e.g., fusion proteins) comprising a first release segment (RSI) sequence having at least 88%, or at least94%, or 100% sequence identity’, when optimally aligned, to a sequence identified in Table 6a, wherein the RSI is a substrate for one or more mammalian proteases. In some embodiments, the RS is further engineered to remove a legumain cleavage site. In some embodiments, the disclosure provides activatable recombinant polypeptides comprising a RSI and a second release segment (RS2) sequence, each having at least 88%, or at least 94%, or 100% sequence identity, when optimally aligned, to a sequence identified herein by the sequences set forth in Table 6a, wherein the RSI and the RS2 each are a substrate for one or more mammalian proteases. In some embodiments, the disclosure provides activatable recombinant polypeptides comprising a RSI, a RS2, a third release segment (RS3) sequence, and a fourth release segment sequence (RS4). each having at least 88%, or at least 94%, or 100% sequence identity, when optimally aligned, to a sequence identified herein by the sequences set forth in Table 6a. wherein the RSI, the RS2, the RS3. and the RS4 each are a substrate for one or more mammalian proteases. In some embodiments, the RSI, the RS2. the RS3, and the RS4 each do not serve as substrates for legumain.

[0238] In some embodiments, disclosure provides activatable recombinant polypeptides comprising a first RS (RSI) sequence having at least 90%, at least 93%, at least 97%, or 100% identity, when optimally aligned, to a sequence identified in Table 6b, wherein the RSI is a substrate for one or more mammalian proteases. In some embodiments, the disclosure provides activatable recombinant polypeptides comprising a RSI and a second release segment (RS2) sequence, each having at least 88%, or at least 94%, or 100% sequence identity', when optimally aligned, to a sequence identified herein by the sequences set forth in Table 6b, wherein the RSI and the RS2 are each a substrate for one or more mammalian proteases (e.g., at one, two. or three cleavage sites within each release segment sequence). In some embodiments, the disclosure provides activatable recombinant polypeptides comprising a RSI. a RS2, a third release segment (RS3) sequence, and a fourth release segment sequence (RS4). each having at least 88%, or at least 94%. or 100% sequence identity, when optimally aligned, to a sequence identified herein by the sequences set forth in Table 6b, wherein the RSI, the RS2, the RS3. and the RS4 are each a substrate for one or more mammalian proteases (e g., at one, two, or three cleavage sites within each release segment sequence). In some embodiments of activatable recombinant polypeptides comprising the RSI, the RS2. the RS3, and the RS4, the four release segments can be identical. In some embodiments of activatable recombinant polypeptides comprising the RSI, the RS2, the RS3, and the RS4 , the four release segments can be different.

[0239] The present disclosure contemplates release segments that are substrates for one, two or three different classes of proteases that are metalloproteinases, cysteine proteases, aspartate proteases, or serine proteases, including the proteases of Table 5. In some embodiments, a fusion protein comprises RSs (e g., RSI, RS2, RS3, and RS4) that serve as substrates for one or more proteases found in close association with or are co-localized with tumors or cancer cells, and upon cleavage of the RSs, the cytokines that are otherwise shielded by ELNNs of the fusion protein (and thus have a lower bindingaffinity for their respective ligands) are released from the ELNNs and regain their full potential to bind target and effector cell ligands. In some embodiments, a fusion protein comprises RSs (e.g., RSI and RS2), that each comprise an amino acid sequence that is a substrate for one or more cellular proteases located within a targeted cell, including but not limited to a protease of Table 5. In some embodiments, RSs are substrates for two or three classes of proteases that cleave different portions of each RS. In some embodiments, each RS that is a substrate for two. three, or more classes of proteases has two, three, or more distinct cleavage sites, but cleavage by a single protease nevertheless results in the release of the cytokines from an ELNN.

[0240] In some embodiments, an RS of the disclosure for incorporation into a fusion protein is a substrate for one or more proteases including but not limited to meprin, neprilysin (CD10), PSMA, BMP-1, A disintegrin and metalloproteinases (ADAMs), ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 (TACE), ADAM19, ADAM28 (MDC-L), ADAM with thrombospondin motifs (AD AMTS), ADAMTS1, ADAMTS4, ADAMTS5, MMP-1 (collagenase 1), matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-2 (MMP-2, gelatinase A), matrix metalloproteinase-3 (MMP-3, stromelysin 1), matrix metalloproteinase-7 (MMP-7, Matrilysin 1), matrix metalloproteinase-8 (MMP-8, collagenase 2). matrix metalloproteinase-9 (MMP-9, gelatinase B), matrix metalloproteinase- 10 (MMP-10, stromelysin 2), matrix metalloproteinase- 11 (MMP-11, stromelysin 3), matrix metalloproteinase- 12 (MMP-12, macrophage elastase), matrix metalloproteinase- 13 (MMP-13, collagenase 3). matrix metalloproteinase- 14 (MMP-14. MT1-MMP), matrix metalloproteinase- 15 (MMP-15. MT2-MMP), matrix metalloproteinase- 19 (MMP-19). matrix metalloproteinase-23 (MMP-23, CA-MMP), matrix metalloproteinase-24 (MMP-24, MT5-MMP). matrix metalloproteinase-26 (MMP-26, matrilysin 2), matrix metalloproteinase-27 (MMP-27, CMMP). legumain. cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin X. cathepsin D, cathepsin E. secretase, urokinase (uPA). tissue-type plasminogen activator (tPA), plasmin, thrombin, prostate-specific antigen (PSA. KLK3). human neutrophil elastase (HNE), elastase, tryptase, Type II transmembrane serine proteases (TTSPs). DESCI, hepsin (HPN), matriptase, matriptase-2, TMPRSS2, TMPRSS3, TMPRSS4 (CAP2), fibroblast activation protein (FAP), kallikrein-related peptidase (KLK family), KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and KLK14. In some embodiments, the RS is a substrate for ADAMI 7. In some embodiments, the RS is a substrate for BMP-1. In some embodiments, the RS is a substrate for cathepsin. In some embodiments, the RS is a substrate for HtrAl. In some embodiments, the RS is a substrate for legumain. In some embodiments, the RS is a substrate for MMP-1. In some embodiments, die RS is a substrate for MMP-2. In some embodiments, the RS is a substrate for MMP-7. In some embodiments, the RS is a substrate for MMP-9. In some embodiments, the RS is a substrate for MMP-11. In some embodiments, the RS is a substrate for MMP-14. In some embodiments, die RS is a substrate for uPA. In some embodiments, die RS is a substrate formatriptase. In some embodiments, the RS is a substrate for MT-SP1. In some embodiments, the RS is a substrate for neutrophil elastase. In some embodiments, the RS is a substrate for thrombin. In some embodiments RS is a substrate for TMPRSS3. In some embodiments, the RS is a substrate for TMPRSS4. In some embodiments, the RS of the subject recombinant polypeptide compositions is a substrate for at least two proteases including but not limited to legumain, MMP-1, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase. In some embodiments, the RS of the subject recombinant polypeptide compositions is a substrate for legumain, MMP-1. MMP-2, MMP-7, MMP- 9. MMP-11. MMP-14. uPA, and matriptase. In specific embodiments, the RS of the subject recombinant polypeptide compositions is not a substrate for legumain. In some embodiments, the RS of the subject recombinant polypeptide compositions is a substrate for uPA. matriptase (also known as MT-SP1 and ST14). MMP2. MMP7, MMP9, and MMP14. In some embodiments, the RS of the subject recombinant polypeptide compositions is substrate for uPA, matriptase, MMP2, MMP7, MMP9. and MMP14 but not legumain.Table 6a: Fusion Protein Release Segment Sequences.Table 6b: Release Segment Sequences

[0241] In some embodiments, a fusion protein comprises an RSI and an RS2 that have different rates of cleavage and different cleavage efficiencies to multiple proteases for which they are substrates. As a given protease may be found in different concentrations in a tumor, compared to healthy tissues or in circulation, the disclosure provides RSs that have a higher or lower cleavage efficiency for a given protease in order to ensure that a fusion protein is preferentially converted from the inactive form to the active form (i.e., by the separation and release of the cytokines and ELNNs from the fusion protein after cleavage of the RSs) when in proximity to the cancer cell or tissue and its co-localized proteases compared to the rate of cleavage of the RSs in healthy tissue or the circulation such that the released cytokine of the fusion proteins have a greater ability to bind to ligands in the tumor compared to the inactive form that remains in circulation. By such selective designs, the therapeutic index of the resulting compositions can be improved, resulting in reduced side effects relative to convention therapeutics that do not incorporate such site-specific activation.

[0242] In some embodiments, cleavage efficiency is the log2 value of the ratio of the percentage of the test substrate comprising the RS cleaved to the percentage of the control substrate AC 1611 cleaved when each is subjected to the protease enzyme in biochemical assays in which reaction in conducted wherein the initial substrate concentration is 6 pM, the reactions are incubated at 37°C for 2 hours before being stopped by adding EDTA. with the amount of digestion products and uncleaved substrate analyzed by non-reducing SDS-PAGE to establish the ratio of the percentage cleaved. TheThus, a cleavage efficiency of -1 means that the amount of test substrate cleaved was 50% compared to that of the control substrate, while a cleavage efficiency of +1 means that the amount of testsubstrate cleaved was 200% compared to that of tire control substrate. A higher rate of cleavage by die test protease relative to the control would result in a higher cleavage efficiency, and a slower rate of cleavage by the test protease relative to the control would result in a lower cleavage efficiency. A control RS sequence AC1611 (RSR-1517), having the amino acid sequence EAGRSANHEPLGLVAT (SEQ ID NO: 7001), was established as having an appropriate baseline cleavage efficiency by the proteases legumain, MMP-2, MMP-7, MMP-9, MMP-14, uPA, and matriptase, when tested in in vitro biochemical assays for rates of cleavage by the individual proteases. By selective substitution of amino acids at individual locations in the RS peptides, libraries of RS were created and evaluated against the panel of the 7 proteases, resulting in profiles that were used to establish guidelines for appropriate amino acid substitutions in order to achieve RS with desired cleavage efficiencies. In some embodiments, in making RSs with desired cleavage efficiencies, substitutions using the hydrophilic amino acids A. E, G. P. S, and T are preferred, however other L-amino acids can be substituted at given positions in order to adjust the cleavage efficiency so long as the RSs retain at least some susceptibility to cleavage by a given protease. Conservative substitutions of amino acids in a peptide to retain or effect activity is well within the knowledge and capabilities of a person within skill in the art. In some embodiments, the disclosure provides an RS in which the RS is cleaved by a protease including but not limited to MMP-2, MMP- 7, MMP-9, MMP-14. uPA, or matriptase (also known as MT-SP1) with at least a 0.2 log2, or 0.4 log2. or 0.8 log2, or 1.0 log2 higher cleavage efficiency in an in vitro biochemical competitive assay compared to tire cleavage by the same protease of a control sequence RSR-1517 having the sequence EAGRSANHEPLGLVAT (SEQ ID NO: 7001). In some embodiments, the disclosure provides an RS in which the RS is cleaved by a protease including but not limited to MMP-2, MMP-7, MMP-9, MMP-11, uPA, or matriptase with at least a 0.2 log2, or 0.4 log2, or 0.8 log2, or 1.0 log2 lower cleavage efficiency in an in vitro biochemical competitive assay compared to the cleavage by the same protease of a control sequence RSR-1517 having the sequence EAGRSANHEPLGLVAT (SEQ ID NO: 7001). In some embodiments, the disclosure provides an RS in which the rate of cleavage of the RS by a protease including but not limited to MMP-2, MMP-7, MMP-9, MMP-14, uPA, or matriptase is at least 2-fold, or at least 4-fold, or at least 8 fold, or at least 16-fold faster compared to the control sequence RSR-1517 having the sequence EAGRSANHEPLGLVAT (SEQ ID NO: 7001). In some embodiments, the disclosure provides an RS in which the rate of cleavage of the RS by a protease including but not limited to MMP-2, MMP-7. MMP-9, MMP-14, uPA. or matriptase is at least 2-fold, or at least 4-fold, or at least 8-fold, or at least 16-fold slower compared to the control sequence RSR-1517 having the sequence EAGRSANHEPLGLVAT (SEQ ID NO: 7001).

[0243] In some embodiments, the RS comprises the amino acid sequence EAGRSAXHTPAGLTGP(SEQ ID NO: 7627), wherein X is any amino acid other than N. In some embodiments, X is S. In some embodiments, X is T. In some embodiments, X is Y. In some embodiments, X is Q. In someembodiments, X is G. In some embodiments, X is A. In some embodiments, X is V. In some embodiments, X is C. In some embodiments, X is P. In some embodiments, X is L. In some embodiments, X is I. In some embodiments, X is M. In some embodiments, X is F. In some embodiments, X is K. In some embodiments, X is R. In some embodiments, X is H. In some embodiments, X is D. In some embodiments, X is E. In some embodiments, the RS is not cleaved by legumain. In some embodiments, the RS is not cleavable by legumain in human blood, plasma, or serum. Advantageously, the RS amino acid sequence EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N, eliminates a glycosylation site on the sequence. Removal of said glycosylation site may improve accessibility of the release site to be acted upon by the tumor associate protease. Removal of said glycosylation site may improve expression of the fusion protein harboring said RS sequence.

