Linker Polypeptides

Abstract

The present disclosure relates to a linker polypeptide. In some embodiments, the linker polypeptide comprises a first targeting sequence; a second targeting sequence; and a first linker between the first targeting sequence and the second targeting sequence, the linker comprising a protease-cleaved polypeptide sequence. In some embodiments, the linker polypeptide comprises a first active domain; a second active domain; a pharmacokinetic modulator; and a first linker between the pharmacokinetic modulator and the first active domain, the first linker comprising a protease-cleaved polypeptide sequence. In some embodiments, the linker polypeptide comprises a first active domain; an inhibitory polypeptide sequence capable of blocking the activity of the first active domain; a first linker between the first active domain and the inhibitory polypeptide sequence, the linker comprising a protease-cleaved polypeptide sequence; and a first targeting sequence.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 224,350, filed July 21, 2021, which is incorporated by reference in its entirety for all purposes.

[0002] Introduction and Overview The present disclosure relates to the field of linker polypeptides that include one or more targeting sequences. The linker polypeptides are useful, for example, for targeting to certain types of extracellular environments. [Background technology]

[0003] It may be beneficial to target protein therapeutics and other polypeptides to specific extracellular environments. It may also be beneficial to modulate activity and / or pharmacokinetics to limit systemic and / or adverse effects.

[0004] For example, immunoglobulin antigen binding domains such as Fv, scFv, Fab, or VHH, as well as various forms of active domains including but not limited to cytokines and chemokines such as IL-2, IL-10, IL-15, TGF-β, CXCL9, CXCL10, play an important role in targeting diseased cells and / or maintaining effective immune cell responses. In some cases, however, systemic administration of such compounds may activate immune cells throughout the body. Systemic activation may result in systemic toxicity and indiscriminate activation of immune cells, including immune cells that respond to various epitopes, antigens, and stimuli. The therapeutic potential of such treatments may be affected by these severe toxicities.

[0005] Peptide, immunoglobulin, and cytokine treatments also suffer from short serum half-lives, in some cases on the order of minutes, and therefore the high doses that may be necessary to achieve optimal efficacy may contribute to severe toxicity.

[0006] Furthermore, in conventional antibodies, the immunoglobulin antigen-binding domain is fixed to a pharmacokinetic modulator such as the Fc region. Thus, the activity of the Fc region is linked to the activity of the immunoglobulin antigen-binding domain, and these regions and domains cannot operate independently even if their activities are required at different locations and / or different times, or have different requirements for Fc function, such as when one region or domain is for target destruction and another region or domain is for immune stimulation. Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, there is a need for polypeptides that overcome the systemic or non-targeting nature of function, severe toxicity, poor pharmacokinetics, and inseparable activity.In addition, cancer cells can be stimulated by the presence of certain growth factors.It would be beneficial if they could interfere with such stimulation while also increasing the immune response to cancer cells.The present disclosure aims to meet one or more of these needs, provide other benefits, or at least provide the public with a useful option. [Means for solving the problem]

[0008] In some aspects, a linker polypeptide is provided that can be targeted to a certain kind of extracellular environment through the use of a targeting sequence.In some embodiments, the linker polypeptide can include a first targeting sequence; a second targeting sequence; and a first linker between the first targeting sequence and the second targeting sequence, the linker comprising a protease-cleaved polypeptide sequence.In some embodiments, the linker polypeptide can include a first active domain; a second active domain; a pharmacokinetic modulator; and a first linker between the pharmacokinetic modulator and the first active domain or between the first active domain and the second active domain, the first linker comprising a protease-cleaved polypeptide sequence.In some embodiments, the linker polypeptide can include a first active domain; an inhibitory polypeptide sequence that can block the activity of the first active domain; a first linker between the first active domain and the inhibitory polypeptide sequence, the linker comprising a protease-cleaved polypeptide sequence; and a first targeting sequence.

[0009] In some embodiments, when one or more protease-cleavable polypeptide sequences are cleaved by one or more proteases, the different functions of the different components of the linker polypeptide may be separated from each other and / or activated. For example, cleavage of the protease-cleavable polypeptide may allow an inhibitory polypeptide sequence to dissociate from a cytokine polypeptide sequence and / or an active domain (which may, for example, have an immune stimulatory function) to dissociate from the remainder of the linker polypeptide (which may, for example, have a target destruction function).

[0010] Many tumors and tumor microenvironments exhibit aberrant expression and activation of proteases. The present disclosure provides linker polypeptides with components that may be cleaved from each other and / or activated via proteolytic cleavage so that they become active when they contact proteases in the tumor or tumor microenvironment. In some cases, for example, this may result in an increase in active domains (e.g., cytokine or immunoglobulin domains) in and near the tumor or tumor microenvironment compared to the rest of the subject's body or healthy tissue. One exemplary advantage that may result is the formation of a gradient of active domains. Such gradients may be formed when linker polypeptides are administered and selectively or preferentially activated in the tumor or tumor microenvironment and then diffuse from these regions to the rest of the body. These gradients may, for example, increase the trafficking of immune cells to the tumor and tumor microenvironment. Immune cells that traffic to the tumor may infiltrate the tumor. Infiltrating immune cells can initiate immune responses against cancer. Infiltrating immune cells can also secrete their own chemokines and cytokines. Cytokines can exert autocrine and / or paracrine effects in tumors and tumor microenvironment. In some cases, immune cells include T cells, such as effector T cells or cytotoxic T cells, or NK cells.

[0011] Also described herein is a method of treatment and a method of administering the linker polypeptide described herein.Such administration can be systemic or local.In some embodiments, the linker polypeptide described herein can be administered systemically or locally to treat cancer.

[0012] The following embodiments are included:

[0013] In embodiment 1, a first targeting sequence; a second targeting sequence; and a first linker between the first targeting sequence and the second targeting sequence, the linker comprising a protease-cleavable polypeptide sequence. is a linker polypeptide comprising:

[0014] Embodiment 2 is a linker polypeptide of the immediately preceding embodiment, further comprising a first active domain, which may be proximal to the first targeting sequence relative to the second targeting sequence.

[0015] Embodiment 3 is a linker polypeptide according to the immediately preceding embodiment, further comprising an additional domain, which may comprise an inhibitory polypeptide sequence capable of blocking activity of the first active domain, a pharmacokinetic modulator, and / or a second active domain, and which may be proximal to the second targeting sequence relative to the first targeting sequence.

[0016] Embodiment 4 is a linker polypeptide according to the immediately preceding embodiment, comprising, in order from N-terminus to C-terminus or from C-terminus to N-terminus, a first active domain, a first targeting sequence, a first linker, a second targeting sequence, and an additional domain.

[0017] In embodiment 5, The first active domain; A second active domain; Pharmacokinetic modulators; and a first linker between the pharmacokinetic modulator and the first active domain, the first linker comprising a protease-cleavable polypeptide sequence. is a linker polypeptide comprising:

[0018] Embodiment 6 is a linker polypeptide according to embodiment 5, further comprising a first targeting sequence.

[0019] In embodiment 7, The first active domain; an inhibitory polypeptide sequence capable of blocking the activity of the first activity domain; a first linker between the first active domain and the inhibitory polypeptide sequence, the linker comprising a protease-cleaved polypeptide sequence; and First targeting sequence is a linker polypeptide comprising:

[0020] Embodiment 8 is a linker polypeptide as described in the immediately preceding embodiment, which comprises a pharmacokinetic modulator.

[0021] Embodiment 9 is a first polypeptide chain comprising a first active domain, a first domain of a pharmacokinetic modulator, and a first linker between the first active domain and the first domain of the pharmacokinetic modulator, the first active domain being C-terminal to the first domain of the pharmacokinetic modulator; a second polypeptide chain comprising a second domain of a pharmacokinetic modulator, an inhibitory polypeptide sequence capable of blocking the activity of the first active domain, and a second linker between the second domain of the pharmacokinetic modulator and the inhibitory polypeptide sequence. Including, the first linker comprises a protease-cleavable polypeptide sequence; The first polypeptide chain or the second polypeptide chain further comprises at least one targeting sequence. It is a linker polypeptide.

[0022] Embodiment 10 is a first polypeptide chain comprising a first active domain, a first domain of a pharmacokinetic modulator, and a first linker between the first active domain and the first domain of the pharmacokinetic modulator, the first active domain being N-terminal to the first domain of the pharmacokinetic modulator; a second polypeptide chain comprising a second domain of a pharmacokinetic modulator, an inhibitory polypeptide sequence capable of blocking the activity of the first active domain, and a second linker between the second domain of the pharmacokinetic modulator and the inhibitory polypeptide sequence. Including, the first linker comprises a protease-cleavable polypeptide sequence; The first polypeptide chain or the second polypeptide chain further comprises at least one targeting sequence. It is a linker polypeptide.

[0023] Embodiment 11 is a linker polypeptide according to embodiment 9 or 10, wherein the inhibitory polypeptide sequence is C-terminal to the second domain of the pharmacokinetic modulator.

[0024] Embodiment 12 is a linker polypeptide according to embodiment 9 or 10, wherein the inhibitory polypeptide sequence is N-terminal to the second domain of the pharmacokinetic modulator.

[0025] Embodiment 13 is a linker polypeptide according to any one of embodiments 9 to 12, wherein the targeting sequence is between the protease-cleavable polypeptide sequence and the first domain of the pharmacokinetic modulator.

[0026] Embodiment 14 is a linker polypeptide according to any one of embodiments 9 to 12, wherein the targeting sequence is between the protease-cleaved polypeptide sequence and the first active domain.

[0027] Embodiment 15 is a linker polypeptide according to any one of embodiments 9 to 12, wherein the targeting sequence is C-terminal to the first active domain.

[0028] Embodiment 16 is a linker polypeptide according to any one of embodiments 9 to 12, wherein the targeting sequence is N-terminal to the first active domain.

[0029] Embodiment 17 is a linker polypeptide according to any one of embodiments 9 to 12, wherein the targeting sequence is C-terminal to the inhibitory polypeptide sequence.

[0030] Embodiment 18 is a linker polypeptide according to any one of embodiments 9 to 12, wherein the targeting sequence is N-terminal to the inhibitory polypeptide sequence.

[0031] Embodiment 19 is a linker polypeptide according to any one of embodiments 9 to 12, wherein the targeting sequence is between the inhibitory polypeptide sequence and the second domain of the pharmacokinetic modulator.

[0032] Embodiment 20 is a linker polypeptide according to any one of embodiments 9 to 19, wherein the targeting sequence binds to heparin, and the targeting sequence may comprise SEQ ID NO:664.

[0033] Embodiment 21 is a linker polypeptide according to any one of embodiments 9 to 19, wherein the targeting sequence binds to type IV collagen, and the targeting sequence may comprise SEQ ID NO: 200.

[0034] Embodiment 22 is a linker polypeptide according to any one of embodiments 9 to 19, wherein the targeting sequence binds to type I collagen, and the targeting sequence may comprise SEQ ID NO:188.

[0035] Embodiment 23 is a linker polypeptide according to any one of embodiments 9 to 19, wherein the targeting sequence binds to fibronectin, and the targeting sequence may comprise SEQ ID NO:653.

[0036] Embodiment 24 is a linker polypeptide according to any one of embodiments 9 to 23, wherein the targeting sequence is a first targeting sequence and the linker polypeptide further comprises a second targeting sequence.

[0037] Embodiment 25 is a linker polypeptide according to the immediately preceding embodiment, wherein the first targeting sequence is part of a first polypeptide chain and the second targeting sequence is part of a second polypeptide chain.

[0038] Embodiment 26 is a linker polypeptide of the immediately preceding embodiment, wherein the first targeting sequence is C-terminal to the first active domain and the second targeting sequence is C-terminal to the inhibitory polypeptide sequence.

[0039] Embodiment 27 is a linker polypeptide according to any one of embodiments 24 to 26, wherein the second targeting sequence binds to heparin, and the targeting sequence may comprise SEQ ID NO:664.

[0040] Embodiment 28 is a linker polypeptide according to any one of embodiments 24 to 26, wherein the second targeting sequence binds to type IV collagen, and the targeting sequence may comprise SEQ ID NO: 200.

[0041] Embodiment 29 is a linker polypeptide according to any one of embodiments 24 to 26, wherein the second targeting sequence binds to type I collagen, and the targeting sequence may comprise SEQ ID NO: 188.

[0042] Embodiment 30 is a linker polypeptide according to any one of embodiments 24 to 26, wherein the second targeting sequence binds to fibronectin, and the targeting sequence may comprise SEQ ID NO:653.

[0043] Embodiment 31 is a linker polypeptide according to any one of embodiments 9 to 30, further comprising a second active domain, which may be part of a second polypeptide chain.

[0044] Embodiment 32 is a linker polypeptide according to any one of embodiments 9 to 31, wherein the inhibitory polypeptide sequence is a first inhibitory polypeptide sequence and the linker polypeptide further comprises a second inhibitory polypeptide sequence.

[0045] Embodiment 33 is a linker polypeptide according to the immediately preceding embodiment, wherein the second inhibitory polypeptide sequence is part of a second polypeptide chain.

[0046] Embodiment 34 is a linker polypeptide according to the immediately preceding embodiment, wherein the second inhibitory polypeptide sequence is C-terminal to the first inhibitory polypeptide sequence.

[0047] Embodiment 35 is a linker polypeptide according to any one of embodiments 32 to 34, wherein the second inhibitory polypeptide sequence is an immunoglobulin inhibitory polypeptide sequence.

[0048] Embodiment 36 is a linker polypeptide according to the immediately preceding embodiment, wherein the first inhibitory polypeptide sequence is an immunoglobulin inhibitory polypeptide sequence.

[0049] Embodiment 37 is a linker polypeptide according to embodiment 35 or 36, wherein one or each of the immunoglobulin inhibitory polypeptide sequences is a VHH.

[0050] Embodiment 38 is a linker polypeptide according to any one of embodiments 8 to 37, wherein the pharmacokinetic modulator comprises a heterodimeric Fc domain or a heterodimeric CH3 domain.

[0051] Embodiment 39 is a linker polypeptide according to the immediately preceding embodiment, wherein the heterodimeric Fc domain or the heterodimeric CH3 domain comprises a knob CH3 domain and a hole CH3 domain.

[0052] Embodiment 40 is a linker polypeptide according to the immediately preceding embodiment, wherein the first domain of the pharmacokinetic modulator is a knob CH3 domain and the second domain of the pharmacokinetic modulator is a hole CH3 domain.

[0053] Embodiment 41 is a linker polypeptide according to embodiment 39, wherein the first domain of the pharmacokinetic modulator is a hole CH3 domain and the second domain of the pharmacokinetic modulator is a knob CH3 domain.

[0054] Embodiment 42 is a linker polypeptide according to any one of embodiments 38 to 41, wherein the pharmacokinetic modulator comprises the sequence of SEQ ID NO:75.