[0244] In some embodiments, the RS is not cleavable upon incubation with about InM or less legumain for about 20 hours. In some embodiments, the RS is cleaved by legumain less quickly or efficiently than RSR-2295 (EAGRSANHTPAGLTGP; SEQ ID NO: 7048) is cleaved by legumain. In some embodiments, the RS is cleaved by legumain at a rate that is less than about 50% of the rate that legumain cleaves RSR-2295 (EAGRSANHTPAGLTGP; SEQ ID NO:7048). In some embodiments, the RS is cleaved by legumain at a rate that is less than about 25% of the rate that legumain cleaves RSR-2295. In some embodiments, the RS is cleaved by legumain at a rate that is less than about 10% of the rate that legumain cleaves RSR-2295. In some embodiments, the RS is cleaved by legumain at a rate that is less than about 5% of the rate that legumain cleaves RSR-2295. In some embodiments, the RS is cleaved by legumain at a rate that is less than about 2.5% of the rate that legumain cleaves RSR-2295.

[0245] In some embodiments, the RS is cleaved by legumain at a rate that is less than about 50% of the rate that legumain cleaves RSR-2295 (EAGRSANHTPAGLTGP: SEQ ID NO: 7048) in human plasma. In some embodiments, the RS is cleaved by legumain at a rate that is less than about 25% of the rate that legumain cleaves RSR-2295 in human plasma. In some embodiments, the RS is cleaved by legumain at a rate that is less than about 10% of the rate that legumain cleaves RSR-2295 in human plasma. In some embodiments, the RS is cleaved by legumain at a rate that is less than about 5% of the rate that legumain cleaves RSR-2295 in human plasma. In some embodiments, the RS is cleaved by legumain at a rate that is less than about 2.5% of the rate that legumain cleaves RSR-2295 in human plasma.

[0246] In some embodiments, the disclosure provides fusion proteins comprising multiple RSs wherein each RS sequence is identified herein by the group of sequences set forth in Table 6a and the RSs are linked to each other by 1 to 6 amino acids that are glycine, serine, alanine, and threonine. In some embodiments, a fusion protein comprises a first RS and a second RS different from the first RS wherein each RS sequence is identified herein by a sequence set forth in Table 6a and the RSs arelinked to each other by 1 to 6 amino acids that are glycine, serine, alanine, and threonine. In some embodiments, the fusion protein comprises a first RS, a second RS different from the first RS, and a third RS different from the first and the second RS wherein each sequence is identified herein by s sequence set forth in Table 6a and tire first and the second and the third RS are linked to each other by 1 to 6 amino acids that are glycine, serine, alanine, and threonine. In some embodiments, multiple RS of the fusion protein can be concatenated to form a sequence that can be cleaved by multiple proteases at different rates or efficiency of cleavage. In some embodiments, the disclosure provides a fusion protein comprising an RSI and an RS2. wherein each has a sequences set forth in Table 6a or 6b and ELNNs (e.g., an ELNN1 and ELNN2), such as those described herein, wherein the RSI is fused between the ELNN1 and the cytokines and the RS2 is fused between the ELNN2 and the cytokines. In some embodiments, a fusion protein is more readily cleaved in target tissues that express multiple proteases (e.g., tumor tissues), compared with healthy tissues or when in the normal circulation, with the result that the resulting fragments bearing the cytokines would more readily penetrate the target tissue; e.g.. a tumor, and have an enhanced ability to bind and link the cancer cell and the effector cell.

[0247] In some embodiments, a fusion protein comprises a first release segment (RSI) positioned between a first ELNN and a cytokine. In some embodiments, the polypeptide further comprises a second release segment (RS2) positioned between the cytokine and a second ELNN. In some embodiments. RSI and RS2 are identical in sequence. In some embodiments, RSI and RS2 are not identical in sequence. In some embodiments, the RSI comprises an amino acid sequence having at least 85%, 90%. 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%. 99% or 100% sequence identity to a sequence identified herein in Table 6a or 6b or a subset thereof. In some embodiments, the RS2 comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence identified herein in Table 6a or 6b or a subset thereof. In some embodiments, the RSI and RS2 are each a substrate for cleavage by multiple proteases at one, two. or three cleavage sites within each release segment sequence.

[0248] In some embodiments, the fusion protein further comprises one or more reference fragments (e.g., barcode fragments) releasable from the fusion protein upon digestion by the protease. In some embodiments, the one or more reference fragments is a single reference fragment that differs in sequence and molecular weight from all other peptide fragments that are releasable from the polypeptide upon digestion of the polypeptide by the protease.Exemplary Fusion Proteins

[0249] In some embodiments, a fusion protein comprises an amino acid sequence having at least (about) 80% sequence identity to a sequence set forth in Table Al and Table A2 (SEQ ID NOs: 1000- 1042) or a subset thereof. In some embodiments, the fusion protein comprises an amino acid sequence having at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%, at least (about) 89%, atleast (about) 90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%, at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity to a sequence set forth in SEQ ID NOs: 1000-1042 or a subset thereof. In some embodiments, the fusion protein comprises an amino acid sequence having at least (about) 90%, at least (about) 91%. at least (about) 92%, at least (about) 93%, at least (about) 94%, at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%. at least (about) 99%, or (about) 100% sequence identity to a sequence set forth in SEQ ID NOs: 1000-1042 or a subset thereof. In some embodiments, the fusion protein comprises an amino acid sequence identical to a sequence set forth in SEQ ID NOs: 1000-1042.

[0250] In some embodiments, a fusion protein comprises a first polypeptide subunit comprising an amino acid sequence having at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%, at least (about) 89%, at least (about) 90%, at least (about) 91%, at least (about) 92%. at least (about) 93%, at least (about) 94%, at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity to a sequence set forth in SEQ ID NO: 1000, and a second polypeptide subunit comprising an amino acid sequence having at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%. at least (about) 87%. at least (about) 88%. at least (about) 89%. at least (about) 90%, at least (about) 91%, at least (about) 92%. at least (about) 93%. at least (about) 94%. at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity to a sequence set forth in SEQ ID NO: 1033.

[0251] In some embodiments, a fusion protein comprises a first polypeptide subunit comprising an amino acid sequence having at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%, at least (about) 89%, at least (about) 90%. at least (about) 91%. at least (about) 92%. at least (about) 93%, at least (about) 94%, at least (about) 95%, at least (about) 96%. at least (about) 97%. at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity to a sequence set forth in SEQ ID NO: 1001, and a second polypeptide subunit comprising an amino acid sequence having at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%, at least (about) 89%, at least (about) 90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%, at least (about) 95%. at least (about) 96%. at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity' to a sequence set forth in SEQ ID NO: 1033.

[0252] In some embodiments, a fusion protein comprises a first polypeptide subunit comprising an amino acid sequence having at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%, atleast (about) 89%, at least (about) 90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%, at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity to a sequence set forth in SEQ ID NO: 1002, and a second polypeptide subunit comprising an amino acid sequence having at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%. at least (about) 89%. at least (about) 90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%. at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity' to a sequence set forth in SEQ ID NO: 1033.

[0253] In some embodiments, a fusion protein comprises a first polypeptide subunit comprising an amino acid sequence having at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%, at least (about) 89%, at least (about) 90%, at least (about) 91%, at least (about) 92%. at least (about) 93%, at least (about) 94%, at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity to a sequence set forth in SEQ ID NO: 1003, and a second polypeptide subunit comprising an amino acid sequence having at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%. at least (about) 87%. at least (about) 88%. at least (about) 89%. at least (about) 90%, at least (about) 91%, at least (about) 92%. at least (about) 93%. at least (about) 94%. at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity to a sequence set forth in SEQ ID NO: 1034.

[0254] In some embodiments, a fusion protein comprises a first polypeptide subunit comprising an amino acid sequence having at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%, at least (about) 89%, at least (about) 90%. at least (about) 91%. at least (about) 92%. at least (about) 93%, at least (about) 94%, at least (about) 95%, at least (about) 96%. at least (about) 97%. at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity to a sequence set forth in SEQ ID NO: 1004, and a second polypeptide subunit comprising an amino acid sequence having at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%, at least (about) 89%, at least (about) 90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%, at least (about) 95%. at least (about) 96%. at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity' to a sequence set forth in SEQ ID NO: 1035.

[0255] In some embodiments, a fusion protein comprises a first polypeptide subunit comprising an amino acid sequence having at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%, atleast (about) 89%, at least (about) 90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%, at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity to a sequence set forth in SEQ ID NO: 1005, and a second polypeptide subunit comprising an amino acid sequence having at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%. at least (about) 89%. at least (about) 90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%. at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity' to a sequence set forth in SEQ ID NO: 1036.

[0256] In some embodiments, a fusion protein comprises a first polypeptide subunit comprising an amino acid sequence having at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%, at least (about) 89%, at least (about) 90%, at least (about) 91%, at least (about) 92%. at least (about) 93%, at least (about) 94%, at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity to a sequence set forth in SEQ ID NO: 1000, and a second polypeptide subunit comprising an amino acid sequence having at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%. at least (about) 87%. at least (about) 88%. at least (about) 89%. at least (about) 90%, at least (about) 91%, at least (about) 92%. at least (about) 93%. at least (about) 94%. at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity to any one sequence set forth in SEQ ID NOs: 1033-1036.

[0257] In some embodiments, a fusion protein comprises a first polypeptide subunit comprising an amino acid sequence having at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%, at least (about) 89%, at least (about) 90%. at least (about) 91%. at least (about) 92%. at least (about) 93%, at least (about) 94%, at least (about) 95%, at least (about) 96%. at least (about) 97%. at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity to a sequence set forth in SEQ ID NO: 1001, and a second polypeptide subunit comprising an amino acid sequence having at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%, at least (about) 89%, at least (about) 90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%, at least (about) 95%. at least (about) 96%. at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity' to any one sequence set forth in SEQ ID NOs: 1033-1036.

[0258] In some embodiments, a fusion protein comprises a first polypeptide subunit comprising an amino acid sequence having at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%, atleast (about) 89%, at least (about) 90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%, at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity to a sequence set forth in SEQ ID NO: 1002, and a second polypeptide subunit comprising an amino acid sequence having at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%. at least (about) 89%. at least (about) 90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%. at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity' to any one sequence set forth in SEQ ID NOs: 1033-1036.

[0259] In some embodiments, a fusion protein comprises a first polypeptide subunit comprising an amino acid sequence having at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%, at least (about) 89%, at least (about) 90%, at least (about) 91%, at least (about) 92%. at least (about) 93%, at least (about) 94%, at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity to a sequence set forth in SEQ ID NO: 1003, and a second polypeptide subunit comprising an amino acid sequence having at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%. at least (about) 87%. at least (about) 88%. at least (about) 89%. at least (about) 90%, at least (about) 91%, at least (about) 92%. at least (about) 93%. at least (about) 94%. at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity to any one sequence set forth in SEQ ID NOs: 1033-1036.

[0260] In some embodiments, a fusion protein comprises a first polypeptide subunit comprising an amino acid sequence having at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%, at least (about) 89%, at least (about) 90%. at least (about) 91%. at least (about) 92%. at least (about) 93%, at least (about) 94%, at least (about) 95%, at least (about) 96%. at least (about) 97%. at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity to a sequence set forth in SEQ ID NO: 1004, and a second polypeptide subunit comprising an amino acid sequence having at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%, at least (about) 89%, at least (about) 90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%, at least (about) 95%. at least (about) 96%. at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity' to any one sequence set forth in SEQ ID NOs: 1033-1036.

[0261] In some embodiments, a fusion protein comprises a first polypeptide subunit comprising an amino acid sequence having at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%, atleast (about) 89%, at least (about) 90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%, at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity to a sequence set forth in SEQ ID NO: 1005, and a second polypeptide subunit comprising an amino acid sequence having at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%. at least (about) 89%. at least (about)90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%. at least(about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or(about) 100% sequence identity to any one sequence set forth in SEQ ID NOs: 1033-1036.

[0262] It is specifically contemplated that the compositions of this disclosure can comprise sequence variants of the amino acid sequences set forth in Table A, such as with linker sequence(s) substituted or inserted or with purification tag sequence(s) attached thereto, so long as the variants exhibit substantially similar or same bioactivity / bioactivities and / or activation mechanism (s).Table Al: Exemplary amino acid sequences of IL-12p35 polypeptidesTable A2: Exemplary amino acid sequences of IL-12p40 polypeptidesRECOMBINANT PRODUCTION

[0263] Also provided are polynucleotides that encode any polypeptide disclosed herein and / or the reverse complements of such polynucleotides.

[0264] The disclosure herein includes an expression vector that comprises a polynucleotide sequence, such as any described in the preceding paragraph, and a regulatory sequence operably linked to the polynucleotide sequence.