[0055] Embodiment 43 is a linker polypeptide according to any one of embodiments 38 to 41, wherein the pharmacokinetic modulator comprises the sequence of SEQ ID NO:76.

[0056] Embodiment 44 is a linker polypeptide according to any one of embodiments 38 to 41, wherein the pharmacokinetic modulator comprises the sequence of SEQ ID NO:756.

[0057] Embodiment 45 is a linker polypeptide according to any one of embodiments 38 to 44, wherein the pharmacokinetic modulator comprises the sequence of SEQ ID NO:77.

[0058] Embodiment 46 is a linker polypeptide according to any one of embodiments 38 to 44, wherein the pharmacokinetic modulator comprises the sequence of SEQ ID NO:78.

[0059] Embodiment 47 is a linker polypeptide according to any one of embodiments 38 to 44, wherein the pharmacokinetic modulator comprises the sequence of SEQ ID NO:757.

[0060] Embodiment 48 is a linker polypeptide according to any one of the previous embodiments, wherein the first active domain comprises a first immunoglobulin antigen-binding domain.

[0061] Embodiment 49 is a linker polypeptide according to any one of the previous embodiments, wherein the second active domain comprises a second immunoglobulin antigen-binding domain.

[0062] Embodiment 50 is a linker polypeptide according to any one of the previous embodiments, wherein one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain independently comprises a VH region and a VL region.

[0063] Embodiment 51 is a linker polypeptide according to any one of the previous embodiments, wherein one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain independently comprises an Fv, scFv, Fab, or VHH.

[0064] Embodiment 52 is a linker polypeptide according to any one of the previous embodiments, wherein one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain is independently a humanized domain or a fully human domain.

[0065] Embodiment 53 is a linker polypeptide according to any one of the previous embodiments, wherein one or each of the first immunoglobulin antigen binding domain and the second immunoglobulin antigen binding domain is independently configured to bind to one or more sequences selected from a cancer cell surface antigen sequence, a growth factor sequence, and a growth factor receptor sequence.

[0066] Embodiment 54 is a linker polypeptide of the immediately preceding embodiment, wherein one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain is configured to independently bind to a HER2 sequence, an EGFR extracellular domain sequence, a PD-1 extracellular domain sequence, a PD-L1 extracellular domain sequence, or a CD3 extracellular domain sequence.

[0067] Embodiment 55 is a linker polypeptide according to any one of the previous embodiments, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain is configured to bind to a HER2 sequence.

[0068] Embodiment 56 is a linker polypeptide as described in the immediately preceding embodiment, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain comprises a VH region comprising hypervariable regions (HVRs) HVR-1, HVR-2, and HVR-3 of the VH region comprising the amino acid sequence of SEQ ID NO:910; and a VL region comprising HVR-1, HVR-2, and HVR-3 of the VL region comprising the amino acid sequence of SEQ ID NO:909.

[0069] Embodiment 57 is a linker polypeptide of the immediately preceding embodiment, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain comprises a VH region comprising the amino acid sequence of SEQ ID NO:910; and a VL region comprising the amino acid sequence of SEQ ID NO:909.

[0070] Embodiment 58 is a linker polypeptide according to embodiment 55 or 56, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain comprises an array having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the array of SEQ ID NO: 909 or 910.

[0071] Embodiment 59 is the linker polypeptide of embodiment 55, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain is the antigen-binding domain of trastuzumab.

[0072] Embodiment 60 is a linker polypeptide according to any one of the previous embodiments, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain is configured to bind to an EGFR extracellular domain sequence.

[0073] Embodiment 61 is a linker polypeptide of the immediately preceding embodiment, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain comprises a VH region comprising HVR-1, HVR-2, and HVR-3 of the VH region comprising the amino acid sequence of SEQ ID NO:914; and a VL region comprising HVR-1, HVR-2, and HVR-3 of the VL region comprising the amino acid sequence of SEQ ID NO:913.

[0074] Embodiment 62 is a linker polypeptide of the immediately preceding embodiment, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain comprises a VH region comprising the amino acid sequence of SEQ ID NO:914; and a VL region comprising the amino acid sequence of SEQ ID NO:913.

[0075] Embodiment 63 is a linker polypeptide according to embodiment 60 or 61, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain comprises an array having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the array of SEQ ID NO: 913 or 914.

[0076] Embodiment 64 is the linker polypeptide of embodiment 60, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain is the antigen-binding domain of cetuximab.

[0077] Embodiment 65 is a linker polypeptide of any one of the previous embodiments, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain is configured to bind to a PD-1 extracellular domain sequence.

[0078] Embodiment 66 is a linker polypeptide of the immediately preceding embodiment, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain comprises a VH region comprising HVR-1, HVR-2, and HVR-3 of the VH region comprising the amino acid sequence of SEQ ID NO:917; and a VL region comprising HVR-1, HVR-2, and HVR-3 of the VL region comprising the amino acid sequence of SEQ ID NO:918.

[0079] Embodiment 67 is a linker polypeptide of the immediately preceding embodiment, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain comprises a VH region comprising the amino acid sequence of SEQ ID NO:917; and a VL region comprising the amino acid sequence of SEQ ID NO:918.

[0080] Embodiment 68 is a linker polypeptide according to embodiment 65 or 66, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain comprises an array having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the array of SEQ ID NO: 917 or 918.

[0081] Embodiment 69 is the linker polypeptide of embodiment 65, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain is the antigen-binding domain of nivolumab.

[0082] Embodiment 70 is a linker polypeptide of any one of the previous embodiments, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain is configured to bind to a PD-L1 extracellular domain sequence.

[0083] Embodiment 71 is a linker polypeptide of the immediately preceding embodiment, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain comprises a VH region comprising HVR-1, HVR-2, and HVR-3 of the VH region comprising the amino acid sequence of SEQ ID NO:921; and a VL region comprising HVR-1, HVR-2, and HVR-3 of the VL region comprising the amino acid sequence of SEQ ID NO:922.

[0084] Embodiment 72 is a linker polypeptide of the immediately preceding embodiment, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain comprises a VH region comprising the amino acid sequence of SEQ ID NO:921; and a VL region comprising the amino acid sequence of SEQ ID NO:922.

[0085] Embodiment 73 is a linker polypeptide according to embodiment 70 or 71, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain comprises an array having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the array of SEQ ID NO: 921 or 922.

[0086] Embodiment 74 is a linker polypeptide according to embodiment 70, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain is the antigen-binding domain of atezolizumab.

[0087] Embodiment 75 is a linker polypeptide according to any one of the previous embodiments, wherein one of the first immunoglobulin antigen binding domain and the second immunoglobulin antigen binding domain is configured to bind to a CD3 extracellular domain sequence.

[0088] Embodiment 76 is a linker polypeptide according to the immediately preceding embodiment, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain comprises a VH region comprising HVR-1, HVR-2, and HVR-3 of the VH region comprising the amino acid sequence of any one of SEQ ID NOs: 925, 929, 933, and 937; and a VL region comprising HVR-1, HVR-2, and HVR-3 of the VL region comprising the amino acid sequence of any one of SEQ ID NOs: 926, 930, 934, and 938.

[0089] Embodiment 77 is a linker polypeptide of the immediately preceding embodiment, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain comprises a VH region comprising the amino acid sequence of any one of SEQ ID NOs: 925, 929, 933, and 937; and a VL region comprising the amino acid sequence of any one of SEQ ID NOs: 926, 930, 934, and 938.

[0090] Embodiment 78 is a linker polypeptide according to embodiment 75 or 76, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain comprises a sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to any one of SEQ ID NOs: 925, 926, 929, 930, 933, 934, 937, and 938.

[0091] Embodiment 79 is the linker polypeptide of embodiment 75, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain is the antigen-binding domain of teplizumab, muromonab, otelixizumab, or visilizumab.

[0092] Embodiment 80 is a linker polypeptide according to any one of the previous embodiments, wherein the first active domain comprises a receptor binding domain.

[0093] Embodiment 81 is a linker polypeptide according to the immediately preceding embodiment, wherein the receptor binding domain comprises a cytokine polypeptide sequence.

[0094] Embodiment 82 is a linker polypeptide according to any one of embodiments 80 to 81, wherein the receptor binding domain comprises a modification that prevents disulfide bond formation, but may otherwise comprise a wild-type sequence.

[0095] Embodiment 83 is a linker polypeptide according to any one of embodiments 80 to 82, wherein the receptor binding domain has at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of the wild-type receptor binding domain or to a receptor binding domain in Table 1.

[0096] Embodiment 84 is the linker polypeptide of the immediately preceding embodiment, wherein the receptor binding domain is a wild-type receptor binding domain.

[0097] Embodiment 85 is a linker polypeptide according to any one of embodiments 80 to 84, wherein the receptor binding domain is a monomeric cytokine or the receptor binding domain is a dimeric receptor binding domain comprising covalently (optionally via a polypeptide linker) or non-covalently associated monomers.

[0098] Embodiment 86 is an inhibitory polypeptide sequence capable of blocking the activity of the receptor binding domain; and a second linker between the receptor binding domain and the inhibitory polypeptide sequence, the second linker comprising a protease-cleaved polypeptide sequence. 86. The linker polypeptide of any one of embodiments 80 to 85, further comprising:

[0099] Embodiment 87 is a linker polypeptide according to any one of embodiments 80 to 86, insofar as it is dependent from any one of embodiments 9 to 24, wherein the inhibitory polypeptide sequence comprises a cytokine binding domain.

[0100] Embodiment 88 is a linker polypeptide according to any one of embodiments 9 to 47 or 86 to 87, wherein the inhibitory polypeptide sequence comprises a cytokine binding domain.

[0101] Embodiment 89 is a linker polypeptide according to embodiment 87 or 88, wherein the cytokine binding domain is a cytokine binding domain of a cytokine receptor or a cytokine binding domain of fibronectin.

[0102] Embodiment 90 is the linker polypeptide of the immediately preceding embodiment, wherein the cytokine binding domain is an immunoglobulin cytokine binding domain.

[0103] Embodiment 91 is a linker polypeptide according to the immediately preceding embodiment, wherein the immunoglobulin cytokine binding domain comprises a VL region and a VH region that binds a cytokine.

[0104] Embodiment 92 is a linker polypeptide according to embodiment 90 or 91, wherein the immunoglobulin cytokine binding domain is an Fv, scFv, Fab, or VHH.

[0105] Embodiment 93 is a linker polypeptide described in any one of embodiments 80 to 92, comprising a targeting sequence, wherein the targeting sequence is between the receptor binding domain and the protease-cleaved polypeptide sequence or one of the multiple protease-cleaved polypeptide sequences.

[0106] Embodiment 94 is a linker polypeptide according to any one of embodiments 80 to 93, wherein the receptor binding domain is an interleukin polypeptide sequence.

[0107] Embodiment 95 is a linker polypeptide according to any one of embodiments 80 to 94, wherein the receptor binding domain is capable of binding to a receptor comprising CD132.

[0108] Embodiment 96 is a linker polypeptide according to any one of embodiments 80 to 95, wherein the receptor binding domain is capable of binding to a receptor comprising CD122.

[0109] Embodiment 97 is a linker polypeptide according to any one of embodiments 80 to 96, wherein the receptor binding domain is capable of binding to a receptor comprising CD25.

[0110] Embodiment 98 is a linker polypeptide according to any one of embodiments 80 to 97, wherein the receptor binding domain is capable of binding to a receptor including IL-10R.

[0111] Embodiment 99 is a linker polypeptide according to any one of embodiments 80 to 98, wherein the receptor binding domain is capable of binding to a receptor including IL-15R.

[0112] Embodiment 100 is a linker polypeptide according to any one of embodiments 80 to 99, wherein the receptor binding domain is capable of binding to a receptor including CXCR3.

[0113] Embodiment 101 is a linker polypeptide according to any one of embodiments 80 to 100, wherein the receptor binding domain is an IL-2 polypeptide sequence.

[0114] Embodiment 102 is the linker polypeptide of the immediately preceding embodiment, wherein the IL-2 polypeptide sequence has at least 80, 85, 90, 95, 97, 98, or 99 percent identity to any one of SEQ ID NOs:1-4.

[0115] Embodiment 103 is the linker polypeptide of the immediately preceding embodiment, wherein the IL-2 polypeptide sequence comprises any one of SEQ ID NOs: 1-4.

[0116] Embodiment 104 is a linker polypeptide according to any one of embodiments 101 to 103, wherein the IL-2 polypeptide sequence is a human IL-2 polypeptide sequence.

[0117] Embodiment 105 is a linker polypeptide according to the immediately preceding embodiment, wherein the IL-2 polypeptide sequence comprises the sequence of SEQ ID NO:1.

[0118] Embodiment 106 is a linker polypeptide according to any one of embodiments 101 to 104, wherein the IL-2 polypeptide sequence comprises the sequence of SEQ ID NO:2.

[0119] Embodiment 107 is a linker polypeptide according to any one of the previous embodiments, wherein the inhibitory polypeptide sequence comprises the IL-2 binding domain of the IL-2 receptor (IL-2R).

[0120] Embodiment 108 is a linker polypeptide according to the previous embodiment, wherein the inhibitory polypeptide sequence comprises an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to any one of SEQ ID NOs: 10-29 and 40-51.

[0121] Embodiment 109 is a linker polypeptide according to embodiment 107 or 108, wherein the IL-2R is a human IL-2R.

[0122] Embodiment 110 is a linker polypeptide according to any one of the previous embodiments, wherein the inhibitory polypeptide sequence comprises an IL-2 binding immunoglobulin domain.

[0123] Embodiment 111 is the linker polypeptide of the immediately preceding embodiment, wherein the IL-2 binding immunoglobulin domain is a human IL-2 binding immunoglobulin domain.

[0124] Embodiment 112 is a linker polypeptide according to embodiment 110 or 111, wherein the IL-2-binding immunoglobulin domain comprises a VH region comprising hypervariable regions (HVRs) HVR-1, HVR-2, and HVR-3 having the sequences of SEQ ID NOs: 37, 38, and 39, respectively; and a VL region comprising HVR-1, HVR-2, and HVR-3 having the sequences of SEQ ID NOs: 34, 35, and 36, respectively.

[0125] Embodiment 113 is a linker polypeptide according to any one of embodiments 110 to 112, wherein the IL-2-binding immunoglobulin domain comprises a VH region comprising an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 33, and a VL region comprising an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 32; or a VH region comprising an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 749, and a VL region comprising an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 748.

[0126] Embodiment 114 is a linker polypeptide according to the previous embodiment, wherein the IL-2-binding immunoglobulin domain comprises a VH region comprising the sequence of SEQ ID NO: 33, and a VL region comprising the sequence of SEQ ID NO: 32; or a VH region comprising the sequence of SEQ ID NO: 749, and a VL region comprising the sequence of SEQ ID NO: 748.