[0265] The disclosure herein includes a host cell comprising an expression vector, such as described any in the preceding paragraph. In some embodiments, the host cell is a prokaryote. In some embodiments, the host cell is E. coli. In some embodiments, the host cell is a mammalian cell. In some embodiments, the mammalian cell is a mammalian cell line, such as a HEK 293 cell line or a CHO cell line.

[0266] In some embodiments, the disclosure provides methods of manufacturing the subject compositions. In some embodiments, such a method comprises culturing a host cell comprising a nucleic acid construct that encodes a fusion protein described herein under conditions that promote the expression of the fusion protein, followed by recovery of the fusion protein using standard purification methods (e.g., column chromatography, HPLC, and the like) wherein the composition is recovered wherein at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 99% of the fusion proteins are correctly folded. In some embodiments of the method of making, die expressed fusion protein is recovered in which at least or at least 90%, or at least 95%, or at least 97%, or at least 99% of the fusion protein is recovered in monomeric, soluble form.

[0267] In some embodiments, the disclosure relates to methods of making a fusion protein at high fermentation expression levels of functional protein using an E. coli or mammalian host cell, as well as providing expression vectors encoding the fusion protein useful in methods to produce the cytotoxically active fusion protein at high expression levels. In some embodiments, the method comprises the steps of 1) preparing a polynucleotide encoding a fusion protein disclosed herein, 2) cloning the polynucleotide into an expression vector, which can be a plasmid or other vector under the control of appropriate transcription and translation sequences for high level protein expression in a biological system, 3) transforming an appropriate host cell with the expression vector, and 4) culturing the host cell in conventional nutrient media under conditions suitable for the expression of die fusion protein. Where desired, the host cell is E. coli. As used herein, the term “correctly folded’’ means that the cytokine component of the composition has the ability to specifically bind the cytokine target ligand (e.g., a cytokine receptor and upon activation). In some embodiments, the disclosure provides a method for producing a fusion protein, the method comprising culturing in a fermentation reaction a host cell that comprises a vector encoding the fusion protein under conditions effective to express the fusion protein product.PHARMACEUTICAL COMPOSITION

[0268] Disclosed herein includes a pharmaceutical composition comprising a polypeptide (such as a fusion protein disclosed herein), and one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition is formulated for intradermal, subcutaneous, intravenous, intra-arterial, intraabdominal, intraperitoneal, intravitreal, intrathecal, or intramuscular administration. In some embodiments, the pharmaceutical composition is formulated for intravenous injection. In some embodiments, the pharmacal composition is in a liquid fonn or frozen. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder to be reconstituted prior to administration.

[0269] The pharmaceutical compositions can be administered for therapy by any suitable route. In some embodiments, the dose is administered intradermally, subcutaneously, intravenously (e.g., by intravenous bolus or intravenous infusion), intra-arterially, intra-abdominally, intraperitoneally, intrathecally, or intramuscularly. In some embodiments, the subject is a mouse, rat, monkey, or human. In preferred embodiments, the subject is a human.

[0270] In some embodiments, the pharmaceutical composition can be administered subcutaneously, intramuscularly, or intravenously. In some embodiments, the pharmaceutical composition is administered at a therapeutically effective amount. In some embodiments, the therapeutically effective amount results in a gain in time spent within a therapeutic window for the fusion protein compared to the corresponding cytokine of the fusion protein not linked to the ELNN and administered at a comparable dose to a subject.

[0271] In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is administered intravenously.

[0272] In some embodiments, the composition may be supplied as a lyophilized powder or cake to be reconstituted prior to administration. In some embodiments, the composition may also be supplied in a liquid form or frozen, which can be administered directly to a subject.PHARMACEUTICAL KITS

[0273] In some embodiments, the present disclosure provides kits to facilitate the use of the fusion proteins disclosed herein. In some embodiments, a kit comprises (a) a first container comprising pharmaceutically effective amount of the fusion proteins disclosed herein in a lyophilized composition; and (b) a second container comprising a diluent for reconstituting the lyophilized formulation. In some embodiments, the kit further comprises instructions for storage of the kit. information regarding a cancer that is treatable with the fusion proteins disclosed herein, instructions for the reconstitution of the lyophilized formulation, and / or administration instructions.METHODS OF TREATMENT

[0274] Disclosed herein are uses of a polypeptide, such as any described herein, in the preparation of a medicament for the treatment of a disease in a subject. In some embodiments, die particular disease to be treated will depend on die choice of the biologically active proteins. In some embodiments, the disease is cancer. Included herein are polypeptides (e.g., a fusion protein comprising a cytokine, a linker comprising a protease-cleavable release segment, and a mask polypeptide) for use in the treatment of cancer. In some embodiments, the cancer or tumor is a solid tumor.

[0275] The present disclosure includes a method of treating a disease in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of die pharmaceutical composition, such as any described herein. In some embodiments, the disease is cancer. In some embodiments, the subject is a mouse, rat, monkey, or human. In some embodiments, die subject is a human.

[0276] In some embodiments, the disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an amount of the fusion protein described herein to the subject, wherein the cancer comprises a solid tumor, and treating the cancer comprises reducing the volume of the solid tumor.

[0277] The following are examples of compositions and evaluations of compositions of the disclosure. It is understood that various some embodiments may be practiced, given the general description provided above.INCORPORATION BY REFERENCE

[0278] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.EXAMPLESExample 1. Design of protease-activatable cytokines

[0279] This example demonstrates the design of protease -activatable cytokine fusion proteins including one to four masks, where each mask is linked to the cytokine by a linker having a protease- cleavable release segment. Fusion proteins including mouse IL-12 (having a p35 subunit and a p40 subunit) were designed having betw een one and four ELNN masks, with various lengths (as shown in amino acid number (AA)), with the following formats:Table 7. Protease activable cytokine descriptions

[0280] The fusion proteins were expressed in Expi293 cells (ThermoFisher) and purified by a one- step histidine tag-based purification method. Briefly, the subunit constructs were co-transfected into Expi293 cells in a transient manner. Supernatants expressing the protein of interest were harvested 6-7 days post transfection. Protein was purified from the supernatants using a single IMAC step to bind via a poly histidine tag (Histrap™ HP 5ml; Cytiva, 17524801). Unmasked versions of were generated proteolytically using MMP9, which cleaves the inbuilt protease activation sites and effects mask release. For MMP9 digestion, the MMP9 (R&D systems) was used at 20 mM, in 20 mM histidine, 154 mM NaCl, 10 mM CaC12, pH 6.5 buffer with 150nM each protein. Incubation was done at 37 C for four hours. Some of the MMP9 digests were noted to be partial (less than 100% unmasking),suggesting that the actual difference in fold masking may be greater than noted based on this experiment.

[0281] The fusion proteins were assayed for their ability to bind to activate the mouse IL- 12 receptor in both their masked and unmasked forms by HEK Blue reporter assay. HEK-Blue IL-12 reporter cells (InvivoGen) were cultured at 37 C, 5% COz in a culture media consisting of DMEM, 4.5 g / 1 glucose, 2 mM L-glutamine, 10% (v / v) heat-inactivated fetal bovine serum, 100 U / ml penicillin, 100 pg / ml streptomycin, 100 pg / ml Normocin, IX HEK-Blue Selection. For the IL-12 activity assay, a test medium was prepared as described in the immediately preceding sentence but without Normocin and selection antibiotics. The test medium and IX PBS were warmed to 37° C in a water bath. Cells were dislodged from the flask by washing the flask with the pre-warmed PBS, followed by a centrifugation at 300xg (1200 rpm) for 5 mins at room temperature, determination of cell viability, and a resuspension of tire cell pellet in the test medium to 0.833xl0e6 cells / mL. Ninety microliters (90 pL) of the cells were aliquoted into each well of a 96-well flat-clear-bottom plate (Costar, cat#3595). IL-12 test articles were prepared at 10X concentration in the test medium with 17 nM being the highest concentration, followed by a serial 10-fold dilution to 1.7 pM. Then, lOuL of the 10X solution were added to the 90 pL of cells, and the plate was incubated for 24h. The next day, a QuantiBlue solution, the detection reagent for secreted embryonic alkaline phosphatase (SEAP), was prepared by diluting QB reagent and QB buffer in room temperature MilliQ water to 1% (v / v) concentration each. The mixture was incubated at room temperature for 10 minutes. Subsequently, 180j.iL were aliquoted to each well of a 96-well flat bottom tissue culture plate, and to each well was added 20pL of the supernatant. The plate was incubated at 37 C, 5% COz for 6h. At different incubation time intervals (15min, 30min, Ih, 2h, 3h), a microplate reader was used to measure the optical density (O.D.) at 650nm. The results were analyzed by Excel software and presented here from the 3hr timepoint. The results of this assay are shown in FIGs. 2-3.

[0282] A single-masked IL-12 (AP2447; X864-P40). a triple-masked IL-12 (AP2450; X288-P40-X288 / P35-X288). and quadruple-masked IL-12 (AP2446; X288~P40~X288 / X288-P35-X288) were further transiently expressed in Expi293 cells and purified as detailed above.

[0283] The fusion proteins, in their masked and unmasked forms, were assayed for their IL- 12 activity according to the HEK Blue reporter assay described above and compared to the activity of WT mouse IL- 12. The masked versions of these fusion proteins provided 8-fold (single mask; AP2447), 11 -fold (triple-mask, AP2447), or 78-fold (quadruple-mask, AP2446) decrease in activity as compared to the unmasked equivalent. The results of this assay are provided in FIG. 4.

[0284] A further experiment was performed to determine the masking effect on IL- 12 activity of a quadruple-masked protease-activatable IL-12 (AP2551, X288-P40-X288 / X288-P35-X288), where the fusion protein was fully purified and fully (100%) unmasked. AP2551 was purified by harvestingand clarification of the conditioned medium; size-exclusion column chromatography (Capto Core 400, Cytiva); anion-exchange (AEX) capture (Q Sepharose Fast Flow (QFF), Cytiva); and AEX polishing (Capto Q ImpRes (Q-IR), Cytiva).

[0285] Quantitative fold masking difference between unmasked and fully masked protein was determined according to the HEK blue assay as detailed above. The quadruple-masked IL- 12 demonstrated 430-fold masking of IL-12 activity relative to its unmasked equivalent (FIG. 5).

[0286] The results of this example demonstrate that fusion of ELNN masks can provide significant masking of IL-12 activity. Additionally. ELNN -dependent masking of IL-12 activity was highest when four ELNN masks were used. Additionally, three or four shorter (288 AA) masks provided better masking as compared to a single, longer (864 AA) mask.Example 2. Selection of ELNN lengths

[0287] Protease-activatable IL- 12 molecules have different mask lengths were compared for masking efficacy as determined by induction of IFNy and for tumor growth inhibition in a mouse tumor model. Anti-tumor activity and mechanism of action were assessed in syngeneic mouse models using murine surrogates (including WT murine IL- 12) as the human IL- 12 does not bind to the mouse IL- 12 receptors. The masking activity of an IL- 12 molecule having four 288 amino acid length ELNN masks (linked by protease -cleavable linkers at C- and N-termini of p35 and p40 subunits) was compared to an equivalent molecule having four 210 amino acid ELNN masks. The molecules were expressed and fully purified as detailed above.

[0288] The mouse surrogate molecules P24M (4 x 288aa ELNNs) and P26M (4 x 210aa ELNNs) were assessed for induction of IFNy by mouse splenocyte assay. The purpose of this assay is to assess the ability of the masked and proteolytic-activated cytokines to engage the IL-12R and induce the subsequent secretion of IFNy. This activity is measured by quantifying IFNy released into the supernatant by the cells. Recombinant mouse IL-12 is included as reference. In brief, spleens were collected from multiple female C57B1 / 6 mice and mechanically dissociated into a single-cell suspension of pooled splenocytes. After elimination of red blood cells, aliquots of 5E5 splenocytes were treated with titrated therapeutic P24M, MMP-activated (unmasked) P24M. therapeutic P26M. MMP-activated (unmasked) P26M, or recombinant murine IL-12. The splenocytes and test articles were incubated for 48 hours following standard tissue culture methods. Supernatants were collected for quantitative analysis of mouse IFNy by ELISA. The results of this assay are provided in FIG. 6. The results demonstrate superior masking by P24M, with the 288aa ELNN masks, as compared to P26M with the shorter 210aa ELNN masks.

[0289] The mouse surrogate molecules P24M (4 x 288aa ELNNs) and P26M (4 x 210aa ELNNs) were also assessed for differential tumor growth inhibition in the B16F10 melanoma C57BL / 6 mouse model. Briefly, for the tumor growth inhibition experiment. 130 mice were subcutaneously implantedIllwith 100k B16F10 cells in the right flank. After 11 days, 24 mice bearing tumors with an average tumor volume of 111 mm3were randomized into 3 groups of 8 mice each. Mice w ere treated intravenously with a weekly dose of diluent control, 3 pg of P24M, or 3 pg of P26M. Mice were measured twice per week for tumor volume and body weight. At day 21, the end of study, mice treated with 3 pg of P24M or P26M had an average tumor growth inhibition of 80% and 61%, respectively, compared to the diluent control group. Treatment with P24M, with the 288aa ELNN masks, had a trend of greater tumor growth inhibition compared to P26M with the shorter 210aa ELNN masks (FIG. 7). Mice did not exhibit signs of toxicity from the treatment.