[0127] Embodiment 115 is a linker polypeptide according to any one of embodiments 110 to 114, wherein the IL-2-binding immunoglobulin domain is an scFv.

[0128] Embodiment 116 is a linker polypeptide according to embodiment 110, 111 or 114, wherein the IL-2-binding immunoglobulin domain comprises the CDRs of the amino acid sequence of SEQ ID NO: 30, 31, 747, 850-856, or 863-870.

[0129] Embodiment 117 is a linker polypeptide according to embodiment 110, 111, 114, or 116, wherein the IL-2-binding immunoglobulin domain comprises an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 30, 31, 747, 850-856, or 863-870.

[0130] Embodiment 118 is the linker polypeptide of the immediately preceding embodiment, wherein the IL-2 binding immunoglobulin domain comprises the sequence of SEQ ID NO: 30, 31, 747, 850-856, or 863-870.

[0131] Embodiment 119 is a linker polypeptide according to any one of the previous embodiments, wherein the receptor binding domain is an IL-10 polypeptide sequence.

[0132] Embodiment 120 is a linker polypeptide according to the immediately preceding embodiment, wherein the IL-10 polypeptide sequence has at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO:900.

[0133] Embodiment 121 is the linker polypeptide of the immediately preceding embodiment, wherein the IL-10 polypeptide sequence comprises the sequence of SEQ ID NO:900.

[0134] Embodiment 122 is a linker polypeptide according to any one of embodiments 119 to 121, wherein the IL-10 polypeptide sequence is a human IL-10 polypeptide sequence.

[0135] Embodiment 123 is a linker polypeptide according to any one of embodiments 118 to 122, wherein the inhibitory polypeptide sequence comprises the IL-10 binding domain of the IL-10 receptor (IL-10R).

[0136] Embodiment 124 is a linker polypeptide according to the immediately preceding embodiment, wherein the inhibitory polypeptide sequence comprises an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO:1011 or 1012.

[0137] Embodiment 125 is a linker polypeptide according to embodiment 123 or 124, wherein the IL-10R is human IL-10R.

[0138] Embodiment 126 is a linker polypeptide according to any one of the previous embodiments, wherein the inhibitory polypeptide sequence comprises an IL-10-binding immunoglobulin domain.

[0139] Embodiment 127 is the linker polypeptide of the immediately preceding embodiment, wherein the IL-10 binding immunoglobulin domain is a human IL-10 binding immunoglobulin domain.

[0140] Embodiment 128 is a linker polypeptide according to embodiment 126 or 127, wherein the IL-10-binding immunoglobulin domain comprises a VH region comprising hypervariable regions (HVRs) HVR-1, HVR-2, and HVR-3 having the sequences of SEQ ID NOs: 946, 947, and 948, respectively; and a VL region comprising HVR-1, HVR-2, and HVR-3 having the sequences of SEQ ID NOs: 942, 943, and 944, respectively.

[0141] Embodiment 129 is a linker polypeptide according to any one of embodiments 126 to 128, wherein the IL-10-binding immunoglobulin domain comprises a VH region comprising an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 945, and a VL region comprising an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 941.

[0142] Embodiment 130 is a linker polypeptide according to the immediately preceding embodiment, wherein the IL-10-binding immunoglobulin domain comprises a VH region comprising the sequence of SEQ ID NO:945, and a VL region comprising the sequence of SEQ ID NO:941.

[0143] Embodiment 131 is a linker polypeptide according to any one of embodiments 126 to 130, wherein the IL-10-binding immunoglobulin domain is an scFv.

[0144] Embodiment 132 is a linker polypeptide according to the immediately preceding embodiment, wherein the IL-10-binding immunoglobulin domain comprises an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 939 or 940.

[0145] Embodiment 133 is a linker polypeptide according to the immediately preceding embodiment, wherein the IL-10-binding immunoglobulin domain comprises the sequence of SEQ ID NO: 939 or 940.

[0146] Embodiment 134 is a linker polypeptide according to any one of the previous embodiments, wherein the receptor binding domain is an IL-15 polypeptide sequence.

[0147] Embodiment 135 is a linker polypeptide according to the immediately preceding embodiment, wherein the IL-15 polypeptide sequence has at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO:901.

[0148] Embodiment 136 is the linker polypeptide of the immediately preceding embodiment, wherein the IL-15 polypeptide sequence comprises the sequence of SEQ ID NO:901.

[0149] Embodiment 137 is a linker polypeptide according to any one of embodiments 134 to 136, wherein the IL-15 polypeptide sequence is a human IL-15 polypeptide sequence.

[0150] Embodiment 138 is a linker polypeptide according to any one of embodiments 133 to 137, wherein the inhibitory polypeptide sequence comprises the IL-15 binding domain of the IL-15 receptor (IL-15R).

[0151] Embodiment 139 is a linker polypeptide according to the previous embodiment, wherein the inhibitory polypeptide sequence comprises an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to any one of SEQ ID NOs: 1016-1019.

[0152] Embodiment 140 is a linker polypeptide according to embodiment 97 or 98, wherein the IL-15R is human IL-15R.

[0153] Embodiment 141 is a linker polypeptide according to any one of the previous embodiments, wherein the inhibitory polypeptide sequence comprises an IL-15-binding immunoglobulin domain.

[0154] Embodiment 142 is the linker polypeptide of the immediately preceding embodiment, wherein the IL-15 binding immunoglobulin domain is a human IL-15 binding immunoglobulin domain.

[0155] Embodiment 143 is a linker polypeptide according to embodiment 141 or 142, wherein the IL-15-binding immunoglobulin domain comprises a VH region comprising HVR-1, HVR-2, and HVR-3 of the VH region comprising the amino acid sequence of any one of SEQ ID NOs: 950, 955, 957, 960, 963, 966, 969, 972, 975, 978, 981, 985, and 988; and a VL region comprising HVR-1, HVR-2, and HVR-3 of the VL region comprising the amino acid sequence of any one of SEQ ID NOs: 952, 954, 958, 961, 964, 967, 970, 973, 976, 979, 982, 984, and 987.

[0156] Embodiment 144 is a linker polypeptide according to any one of embodiments 141 to 143, wherein the IL-15 binding immunoglobulin domain comprises a VH region comprising an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to any one of SEQ ID NOs: 950, 955, 957, 960, 963, 966, 969, 972, 975, 978, 981, 985, and 988; and a VL region comprising an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to any one of SEQ ID NOs: 952, 954, 958, 961, 964, 967, 970, 973, 976, 979, 982, 984, and 987.

[0157] Embodiment 145 is a linker polypeptide according to the immediately preceding embodiment, wherein the IL-15-binding immunoglobulin domain comprises a VH region comprising the sequence of any one of SEQ ID NOs: 950, 955, 957, 960, 963, 966, 969, 972, 975, 978, 981, 985, and 988; and a VL region comprising the sequence of any one of SEQ ID NOs: 952, 954, 958, 961, 964, 967, 970, 973, 976, 979, 982, 984, and 987.

[0158] Embodiment 146 is a linker polypeptide according to any one of embodiments 141 to 145, wherein the IL-15-binding immunoglobulin domain is an scFv.

[0159] Embodiment 147 is a linker polypeptide according to the immediately preceding embodiment, wherein the IL-15-binding immunoglobulin domain comprises an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to any one of SEQ ID NOs: 953, 956, 959, 962, 965, 968, 971, 974, 977, 980, 983, and 986.

[0160] Embodiment 148 is a linker polypeptide according to the immediately preceding embodiment, wherein the IL-15 binding immunoglobulin domain comprises the sequence of any one of SEQ ID NOs: 953, 956, 959, 962, 965, 968, 971, 974, 977, 980, 983, and 986.

[0161] Embodiment 149 is a linker polypeptide according to any one of the previous embodiments, wherein the receptor binding domain is a CXCL9 polypeptide sequence.

[0162] Embodiment 150 is a linker polypeptide according to the immediately preceding embodiment, wherein the CXCL9 polypeptide sequence has at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO:902.

[0163] Embodiment 151 is the linker polypeptide of the immediately preceding embodiment, wherein the CXCL9 polypeptide sequence comprises the sequence of SEQ ID NO:902.

[0164] Embodiment 152 is a linker polypeptide according to any one of embodiments 149 to 150, wherein the CXCL9 polypeptide sequence is a human CXCL9 polypeptide sequence.

[0165] Embodiment 153 is a linker polypeptide according to any one of embodiments 148 to 152, wherein the inhibitory polypeptide sequence comprises the CXCL9-binding domain of CXCR3.

[0166] Embodiment 154 is a linker polypeptide according to the immediately preceding embodiment, wherein the inhibitory polypeptide sequence comprises an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO:1020 or 1021.

[0167] Embodiment 155 is a linker polypeptide according to embodiment 153 or 154, wherein the CXCR3 is human CXCR3.

[0168] Embodiment 156 is a linker polypeptide according to any one of the previous embodiments, wherein the inhibitory polypeptide sequence comprises a CXCL9-binding immunoglobulin domain.

[0169] Embodiment 157 is the linker polypeptide of the immediately preceding embodiment, wherein the CXCL9-binding immunoglobulin domain is a human CXCL9-binding immunoglobulin domain.

[0170] Embodiment 158 ​​is a linker polypeptide according to any one of the previous embodiments, wherein the receptor binding domain is a CXCL10 polypeptide sequence.

[0171] Embodiment 159 is a linker polypeptide according to the immediately preceding embodiment, wherein the CXCL10 polypeptide sequence has at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO:903.

[0172] Embodiment 160 is the linker polypeptide of the immediately preceding embodiment, wherein the CXCL10 polypeptide sequence comprises the sequence of SEQ ID NO:903.

[0173] Embodiment 161 is a linker polypeptide according to any one of embodiments 158 to 160, wherein the CXCL10 polypeptide sequence is a human CXCL10 polypeptide sequence.

[0174] Embodiment 162 is a linker polypeptide according to any one of embodiments 156 to 161, wherein the inhibitory polypeptide sequence comprises the CXCL10-binding domain of CXCR3.

[0175] Embodiment 163 is a linker polypeptide according to the immediately preceding embodiment, wherein the inhibitory polypeptide sequence comprises an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO:1020 or 1021.

[0176] Embodiment 164 is a linker polypeptide according to embodiment 162 or 163, wherein the CXCR3 is human CXCR3.

[0177] Embodiment 165 is a linker polypeptide according to any one of the previous embodiments, wherein the inhibitory polypeptide sequence comprises a CXCL10-binding immunoglobulin domain.

[0178] Embodiment 166 is the linker polypeptide of the immediately preceding embodiment, wherein the CXCL10-binding immunoglobulin domain is a human CXCL10-binding immunoglobulin domain.

[0179] Embodiment 167 is a linker polypeptide described in embodiment 165 or 166, wherein the CXCL10-binding immunoglobulin domain comprises a VH region comprising hypervariable regions (HVRs) HVR-1, HVR-2, and HVR-3 having the sequences of SEQ ID NOs: 993, 994, and 995, respectively; and a VL region comprising HVR-1, HVR-2, and HVR-3 having the sequences of SEQ ID NOs: 996, 997, and 998, respectively.

[0180] Embodiment 168 is a linker polypeptide described in any one of embodiments 165 to 167, wherein the CXCL10-binding immunoglobulin domain comprises a VH region comprising an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 991, and a VL region comprising an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 992.

[0181] Embodiment 169 is a linker polypeptide according to the immediately preceding embodiment, wherein the CXCL10-binding immunoglobulin domain comprises a VH region comprising the sequence of SEQ ID NO:991, and a VL region comprising the sequence of SEQ ID NO:992.

[0182] Embodiment 170 is a linker polypeptide according to any one of embodiments 165 to 169, wherein the CXCL10-binding immunoglobulin domain is an scFv.

[0183] Embodiment 171 is a linker polypeptide according to the immediately preceding embodiment, wherein the CXCL10-binding immunoglobulin domain comprises an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 989 or 990.

[0184] Embodiment 172 is a linker polypeptide according to the immediately preceding embodiment, wherein the CXCL10-binding immunoglobulin domain comprises the sequence of SEQ ID NO: 989 or 990.

[0185] Embodiment 173 is a linker polypeptide according to any one of the previous embodiments, wherein the inhibitory polypeptide sequence interferes with binding of the first active domain to a receptor for the first active domain and / or binding of the second active domain to a receptor for the second active domain.

[0186] Embodiment 174 is a linker polypeptide according to any one of the previous embodiments, wherein the inhibitory polypeptide sequence and the pharmacokinetic modulator are different elements of the linker polypeptide.

[0187] Embodiment 175 is a linker polypeptide according to any one of the previous embodiments, wherein the inhibitory polypeptide sequence comprises a steric blocker.

[0188] Embodiment 176 is a linker polypeptide according to any one of the previous embodiments, wherein the inhibitory polypeptide sequence comprises at least a portion of a pharmacokinetic modulator.

[0189] Embodiment 177 is a linker polypeptide according to any one of the previous embodiments, wherein the pharmacokinetic modulator comprises at least a portion of an immunoglobulin constant domain.

[0190] Embodiment 178 is a linker polypeptide as described in the immediately preceding embodiment, wherein the pharmacokinetic modulator comprises at least a portion of an immunoglobulin Fc region.

[0191] Embodiment 179 is a linker polypeptide as described in the immediately preceding embodiment, wherein the pharmacokinetic modulator comprises an immunoglobulin Fc region.

[0192] Embodiment 180 is a linker polypeptide according to any one of embodiments 177 to 179, wherein the immunoglobulin is a human immunoglobulin.

[0193] Embodiment 181 is a linker polypeptide according to any one of embodiments 177 to 180, wherein the immunoglobulin is an IgG.

[0194] Embodiment 182 is a linker polypeptide according to the immediately preceding embodiment, wherein the IgG is an IgG1, IgG2, IgG3, or IgG4.

[0195] Embodiment 183 is a linker polypeptide according to any of the previous embodiments, further comprising a growth factor binding polypeptide sequence, or a growth factor receptor binding polypeptide sequence.

[0196] Embodiment 184 is a linker polypeptide according to the immediately preceding embodiment, wherein the growth factor binding polypeptide sequence comprises a TGF-βR extracellular domain sequence.

[0197] Embodiment 185 is a linker polypeptide according to the immediately preceding embodiment, wherein the TGF-βR extracellular domain sequence comprises an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO:1022 or 1023.

[0198] Embodiment 186 is a linker polypeptide according to embodiments 142 to 144, wherein the growth factor binding polypeptide sequence comprises a growth factor binding immunoglobulin domain.

[0199] Embodiment 187 is a linker polypeptide according to the immediately preceding embodiment, wherein the growth factor binding immunoglobulin domain is configured to bind to TGF-β.

[0200] Embodiment 188 is a linker polypeptide described in embodiment 145 or 146, wherein the growth factor binding immunoglobulin domain comprises a VH region comprising HVR-1, HVR-2, and HVR-3 of the VH region comprising the amino acid sequence of SEQ ID NO: 1008; and a VL region comprising HVR-1, HVR-2, and HVR-3 of the VL region comprising the amino acid sequence of SEQ ID NO: 1010.

[0201] Embodiment 189 is a linker polypeptide according to the immediately preceding embodiment, wherein the growth factor binding immunoglobulin domain comprises a VH region comprising the amino acid sequence of SEQ ID NO:1008; and a VL region comprising the amino acid sequence of SEQ ID NO:1010.

[0202] Embodiment 190 is a linker polypeptide according to embodiments 185 to 189, wherein the growth factor binding immunoglobulin domain comprises a sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 1007 or 1009.

[0203] Embodiment 191 is a linker polypeptide according to embodiments 183 to 190, wherein the growth factor receptor-binding polypeptide sequence comprises a TGF-β sequence.

[0204] Embodiment 192 is a linker polypeptide according to the immediately preceding embodiment, wherein the TGF-β sequence comprises an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to any one of SEQ ID NOs: 904-906.

[0205] Embodiment 193 is a linker polypeptide according to embodiments 183 to 192, wherein the growth factor receptor-binding polypeptide sequence comprises a growth factor receptor-binding immunoglobulin domain.

[0206] Embodiment 194 is a linker polypeptide according to the immediately preceding embodiment, wherein the growth factor receptor-binding immunoglobulin domain is configured to bind to a TGF-βR extracellular domain sequence.

[0207] Embodiment 195 is a linker polypeptide according to embodiment 193 or 194, wherein the growth factor receptor binding immunoglobulin domain comprises a VH region comprising HVR-1, HVR-2, and HVR-3 of the VH region comprising the amino acid sequence of SEQ ID NO: 999 or 1003; and a VL region comprising HVR-1, HVR-2, and HVR-3 of the VL region comprising the amino acid sequence of SEQ ID NO: 1000 or 1004.

[0208] Embodiment 196 is a linker polypeptide according to the previous embodiment, wherein the growth factor receptor-binding immunoglobulin domain comprises a VH region comprising the amino acid sequence of SEQ ID NO: 999 or 1003; and a VL region comprising the amino acid sequence of SEQ ID NO: 1000 or 1004.

[0209] Embodiment 197 is a linker polypeptide described in embodiments 152 to 155, wherein the growth factor receptor binding immunoglobulin domain comprises an array having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to any one of the arrays of SEQ ID NOs: 1001, 1002, 1005, and 1006.

[0210] Embodiment 198 is a linker polypeptide according to any one of the previous embodiments, comprising multiple protease-cleavable polypeptide sequences.

[0211] Embodiment 199 is a linker polypeptide according to any one of the previous embodiments, wherein the protease-cleaved polypeptide sequence is C-terminal to the VH region, C-terminal to at least a portion of the CH1 domain, between the CH1 and CH2 domains, N-terminal to at least a portion of the CH2 domain, N-terminal to the inter-heavy chain disulfide bond, N-terminal to the disulfide bond within the CH2 domain, or N-terminal to the hinge region, or within the hinge region.

[0212] Embodiment 200 is a linker polypeptide according to any one of the previous embodiments, wherein the protease-cleavable polypeptide sequence is C-terminal to the first targeting sequence and the second targeting sequence.

[0213] Embodiment 201 is a linker polypeptide according to any one of the previous embodiments, wherein the protease-cleavable polypeptide sequence is N-terminal to the first targeting sequence and the second targeting sequence.

[0214] Embodiment 202 is a linker polypeptide according to any one of the previous embodiments, wherein the protease-cleavable polypeptide sequence is C-terminal to the first plurality of targeting sequences and N-terminal to the second plurality of targeting sequences.

[0215] Embodiment 203 is a linker polypeptide according to any one of the previous embodiments, wherein the protease-cleavable polypeptide sequence is C-terminal to a plurality of targeting sequences and N-terminal to at least one targeting sequence.

[0216] Embodiment 204 is a linker polypeptide according to any one of the previous embodiments, wherein the protease-cleavable polypeptide sequence is N-terminal to a plurality of targeting sequences and C-terminal to at least one targeting sequence.

[0217] Embodiment 205 is a linker polypeptide according to any one of the previous embodiments, wherein the protease-cleavable polypeptide sequence is C-terminal to the first targeting sequence and the second targeting sequence and is not N-terminal to the targeting sequence.

[0218] Embodiment 206 is a linker polypeptide according to any one of the previous embodiments, wherein the protease-cleavable polypeptide sequence is N-terminal to the first targeting sequence and the second targeting sequence and is not C-terminal to the targeting sequence.

[0219] Embodiment 207 is a linker polypeptide according to any one of the previous embodiments, configured to release the first active domain from the remainder of the linker polypeptide upon cleavage of the protease-cleavable polypeptide sequence.

[0220] Embodiment 208 is a linker polypeptide as described in the immediately preceding embodiment, wherein the first active domain is configured to maintain connection to one of the first targeting sequence and one of the second targeting sequence, one of the at least one targeting sequence, one of the first plurality of targeting sequences, one of the second plurality of targeting sequences, one of the plurality of targeting sequences, and one or more of the pharmacokinetic modulator upon cleavage of the protease-cleaved polypeptide sequence.

[0221] Embodiment 209 is a linker polypeptide according to any one of the previous embodiments, configured to release the second active domain from the remainder of the linker polypeptide upon cleavage of the protease-cleavable polypeptide sequence.

[0222] Embodiment 210 is a linker polypeptide as described in the immediately preceding embodiment, wherein the second active domain is configured to maintain connection to one of the first targeting sequence and the second targeting sequence, one of the at least one targeting sequence, one of the first plurality of targeting sequences, one of the second plurality of targeting sequences, one of the plurality of targeting sequences, and one or more of the pharmacokinetic modulator upon cleavage of the protease-cleaved polypeptide sequence.

[0223] Embodiment 211 is a further embodiment of the present invention, wherein the protease-cleaved polypeptide sequence is selected from the group consisting of metalloproteases, serine proteases, cysteine ​​proteases, aspartic acid proteases, threonine proteases, glutamic acid proteases, gelatinases, aspartic peptide lyases, cathepsins, kallikreins, plasmins, collagenases, hKl, hK10, hK15, stromelysins, factor Xa, chymotrypsin-like proteases, trypsin-like proteases, elastase-like proteases, subtilisin-like proteases, actinidains, bromelain, calpains, caspases, Mir1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matrosin, cysteine ​​... The linker polypeptide of any one of the previous embodiments is recognized by liptase, legumain, plasmepsin, nepenthesin, metalloexopeptidase, metalloendopeptidase, ADAM10, ADAM17, ADAM12, urokinase plasminogen activator (uPA), enterokinase, prostate-specific target (PSA, hK3), interleukin 1b converting enzyme, thrombin, FAP (FAP-a), dipeptidyl peptidase or dipeptidyl peptidase IV (DPPIV / CD26), type II transmembrane serine protease (TTSP), neutrophil elastase, proteinase 3, mast cell chymase, mast cell tryptase, or dipeptidyl peptidase.

[0224] Embodiment 212 is a linker polypeptide according to any one of the previous embodiments, wherein the protease-cleaved polypeptide sequence comprises any one of SEQ ID NOs: 701-742, or a variant having one or two mismatches compared to any one of SEQ ID NOs: 701-742.

[0225] Embodiment 213 is a linker polypeptide according to any one of the previous embodiments, wherein the protease-cleavable polypeptide sequence is recognized by a matrix metalloprotease.

[0226] Embodiment 214 is a linker polypeptide according to any one of the previous embodiments, wherein the protease-cleaved polypeptide sequence is recognized by MMP1.

[0227] Embodiment 215 is a linker polypeptide according to any one of the previous embodiments, wherein the protease-cleaved polypeptide sequence is recognized by MMP2.

[0228] Embodiment 216 is a linker polypeptide according to any one of the previous embodiments, wherein the protease-cleaved polypeptide sequence is recognized by MMP3.

[0229] Embodiment 217 is a linker polypeptide according to any one of the previous embodiments, wherein the protease-cleaved polypeptide sequence is recognized by MMP7.

[0230] Embodiment 218 is a linker polypeptide according to any one of the previous embodiments, wherein the protease-cleaved polypeptide sequence is recognized by MMP8.

[0231] Embodiment 219 is a linker polypeptide according to any one of the previous embodiments, wherein the protease-cleaved polypeptide sequence is recognized by MMP9.

[0232] Embodiment 220 is a linker polypeptide according to any one of the previous embodiments, wherein the protease-cleaved polypeptide sequence is recognized by MMP12.

[0233] Embodiment 221 is a linker polypeptide according to any one of the previous embodiments, wherein the protease-cleaved polypeptide sequence is recognized by MMP13.

[0234] Embodiment 222 is a linker polypeptide according to any one of the previous embodiments, wherein the protease-cleaved polypeptide sequence is recognized by MMP14.

[0235] Embodiment 223 is a linker polypeptide according to any one of the previous embodiments, wherein the protease-cleaved polypeptide sequence is recognized by more than one MMP.

[0236] Embodiment 224 is a linker polypeptide according to any one of the previous embodiments, wherein the protease-cleaved polypeptide sequence is recognized by two, three, four, five, six, or seven of MMP2, MMP7, MMP8, MMP9, MMP12, MMP13, and MMP14.

[0237] Embodiment 225 is a linker polypeptide according to any one of the previous embodiments, wherein the protease-cleaved polypeptide sequence comprises any one of SEQ ID NOs: 80-94, or a variant sequence having one or two mismatches compared to any one of SEQ ID NOs: 80-90.

[0238] Embodiment 226 is a linker polypeptide according to any one of the previous embodiments, wherein the protease-cleaved polypeptide sequence comprises the sequence of SEQ ID NO: 80 or a variant sequence having one or two mismatches compared thereto.

[0239] Embodiment 227 is a linker polypeptide according to any one of embodiments 1 to 225, wherein the protease-cleaved polypeptide sequence comprises the sequence of SEQ ID NO: 81 or a variant sequence having one or two mismatches compared thereto.

[0240] Embodiment 228 is a linker polypeptide according to any one of embodiments 1 to 225, wherein the protease-cleaved polypeptide sequence comprises the sequence of SEQ ID NO: 82 or a variant sequence having one or two mismatches compared thereto.

[0241] Embodiment 229 is a linker polypeptide according to any one of embodiments 1 to 225, wherein the protease-cleaved polypeptide sequence comprises the sequence of SEQ ID NO: 83 or a variant sequence having one or two mismatches compared thereto.

[0242] Embodiment 230 is a linker polypeptide according to any one of embodiments 1 to 225, wherein the protease-cleaved polypeptide sequence comprises the sequence of SEQ ID NO: 84 or a variant sequence having one or two mismatches compared thereto.

[0243] Embodiment 231 is a linker polypeptide according to any one of embodiments 1 to 225, wherein the protease-cleaved polypeptide sequence comprises the sequence of SEQ ID NO: 85 or a variant sequence having one or two mismatches compared thereto.

[0244] Embodiment 232 is a linker polypeptide according to any one of embodiments 1 to 225, wherein the protease-cleaved polypeptide sequence comprises the sequence of SEQ ID NO: 86 or a variant sequence having one or two mismatches compared thereto.

[0245] Embodiment 233 is a linker polypeptide according to any one of embodiments 1 to 225, wherein the protease-cleaved polypeptide sequence comprises the sequence of SEQ ID NO: 87 or a variant sequence having one or two mismatches compared thereto.

[0246] Embodiment 234 is a linker polypeptide according to any one of embodiments 1 to 225, wherein the protease-cleaved polypeptide sequence comprises the sequence of SEQ ID NO: 88 or a variant sequence having one or two mismatches compared thereto.

[0247] Embodiment 235 is a linker polypeptide according to any one of embodiments 1 to 225, wherein the protease-cleaved polypeptide sequence comprises the sequence of SEQ ID NO: 89 or a variant sequence having one or two mismatches compared thereto.

[0248] Embodiment 236 is a linker polypeptide according to any one of embodiments 1 to 225, wherein the protease-cleaved polypeptide sequence comprises the sequence of SEQ ID NO: 90 or a variant sequence having one or two mismatches compared thereto.

[0249] Embodiment 237 is a linker polypeptide according to any one of embodiments 1 to 225, wherein the protease-cleaved polypeptide sequence comprises any one of SEQ ID NOs: 80 to 90.

[0250] Embodiment 238 is a linker polypeptide according to any one of embodiments 1 to 225, wherein the protease-cleaved polypeptide sequence comprises the sequence of SEQ ID NO:91.

[0251] Embodiment 239 is a linker polypeptide according to any one of embodiments 1 to 225, wherein the protease-cleaved polypeptide sequence comprises the sequence of SEQ ID NO:92.

[0252] Embodiment 240 is a linker polypeptide according to any one of embodiments 1 to 225, wherein the protease-cleaved polypeptide sequence comprises the sequence of SEQ ID NO:93.

[0253] Embodiment 241 is a linker polypeptide according to any one of embodiments 1 to 225, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO:94.

[0254] Embodiment 242 is a linker polypeptide according to any one of the previous embodiments, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences are independently configured to bind to an extracellular matrix component, heparin, integrin, or syndecan; or configured to bind to an extracellular matrix component, heparin, IgB (CD79b), integrin, cadherin, heparan sulfate proteoglycan, syndecan, or fibronectin in a pH-sensitive manner; or wherein the targeting sequence comprises any one of SEQ ID NOs: 179-665, or a variant having one or two mismatches compared to any one of SEQ ID NOs: 179-665.

[0255] Embodiment 243 is a linker polypeptide according to any one of the previous embodiments, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences independently comprises any one of SEQ ID NOs: 179-665, or a variant having one or two mismatches compared to any one of SEQ ID NOs: 179-665.

[0256] Embodiment 244 is a linker polypeptide according to any one of the preceding embodiments, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences independently comprises any one of SEQ ID NOs: 179-665.

[0257] Embodiment 245 is a linker polypeptide according to any one of the previous embodiments, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences independently comprises any one of SEQ ID NOs: 200, 330, 619, 653, and 663-665, or a variant having one or two mismatches compared to any one of SEQ ID NOs: 200, 330, 619, 653, and 663-665.

[0258] Embodiment 246 is a linker polypeptide according to any one of the preceding embodiments, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences independently comprises any one of SEQ ID NOs: 200, 330, 619, 653, and 663-665.

[0259] Embodiment 247 is a linker polypeptide according to any one of the previous embodiments, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences are independently configured to bind to denatured collagen.

[0260] Embodiment 248 is a linker polypeptide according to any one of the previous embodiments, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences are independently configured to bind to collagen.

[0261] Embodiment 249 is a linker polypeptide according to embodiment 247 or 248, wherein the collagen is type I collagen.

[0262] Embodiment 250 is a linker polypeptide according to embodiment 247 or 248, wherein the collagen is type II collagen.