[0290] Quadruple masks of 288 amino acids each provided better masking in in vitro mouse splenocyte activity assays and in vivo efficacy assays done in mouse tumor models.Example 3. Design of barcoded ELNNs

[0291] ELNN polypeptide sequences can optionally contain a barcode fragment releasable from the polypeptide upon digestion by a protease. A barcode fragment may be, e.g., (1) a portion of the ELNN that includes at least part of a (non-recurring, non-overlapping) sequence motif that occurs only once within the ELNN; and (2) differs in sequence and molecular weight from all other peptide fragments that are releasable from the polypeptide containing them upon complete digestion of the polypeptide by a protease. The term “barcode fragment” (“barcode,” or “barcode sequence”) can refer to either the portion of the ELNN clcavably fused w ithin the polypeptide, or the resulting peptide fragment released from the polypeptide. Previous barcode sequences (see, e.g., PCT International Patent Publication No. WO2021 / 263058, die entire content of which is incorporated herein by reference) were designed with the intention of creating unique barcode polypeptide sequences with as minimal mutations in the original ELNN sequence as possible. However, such barcode sequences required 1 OOOpg / mL of Glu-C and an overnight digest to release them from peptides containing them. The barcode polypeptide sequences described in this Example were designed and tested to perform against a second criteria: That the barcode polypeptide is releasable from the ELNN polypeptide rapidly (in approximately two hours vs an overnight digest) by a low concentration of protease (less than 30pg / mL protease); in addition to the criteria of introducing the fewest mutations to the original ELNN sequence as possible.

[0292] In order to determine w hich peptide sequences were most favorably cleaved by Glu-C protease in a two-hour protease digest, a library of approximately 1000 peptides w as constructed with each peptide containing a different cleavage sequence for the protease Glu-C. Equimolar concentrations of these Glu-C site-containing peptides were tested in a 2-hour digest against a range of Glu-C protease concentrations from 0.05 pg / mL to 1000 pg / mL of protease. After digestion the peptides were analyzed by liquid chromatography mass spectrometry. The Glu-C cleavage site sequences that were cleaved by the lowest concentrations of protease were cataloged. From this list of the fastest sequences, a select few were selected that were most compatible with ELNNpolypeptides. These sequences were then implemented to flank new “Generation 2” barcode sequences.

[0293] A selection of Generation 2 barcode sequences was cloned into ELNN sequences and their performance as barcode peptides was tested by Glu-C digestion and subsequent liquid chromatography mass spectrometry (LC-MS) analyses. Successful barcode sequences from this experiment had 3 criteria: 1.) The barcode peptide was fully releasable from the ELNN polypeptide in a 2-hour digest by a concentration of 40 pg / mL of protease. 2.) The barcode peptide was not cleaved or otherwise degraded by much higher concentrations of protease, and 3.) The barcode peptide that met conditions 1 and 2 contained the fewest mutations from the original ELNN polypeptide sequence. Table 8 provides examples of successful Generation 2 barcode sequences according to die criteria of the aforementioned selection process.Table 8. Exemplary Generation 2 Barcode Sequences

[0294] Table 9 provides Generation 3 barcode sequences selected based on rate of Glu-C cleavage and based on separation and quantification by LC-MS. FIG. 8 shows the incorporation of barcode fragments into the ELNNs of an IL-12 fusion protein.Table 9. Exemplary Generation 3 Barcode SequencesExample 4. Release Site Engineering

[0295] Release site engineering was performed to ( 1) remove a glycosylation site, and (2) reduce the rate of cleavage in plasma which was surprisingly attributed to legumain.Glycosylation of release site

[0296] To assess glycosylation of the IL-12 fusion protein, a modified peptide-mapping protocol was performed. Briefly, IL-12 fusion protein samples were digested with Trypsin / LysC and run via LC / MS. Data was processed with ProteinMetrics softw are searching for N-glycosylated peptides. Observed glycosylation events were manually validated in ProteinMetrics software and a report was generated. The report contains the site of glycosylation and the site occupancy of glycosylation (intensity of glycosylated peptide / intensity of glycosylated peptide + intensity of un-glycosylated wild-type peptide).

[0297] An IL-12 fusion protein including RSR-2295 (EAGRSANHTPAGLTGP; SEQ ID NO:7048) was expressed in a HEK 293 cell line. RSR-2295 was found to be 50% N-glycosylated. N- glycosylation and the resulting heterogeneity of the release site is undesirable as it may affect protease cleavage rate and specificity. RSR-2295 was mutated to remove the N-glycosylation site. RSR-3213 (EAGRSASHTPAGLTGP; SEQ ID NO:7628) lacks an N-glycosylation site and therefore is not expected to be glycosylated.Legumain cleavage in plasma

[0298] Incubation of a fusion protein including RSR-2295 in human plasma showed some cleavage that, though not high, was unexpected. Further investigation revealed that the cleavage in human plasma was surprisingly due to legumain, which has previously believed to be specifically present in tumor tissues. Additionally, it was previously believed that legumain cleavage provided meaningful levels of unmasking and fusion protein activation in tumor tissues.

[0299] RSR-3213 was designed to avoid cleavage by legumain. Surprisingly, a protease-cleavable linker including the RSR-3213 release sequence was cleaved less in plasma but at comparable amounts to a corresponding protease-cleavable linker including the RSR-2295 release sequences in multiple tumor types (including gastric carcinoma (NCI-N87), colorectal adenocarcinoma (HT-29), colon carcinoma (HT-55) tumors). Thus, fusion proteins comprising RSR-3213 have enhanced specificity for tumor tissues without a significant loss of activation in tumor tissues.In vitro digest:

[0300] In vitro digest assays were performed to demonstrate that RSR-3213 is cleaved by MMP and ST14 / matriptase. but not legumain. Protease activatable T cell engager (paTCE) fusion proteins were designed, each including a bispecific TCE core that binds to EpCAM and CD3, and two protease- cleavable release sites where each release site is bound to a polypeptide mask such that cleavage of the release site release the polypeptide mask. A first paTCE fusion protein included RSR-2295 in both release sites flanking the TCE core. A second paTCE fusion protein included RSR-3213 in both release sites flanking the TCE core. The paTCEs were expressed in E. coli. The paTCEs (RSR-2295- containing and RSR-3213 containing) were digested with 5-fold dilutions of MMP9, legumain, or ST14 / matriptase. Similar banding patterns were observed for both MMP9 and matriptase, suggesting die mutation of the legumain cleavage site did not affect cleavability of the MMP and serine protease cleavage sites. Unmasked TCE (uTCE) was observed for the paTCE containing RSR-2295 after digestion with legumain, indicating cleavage at the protease cleavable linker by legumain. uTCE was not observed for the paTCE containing RSR-3213 after digestion with legumain, indicating the mutation successfully prevented cleavage at the protease cleavable linker by legumain (FIG. 9A and FIG. 9B).Plasma Stability -In Vivo Cleavabilitv

[0301] Fluorescently labeled variants of a paTCE containing either RSR-2295 or RSR-3213 were labeled with Sulfo-Cy5.5 or Sulfo-Cy7.5. Opposite colors were co-injected into mice containing NCI- N87, HT-29. or HT-55 xenograft tumors. 48 horns after injection, tumors were harvested, homogenized, and protein extracts were analyzed by SDS-PAGE and LI-COR. Relative abundances for paTCE, a metabolite including a single C-terminal mask (Ix-C), a metabolite including a single N- terminal mask (Ix-N), and uTCE were quantified. No significant differences were observed in uTCE and Ix-C between the two protease cleavable linkers. paTCE containing RSR-2295 showed a small but statistically significant increase (average 2.19% more) in Ix-N than the corresponding paTCE containing RSR-3213. (FIG. 10A and FIG.10B).

[0302] The observed cleavability in vivo from tumor homogenates was also determined from 3 different mouse tumor models. The % abundance for metabolites Ix-C, Ix-N, and uTCE was measured with results depicted in FIG. 10C. Finally, FIG. 10D depicts the % of total for paTCE plus the 3 metabolites (Ix-N, Ix-C, and uTCE) when employing RSR-2295 or RSR-3213.

[0303] Overall, these data suggest that differences between in vivo cleavability of RSR-2295 and RSR-3213 are minor across 3 different tumor models.Tumor Uptake:

[0304] Tumor uptake between paTCEs containing either RSR-2295 or RSR-3213 were compared using the ratio of calculated concentrations of total drug (paTCE, Ix-C, Ix-N, and uTCE). While differences in tumor uptake were observed across 3 different tumor models, no significant differences were observed between RSR-2295 and RSR-3213 within each model. This indicates that the changes to the protease cleavable linkers between RSR-2295 and RSR-3213 do not affect tumor uptake of paTCE (FIG. 11).Efficacy of protease-activatable cytokine including RSR-3213

[0305] Protease-activatable IL-12 molecules having either RSR-2295 or RSR-3213 release segments were compared for masking efficacy as determined by induction of IFNy and for tumor growth inhibition in a mouse tumor model. Anti-tumor activity and mechanism of action were assessed in syngeneic mouse models using murine surrogates (including WT murine IL- 12) as the human IL- 12 does not bind to the mouse IL-12 receptors. A quadruple ELNN-masked IL-12 molecule having an RSR-3213 release site linking IL- 12 to each of ELNN masks (P24M) was compared to a quadruple ELNN-masked IL-12 molecule having an RSR-2295 release site linking IL-12 to each of ELNN masks. The molecules were expressed and fully purified as detailed above.

[0306] The mouse surrogate molecules P24M (4 x 288aa ELNNs; 4 x RSR-3213) and P01M (4 x 288aa ELNNs; 4 x RSR-2295) were assessed for induction of IFNy by mouse splenocyte assay. The purpose of this assay is to assess the ability of the masked and proteolytic-activated cytokines to engage the IL-12R and induce the subsequent secretion of IFNy. This activity is measured by quantifying IFNy released into the supernatant by the cells. Recombinant mouse IL- 12 is included as reference. In brief, spleens were collected from multiple female C57B1 / 6 mice and mechanically dissociated into a single-cell suspension of pooled splenocytes. After elimination of red blood cells, aliquots of 5E5 splenocytes were treated with titrated therapeutic P24M. MMP-activated (unmasked) P24M. therapeutic P01M. MMP-activated (unmasked) P01M, or recombinant murine IL-12. The splenocytes and test articles were incubated for 48 hours following standard tissue culture methods. Supernatants were collected for quantitative analysis of mouse IFNy by ELISA. The results of this assay are provided in FIG. 12.

[0307] The mouse surrogate molecules P24M (4 x 288aa ELNNs; 4 x RSR-3213) and P01M (4 x 288aa ELNNs; 4 x RSR-2295) were also assessed for differential tumor growth inhibition in the B16F10 melanoma C57BL / 6 mouse model. Briefly, for the tumor growth inhibition experiment. 130 mice were subcutaneously implanted with 100k B16F10 cells in the right flank. After 11 days, 24 mice bearing tumors with an average tumor volume of 111 mm3were randomized into 3 groups of 8 mice each. Mice were treated intravenously with a weekly dose of diluent control, 3 pg of P24M. or 3 pg of P01 M. Mice were measured twice per week for tumor volume and body weight. At day 21. theend of study, mice treated with 3 pg of P24M or P01M had an average tumor growth inhibition of 80% and 44%, respectively, compared to the diluent control group. Treatment with P24M, with RSR- 3213, had a trend of greater tumor growth inhibition compared to P01M with RSR-2295 (FIG. 13). Mice did not exhibit signs of toxicity from the treatment.Example 5. Glycosylation of human IL-12

[0308] To assess glycosylation of human IL-12 p35 and p40 subunits in the context of a quadruple- masked fusion protein, a modified peptide-mapping protocol was performed. Briefly, fusion protein samples were digested with Trypsin / LysC and rim via LC / MS. Data was processed with ProtcinMctrics software searching for N-glycosylatcd peptides. Observed glycosylation events were manually validated in ProteinMetrics software and a report was generated. The report contains the site of glycosylation and the site occupancy of glycosylation (intensity of glycosylated peptide / intensity of glycosylated peptide + intensity of un-glycosylated wild-type peptide).

[0309] Human and mouse protease-activatable IL- 12 fusion proteins and human IL- 12 (not in a fusion protein) were each produced in HEK 293 cells and were assayed for glycosylation. The results are provided in Table 10.Table 10. N-Glycosylation of human IL-12n / a = no glycosylation site at this position in mouse IL-12 n.d. = no data

[0310] N 195 of the p35 subunit of huIL-12 in protease-activatable fusion protein with four ELNN masks was 99% glycosylated but glycosylation of N 195 was not observed in recombinant huIL-12 (not masked). It is hypothesized that N-glycosylation of N195 was created in the IL-12 fusion proteins by the addition of a polypeptide (e.g., a linker and ELNN mask polypeptide) to the C-terminus of the p35 subunit. In endogenous or recombinantly produced IL-12, N195 is not glycosylated as it is 3 amino acids from the C-terminus of the polypeptide chain and is unable to be glycosylated. Protease- activatable IL-12 fusion proteins were designed to remove the p35 N 195 glycosylation site. When mutating N 195, amino acid substitutions that did not increase potential immunogenicity’ of the fusion protein were selected, for example N195Q.Example 6. P72H and P74H, exemplary protease-activatable IL-12 fusion proteins

[0311] This example provides data relating to exemplary' protease-activatable IL- 12 fusion proteins, P72H and P74H.