[0263] Embodiment 251 is a linker polypeptide according to embodiment 247 or 248, wherein the collagen is type III collagen.

[0264] Embodiment 252 is a linker polypeptide according to embodiment 247 or 248, wherein the collagen is type IV collagen.

[0265] Embodiment 253 is a linker polypeptide according to any one of the previous embodiments, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences are independently configured to bind to an integrin.

[0266] Embodiment 254 is a linker polypeptide according to the immediately preceding embodiment, wherein the integrin is one or more of α1β1 integrin, α2β1 integrin, α3β1 integrin, α4β1 integrin, α5β1 integrin, α6β1 integrin, α7β1 integrin, α9β1 integrin, α4β7 integrin, αvβ3 integrin, αvβ5 integrin, αIIbβ3 integrin, αIIIbβ3 integrin, αMβ2 integrin, or αIIbβ3 integrin.

[0267] Embodiment 255 is a linker polypeptide according to any one of the previous embodiments, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences are independently configured to bind to von Willebrand factor.

[0268] Embodiment 256 is a linker polypeptide according to any one of the previous embodiments, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences are independently configured to bind IgB.

[0269] Embodiment 257 is a linker polypeptide according to any one of the previous embodiments, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences are independently configured to bind to heparin.

[0270] Embodiment 258 is a linker polypeptide according to any one of the previous embodiments, wherein the first targeting sequence is configured to bind to heparin and the second targeting sequence is configured to bind to heparin, or the first targeting sequence is configured to bind to type IV collagen and the second targeting sequence is configured to bind to heparin, or the first targeting sequence is configured to bind to heparin and the second targeting sequence is configured to bind to type IV collagen.

[0271] Embodiment 259 is a linker polypeptide according to any one of the previous embodiments, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences are independently configured to bind to heparin and syndecan, heparan sulfate proteoglycan, or integrin, and the integrin may be one or more of α1β1 integrin, α2β1 integrin, α3β1 integrin, α4β1 integrin, α5β1 integrin, α6β1 integrin, α7β1 integrin, α9β1 integrin, α4β7 integrin, αvβ3 integrin, αvβ5 integrin, αIIbβ3 integrin, αIIIbβ3 integrin, αMβ2 integrin, or αIIbβ3 integrin.

[0272] Embodiment 260 is a linker polypeptide according to the immediately preceding embodiment, wherein the syndecan is one or more of syndecan-1, syndecan-4, and syndecan-2(w).

[0273] Embodiment 261 is a linker polypeptide according to any one of the previous embodiments, wherein one or each of the first targeting sequence and the second targeting sequence, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences are independently configured to bind to a heparan sulfate proteoglycan.

[0274] Embodiment 262 is a linker polypeptide according to any one of the previous embodiments, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences are independently configured to bind to a sulfated glycoprotein.

[0275] Embodiment 263 is a linker polypeptide according to any one of the previous embodiments, wherein one or each of the first targeting sequence and the second targeting sequence, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences are independently configured to bind to hyaluronic acid.

[0276] Embodiment 264 is a linker polypeptide according to any one of the previous embodiments, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences are independently configured to bind to fibronectin.

[0277] Embodiment 265 is a linker polypeptide according to any one of the previous embodiments, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences are independently configured to bind to a cadherin.

[0278] Embodiment 266 is a linker polypeptide according to any one of the previous embodiments, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences is independently configured to bind to its target in a pH-sensitive manner.

[0279] Embodiment 267 is a linker polypeptide according to any one of the previous embodiments, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences, independently, has a higher affinity for its target at a pH below normal physiological pH than normal physiological pH, and the pH below normal physiological pH may be below 7 or below 6.

[0280] Embodiment 268 is a linker polypeptide according to any one of the preceding embodiments, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences, independently, has a higher affinity for its target at a pH in the range of 5 to 7, e.g., 5 to 5.5, 5.5 to 6, 6 to 6.5, or 6.5 to 7, than normal physiological pH.

[0281] Embodiment 269 is a linker polypeptide according to any one of the previous embodiments, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences independently comprises one or more histidines, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 histidines.

[0282] Embodiment 270 is a linker polypeptide according to any one of the previous embodiments, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences independently comprises any one of SEQ ID NOs: 641-663, or a variant having one or two mismatches compared to any one of SEQ ID NOs: 641-663.

[0283] Embodiment 271 is a linker polypeptide according to any one of the preceding embodiments, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences independently comprises any one of SEQ ID NOs: 641-665.

[0284] Embodiment 272 is a linker polypeptide according to any one of the preceding embodiments, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences are independently configured to bind in a pH-sensitive manner to an extracellular matrix component, IgB (CD79b), an integrin, a cadherin, a heparan sulfate proteoglycan, a syndecan, or a fibronectin.

[0285] Embodiment 273 is a linker polypeptide according to the immediately preceding embodiment, wherein the extracellular matrix component is hyaluronic acid, heparin, heparan sulfate, or a sulfated glycoprotein.

[0286] Embodiment 274 is a linker polypeptide according to any one of the previous embodiments, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences are independently configured to bind to fibronectin in a pH-sensitive manner.

[0287] Embodiment 275 is a linker polypeptide according to any one of the previous embodiments, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences is independently configured to bind to its target with an affinity of between 0.1 nM and 1 nM, between 1 nM and 10 nM, between 10 nM and 100 nM, between 100 nM and 1 μM, between 1 μM and 10 μM, or between 10 μM and 100 μM.

[0288] Embodiment 276 is a linker polypeptide as described in the immediately preceding embodiment, wherein one or each of the first targeting sequence and the second targeting sequence, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences is independently configured to bind to its target with an affinity of 0.1 nM to 1 nM.

[0289] Embodiment 277 is a linker polypeptide according to embodiment 275, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences are independently configured to bind to their target with an affinity of 1 nM to 10 nM.

[0290] Embodiment 278 is a linker polypeptide according to embodiment 275, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences are independently configured to bind to their target with an affinity of 10 nM to 100 nM.

[0291] Embodiment 279 is a linker polypeptide according to embodiment 275, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences are independently configured to bind to their target with an affinity of 100 nM to 1 μM.

[0292] Embodiment 280 is a linker polypeptide described in embodiment 275, wherein one or each of the first targeting sequence and the second targeting sequence, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences are independently configured to bind to their target with an affinity of 1 μM to 10 μM.

[0293] Embodiment 281 is a linker polypeptide described in embodiment 275, wherein one or each of the first targeting sequence and the second targeting sequence, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences are independently configured to bind to their target with an affinity of 10 μM to 100 μM.

[0294] Embodiment 282 is a linker polypeptide according to any one of the previous embodiments, wherein at least one of the first linker and the second linker comprises one of a first targeting sequence and a second targeting sequence, one of at least one targeting sequence, one of a first plurality of targeting sequences, one of a second plurality of targeting sequences, or one of a plurality of targeting sequences.

[0295] Embodiment 283 is a linker polypeptide of the immediately preceding embodiment, wherein the protease-cleavable polypeptide sequence comprises one of a first targeting sequence and a second targeting sequence, one of at least one targeting sequence, one of a first plurality of targeting sequences, one of a second plurality of targeting sequences, or one of a plurality of targeting sequences.

[0296] Embodiment 284 is a linker polypeptide according to any one of the previous embodiments, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences increases the serum half-life of the linker polypeptide.

[0297] Embodiment 285 is a linker polypeptide according to any one of the previous embodiments, wherein one or each of the first and second targeting sequences, one or each of at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the multiple targeting sequences synergistically increases the serum half-life of the linker polypeptide in combination with a pharmacokinetic modulator, or in combination with another one of the first and second targeting sequences, another one of at least one targeting sequence, another one of the first plurality of targeting sequences, another one of the second plurality of targeting sequences, or another one of the multiple targeting sequences.

[0298] Embodiment 286 is a linker polypeptide according to any one of the previous embodiments, wherein one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences independently increases the serum half-life of the linker polypeptide.

[0299] Embodiment 287 is a linker polypeptide according to any one of the previous embodiments, further comprising a blocking agent conjugated to one or each of the first active domain and the second active domain.

[0300] Embodiment 288 is a linker polypeptide according to the previous embodiment, wherein the blocking agent is conjugated to one or each of the first and second active domains via a protease-cleavable polypeptide sequence.

[0301] Embodiment 289 is a linker polypeptide according to embodiment 287 or 288, wherein the blocking agent is albumin.

[0302] Embodiment 290 is a linker polypeptide according to any one of embodiments 287 to 289, wherein the blocking agent is serum albumin.

[0303] Embodiment 291 is a linker polypeptide according to any one of embodiments 287 to 290, wherein the blocking agent is human albumin.

[0304] Embodiment 292 is a linker polypeptide according to any one of the previous embodiments, further comprising a chemotherapeutic agent.

[0305] Embodiment 293 is a linker polypeptide according to the immediately preceding embodiment, wherein the chemotherapeutic agent is conjugated to a pharmacokinetic modulator.

[0306] Embodiment 294 is an embodiment in which the chemotherapeutic agent is selected from the group consisting of altretamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, mechlorethamine, melphalan, oxaliplatin, temozolomide, thiotepa, trabectedin, carmustine, lomustine, streptozocin, azacitidine, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cytarabine, decitabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, nelarabine, pemetrexed, pentostatin, pralatrexate, thioguanine, trifluridine. , tipiracil, daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, bleomycin, dactinomycin, mitomycin c, mitoxantrone, irinotecan, topotecan, etoposide, mitoxantrone, teniposide, cabazitaxel, docetaxel, paclitaxel, vinblastine, vincristine, vinorelbine, prednisone, methylprednisolone, dexamethasone, retinoic acid, arsenic trioxide, asparaginase, eribulin, hydroxyurea, ixabepilone, mitotane, omacetaxine, pegaspargase, procarbazine, romidepsin, and vorinostat.

[0307] Embodiment 295 is a linker polypeptide according to any of the previous embodiments, wherein the molecular weight of one or each of the first active domain and the second active domain is independently about 14 kDa or less.

[0308] Embodiment 296 is a linker polypeptide according to the immediately preceding embodiment, wherein the molecular weight is from about 12 kDa to about 14 kDa.

[0309] Embodiment 297 is a linker polypeptide according to embodiment 295, having a molecular weight of about 10 kDa to about 12 kDa.

[0310] Embodiment 298 is a linker polypeptide according to embodiment 295, having a molecular weight of about 8 kDa to about 10 kDa.

[0311] Embodiment 299 is a linker polypeptide according to embodiment 295, having a molecular weight of about 6 kDa to about 8 kDa.

[0312] Embodiment 300 is a linker polypeptide according to embodiment 295, having a molecular weight of about 4 kDa to about 6 kDa.

[0313] Embodiment 301 is a linker polypeptide according to embodiment 295, having a molecular weight of about 2 kDa to about 4 kDa.

[0314] Embodiment 302 is a linker polypeptide according to embodiment 295, having a molecular weight of about 800 Da to about 2 kDa.

[0315] Embodiment 303 is a linker polypeptide according to any of embodiments 1 to 294, wherein the molecular weight of one or each of the first active domain and the second active domain is, independently, about 16 kDa or greater than 16 kDa.

[0316] Embodiment 304 is a linker polypeptide according to the immediately preceding embodiment, wherein the molecular weight is from about 16 kDa to about 18 kDa.

[0317] Embodiment 305 is a linker polypeptide according to embodiment 303, having a molecular weight of about 18 kDa to about 20 kDa.

[0318] Embodiment 306 is a linker polypeptide according to embodiment 303, having a molecular weight of about 20 kDa to about 22 kDa.

[0319] Embodiment 307 is a linker polypeptide according to embodiment 303, having a molecular weight of about 22 kDa to about 24 kDa.

[0320] Embodiment 308 is a linker polypeptide according to embodiment 303, having a molecular weight of about 24 kDa to about 26 kDa.

[0321] Embodiment 309 is a linker polypeptide according to embodiment 303, having a molecular weight of about 26 kDa to about 28 kDa.

[0322] Embodiment 310 is a linker polypeptide according to embodiment 303, having a molecular weight of about 28 kDa to about 30 kDa.

[0323] Embodiment 311 is a linker polypeptide according to embodiment 303, having a molecular weight of about 30 kDa to about 50 kDa.

[0324] Embodiment 312 is a linker polypeptide according to embodiment 303, having a molecular weight of about 50 kDa to about 100 kDa.

[0325] Embodiment 313 is a linker polypeptide according to embodiment 303, having a molecular weight of about 100 kDa to about 150 kDa.

[0326] Embodiment 314 is a linker polypeptide according to embodiment 303, having a molecular weight of about 150 kDa to about 200 kDa.

[0327] Embodiment 315 is a linker polypeptide according to embodiment 303, having a molecular weight of about 200 kDa to about 250 kDa.

[0328] Embodiment 316 is a linker polypeptide according to embodiment 303, having a molecular weight of about 250 kDa to about 300 kDa.

[0329] Embodiment 317 is a linker polypeptide described in any one of the previous embodiments, comprising a combination of a targeting sequence and a protease-cleavable sequence, wherein the combination of the targeting sequence and the protease-cleavable sequence is any one of SEQ ID NOs: 667-673.

[0330] Embodiment 318 is a linker polypeptide comprising an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to any one of SEQ ID NOs: 800-848 or 1024-1041.

[0331] Embodiment 319 is a linker polypeptide according to the previous embodiment, comprising any one of SEQ ID NOs: 800-848 or 1024-1041.

[0332] Embodiment 320 is a pharmaceutical composition comprising a linker polypeptide according to any one of the previous embodiments.

[0333] Embodiment 321 is a linker polypeptide or a pharmaceutical composition according to any one of the previous embodiments for use in therapy.

[0334] Embodiment 322 is a linker polypeptide or a pharmaceutical composition according to any one of the previous embodiments for use in the treatment of cancer.

[0335] Embodiment 323 is a method of treating cancer, comprising administering to a subject in need thereof a linker polypeptide or a pharmaceutical composition according to any one of the previous embodiments.

[0336] Embodiment 324 is the use of a linker polypeptide or a pharmaceutical composition according to any one of embodiments 1 to 321 for the manufacture of a medicament for treating cancer.

[0337] Embodiment 325 is a method, use, or linker polypeptide for use according to any one of embodiments 322 to 324, wherein the cancer is a solid tumor.

[0338] Embodiment 326 is a method, use, or linker polypeptide for use according to the immediately preceding embodiment, wherein the solid tumor is metastatic and / or unresectable.

[0339] Embodiment 327 is a method, use, or linker polypeptide for use according to any one of embodiments 322 to 326, wherein the cancer is a PD-L1 expressing cancer.

[0340] Embodiment 328 is a linker polypeptide for use according to any one of embodiments 322 to 327, wherein the cancer is melanoma, colorectal cancer, breast cancer, pancreatic cancer, lung cancer, prostate cancer, ovarian cancer, cervical cancer, gastric or gastrointestinal cancer, lymphoma, colon or colorectal cancer, endometrial cancer, thyroid cancer, or bladder cancer.