[0312] P72H comprises a first polypeptide subunit comprising IL-12p35 with the amino acid sequence set forth as SEQ ID NO: 1000 and a second polypeptide subunit comprising IL-12p40 with the amino acid sequence set forth as SEQ ID NO: 1033. The annotated amino acid sequence for P72H is provided below in Table 11:Table 11. P72H Sequences*may optionally include a C-tenninal "proline" residue on N-ELNN of each subunit

[0313] P74H comprises a first polypeptide subunit comprising IL-12p35 with the amino acid sequence set forth as SEQ ID NO: 1002 and a second polypeptide subunit comprising IL-12p40 with the amino acid sequence set forth as SEQ ID NO: 1033. P74H includes a p35 variant subunit drat is aglycosylated at N195 (N 195Q). The annotated amino acid sequence for P74H is provided below in Table 12:Table 12. P74H Sequences*may optionally include a C-terminal “proline” residue on N-ELNN of each subunit

[0314] Methods for producing protease-activatable cytokine fusion proteins are known in the art, e.g.. as described in PCT International Patent Publication No. WO2021 / 262985.

[0315] In some of the experiments below, mouse surrogates for P72H and P74H are used. The antitumor activity and mechanism of action were assessed in syngeneic mouse models using murine surrogates as the human IL-12 does not bind to the mouse IL-12 receptors.

[0316] P71M is die mouse surrogate for P72H. P71M is identical to P72H except that human IL-12 has been replaced with mouse IL-12 in the P71M mouse surrogate. Additionally, mouse IL-12 of P71M has been mutated to introduce an N195 glycosylation site (not present in mouse, introduced to better mimic glycosylation pattern of human IL-12 in P72H).

[0317] P70M is the mouse surrogate for P74H except that human IL- 12 has been replaced with mouse IL-12 in the P70M mouse surrogate. P74H contains an N-195 aglycosylated version of human IL-12 and therefore WT mouse IL-12, which naturally lacks N195, can be used as a surrogate.In vitro characterization of P71M and P70M surrogate mouse fusion proteins

[0318] P71M is the mouse surrogate for P72H. as described above. P70M is the mouse surrogate for P74H, as described above.

[0319] Fusion of an ELNN to the C-terminal end of human IL-12 P35 subunit results in additional glycosylation of the N195 site as described above. The C-terminal end of the mouse IL-12 P35 is differs in sequence from the corresponding human sequence and does not exhibit this extra glycosylation. Equivalent mouse surrogate variant P71M was designed to have a similar glycosylation pattern to human P72H to investigate if there are any in vitro or in vivo effects of N 195 glycosylation in human P72H on IL-12 activity and / or the pharmacokinetic profile.

[0320] The mouse surrogate molecules P71M (glycosylated at N195 of P35; surrogate for P72H) and P70M (lacking glycosylation position at N195; surrogate for P74H which contains a P35 N195Q mutation) were assessed for binding to the mouse IL-12 receptor (mIL-12R) by SPR. The SPR assay was run on Biacore T200 at 25 degrees Celsius. The chip type that was used was Series S Sensor Chip CM5. For each antigen, a separate chip was made. For ML-12RB2, the anti-His antibody was immobilized on the CM5 chip surface. For mIL-12RB2 and cIL-12RB2, the anti-mouse Fc antibody was immobilized on the CM5 chip surface. The running buffer that was used was PBS-P+ from Cytiva. Chips were regenerated by 10 mM Glycine HC1, pH 1.5. Sample flow was 30|xl / min, while capture flow was lOpl / min. The association time was 100 seconds. The dissociation time was 360 seconds. Each sample was assayed as a titration ranging from 500nM to O.6811M. Sersorgram was fitted globally as a 1 : 1 kinetic interaction model using Biacore T200 Evaluation software.

[0321] Binding affinity data is shown in Table 13. A biphasic binding curve observed with mlL- 12R(31. Affinity trends were similar to that determined for mIL-12Rp2.Table 13. Affinity of mouse surrogate molecules to mIL-12Rp2

[0322] P71M and P70M were also assessed for induction of IFNy by mouse splenocyte assay. The purpose of this assay is to assess the ability of the masked and proteolytic-activated cytokines to engage the IL-12R and induce the subsequent secretion of IFNy. This activity is measured by quantifying IFNy released into the supernatant by the cells. Recombinant mouse IL- 12 is included as reference.

[0323] Spleens were collected from multiple female C57B1 / 6 mice and mechanically dissociated into a single-cell suspension of pooled splenocytes. After elimination of red blood cells, aliquots of 5E5 splenocytes were treated with titrated therapeutic P70M, MMP-activated (unmasked) P70M, therapeutic P71M, MMP-activated (unmasked) P71M, or recombinant murine IL-12. The splenocytes and test articles were incubated for 48 hours following standard tissue culture methods. Supernatants were collected for quantitative analysis of mouse IFNy by ELISA. The results are provided in FIG.14.

[0324] The mouse surrogates, P70M (N195 aglycosylated) and P71M (N195 glycosy lated), exhibited similar EC50 values of 2.6 nM and 2.8 nM, respectively. Their proteolytic-activated counterparts were more likewise active at 0.0031 nM and 0.0013 nM, respectively. Both were comparable to recombinant mouse IL-12 (EC50 0.0017 pM). The fold protection conferred by the masked constructs relative to the MMP-activated counterparts demonstrates a reduced potency ranging from 100-fold to 1000-fold.Pharmacokinetics of P71M and P70M surrogate mouse fusion proteins

[0325] P71M is the mouse surrogate for P72H, as described above. P70M is the mouse surrogate for P74H. as described above.

[0326] The impact of glycosylation of N195 in the P35 domain on the pharmacokinetics of masked and unmasked murine IL-12 was evaluated in non-tumor-bearing C57BL / 6 mice. Twelve mice each were administered a single bolus intravenous dose of 70 pg unmasked P70M or P71M (24 total mice). Plasma samples were collected from 3 mice in each dose group prior to IV administration and at 0.083, 0.25. 0.5. 1. 2, 4, 8, 24, and 48 hours post-dose. Additionally, 24 mice each were administered a single bolus intravenous dose of 30 pg masked P70M and P71M. Plasma samples were collected from 3 mice in each dose group prior to administration and at 0.083. 0.25. 0.5. 1. 2, 4, 8, 24 (n=6), 48 (n=6). 72, 96, 168, and 240 hours post-dose. The concentration of masked and unmasked P70M and P71M was quantified using qualified ligand binding assays. Pharmacokinetic parameters were determined for each test item by non-compartment analysis using composite profiles.

[0327] As shown in FIG. 15, the unmasked murine IL-12 was cleared approximately 100-fold faster than the masked murine IL-12, supporting the use of the masks to prolong half-life. The addition of the N195 glycosylation site in P71M resulted in a modestly shorter elimination half-life of the unmasked murine IL- 12. suggesting that glycosylation of N 195 has an impact on the pharmacokinetics of unmasked murine IL-12. In contrast, the addition of the N195 glycosylation site had no impact on the pharmacokinetics of masked murine IL- 12, suggesting that the presence of the masks may interfere with the glycosylation-specific elimination of murine IL-12. Clearance values (CL) and terminal half-life (Tl / 2) are provided in Table 14.Table 14. Clearance values (CL) and terminal half-life (Tl / 2) of mouse surrogate moleculesEfficacy and toxicity of P71M and P70M surrogate mouse fusion proteins

[0328] P71M is the mouse surrogate for P72H, as described above. P70M is the mouse surrogate for P74H, as described above.

[0329] P70M (native mouse IL-12 sequence) and P71M (added N195 glycosylation site on the P35 subunit) were assessed for differential tumor growth inhibition and toxicity in the MC38 colon carcinoma C57BL / 6 mouse model. The results are provided in FIG. 16.

[0330] Briefly, for the tumor growth inhibition experiment, 120 mice were subcutaneously implanted with 250k MC38 cells in the right flank. After 14 days, 40 mice bearing tumors with an average tumor volume of 101 mm3were randomized into 5 groups of 8 mice each. Mice were treated intravenously with a weekly dose of diluent control, 3 pg of P70M, 10 pg of P70M, 3 pg of P71M, or 10 pg of P71M. Mice were measured twice per week for tumor volume and body weight. At day 31, the end of study, mice treated with 3 pg of P70M or P71M had an average tumor growth inhibition of 56% and 45%, respectively, compared to the diluent control group. Mice treated with 10 pg of P70M or P71 M had an average tumor growth inhibition of 91% and 86%, respectively. These data indicate that P70M and P71M exhibit similar dose-dependent anti-tumor efficacy when using P70M or P71M. Mice did not exhibit signs of toxicity from the treatment.

[0331] For the toxicity experiment, 130 mice were subcutaneously implanted with 250k MC38 cells in the right flank. After 14 days, 54 mice bearing tumors with an average tumor volume of 80 mm3were randomized into 3 groups of 18 mice each. Mice were treated intravenously twice weekly with diluent control. 100 pg of P70M, or 100 pg P71M. Mice were assessed daily for signs of overt toxicity, which was defined by death, morbidity, or body weight percentage loss greater than 15%. For the last 5 days of the study (day 1 1 - and 15-days post-dosing start), P70M and P71M had similar overt toxicity of 44% and 0% of animals, respectively.In vitro characterization of P72H and P74H fusion proteins

[0332] In vitro assays were performed to characterize the IL-12 receptor (IL-12R) binding and activity of human P72H and P74H. Binding affinity of P72H and P74H were measured against human and cynomolgus monkey IL-12R0, by SPR. The assay was run on Biacore T200 at 25 degrees Celsius. The chip type that was used was Series S Sensor Chip CM5. For each antigen, a separate chip was made. For ML-12RB2, the anti-His antibody was immobilized on the CM5 chip surface. For mIL-12RB2 and cIL-12RB2. the anti-mouse Fc antibody was immobilized on the CM5 chip surface. The running buffer that was used was PBS-P+ from Cytiva. Chips were regenerated by 10 mM Glycine HC1, pH 1.5. Sample flow was 30pl / min, while capture flow was lOpl / min. The associationtime was 100 seconds. The dissociation time was 360 seconds. Each sample was assayed as a titration ranging from 500nM to O.6811M. Sersorgram was fitted globally as a 1:1 kinetic interaction model using Biacore T200 Evaluation software.

[0333] Affinity7data is shown in Table 15. A biphasic binding curve was observed with IL-12R 1. Affinity trends were similar to that determined for IL-12R 2.Table 15. Affinity of P72H and P74Hto human and cynomolgus monkey IL-12Rp

[0334] Activity7was determined by their respective ability to induce the downstream secretion of IFNy into the supernatant by cells. Frozen PBMCs from a single human donor were quickly thawed, washed, and resuspended. Aliquots of 5E6 PBMCs are activated with 5pg / ml anti-CD3 monoclonal antibody (clone OKT3) in 3 ml of media for 48 hours following standard tissue culture methods. Activated PBMCs were aliquoted in 1E5 cells and combined with titrated therapeutic P72H, MMP- activated (unmasked) P72H, therapeutic P74H, MMP-activated (unmasked) P74H, or recombinant human IL- 12. The cells and therapeutic reagents are incubated for 48 horns. Supernatants were collected for quantitative analysis of human IFNy by ELISA. This experiment was repeated in triplicate, each utilizing a unique human PBMC donor. The results are provided in FIG. 17.

[0335] The human constructs, P72H and P74H, elicited a median EC50 values of 1 nM and 1.4 nM, respectively, across the three donors making. Similarly, the pro teolytically -activated counterparts were more active, 0.0055 nM and 0.0093 nM, respectively. This is comparable to the activity exhibited by recombinant human IL-12 (EC50 0.0065 nM). However, the masks conferred a reduced potency of 100-fold.

[0336] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from die invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A fusion protein comprising: a cytokine, a linker (Linkerl) comprising a protease-cleavable release segment (RSI), and a mask polypeptide (Maskl), wherein the linker (Linkerl) comprising the release segment (RSI) is positioned between the cytokine and the mask polypeptide (Maskl); wherein the release segment is capable of being cleaved by at least one protease that is present in a tumor: and wherein the release segment (RSI) (i) is not capable of being cleaved by legumain in human plasma, (ii) is cleaved by legumain in human plasma at a rate that is less than 25% of the rate that EAGRSANHTPAGLTGP (RSR-2295; SEQ ID NO: 7048) is cleaved by legumain in human plasma, or (iii) comprises an amino acid sequence comprising the sequence EAGRSAXHTPAGLTGP, wherein X is any amino acid other than N (SEQ ID NO:7627).