[0341] Embodiment 329 is a method, use, or linker polypeptide for use according to any one of embodiments 322 to 328, wherein the cancer is a microsatellite instability high cancer.

[0342] Embodiment 330 is a method, use, or linker polypeptide for use according to any one of embodiments 322 to 329, wherein the cancer is mismatch repair deficient.

[0343] Embodiment 331 is a nucleic acid encoding a linker polypeptide according to any one of embodiments 1 to 319.

[0344] Embodiment 332 is an expression vector comprising a nucleic acid according to the immediately preceding embodiment.

[0345] Embodiment 333 is a host cell comprising the nucleic acid of embodiment 331 or the vector of embodiment 332.

[0346] Embodiment 334 is a method of producing a linker polypeptide, comprising culturing a host cell as described in the immediately preceding embodiment under conditions such that the linker polypeptide is produced.

[0347] Embodiment 335 is the method of the immediately preceding embodiment, further comprising isolating the linker polypeptide. [Brief description of the drawings]

[0348] [Figure 1A] FIG 1A shows an exemplary linker polypeptide structure and an SDS-PAGE gel (with Coomassie staining) characterizing a number of purified linker polypeptides. FIG 1B-1C show SDS-PAGE gels (with Coomassie staining), respectively, characterizing a number of purified linker polypeptides. FIG 1D shows another exemplary linker polypeptide structure and an SDS-PAGE gel (with Coomassie staining) characterizing a number of purified linker polypeptides. [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 1D] Same as above. [Figure 2A] 2A-2F each show one or more SDS-PAGE gels followed by immunoblotting characterizing multiple linker polypeptides with and without treatment with matrix metallopeptidase 9 (MMP9). [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 2D] Same as above. [Figure 2E] Same as above. [Figure 2F] Same as above. [Figure 3A]3A-3BB show the results of a HEK Blue IL-2 assay measuring the IL-2 and IL-15 activities, respectively, of specific linker polypeptides with and without treatment with MMPs. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above. [Figure 3E] Same as above. [Figure 3F] Same as above. [Figure 3G] Same as above. [Figure 3H] Same as above. [Figure 3I] Same as above. [Figure 3J] Same as above. [Figure 3K] Same as above. [Figure 3L] Same as above. [Figure 3M] Same as above. [Figure 3N] Same as above. [Figure 3O] Same as above. [Figure 3P] Same as above. [Figure 3Q] Same as above. [Figure 3R] Same as above. [Figure 3S] Same as above. [Figure 3T] Same as above. [Figure 3U] Same as above. [Figure 3V] Same as above. [Figure 3W] Same as above. [Figure 3X] Same as above. [Figure 3Y] Same as above. [Figure 3Z] Same as above. [Figure 3AA] Same as above. [Figure 3BB] Same as above. [Figure 4A]FIG. 4A shows exemplary structures of different MMP linker peptides within a linker polypeptide, particularly within a linker peptide that binds heparin. FIG. 4B shows the results of an assay measuring binding of the linker peptide of FIG. 4A to heparin. FIG. 4C shows exemplary structures of different MMP linker peptides within a linker polypeptide, particularly within a linker peptide that binds fibronectin, and also shows the results of an assay measuring binding of the linker peptide to fibronectin. FIG. 4D shows exemplary structures of different MMP linker peptides within a linker polypeptide, particularly within a linker peptide that binds collagen, and also shows the results of an assay measuring binding of the linker peptide to collagen. FIG. 4E shows exemplary structures of different linker polypeptides, and also shows the results of an assay measuring binding of the linker polypeptide to heparin. FIG. 4F shows the results of an assay measuring binding of heparin by different linker polypeptides, including linker polypeptides that share the same heparin-binding motif as the linker polypeptide construct CC in FIG. 4E. An asterisk (*) indicates that for construct NN, the software was unable to calculate an EC50 based on the fit; however, the binding curve for construct NN mimicked the binding profile of construct CC. FIG. 4G shows the results of an assay measuring binding to heparin with different linker polypeptides, including a linker polypeptide that shares the same heparin-binding motif as linker polypeptide construct CC in FIG. 4E. FIG. 4H shows the results of an assay measuring binding to heparin with different linker polypeptides, including a linker polypeptide that shares the same heparin-binding motif as linker polypeptide construct Y in FIG. 4E. FIG. 4I shows the results of an assay measuring binding to heparin with different linker polypeptides, including a linker polypeptide that shares the same heparin-binding motif as linker polypeptide construct Y in FIG. 4E. FIG. 4J shows the results of an assay measuring binding to heparin with different IL-15Rα-IL-15 linker polypeptides.Figure 4K shows the results of an assay measuring binding to fibronectin by different linker polypeptides, Figure 4L shows the results of a pull-down assay measuring binding to collagen by different linker polypeptides, and Figure 4M shows the results of an assay measuring binding to heparin by different linker polypeptides with or without a heparin-binding site. [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above. [Figure 4E] Same as above. [Figure 4F] Same as above. [Figure 4G] Same as above. [Figure 4H] Same as above. [Figure 4I] Same as above. [Figure 4J] Same as above. [Figure 4K] Same as above. [Figure 4L] Same as above. [Figure 4M] Same as above. [Figure 5A] Figure 5A shows the results of real-time whole-body imaging to measure intratumoral levels of IL-2 fusion proteins in vivo using fluorescently labeled proteins, and Figure 5B shows the levels of the fusion proteins in Figure 5A. [Figure 5B] Same as above. [Figure 6] FIG. 6 shows measurements of tumor volume in C57BL / 6 mice inoculated with B16F10 melanoma cells and treated with different linker polypeptides, and also shows a schematic ranking the antitumor activity of the different linker polypeptides. [Figure 7A] 7A-7D show the results of assays measuring intratumoral levels of full-length fusion protein (FIG. 7A), intratumoral levels of IL-2 (FIG. 7B), intratumoral levels of IFN-γ (FIG. 7C), and serum levels of full-length fusion protein (FIG. 7D), respectively. [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 7D] Same as above. [Figure 8A] Figures 8A-8B show the results of assays measuring serum levels of TNF-α (Figure 8A) and IL-6 (Figure 8B), respectively, after animals were treated with different linker polypeptides, and Figure 8C shows the results of an AST activity assay after animals were treated with different linker polypeptides. [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 9A] 9A-9D each illustrate a linker polypeptide according to certain embodiments of the present disclosure (AD: active domain; PM: pharmacokinetic modulator; CL: protease-cleavable polypeptide sequence, which may be a targeting sequence; IBD: immunoglobulin antigen-binding domain; D: chemotherapeutic drug). [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 9D] Same as above. [Figure 10A] 10A-10B each illustrate a linker polypeptide according to certain embodiments of the present disclosure (AD: active domain; PM: pharmacokinetic modulator; CL: protease-cleavable polypeptide sequence, which may be a targeting sequence; IBD: immunoglobulin antigen-binding domain; RBD: receptor-binding domain; CY: cytokine polypeptide sequence). [Figure 10B] Same as above. [Figure 11A] 11A-11B each illustrate the release of a first active domain from the remainder of the linker polypeptide after one or more protease-cleavable polypeptide sequences have been cleaved (AD: active domain; PM: pharmacokinetic modulator; CL: protease-cleavable polypeptide sequence, which may be a targeting sequence; IBD: immunoglobulin antigen-binding domain; D: chemotherapeutic drug). [Figure 11B] Same as above. [Figure 12A] 12A-12B each illustrate the release of a first active domain from the remainder of the linker polypeptide after one or more protease-cleavable polypeptide sequences have been cleaved (AD: active domain; PM: pharmacokinetic modulator; CL: protease-cleavable polypeptide sequence, which may be a targeting sequence; IBD: immunoglobulin antigen-binding domain; RBD: receptor-binding domain; CY: cytokine polypeptide sequence). [Figure 12B] Same as above. [Figure 13A] Figures 13A-13C show the effect of treatment with different fusion proteins on tumor xenografts. Mean tumor volumes are shown in Figures 13A-13B, and inhibition on tumor volume is shown in Figure 13C. Figure 13D shows the levels of IFN-γ in mice bearing tumor xenografts and treated with different fusion proteins. [Figure 13B] Same as above. [Figure 13C] Same as above. [Figure 13D] Same as above. [Figure 14A] 14A-14E show results from flow cytometry analysis of select immune cell populations within tumors harvested in a mouse syngeneic model. [Figure 14B] Same as above. [Figure 14C] Same as above. [Figure 14D] Same as above. [Figure 14E] Same as above. [Figure 15A]FIG. 15A shows a schematic diagram for asymmetric IL-2Fc fusion proteins containing an ECM targeting sequence and single or double masks. FIG. 15B shows the results of SDS-PAGE analysis for asymmetric IL-2Fc fusion proteins. FIG. 15C-15U show the results of a HEK Blue IL-2 assay measuring the IL-2 activity of specific asymmetric IL-2Fc fusion proteins with and without MMP treatment, respectively. FIG. 15V-15X show the results from an assay measuring binding to heparin and fibronectin by different asymmetric IL-2Fc fusion proteins with or without heparin or fibronectin binding sites. FIG. 15Y shows the results from an assay measuring binding to collagen by different asymmetric IL-2Fc fusion proteins with or without collagen binding sites. [Figure 15B] Same as above. [Figure 15C] Same as above. [Figure 15D] Same as above. [Figure 15E] Same as above. [Figure 15F] Same as above. [Figure 15G] Same as above. [Figure 15H] Same as above. [Figure 15I] Same as above. [Figure 15J] Same as above. [Figure 15K] Same as above. [Figure 15L] Same as above. [Figure 15M] Same as above. [Figure 15N] Same as above. [Figure 15O] Same as above. [Figure 15P] Same as above. [Figure 15Q] Same as above. [Figure 15R] Same as above. [Figure 15S] Same as above. [Figure 15T] Same as above. [Figure 15U]Same as above. [Figure 15V] Same as above. [Figure 15W] Same as above. [Fig. 15X] Same as above. [Figure 15Y] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0349] This specification describes exemplary embodiments and applications of the present disclosure. However, this disclosure is not limited to these exemplary embodiments and applications or the manner in which the exemplary embodiments and applications are implemented or described herein. The term "or" is used in an inclusive sense, i.e., synonymous with "and / or," unless the context dictates otherwise. It is noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the," as well as the use of any singular form of any word, include multiple referents unless expressly and unambiguously limited to one referent. As used herein, the terms "comprise," "include," and grammatical variations thereof are intended to be non-limiting, such that the enumeration of items in a list is not a enumeration to the exclusion of other similar items that may be replaced by them or added to the listed items. The division of sections within this specification is provided solely for the convenience of the reader and does not limit any combination of elements discussed. In the event of a conflict or inconsistency between the material incorporated by reference and the expressly set forth content provided herein, the expressly set forth content shall control.

[0350] Overview Provided herein is a linker polypeptide comprising a first targeting sequence; a second targeting sequence; and a first linker between the first targeting sequence and the second targeting sequence, the linker comprising a protease-cleaved polypeptide sequence.In some embodiments, the linker polypeptide comprises a first active domain; a second active domain; a pharmacokinetic modulator; and a first linker between the pharmacokinetic modulator and the first active domain, the first linker comprising a protease-cleaved polypeptide sequence.In some embodiments, the linker polypeptide comprises a first active domain; an inhibitory polypeptide sequence capable of blocking the activity of the first active domain; a first linker between the first active domain and the inhibitory polypeptide sequence, the linker comprising a protease-cleaved polypeptide sequence; and a first targeting sequence.

[0351] Proteolysis by the protease-cleaved polypeptide sequence may release the first binding domain and / or the second binding domain so that it may, for example, neutralize tumor antigens and / or activate immune cells. In addition, in some embodiments, each of the active domains may bind to a growth factor and reduce the extent to which the growth factor exerts an activity in vivo, such as stimulating cancer cell growth.

[0352] In some embodiments, the protease-cleavable polypeptide sequence is cleavable by a protease that is expressed at a higher level in the tumor microenvironment (TME) than in healthy tissue of the same type. In some embodiments, the protease-cleavable polypeptide sequence is a matrix metalloprotease (MMP)-cleavable linker, such as any of the MMP-cleavable linkers described herein. Without being bound to any particular theory, increased expression and / or activity of proteases, including but not necessarily limited to MMPs, in the tumor microenvironment (TME) may provide a mechanism for achieving selective or preferential activation of the linker polypeptide at or near the tumor site. Certain protease-cleavable polypeptide sequences described herein are believed to be particularly suitable for achieving such selective or preferential activation.

[0353] In other embodiments, the first targeting sequence and / or the second targeting sequence is configured to bind to an extracellular matrix component, an integrin, or a syndecan, or to bind pH-sensitively to fibronectin. In some embodiments, the targeting sequence is a targeting sequence described herein, such as a targeting sequence configured to bind to an extracellular matrix component, a heparin, an integrin, or a syndecan; or a targeting sequence configured to bind pH-sensitively to an extracellular matrix component, a heparin, an IgB (CD79b), an integrin, a cadherin, a heparan sulfate proteoglycan, a syndecan, or a fibronectin; or a targeting sequence comprising any one of the sequences of SEQ ID NOs: 179-665. The targeting sequence may promote the accumulation and / or increased residence time of the linker polypeptide and / or the released active domain in the extracellular matrix (ECM). In some embodiments, the targeting sequence is combined with a protease-cleavable polypeptide sequence that is expressed at high levels in the TME and / or is cleavable by an MMP.

[0354] In some embodiments, the pharmacokinetic modulator may, for example, increase the half-life of the linker polypeptide.

[0355] Exemplary component sequences of linker polypeptides are shown in Tables 1 and 2. In Table 1, "X Hy " represents a hydrophobic amino acid residue. In some embodiments, the hydrophobic amino acid residue is any one of glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan (Trp). In some embodiments, the hydrophobic amino acid residue is any one of Ala, Leu, Val, Ile, Pro, Phe, Met, and Trp. In some embodiments, the hydrophobic amino acid residue is any one of Leu, Val, Ile, Pro, Phe, Met, and Trp. In some embodiments, the hydrophobic amino acid residue is any one of Ala, Leu, Val, Ile, Phe, Met, and Trp. In some embodiments, the hydrophobic amino acid residue is any one of Leu, Val, Ile, Phe, Met, and Trp. "(Pip)" represents piperidine. "(Hof)" stands for homophenylalanine. "(Cit)" stands for citrulline. "(Et)" stands for ethionine. "C(me)" stands for methylcysteine. Within a particular sequence, underlines are used to indicate mutation positions.

[0356] The present disclosure further provides for the use of these linker polypeptides, for example, for the treatment of cancer. In some embodiments, the linker polypeptide is selectively or preferentially cleaved within the tumor microenvironment, which may result in beneficial effects, such as improved recruitment and / or activation of immune cells in the vicinity of the tumor and / or reduced systemic exposure to certain components of the linker polypeptide.