2. The fusion protein of claim 1. wherein the cytokine is an interleukin, a transforming growth factor, an interferon, a tumor necrosis factor, a chemokine, or granulocyte macrophage-colony stimulating factor.

3. The fusion protein of claim 2. wherein the cytokine is an interleukin.

4. The fusion protein of claim 3, wherein the interleukin is selected from IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, and IL-25.

5. The fusion protein of claim 4, wherein the interleukin is IL-12.

6. The fusion protein of claim 5, wherein the IL-12 is a disulfide-linked heterodimer comprising a p35 polypeptide and a p40 polypeptide, and wherein the linker and mask are covalently joined to (i) the N-terminus or C-terminus of the p35 polypeptide, or (ii) the N-terminus or C-tenninus of the p40 polypeptide.

7. A fusion protein comprising:(a) a first polypeptide subunit comprising the following elements in an N-to-C or C-to-N terminal orientation:i. a first mask polypeptide (Maskl), ii. a first linker (Linker 1) comprising a first protease-cleavable release segment (RSI), iii. an IL- 12 p35 polypeptide, iv. a second linker (Linker2) comprising a second protease -cleavable release segment (RS2), and v. a second mask polypeptide (Mask2); and(b) a second polypeptide subunit comprising the following elements in an N-to-C or C- to-N terminal orientation: i. a third mask polypeptide (Mask3), ii. a third linker (Linker3) comprising a third protease-cleavable release segment (RS3). iii. an IL- 12 p40 polypeptide, iv. a fourth linker (Linker4) comprising a fourth protease-cleavable release segment (RS4). and v. a fourth mask polypeptide (Mask4); wherein the IL-12 p35 polypeptide of the first polypeptide subunit and the IL-12 p40 polypeptide of the second polypeptide subunit are disulfide-linked thereby forming a disulfide-linked heterodimer; wherein each release segment (RSI. RS2, RS3, and RS4) is capable of being cleaved by at least one protease that is present in a tumor; and wherein each release segment (RSI. RS2, RS3, and RS4) (i) is not capable of being cleaved by legumain in human plasma, (ii) is cleaved by legumain in human plasma at a rate that is less than 25% of the rate that RSR-2295 (EAGRSANHTPAGLTGP; SEQ ID NO:7048) is cleaved by legumain in human plasma, or (iii) comprises an amino acid sequence comprising the sequence EAGRSAXHTPAGLTGP, wherein X is any amino acid other than N (SEQ ID NO: 7627).

8. The fusion protein of claim 7, wherein the first mask polypeptide (Maskl) comprises a first a first barcode fragment (Barcode 1), the second mask polypeptide (Mask2) comprises a second barcode fragment (Barcode2), the third mask polypeptide (Mask3) comprises a third barcode fragment (Barcode3), and the fourth mask polypeptide (Mask4) comprises a fourth barcode fragment (Barcode4); and wherein each barcode fragment (Barcode 1, Barcode2, Barcode3, and Barcode4) is releasable from the fusion protein upon digestion with a non-mammalian protease, and each barcode fragment differs in both sequence and molecular weight from all other barcode fragments or peptide fragments that are releasable from the fusion protein upon complete digestion of the fusion protein by the non-mammalian protease.

9. A fusion protein comprising:(a) a first polypeptide subunit comprising the following elements in an N-to-C or C-to-N terminal orientation: i. a first mask polypeptide (Maskl) comprising a first barcode fragment (Barcode 1), ii. a first linker (Linker 1) comprising a first protease-cleavable release segment (RSI). iii. an IL- 12 p35 polypeptide, iv. a second linker (Linker2) comprising a second protease -cleavable release segment (RS2), and v. a second mask polypeptide (Mask2) comprising a second barcode fragment (Barcode2); and(b) a second polypeptide subunit comprising the following elements in an N-to-C or C- to-N terminal orientation: i. a third mask polypeptide (Mask3) comprising a third barcode fragment (Barcode3), ii. a third linker (Linker3) comprising a third protease-cleavable release segment (RS3), iii. an IL- 12 p40 polypeptide. iv. a fourth linker (Linker4) comprising a fourth protease-cleavable release segment (RS4). and v. a fourth mask polypeptide (Mask4) comprising a fourth barcode fragment (Barcode4); wherein the IL-12 p35 poly peptide of the first subunit and the IL-12 p40 polypeptide of the second unit are disulfide-linked thereby forming a disulfide-linked heterodimer; wherein each release segment (RSI, RS2, RS3, and RS4) is capable of being cleaved by at least one protease that is present in a tumor; and wherein each barcode fragment (Barcode 1, Barcode2, Barcode3, and Barcode4) is releasable from the fusion protein upon digestion with a non-mammalian protease, and each barcode fragment differs in both sequence and molecular weight from all other barcode fragments or peptide fragments that are releasable from the fusion protein upon complete digestion of the fusion protein by the non-mammalian protease.

10. The fusion protein of claim 9, wherein each release segment comprises an amino acid sequence having at least 85%, 90%, 91%. 92%, 93%, 94%, 95%. 96%. 97%, 98%, or 99% identity, or 100% identity, to a sequence of Table 6a.

11. The fusion protein of claim 9, wherein each release segment (RSI, RS2, RS3, and RS4) (i) is not capable of being cleaved by legumain in human plasma, (ii) is cleaved by legumain in human plasma at a rate that is less than 25% of the rate that RSR-2295 (EAGRSANHTPAGLTGP; SEQ ID NO: 7048) is cleaved by legumain in human plasma, or (iii) comprises an amino acid sequence comprising the sequence EAGRSAXHTPAGLTGP, wherein X is any amino acid other than N (SEQ ID NO:7627).

12. The fusion protein of any one of claims 1-11, wherein each release segment is a substrate for at least one protease of Table 5.

13. The fusion protein of any one of claims 1-12, wherein each release segment is capable of being cleaved by uPA. ST 14. MMP2. MMP7. MMP9. and MMP14.

14. The fusion protein of any one of claims 1-13. wherein each release segment is not capable of being cleaved by legumain in human plasma.

15. The fusion protein of any one of claims 1-14, wherein each release segment is not capable of being cleaved by legumain in human blood, plasma, or serum.

16. The fusion protein of any one of claims 1-15, wherein each release segment is not capable of being cleaved upon incubation with about InM or less legumain for about 20 horns.

17. The fusion protein of any one of claims 1-16, wherein each release segment is cleaved by legumain in human plasma at a rate that is less than 25%, less than 15%, less than 10%, less than 5%, or less than 2.5% of the rate that RSR-2295 (EAGRSANHTPAGLTGP; SEQ ID NO:7048) is cleaved by legumain in human plasma.

18. The fusion protein of any one of claims 1-17, wherein each release segment comprises the amino acid sequence of RSR-3213 (EAGRSASHTPAGLTGP; SEQ ID NO:7628)19. The fusion protein of any one of claims 1-18, wherein each mask polypeptide is an extended length non-natural polypeptide (ELNN).

20. The fusion protein of claim 19. wherein each ELNN is, independently, characterized in that: at least 90% of the amino acid residues of each ELNN are selected from glycine (G), alanine (A), serine (S). threonine (T). glutamate (E). proline (P). or any combination thereof; andeach ELNN comprises at least 3 types of amino acid residues selected from the group consisting of G, A, S, T, E, and P.

21. The fusion protein of claim 20, wherein each ELNN. independently, comprises a plurality of non-overlapping sequence motifs that are each from 9 to 14 amino acid residues in length, wherein the plurality of non-overlapping sequence motifs comprises a set of non-overlapping sequence motifs, wherein each non-overlapping sequence motif of the set of non-overlapping sequence motifs is repeated at least two times in the ELNN.

22. The fusion protein of claim 21, wherein the plurality of non-overlapping sequence motifs comprises at least one non-overlapping sequence motif that occurs only once within the ELNN.

23. The fusion protein of claim 21 or 22, wherein the non-overlapping sequence motifs comprise one of or any combination of the sequence motifs listed in Table lb.

24. The fusion protein of any one of claims 21-23, wherein the non-overlapping sequence motifs comprise at least 2. 3, or 4 of the sequence motifs listed in Table lb.

25. The fusion protein of any one of claims 21-24, wherein the non-overlapping sequence motifs comprise any one of or any combination of GTSTEPSEGSAP (SEQ ID NO: 189), GTSESATPESGP (SEQ ID NO: 188). GSGPGTSESATP (SEQ ID NO:201), GSEPATSGSETP (SEQ ID NO: 187), GSPAGSPTSTEE (SEQ ID NO: 186). and GTSPSATPESGP (SEQ ID NO:202).

26. The fusion protein of any one of claims 19-25, wherein each ELNN comprises at least 4 types of amino acid residues selected from the group consisting of G, A, S, T, E, and P.

27. The fusion protein of any one of claims 19-26, wherein the amino acid residues of each ELNN consists of A. E, G, S, P, and / or T.

28. The fusion protein of any one of claims 19-27, wherein each ELNN, independently, has a length of least 100 amino acid residues, at least 200 amino acid residues, or at least 250 amino acid residues.

29. The fusion protein of any one of claims 19-28, wherein each ELNN, independently, has a length of 100 to 1000 amino acid residues, 100 to 500 amino acid residues, 200 to 100 amino acid residues, 200 to 500 amino acid residues, 200 to 300 amino acid residues. 250 to 1000 amino acid residues, 250 to 500 amino acid residues, or 250 to 300 amino acid residues.

30. The fusion protein of claim 23, wherein each ELNN has a length of between 250 and 300 amino acid residues.

31. The fusion protein of claim 30, wherein each ELNN has a length of 287 or 288 amino acid residues.

32. The fusion protein of any one of claims 19-31, wherein each ELNN comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%. 94%, 95%, 96%, 97%. 98%, 99%, or 100% sequence identity to an amino acid sequence listed in Table 3a or 3b.

33. The fusion protein of any one of claims 7-32, wherein the first polypeptide mask is a first ELNN (ELNN 1), and wherein ELNN 1 comprises an amino sequence that has at least 85%, 90%, 91%, 92%. 93%. 94%, 95%, 96%. 97%. 98%, or 99% identity, or 100% identity to:ASGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSESATPESGPGSEPATSG SETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATP ESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGT STEPSEGSAPGSEPATSGSETPGTSESAT (SEQ ID NO: 8026).

34. The fusion protein of any one of claims 7-33, wherein the second polypeptide mask is a second ELNN (ELNN2), and wherein ELNN2 comprises an amino sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to:ATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESA TPESGPGTSASATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGEPEA (SEQ ID NO: 8028).

35. The fusion protein of any one of claims 7-34, wherein the third polypeptide mask is a third ELNN (ELNN3), and wherein ELNN3 comprises an amino sequence that has at least 85%, 90%. 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%. or 99% identity, or 100% identity to:ASGPGTSTEPSEGSAPGTSESATPESGPGSGPATSESATPGTSESATPESGPGSEPATSG SETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATP ESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGT STEPSEGSAPGSEPATSGSETPGTSESAT (SEQ ID NO: 8023).

36. The fusion protein of any one of claims 7-35, wherein the fourth polypeptide mask is a fourth ELNN (ELNN4), and wherein ELNN4 comprises an amino sequence that has at least 85%, 90%.91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity to:ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGS PTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSPSATPESGP GSEPATSGSETPGSEPATSGSETPGSPAG (SEQ ID NO: 8025).

37. The fusion protein of any one of claims 6-36 wherein the IL-12 p35 polypeptide is a human IL-12 p35 polypeptide and the IL12 p40 polypeptide is a human IL-12 p40 polypeptide.

38. The fusion protein of claim 37, wherein the human IL-12 p35 polypeptide comprises an amino acid sequence that has at least 85%. 90%, 91%, 92%, 93%. 94%. 95%, 96%, 97%. 98%. or 99% identity, or 100% identity to:RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTS TVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNA KLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRA VTIDRVMSYLNAS (SEQ ID NO: 8050).

39. The fusion protein of claim 38, wherein the human IL-12 p35 polypeptide comprises the amino acid sequence of:RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTS TVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNA KLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRA VTIDRVMSYLNAS (SEQ ID NO: 8050).

40. The fusion protein of claim 37 or 38, wherein the human IL-12 p35 polypeptide is an N195 aglycosylated variant.

41. The fusion protein of claim 40, wherein the human IL- 12 p35 polypeptide comprises the amino acid sequence of:RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTS TVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNA KLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRA VTIDRVMSYLXAS (SEQ ID NO: 8055),wherein X is any amino acid residue other than asparagine (N).

42. The fusion protein of claim 40 or 41, wherein the human IL-12 p35 polypeptide comprises an amino acid sequence selected from:RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTS TVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNA KLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRA VTIDRVMSYLSSA (SEQ ID NO: 8052);RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTS TVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNA KLLMDPKRQ1FLDQNMLAV1DELMQALNFNSETVPQKSSLEEPDFYKTK1KLCILLHAFRIRA VTIDRVMSYLQAS (SEQ ID NO: 8053); andRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTS TVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNA KLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRA VTIDRVMSYLDAS (SEQ ID NO: 8054).