[0357]

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

Table 1-26

Table 1-27

Table 1-28

Table 1-29

Table 1-30

Table 1-31

Table 1-32

Table 1-33

Table 1-34

Table 1-35

Table 1-36

Table 1-37

Table 1-38

Table 1-39

Table 1-40

Table 1-41

Table 1-42

Table 1-43

Table 1-44

Table 1-45

Table 1-46

Table 1-47

Table 1-48

Table 1-49

Table 1-50

Table 1-51

Table 1-52

Table 1-53

Table 1-54

Table 1-55

Table 1-56

Table 1-57

Table 1-58

Table 1-59

Table 1-60

Table 1-61

Table 1-62

Table 1-63

Table 1-64

Table 1-65

Table 1-66

Table 1-67

Table 1-68

Table 1-69

Table 1-70

Table 1-71

Table 1-72

Table 1-73

Table 1-74

Table 1-75

Table 1-76

Table 1-77

Table 1-78

Table 1-79

Table 1-80

Table 1-81

Table 1-82

Table 1-83

Table 1-84

Table 1-85

Table 1-86

Table 1-87

Table 1-88

Table 1-89

Table 1-90

Table 1-91

Table 1-92

Table 1-93

Table 1-94

Table 1-95

Table 1-96

Table 1-97

Table 1-98

Table 1-99

Table 1-100

Table 1-101

Table 1-102

[0358]

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 2-18

Table 2-19

Table 2-20

Table 2-21

Table 2-22

Table 2-23

[0359] I. Definition As used herein, an "active domain" refers to a polypeptide or collection of polypeptides that has affinity for a target, which may be one or more polypeptides, nucleic acids, sugars, and / or combinations thereof. In some embodiments, the active domain will be an agonist or antagonist of the target, or will effect and / or inhibit signal transduction with respect to the target. An active domain need not have exclusive affinity for the target, but only need have an affinity for the target that is significantly greater (e.g., 10-fold or more) than the affinity of the domain for non-targets. The dissociation constant (K D ) can be in the pM, nM, μM, or mM range. An active domain can contain one or more subdomains or subunits, each with a significantly different function, that together have the function of the active domain. For example, an active domain that contains an IL-12 polypeptide sequence can contain two subunits.

[0360] As used herein, an "immunoglobulin antigen-binding domain" refers to a domain that is an immunoglobulin or a fragment thereof, such as an Fv, scFv, Fab, or VHH. Exemplary immunoglobulin antigen-binding domains are provided in Table 1.

[0361] As used herein, "receptor binding domain" refers to an active domain, such as a cytokine polypeptide sequence, that is not an immunoglobulin antigen binding domain.

[0362] As used herein, a "cytokine polypeptide sequence" refers to a polypeptide sequence (which may be part of a larger sequence, e.g., a fusion polypeptide) that has significant sequence identity to a wild-type cytokine and can bind to and activate a cytokine receptor (e.g., when separated from an inhibitory polypeptide sequence). In some embodiments, the cytokine polypeptide sequence has at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of a wild-type cytokine, e.g., a wild-type human cytokine. In some embodiments, the cytokine polypeptide sequence has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid differences from a wild-type cytokine, e.g., a wild-type human cytokine. Cytokines include, but are not limited to, chemokines. Exemplary cytokine polypeptide sequences are provided in Table 1. This definition applies to the IL-2 polypeptide sequence, with "IL-2" replaced by "cytokine".

[0363] As used herein, "inhibitory polypeptide sequence" refers to a polypeptide or collection of polypeptides that inhibits the activity of the active domain in the linker polypeptide. The inhibitory polypeptide sequence may bind to the active domain or may sterically hinder it. In some embodiments, such binding is reduced or eliminated by the action of a suitable protease on the protease-cleaved polypeptide sequence of the linker polypeptide. Exemplary inhibitory polypeptide sequences are provided in Table 1. The inhibitory polypeptide sequence may include, for example, a polypeptide with significant sequence identity to a portion of the wild-type target of the active domain, or an immunoglobulin, or a portion thereof, such as an Fv, scFv, Fab, or VHH.

[0364] As used herein, a "protease-cleavable polypeptide sequence" is a sequence that is a substrate for cleavage by a protease. The protease-cleavable polypeptide sequence is positioned within a linker polypeptide such that cleavage thereof releases one or more elements of the linker polypeptide from the remainder of the linker polypeptide or reduces or eliminates binding of an inhibitory polypeptide sequence to an active domain.

[0365] As used herein, a protease-cleaved polypeptide sequence is "recognized by" a given protease or class thereof if exposure of a polypeptide comprising the protease-cleaved polypeptide sequence to a protease under conditions permissive for cleavage by the protease results in a significantly greater amount of cleavage than is seen for a control polypeptide having an unrelated sequence, and / or the protease-cleaved polypeptide sequence corresponds to a known recognition sequence for the protease (e.g., as described for various exemplary proteases elsewhere herein).

[0366] As used herein, a "pharmacokinetic modulator" is a moiety that extends the half-life of a linker polypeptide or an element of a linker polypeptide in vivo. A pharmacokinetic modulator can be a fusion domain within a linker polypeptide or a chemical entity that is post-translationally attached. The attachment can be, but is not necessarily, a covalent attachment. Polypeptide sequences of exemplary pharmacokinetic modulators are provided in Table 1. Exemplary non-polypeptide pharmacokinetic modulators are described elsewhere herein.

[0367] As used herein, a "targeting sequence" is a sequence that results in the localization of the majority of the linker polypeptide to a region of interest, such as the tumor microenvironment. The targeting sequence may bind to an extracellular matrix component or other entity, such as an integrin or syndecan, found in the region of interest. Exemplary targeting sequences are provided in Table 2.

[0368] As used herein, "extracellular matrix components" refers to extracellular proteins or polysaccharides found in vivo. Integral membrane proteins and peripheral membrane proteins on cells, including fibronectin, cadherins, integrins, and syndecans, are not considered extracellular matrix components.

[0369] As used herein, "immunoglobulin constant domain" refers to a domain that occurs within or has significant sequence identity to the domain of the constant region of an immunoglobulin, such as IgG. Exemplary constant domains include the C H 2 domain and C H 3 domains. Unless otherwise indicated, a linker polypeptide comprising an immunoglobulin constant domain may comprise more than one immunoglobulin constant domain. In some embodiments, the immunoglobulin constant domain has at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of a wild-type immunoglobulin constant domain, e.g., a wild-type human immunoglobulin constant domain. In some embodiments, the immunoglobulin constant domain has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid differences from a wild-type immunoglobulin constant domain, e.g., a wild-type human immunoglobulin constant domain. In some embodiments, the immunoglobulin constant domain has a sequence that is identical to a wild-type immunoglobulin constant domain, e.g., a wild-type human immunoglobulin constant domain. Exemplary immunoglobulin constant domains are contained within the sequences provided in Table 1. This definition is based on the C H The two-domain sequence is CH For the wild-type sequence of an immunoglobulin constant domain other than 2, H 2 domain wild-type sequence, and H The three-domain sequence is C H For the wild-type sequence of an immunoglobulin constant domain other than 3, H provided that the percent identity of the wild-type sequence of the three domains is no greater than that of the "C H 2" or "C H 3" was replaced by "immunoglobulin constant"; C H 2 domain and C H These definitions also include domains that have rare truncations relative to the wild-type sequence, to the extent that the truncations do not substantially abolish normal domain folding.

[0370] As used herein, an "immunoglobulin Fc region" refers to a C region, as defined above. H 2 domain and C H The Fc region refers to the region of an immunoglobulin heavy chain that contains three domains. The Fc region refers to the variable domain or C H Does not include one domain.

[0371] As used herein, a given component is "between" a first component and a second component, for example, in the primary sequence of a polypeptide, when the first component is on one side of the given component and the second component is on the other side of the given component. The term does not require direct adjacency. Thus, in the structure 1-2-3-4, 2 is between 1 and 4, but also between 1 and 3.

[0372] As used herein, a "domain" can refer, depending on the context, to a structural domain of a polypeptide, or to a functional assembly of at least one domain (but possibly multiple structural domains). For example, HThe term "domain" refers to the portion of the sequence considered in this manner. An immunoglobulin cytokine binding domain may comprise a VH structural domain and a VL structural domain.

[0373] As used herein, "denatured collagen" includes gelatin and cleavage products resulting from the action of MMPs on collagen, and more generally refers to forms of collagen or fragments thereof that are not present within the native structure of full-length collagen.

[0374] As used herein, "configured for pH-sensitive binding to" means that a polypeptide sequence (e.g., a targeting sequence) exhibits differential binding affinity for its binding partner as a function of pH. For example, a polypeptide sequence may have a high affinity at a relatively acidic pH compared to normal physiological pH (about 7.4). High affinity may occur at a pH below 7, e.g., pH 5.5-7, 6-7, or 5.5-6.5, or in the range below pH 6.

[0375] As used herein, a "cytokine-binding domain of a cytokine receptor" refers to an extracellular portion of a cytokine receptor, or a fragment or truncation thereof, capable of binding to a cytokine polypeptide sequence. In some embodiments, the sequence of the cytokine-binding domain of a cytokine receptor has at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the cytokine-binding domain of a wild-type cytokine receptor, e.g., the sequence of the cytokine-binding domain of a wild-type human cytokine receptor. Exemplary sequences of cytokine-binding domains of cytokine receptors are provided in Table 1. This definition applies to IL-2, IL-10, IL-15, CXCL9, CXCL10, and the TGF-β binding domains of IL-2, IL-10, IL-15, CXCL9, CXCL10, and TGF-β receptors, with "cytokine" replacing each of "IL-2", "IL-10", "IL-15", "CXCL9", "CXCL10", and "TGF-β".

[0376] As used herein, an "immunoglobulin cytokine binding domain" refers to one or more immunoglobulin variable domains (e.g., VH and VL regions) capable of binding to a cytokine polypeptide sequence. Exemplary sequences of cytokine binding immunoglobulin domains are provided in Table 1. This definition applies to IL-2, IL-10, IL-15, CXCL9, CXCL10, and the TGF-β binding domains of IL-2, IL-10, IL-15, CXCL9, CXCL10, and the TGF-β receptor, with each of "IL-2," "IL-10," "IL-15," "CXCL9," "CXCL10," and "TGF-β" replaced by "cytokine."

[0377] As used herein, a first element of a linker polypeptide being "proximal to" a second element relative to a third element means that the first element is closer to the second element than to the third element within the primary polypeptide sequence of the linker polypeptide, regardless of whether the first element is spatially closer to the second element than to the third element when the linker polypeptide is folded.

[0378] As used herein, "substantially" and other grammatical forms mean sufficient to serve the intended purpose. Thus, the term "substantially" allows for slight, non-significant variations from an absolute or complete state, dimension, measurement, result, etc., such as variations that would be expected by one of ordinary skill in the art and that have no appreciable effect on overall performance. When used in reference to a numerical value or parameter, or a characteristic that can be expressed as a numerical value, "substantially" means within 10 percent.

[0379] As used herein, the term "plurality" can be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

[0380] As used herein, if the alignment of a first sequence to a second sequence shows that X% or more of the positions of the second sequence in its entirety match with the first sequence, the first sequence is considered to "comprise a sequence with at least X% identity to" the second sequence.For example, alignment results in 100% identity when there is a match to all three positions of the second sequence, so the sequence QLYV (SEQ ID NO: 1168) comprises a sequence with 100% identity to the sequence QLY.Exemplary alignment algorithms are the Smith-Waterman algorithm and the Needleman-Wunsch algorithm, which are well known in the art. Those skilled in the art will understand what choice of algorithm and parameter settings is appropriate for a given pair of sequences to be aligned; generally, for sequences of similar length and with predicted amino acid identity >50% or nucleotide identity >75%, the Needleman-Wunsch algorithm, with default settings in the Needleman-Wunsch algorithm interface provided by the EBI at its web server at www.ebi.ac.uk, is generally appropriate.

[0381] As used herein, a "subject" refers to any member of the animal kingdom. In some embodiments, a "subject" refers to a human. In some embodiments, a "subject" refers to a non-human animal. In some embodiments, a "subject" refers to a primate. In some embodiments, a subject includes, but is not limited to, a mammal, a bird, a reptile, an amphibian, a fish, an insect, and / or a worm. In certain embodiments, a non-human subject is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a cow, a primate, and / or a pig). In some embodiments, a subject may be a transgenic animal, a genetically engineered animal, and / or a clone. In certain embodiments of the invention, a subject is an adult, an adolescent, or a child. In some embodiments, the terms "individual" or "patient" are used and are intended to be interchangeable with "subject."

[0382] II. Linker Polypeptides The linker polypeptide may comprise a first targeting sequence; a second targeting sequence; and a first linker between the first targeting sequence and the second targeting sequence, the linker comprising a protease-cleavable polypeptide sequence. In some embodiments, the first targeting sequence and / or the second targeting sequence may each comprise two or more targeting moieties, each binding to a target. In some embodiments, some or all of the two or more targeting moieties may bind to the same target (e.g., tandem repeats). In some embodiments, the linker polypeptide comprises a first active domain; a second active domain; a pharmacokinetic modulator; and a first linker between the pharmacokinetic modulator and the first active domain, the first linker comprising a protease-cleavable polypeptide sequence. In some embodiments, the linker polypeptide comprises a first active domain; an inhibitory polypeptide sequence capable of blocking the activity of the first active domain; a first linker between the first active domain and the inhibitory polypeptide sequence, the linker comprising a protease-cleavable polypeptide sequence; and a first targeting sequence.

[0383] These elements of a linker polypeptide may be covalently connected, non-covalently linked, or may be present in multiple associated polypeptide chains that may be covalently linked (e.g., via one or more disulfide bonds) to form a single polypeptide chain.

[0384] In some embodiments, the linker polypeptide comprises a first polypeptide chain comprising a first active domain, a first domain of a pharmacokinetic modulator, and a first linker between the first active domain and the first domain of the pharmacokinetic modulator, wherein the first active domain is C-terminal to the first domain of the pharmacokinetic modulator; a second polypeptide chain comprising a second domain of the pharmacokinetic modulator, an inhibitory polypeptide sequence capable of blocking activity of the first active domain, and a second linker between the second domain of the pharmacokinetic modulator and the inhibitory polypeptide sequence; in which the first linker comprises a protease-cleavable polypeptide sequence; and the first polypeptide chain or the second polypeptide chain further comprises at least one targeting sequence.