43. The fusion protein of any one of claims 37-42, wherein the human IL-12 p40 polypeptide comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%. 93%, 94%, 95%, 96%. 97%, 98%, or 99% identity, or 100% identity to:IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQ VKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRF TCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAA EESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTP HSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPC S (SEQ ID NO: 8051).

44. The fusion protein of claim 43, wherein the human IL-12 p40 polypeptide comprises the amino acid sequence of:IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQ VKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRF TCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAA EESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTP HSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPC S (SEQ ID NO: 8051).

45. The fusion protein of any one of claims 6-36, wherein the IL-12 p35 polypeptide is a mouse IL-12 p35 polypeptide and the IL-12 p40 polypeptide is a mouse IL-12 p40 polypeptide.

46. The fusion protein of any one of claims 1-45, wherein each linker further comprises a spacer.

47. The fusion protein of claim 46, wherein each spacer is positioned between a release segment and a cytokine, an IL-12 p35 polypeptide, or an IL-12 p40 polypeptide.

48. The fusion protein of claim 46 or 47, wherein each spacer is, independently, characterized in that: at least 90% of the amino acid residues of each spacer are selected from glycine (G). alanine (A), serine (S), threonine (T), glutamate (E), proline (P). or any combination thereof; and each spacer comprises at least 3 types of amino acid residues selected from the group consisting of G, A. S. T, E, and P.

49. The fusion protein of any one of claims 46-48, wherein each spacer is. independently, 5 to 14 amino acid residues in length.

50. The fusion protein of any one of claims 46-49, wherein each spacer comprises at least 4 types of amino acid residues selected from the group consisting of G. A, S, T. E, and P.

51. The fusion protein of any one of claims 46-50, wherein the amino acid residues of each spacer comprise only amino acid residues selected from G, A, S, T, E, and / or P.

52. The fusion protein of any one of claims 46-51, wherein each spacer is cleavable by a nonmammalian protease.

53. The fusion protein of claim 52, wherein the non-mammalian protease is Glu-C.

54. The fusion protein of any one of claims 46-53, wherein each spacer, independently, comprises an amino acid sequence having at least 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table C.

55. The fusion protein of any one of claims 46-54, wherein each spacer, independently, comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%. 93%. 94%, 95%, 96%, 97%. 98%, or 99% identity, or 100% identity to TSESATPES (SEQ ID NO:98), GTATPESGPG (SEQ ID NO:97). GTPESATPES (SEQ ID NO:99), or GTATPESAP (SEQ ID NO: 100).

56. The fusion protein of any one of claims 46-55, wherein no spacer shares 100% sequence identity with any other spacer.

57. The fusion protein of any one of claims 1-56, wherein the fusion protein does not comprise a His-tag, wherein a His-tag is a polypeptide sequence comprising an uninterrupted chain of between 5 and 12 histidine residues.

58. The fusion protein of any one of claims 8-57, wherein each barcode fragment is releasable from the fusion protein upon digestion with a non-mammalian protease and the non-mammalian protease is Glu-C.

59. The fusion protein of any one of claims 8-58, wherein none of the barcode fragments include any of the following: the N-terminal amino acid of the first polypeptide subunit, the C-terminal acid of the first polypeptide subunit, the N-terminal amino acid of the second polypeptide subunit, and the C-terminal amino acid of the polypeptide subunit.

60. The fusion protein of any one of claims 8-59, wherein the entirety of each barcode fragment is 5 to 100, 5 to 50. 10 to 100, or 10 to 50 amino acid residues from any of the following: the N-terminal amino acid of the first polypeptide subunit, the C-terminal acid of the first polypeptide subunit, the N- terminal amino acid of the second polypeptide subunit, and the C-terminal amino acid of the polypeptide subunit.

61. The fusion protein of any one of claims 8-60, wherein each barcode fragment is, independently , at least 4 amino acid residues in length.

62. The fusion protein of any one of claims 8-61, wherein each barcode fragment is, independently, 4 to 20. 5 to 15, 6 to 12, 7 to 10, 8 to 13, 10 to 13. or 11 to 12 amino acid residues in length.

63. The fusion protein of any one of claims 8-62, wherein each barcode fragment comprises a glutamate at the C-terminus thereof.

64. The fusion protein of claim 63, wherein each barcode fragment does not include a second glutamate at a position other than the C-terminus of the barcode fragment unless the second glutamate is immediately followed by a proline.

65. The fusion protein of any one of claims 8-641, wherein the amino acid residue that is directlyN-tenninal to each barcode fragment in the primary amino acid sequence of the fusion protein is a glutamate.

66. The fusion protein of claim 65. wherein the glutamate that is directly N-tenninal to each barcode fragment is not immediately adjacent to another glutamate.

67. The fusion protein of any one of claims 8-66, wherein none of the barcode fragments includes a glutamate that is immediately adjacent to another glutamate, if present, in the mask polypeptide that contains the barcode fragment.

68. The fusion protein of any one of claims 8-67, wherein each barcode fragment is. independently, selected from Table 2 or Table 3a.

69. The fusion protein of any one of claims 8-68, wherein each barcode fragment comprises only amino acid residues selected from A, E, G, S, P, and T.

70. The fusion protein of any one of claims 8-69, wherein each barcode fragment comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%. or 99% identity, or 100% identity, to SGPGSGPGTSE (SEQ ID NO: 78), SGPGTSASATPE (SEQ ID NO: 1029), SGPGSGPATSE (SEQ ID NO: 1028), or SGPGTSPSATPE (SEQ ID NO:79).

71. The fusion protein of any one of claims 8-70, wherein each barcode fragment is selected from die following four amino acid sequences: SGPGSGPGTSE (SEQ ID NO:78), SGPGTSASATPE (SEQ ID NO: 1029), SGPGSGPATSE (SEQ ID NO: 1028), or SGPGTSPSATPE (SEQ ID NO:79).

72. The fusion protein of any one of claims 8-71, wherein the first barcode fragment (Barcode 1) comprises the amino acid sequence of SGPGSGPGTSE (SEQ ID NO:78).

73. The fusion protein of any one of claims 8-72, wherein the second barcode fragment (Barcode2) comprises the amino acid sequence of SGPGTSASATPE (SEQ ID NO: 1029).

74. The fusion protein of any one of claims 8-73, wherein the third barcode fragment (Barcode3) comprises the amino acid sequence of SGPGSGPATSE (SEQ ID NO: 1028).

75. The fusion protein of any one of claims 8-74, wherein the fourth barcode fragment (Barcode4) comprises the amino acid sequence of SGPGTSPSATPE (SEQ ID NO:79).

76. The fusion protein of any one of claims 1-75, comprising a Glu-C cleavage site comprising one of the following amino acid sequences: ATPESGPG (SEQ ID NO:2223), SGSETPGT (SEQ ID NO:2242), and GTSESATP (SEQ ID NO:204).

77. The fusion protein of any one of claims 1-76, comprising at least one of the following amino acid sequences: SGPE.SGPGXnSGPE.SGPG (SEQ ID NO:2001), SGPE.SGPGXnATPE.SGPG (SEQ ID N0:2002), SGPE.SGPGXnGTSE.SATP (SEQ ID N0:2003), SGPE.SGPGXnTTPE.SGPG (SEQ ID N0:2004), SGPE.SGPGXnSTPE.SGPG (SEQ ID N0:2005), SGPE.SGPGXnGTPE.SGPG (SEQ ID N0:2006), SGPE.SGPGXnGTPE.TPGS (SEQ ID N0:2007), SGPE.SGPGXnSGSE.TGTP (SEQ ID N0:2008), SGPE.SGPGXnGTPE.GSAP (SEQ ID N0:2009). SGPE.SGPGXnEPSE.SATP (SEQ ID NQ:2010). ATPE.SGPGXnSGPE.SGPG (SEQ ID NO:2011). ATPE.SGPGXnATPE.SGPG (SEQ ID NQ:2012). ATPE.SGPGXnGTSE.SATP (SEQ ID NQ:2013). ATPE.SGPGXnATSE.SATP (SEQ ID NQ:2014), ATPE.SGPGXnTTPE.SGPG (SEQ ID NQ:2015), ATPE.SGPGXnSTPE.SGPG (SEQ ID NQ:2016), ATPE.SGPGXnGTPE.SGPG (SEQ ID NQ:2017), ATPE.SGPGXnGTPE.TPGS (SEQ ID NO:2018), ATPE.SGPGXnSGSE.TGTP (SEQ ID NO:2019), ATPE.SGPGXnGTPE.GSAP (SEQ ID N0:2020), ATPE.SGPGXnEPSE.SATP (SEQ ID NO:2021), GTSE.SATPXnSGPE.SGPG (SEQ ID NO:2022), GTSE.SATPXnATPE.SGPG (SEQ ID NO:2023), GTSE.SATPXnGTSE.SATP (SEQ ID NO:2024), GTSE.SATPXnTTPE.SGPG (SEQ ID NO:2025), GTSE.SATPXnSTPE.SGPG (SEQ ID NO:2026), GTSE.SATPXnGTPE.SGPG (SEQ ID NO:2027), GTSE.SATPXnGTPE.TPGS (SEQ ID NO:2028), GTSE.SATPXnSGSE.TGTP (SEQ ID NO:2029), GTSE.SATPXnGTPE.GSAP (SEQ ID N0:2030), GTSE.SATPXnEPSE.SATP (SEQ ID NO:2031), TTPE.SGPGXnSGPE.SGPG (SEQ ID NO:2032), TTPE.SGPGXnATPE.SGPG (SEQ ID NO:2033), TTPE.SGPGXnGTSE.SATP (SEQ ID NO:2034), TTPE.SGPGXnTTPE.SGPG (SEQ ID NO:2035), TTPE.SGPGXnSTPE.SGPG (SEQ ID NO:2036), TTPE.SGPGXnGTPE.SGPG (SEQ ID NO:2037), TTPE.SGPGXnGTPE.TPGS (SEQ ID NO:2038), TTPE.SGPGXnSGSE.TGTP (SEQ ID NO:2039), TTPE.SGPGXnGTPE.GSAP (SEQ ID N0:2040), TTPE.SGPGXnEPSE.SATP (SEQ ID NO:2041), STPE.SGPGXnSGPE.SGPG (SEQ ID NO:2042), STPE.SGPGXnATPE.SGPG (SEQ ID NO:2043), STPE.SGPGXnGTSE.SATP (SEQ ID NO:2044), STPE.SGPGXnTTPE.SGPG (SEQ ID NO:2045), STPE.SGPGXnSTPE.SGPG (SEQ ID NO:2046), STPE.SGPGXnGTPE.SGPG (SEQ ID NO:2047), STPE.SGPGXnGTPE.TPGS (SEQ ID NO:2048), STPE.SGPGXnSGSE.TGTP (SEQ ID NO:2049), STPE.SGPGXnGTPE.GSAP (SEQ ID N0:2050), STPE.SGPGXnEPSE.SATP (SEQ ID NO:2051), GTPE.SGPGXnSGPE.SGPG (SEQ ID NO:2052), GTPE.SGPGXnATPE.SGPG (SEQ ID NO:2053). GTPE.SGPGXnGTSE.SATP (SEQ ID NO:2054), GTPE.SGPGXnTTPE.SGPG (SEQ ID NO:2055), GTPE.SGPGXnSTPE.SGPG (SEQ ID NO:2056), GTPE.SGPGXnGTPE.SGPG (SEQ ID NO:2057). GTPE.SGPGXnGTPE.TPGS (SEQ ID NO:2058). GTPE.SGPGXnSGSE.TGTP (SEQ ID NO:2059). GTPE.SGPGXnGTPE.GSAP (SEQ ID NQ:2060), GTPE.SGPGXnEPSE.SATP (SEQ ID NQ:2061), GTPE.TPGSXnSGPE.SGPG(SEQ ID NO:2062), GTPE.TPGSXnATPE.SGPG (SEQ ID NO:2063), GTPE.TPGSXnGTSE.SATP (SEQ ID NO:2064), GTPE.TPGSXnTTPE.SGPG (SEQ ID NO:2065), GTPE.TPGSXnSTPE.SGPG (SEQ ID NO:2066), GTPE.TPGSXnGTPE.SGPG (SEQ ID NO:2067), GTPE.TPGSXnGTPE.TPGS (SEQ ID NO:2068), GTPE.TPGSXnSGSE.TGTP (SEQ ID NO:2069), GTPE.TPGSXnGTPE.GSAP (SEQ ID N0:2070), GTPE.TPGSXnEPSE.SATP (SEQ ID NO:2071), SGSE.TGTPXnSGPE.SGPG (SEQ ID NO:2072), SGSE.TGTPXnATPE.SGPG (SEQ ID NO:2073), SGSE.TGTPXnGTSE.SATP (SEQ ID NO:2074), SGSE.TGTPXnTTPE.SGPG (SEQ ID NO:2075), SGSE.TGTPXnSTPE.SGPG (SEQ ID NO:2076), SGSE.TGTPXnGTPE.SGPG (SEQ ID NO:2077), SGSE.TGTPXnGTPE.TPGS (SEQ ID NO:2078), SGSE.TGTPXnSGSE.TGTP (SEQ ID NO:2079), SGSE.TGTPXnGTPE.GSAP (SEQ ID N0:2080), SGSE.TGTPXnEPSE.SATP (SEQ ID NO:2081), GTPE.GSAPXnSGPE.SGPG (SEQ ID NO:2082), GTPE.GSAPXnATPE.SGPG (SEQ ID NO:2083). GTPE.GSAPXnGTSE.SATP (SEQ ID NO:2084). GTPE.GSAPXnTTPE.SGPG (SEQ ID NO:2085). GTPE.GSAPXnSTPE.SGPG (SEQ ID NQ:2086). GTPE.GSAPXnGTPE.SGPG (SEQ ID NQ:2087). GTPE.GSAPXnGTPE.TPGS (SEQ ID NQ:2088), GTPE.GSAPXnSGSE.TGTP (SEQ ID NQ:2089), GTPE.GSAPXnGTPE.GSAP (SEQ ID NQ:2090), GTPE.GSAPXnEPSE.SATP (SEQ ID NQ:2091), EPSE.SATPXnSGPE.SGPG (SEQ ID NO:2092), EPSE.SATPXnATPE.SGPG (SEQ ID NO:2093), EPSE.SATPXnGTSE.SATP (SEQ ID NO:2094), EPSE.SATPXnTTPE.SGPG (SEQ ID NO:2095), EPSE.SATPXnSTPE.SGPG (SEQ ID NO:2096), EPSE.SATPXnGTPE.SGPG (SEQ ID NO:2097), EPSE.SATPXnGTPE.TPGS (SEQ ID NO:2098), EPSE.SATPXnSGSE.TGTP (SEQ ID NO:2099), EPSE.SATPXnGTPE.GSAP (SEQ ID N0:2100), or EPSE.SATPXnEPSE.SATP (SEQ ID NQ:2101), wherein each is a Glu-C cleavage site and n is any integer from 0 to 50.

78. The fusion protein of claim 77, comprising at least one of the following amino acid sequences: ATPE.SGPGXnATPE.SGPG (SEQ ID NO:2102), ATPE.SGPGXnGTSE.SATP (SEQ ID NO:2103), ATPE.SGPGXnATSE.SATP (SEQ ID NO:2104), GPE.SGPGXnATPE.SGPG (SEQ ID NO:2116), ATPE.SGPGXnTTPE.SGPG (SEQ ID NO:2106), ATPE.SGPGXnSTPE.SGPG (SEQ ID NO:2107), ATPE.SGPGXnGTPE.SGPG (SEQ ID NO:2108), GTPE.SGPGXnGTPE.SGPG (SEQ ID NO:2109), GTPE.SGPGXnSTPE.SGPG (SEQ ID NO:2HO), GTPE.SGPGXnTTPE.SGPG (SEQ ID NO:2111), GTPE.TPGSXnSGSE.TGTP (SEQ ID NO:2112), GTPE.GSAPXnEPSE.SATP (SEQ ID NO:2113), TTPE.SGPGXnTTPE.SGPG (SEQ ID NO:2114), or STPE.SGPGXnSTPE.SGPG (SEQ ID NO:2115), wherein each is a Glu-C cleavage site and n is any integer from 0 to 30.

79. The fusion protein of claim 78, comprising at least one of the following amino acid sequence ATPE.SGPGXnATSE.SATP, wherein is a Glu-C cleavage site and n is any integer from 0 to 30 (SEQ ID NO:2104).

80. The fusion protein of any one of claims 77-79, wherein n is any integer from 1 to 20, 5 to 15, 3 to 7, 2 to 5, or 5 to 10.

81. The fusion protein of any one of claims 77-79, wherein n is 9.

82. The fusion protein of any one of claims 77-79, wherein n is 4.

83. The fusion protein of any one of claims 77-79wherein n is 3.

84. The fusion protein of any one of claims 77-79, wherein Xn is PGTGTSAT (SEQ IDNO:2233), PGSGPGT (SEQ ID NO:2221), PGTTPGTT (SEQ ID NO:2241). PGTPPTST (SEQ ID NO:2235), PGTSPSAT (SEQ ID NO:2238), PGTGSAGT (SEQ ID NO:2230), PGTGGAGT (SEQ ID NO:2228), PGTSPGAT (SEQ ID NO:2237), PGTSGSGT (SEQ ID NO:2236), PGTSSAST (SEQ ID NO:2239), PGTGAGTT (SEQ ID NO:2227), PGTGSTST (SEQ ID NO:2232), GSEPATSG (SEQ ID NO:2224). APGTSTEP (SEQ ID NO:2222). PGTAGSGT (SEQ ID NO:2226), PGTSSGGT (SEQ ID NQ:2240). PGTAGPAT (SEQ ID NO:2225), PGTPGTGT (SEQ ID NO:2234), PGTGGPTT (SEQ ID NO:2229). or PGTGSGST (SEQ ID NO:2231).

85. The fusion protein of any one of claims 77-79, wherein Xn is TSAS (SEQ ID NO:2214), TGTS (SEQ ID NO:2211), SGP, TTPG (SEQ ID NQ:2220), TPPT (SEQ ID NO:2213), TSPS (SEQ ID NO:2217), TGSA (SEQ ID NO:2208), TGGA (SEQ ID NQ:2206), TSPG (SEQ ID NO:2216), TSGS (SEQ ID NO:2215), TSSA (SEQ ID NO:2218), TGAG (SEQ ID NQ:2205), TGST (SEQ ID NO:2210), EPAT (SEQ ID NO:2201), GTST (SEQ ID NQ:2202), TAGS (SEQ ID NO:2204), TSSG (SEQ ID NO:2219), TAGP (SEQ ID NO:2203), TPGT (SEQ ID NO:2212), TGGP (SEQ ID NO:2207), or TGSG (SEQ ID NO:2209).

86. The fusion protein of any one of claim 77-79, wherein Xn is SGP,87. The fusion protein of any one of claims 7-86, wherein the first polypeptide subunit comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table Al (SEQ ID NOs: 1000-1005).

88. The fusion protein of any one of claims 7-86, wherein the first polypeptide subunit comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%. 98%, or 99% identity, or 100% identity, to:ASGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSESATPESGPGSEPATSGSETPGT SESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGT SESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE GSAPGSEPATSGSETPGTSESATEAGRSASHTPAGLTGPGTPESATPESRNLPVATPDPGMFP CLHHSQNLLRAVSNMLOKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNS RETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYOVEFKTMNAKLLMDPKRQIFLDQNMLA VIDELMOALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGTATPE SAPEAGRSASHTPAGLTGPATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGT SESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGT SESATPESGPGTSESATPESGPGTSASATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT STEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGEPEA (SEQ 1D NO: 1000).

89. The fusion protein of any one of claims 7-88, wherein the second polypeptide subunit comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. or 99% identity, or 100% identity, to a sequence listed in Table A2 (SEQ ID NOs: 1033- 1036).

90. The fusion protein of any one of claims 7-89, wherein the second polypeptide subunit comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%. 93%, 94%, 95%, 96%. 97%, 98%, or 99% identity, or 100% identity, to:ASGPGTSTEPSEGSAPGTSESATPESGPGSGPATSESATPGTSESATPESGPGSEPATSGSETPGT SESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGT SESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE GSAPGSEPATSGSETPGTSESATEAGRSASHTPAGLTGPTSESATPESIWELKKDVYVVELD WYPDAPGEMVVLTCDTPEEDGITWTLDOSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVL SHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSR GSSDPOGVTCGAATLSAERVRGDNKEYEYSVECOEDSACPAAEESLPIEVMVDAVHKLKYE NYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVOVOGKSKRE KKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGTATPESGPGEAGRSASH TPAGLTGPATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGT STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSPSATP ESGPGSEPATSGSETPGSEPATSGSETPGSPAG (SEQ ID NO: 1033).

91. The fusion protein of any one of claims 7-87, wherein the first polypeptide subunit comprises die ammo acid sequence of: ASGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSESATPESGPGSEPATSGSETPGT SESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGT SESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE GSAPGSEPATSGSETPGTSESATEAGRSASHTPAGLTGPGTPESATPESRNLPVATPDPGMFP CLHHSQNLLRAVSNMLOKARQTLEFYPCTSEEIDHEDITK.DKTSTVEACLPLELTKNESCLNS RETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYOVEFKTMNAKLLMDPKRQIFLDQNMLA VIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGTATPE SAPEAGRSASHTPAGLTGPATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGT SESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGT SESATPESGPGTSESATPESGPGTSASATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT STEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGEPEA (SEQ ID NO: 1000). and wherein the second polypeptide subunit comprises the amino acid sequence of:ASGPGTSTEPSEGSAPGTSESATPESGPGSGPATSESATPGTSESATPESGPGSEPATSGSETPGT SESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGT SESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE GSAPGSEPATSGSETPGTSESATEAGRSASHTPAGLTGPTSESATPESIWELKKDVYVVELD WYPDAPGEMVVLTCDTPEEDGITWTLDOSSEVLGSGKTLTIQVKEFGDAGOYTCHKGGEVL SHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSR GSSDPOGVTCGAATLSAERVRGDNKEYEYSVECOEDSACPAAEESLPIEVMVDAVHKLKYE NYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKRE KKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGTATPESGPGEAGRSASH TPAGLTGPATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGT STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSPSATP ESGPGSEPATSGSETPGSEPATSGSETPGSPAG (SEQ ID NO: 1033).

92. A fusion protein comprising a first polypeptide subunit and a second polypeptide subunit, wherein the first polypeptide subunit and the second polypeptide subunit are disulfide-linked thereby forming a disulfide-linked heterodimer, wherein the first polypeptide subunit comprises an amino acid sequence having 85%. 90%, 91%, 92%. 93%. 94%, 95%, 96%, 97%. 98%, or 99% identity, or 100% identity, to:ASGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSESATPESGPGSEPATSGSETPGT SESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGT SESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE GSAPGSEPATSGSETPGTSESATEAGRSASHTPAGLTGPGTPESATPESRNLPVATPDPGMFPC LHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSR ETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAV IDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGTATPES APEAGRSASHTPAGLTGPATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSE SATPESGPGTSESATPESGPGTSASATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTST EEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGEPEA (SEQ ID NO: 1000). and wherein the second polypeptide submit comprises an amino acid sequence having 85%.90%. 91%. 92%, 93%, 94%, 95%. 96%, 97%, 98%, or 99% identity, or 100% identity, to: ASGPGTSTEPSEGSAPGTSESATPESGPGSGPATSESATPGTSESATPESGPGSEPATSGSETPGT SESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGT SESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE GSAPGSEPATSGSETPGTSESATEAGRSASHTPAGLTGPTSESATPESIWELKKDVYVVELD WYPDAPGEMVVLTCDTPEEDGITWTLDOSSEVLGSGKTLTIQVKEFGDAGOYTCHKGGEVL SHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSR GSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYE NYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVOVQGKSKRE KKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGTATPESGPGEAGRSASH TPAGLTGPATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGT STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSPSATP ESGPGSEPATSGSETPGSEPATSGSETPGSPAG (SEQ ID NO: 1033).

93. The fusion protein of claim 92, wherein the first polypeptide subunit comprises the amino acid sequence of:ASGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSESATPESGPGSEPATSGSETPGT SESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGT SESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATEAGRSASHTPAGLTGPGTPESATPESRNLPVATPDPGMFPCLHHSONLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNS RETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYOVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMOALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGTATPESAPEAGRSASHTPAGLTGPATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSASATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT STEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGEPEA (SEQ ID NO: 1000), and wherein the second polypeptide subunit comprises the amino acid sequence of:ASGPGTSTEPSEGSAPGTSESATPESGPGSGPATSESATPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATEAGRSASHTPAGLTGPTSESATPESIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDOSSEVLGSGKTLTIQVKEFGDAGOYTCHKGGEVL SHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLOLKPLKNSROVEVSWEYPDTWSTPHSYFSLTFCVOVOGKSKRE KKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGTATPESGPGEAGRSASH TPAGLTGPATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSPSATP ESGPGSEPATSGSETPGSEPATSGSETPGSPAG (SEQ ID NO: 1033).

94. A pharmaceutical composition comprising the fusion of any one of claims 1-93 and at least one pharmaceutically acceptable excipient.

95. A polynucleotide sequence encoding the fusion protein of any one of claims 1-93.

96. An expression vector comprising the polynucleotide sequence of claim 95.

97. A host cell comprising the expression vector of claim 96.

98. A method of producing the fusion protein of any one of claims 1-93.

99. The method of claim 98, further comprising isolating the fusion protein from a host cell.

100. A method of treating cancer in a subject in need thereof, the method comprising administering an effective amount of the fusion protein of any one of claims 1-93 or the pharmaceutical composition of claim 94 to the subject.

101. The method of claim 100, wherein the cancer comprises a solid tumor.

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