[0385] In some embodiments, the linker polypeptide comprises a first polypeptide chain comprising a first active domain, a first domain of a pharmacokinetic modulator, and a first linker between the first active domain and the first domain of the pharmacokinetic modulator, wherein the first active domain is N-terminal to the first domain of the pharmacokinetic modulator; a second polypeptide chain comprising a second domain of the pharmacokinetic modulator, an inhibitory polypeptide sequence capable of blocking activity of the first active domain, and a second linker between the second domain of the pharmacokinetic modulator and the inhibitory polypeptide sequence; wherein the first linker comprises a protease-cleavable polypeptide sequence; and the first polypeptide chain or the second polypeptide chain further comprises at least one targeting sequence.

[0386] A. Active Domain 1. Immunoglobulin antigen-binding domain In some embodiments, the first active domain comprises an immunoglobulin antigen-binding domain. In some embodiments, the second active domain comprises an immunoglobulin antigen-binding domain.

[0387] In some embodiments, the immunoglobulin antigen-binding domain comprises a VH region and a VL region. In some embodiments, the immunoglobulin antigen-binding domain comprises an Fv, scFv, Fab, or VHH. The immunoglobulin antigen-binding domain may be a humanized domain or a fully human domain.

[0388] In some embodiments, the immunoglobulin antigen binding domain binds to one or more sequences selected from a cancer cell surface antigen sequence, a growth factor sequence, and a growth factor receptor sequence.

[0389] Under physiological conditions, cells receive signals from surrounding tissues in the form of growth factors. Growth factors can affect normal cell differentiation as well as constitutively activate growth-promoting pathways in cancer cells. The linker polypeptides disclosed herein can bind to growth factors and facilitate neutralization of growth factor activity, at least to some extent, for example, in the vicinity of tumors. Thus, in some embodiments, the linker polypeptides disclosed herein can reduce growth-promoting signaling received by cancer cells and stromal cells, including fibroblasts and endothelial cells, via the immunoglobulin antigen-binding domain, while also activating or recruiting immune cells to tumors. In some embodiments, the immunoglobulin antigen-binding domains can also facilitate localization of the linker polypeptide to tissues that specifically express a particular growth factor or that express a high amount of a particular growth factor, for example, tissues within and near tumors.

[0390] Growth factor receptors are generally transmembrane proteins that bind to specific growth factors and send instructions from the factors on the extracellular surface to the intracellular lumen. Generally, growth factor receptors include an extracellular domain, a transmembrane domain, and a cytoplasmic domain. In some embodiments, the linker polypeptide disclosed herein, the immunoglobulin antigen-binding domain, can inhibit the binding of growth factor to growth factor receptor. This can facilitate at least some reduction in signal transduction by growth factor, for example, in the vicinity of tumor.

[0391] In some embodiments, one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain of the linker polypeptide is independently configured to bind to a HER2 sequence. In some embodiments, one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain of the linker polypeptide independently comprises a hypervariable region (HVR) HVR-1, HVR-2, and HVR-3 of the VH region comprising the amino acid sequence of SEQ ID NO: 910; and a VL region comprising the HVR-1, HVR-2, and HVR-3 of the VL region comprising the amino acid sequence of SEQ ID NO: 909. In general, those skilled in the art will be familiar with the methods described in, for example, Kabat et al. in Sequences of Proteins of Immunological Interest, 5 thHVRs within the VH and VL sequences may be identified by assigning amino acids to framework and HVR domains within the VH and VL sequences according to the definitions in US Pat. No. 6,333,623, Ed., US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991. Other numbering systems for amino acids within immunoglobulin chains include IMGT™ (international ImMunoGeneTics information system; Lefranc et al, Dev. Comp. Immunol. 29:185-203; 2005), and AHo [Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001]. In some embodiments, one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain independently comprises a VH region comprising the amino acid sequence of SEQ ID NO: 910; and a VL region comprising the amino acid sequence of SEQ ID NO: 909. In some embodiments, one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain independently comprises a sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 909 or 910. In some embodiments, one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain independently is an antigen-binding domain of trastuzumab.

[0392] In some embodiments, one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain of the linker polypeptide is independently configured to bind to an EGFR extracellular domain sequence. In some embodiments, each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain independently comprises a VH region comprising HVR-1, HVR-2, and HVR-3 of the VH region comprising the amino acid sequence of SEQ ID NO: 914; and a VL region comprising HVR-1, HVR-2, and HVR-3 of the VL region comprising the amino acid sequence of SEQ ID NO: 913. In general, those skilled in the art will be familiar with the methods described in, for example, Kabat et al. in Sequences of Proteins of Immunological Interest, 5 thHVRs within the VH and VL sequences may be identified by assigning amino acids to framework and HVR domains within the VH and VL sequences according to the definitions in US Pat. No. 6,333,623, Ed., US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991. Other numbering systems for amino acids within immunoglobulin chains include IMGT™ (international ImMunoGeneTics information system; Lefranc et al, Dev. Comp. Immunol. 29:185-203; 2005), and AHo [Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001]. In some embodiments, one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain independently comprises a VH region comprising the amino acid sequence of SEQ ID NO: 914; and a VL region comprising the amino acid sequence of SEQ ID NO: 913. In some embodiments, one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain independently comprises a sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 913 or 914. In some embodiments, one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain is the antigen-binding domain of cetuximab.

[0393] In some embodiments, one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain of the linker polypeptide is independently configured to bind to a PD-1 extracellular domain sequence. In some embodiments, one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain independently comprises a VH region comprising HVR-1, HVR-2, and HVR-3 of the VH region comprising the amino acid sequence of SEQ ID NO: 917; and a VL region comprising HVR-1, HVR-2, and HVR-3 of the VL region comprising the amino acid sequence of SEQ ID NO: 918. In general, those skilled in the art will be familiar with the methods described in, for example, Kabat et al. in Sequences of Proteins of Immunological Interest, 5 thHVRs within the VH and VL sequences may be identified by assigning amino acids to framework and HVR domains within the VH and VL sequences according to the definitions in US Pat. No. 6,333,623, Ed., US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991. Other numbering systems for amino acids within immunoglobulin chains include IMGT™ (international ImMunoGeneTics information system; Lefranc et al, Dev. Comp. Immunol. 29:185-203; 2005), and AHo [Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001]. In some embodiments, one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain independently comprises a VH region comprising the amino acid sequence of SEQ ID NO: 917; and a VL region comprising the amino acid sequence of SEQ ID NO: 918. In some embodiments, one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain independently comprises a sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 917 or 918. In some embodiments, one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain independently is the antigen-binding domain of nivolumab.

[0394] In some embodiments, one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain of the linker polypeptide is independently configured to bind to a PD-L1 extracellular domain sequence. In some embodiments, one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain independently comprises a VH region comprising HVR-1, HVR-2, and HVR-3 of the VH region comprising the amino acid sequence of SEQ ID NO: 921; and a VL region comprising HVR-1, HVR-2, and HVR-3 of the VL region comprising the amino acid sequence of SEQ ID NO: 922. In general, those skilled in the art will be familiar with the methods and techniques described in, for example, Kabat et al. in Sequences of Proteins of Immunological Interest, 5 thHVRs within the VH and VL sequences may be identified by assigning amino acids to framework and HVR domains within the VH and VL sequences according to the definitions in US Pat. No. 6,333,623, Ed., US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991. Other numbering systems for amino acids within immunoglobulin chains include IMGT™ (international ImMunoGeneTics information system; Lefranc et al, Dev. Comp. Immunol. 29:185-203; 2005), and AHo [Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001]. In some embodiments, one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain independently comprises a VH region comprising the amino acid sequence of SEQ ID NO: 921; and a VL region comprising the amino acid sequence of SEQ ID NO: 922. In some embodiments, one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain independently comprises a sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 921 or 922. In some embodiments, one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain independently is an antigen-binding domain of atezolizumab.

[0395] In some embodiments, one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain of the linker polypeptide is independently configured to bind to a CD3 extracellular domain sequence. In some embodiments, one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain independently comprises a VH region comprising HVR-1, HVR-2, and HVR-3 of the VH region comprising any one of the amino acid sequences of SEQ ID NOs: 925, 929, 933, and 937; and a VL region comprising HVR-1, HVR-2, and HVR-3 of the VL region comprising any one of the amino acid sequences of SEQ ID NOs: 926, 930, 934, and 938. In general, those skilled in the art will be familiar with the methods and techniques described in, for example, Kabat et al. in Sequences of Proteins of Immunological Interest, 5 thHVRs within the VH and VL sequences may be identified by assigning amino acids to framework and HVR domains within the VH and VL sequences according to the definitions in US Pat. No. 6,333,623, Ed., US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991. Other numbering systems for amino acids within immunoglobulin chains include IMGT™ (international ImMunoGeneTics information system; Lefranc et al, Dev. Comp. Immunol. 29:185-203; 2005), and AHo [Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001]. In some embodiments, one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain independently comprises a VH region comprising the amino acid sequence of any one of SEQ ID NOs: 925, 929, 933, and 937; and a VL region comprising the amino acid sequence of any one of SEQ ID NOs: 926, 930, 934, and 938. In some embodiments, one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain independently comprises a sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of any one of SEQ ID NOs: 925, 926, 929, 930, 933, 934, 937, and 938. In some embodiments, one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain is independently an antigen-binding domain of teplizumab, muromonab, otelixizumab, or visilizumab.

[0396] 2. Receptor-binding domain In some embodiments, the first active domain comprises a receptor binding domain. The receptor binding domain can comprise, for example, a cytokine polypeptide sequence.

[0397] The receptor binding domain may be a wild-type receptor binding domain or a sequence with one or more differences from the wild-type receptor binding domain. In some embodiments, the receptor binding domain is a human receptor binding domain (whether wild-type or with one or more differences). In some embodiments, the receptor binding domain may include modifications that prevent disulfide bond formation and may otherwise include a wild-type sequence. In some embodiments, the receptor binding domain has at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of the wild-type receptor binding domain or to a receptor binding domain in Table 1. In some embodiments, the receptor binding domain is a dimeric receptor binding domain, e.g., a heterodimeric cytokine. In some embodiments, the receptor binding domain is a homodimeric receptor binding domain, e.g., a homodimeric cytokine. The monomers may be linked as a fusion protein, covalently (e.g., disulfide bonds), or non-covalently, e.g., by a linker. In some embodiments, the receptor binding domain is an interleukin polypeptide sequence. In some embodiments, the receptor binding domain is capable of binding to a receptor comprising CD132. In some embodiments, the receptor binding domain is capable of binding to a receptor comprising CD122. In some embodiments, the receptor binding domain is capable of binding to a receptor comprising CD25.

[0398] In some embodiments, the receptor binding domain is an IL-2 polypeptide sequence. The IL-2 polypeptide sequence may be a wild-type IL-2 polypeptide sequence or a sequence with one or more differences from the wild-type IL-2 polypeptide sequence. In some embodiments, the IL-2 polypeptide sequence is a human IL-2 polypeptide sequence (which may be wild-type or have one or more differences). In some embodiments, the IL-2 may contain modifications that prevent disulfide bond formation (e.g., the sequence of aldesleukin, commercially available as Proleukin®), but may otherwise contain the wild-type sequence. In some embodiments, the IL-2 polypeptide sequence has at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of a wild-type IL-2 polypeptide sequence or to an IL-2 polypeptide sequence in Table 1.

[0399] In some embodiments, the IL-2 polypeptide sequence comprises the sequence of any one of SEQ ID NOs: 1-4. In some embodiments, the IL-2 polypeptide sequence comprises the sequence of SEQ ID NO: 1. In some embodiments, the IL-2 polypeptide sequence comprises the sequence of SEQ ID NO: 2.

[0400] In some embodiments, the receptor binding domain is an IL-10 polypeptide sequence. The IL-10 polypeptide sequence may be a wild-type IL-10 polypeptide sequence or a sequence with one or more differences from the wild-type IL-10 polypeptide sequence. In some embodiments, the IL-10 polypeptide sequence is a human IL-10 polypeptide sequence (whether wild-type or with one or more differences). In some embodiments, the IL-10 may include modifications that prevent disulfide bond formation and may otherwise include the wild-type sequence. In some embodiments, the IL-10 polypeptide sequence has at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of the wild-type IL-10 polypeptide sequence or to an IL-10 polypeptide sequence in Table 1. In some embodiments, the IL-10 polypeptide sequence includes the sequence of SEQ ID NO: 900.

[0401] In some embodiments, the receptor binding domain is an IL-15 polypeptide sequence. The IL-15 polypeptide sequence may be a wild-type IL-15 polypeptide sequence or a sequence with one or more differences from the wild-type IL-15 polypeptide sequence. In some embodiments, the IL-15 polypeptide sequence is a human IL-15 polypeptide sequence (which may be wild-type or have one or more differences). In some embodiments, the IL-15 may include modifications that prevent disulfide bond formation and may otherwise include the wild-type sequence. In some embodiments, the IL-15 polypeptide sequence has at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of the wild-type IL-15 polypeptide sequence or to an IL-15 polypeptide sequence in Table 1. In some embodiments, the IL-15 polypeptide sequence includes the sequence of SEQ ID NO:901.

[0402] In some embodiments, the receptor binding domain is a CXCL9 polypeptide sequence. The CXCL9 polypeptide sequence may be a wild-type CXCL9 polypeptide sequence or a sequence with one or more differences from the wild-type CXCL9 polypeptide sequence. In some embodiments, the CXCL9 polypeptide sequence is a human CXCL9 polypeptide sequence (whether wild-type or with one or more differences). In some embodiments, the CXCL9 may include modifications that prevent disulfide bond formation and may otherwise include the wild-type sequence. In some embodiments, the CXCL9 polypeptide sequence has at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of a wild-type CXCL9 polypeptide sequence or a CXCL9 polypeptide sequence in Table 1. In some embodiments, the CXCL9 polypeptide sequence comprises the sequence of SEQ ID NO: 902.

[0403] In some embodiments, the receptor binding domain is a CXCL10 polypeptide sequence. The CXCL10 polypeptide sequence may be a wild-type CXCL10 polypeptide sequence or a sequence with one or more differences from the wild-type CXCL10 polypeptide sequence. In some embodiments, the CXCL10 polypeptide sequence is a human CXCL10 polypeptide sequence (whether wild-type or with one or more differences). In some embodiments, the CXCL10 may include modifications that prevent disulfide bond formation and may otherwise include the wild-type sequence. In some embodiments, the CXCL10 polypeptide sequence has at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of a wild-type CXCL10 polypeptide sequence or a CXCL10 polypeptide sequence in Table 1. In some embodiments, the CXCL10 polypeptide sequence comprises the sequence of SEQ ID NO: 903.

[0404] 3. Size of the active domain In some embodiments, the molecular weight of one or each of the first active domain and the second active domain is independently about 14 kDa or less. In some embodiments, the molecular weight is about 12 kDa to about 14 kDa. In some embodiments, the molecular weight is about 10 kDa to about 12 kDa. In some embodiments, the molecular weight is about 8 kDa to about 10 kDa. In some embodiments, the molecular weight is about 6 kDa to about 8 kDa. In some embodiments, the molecular weight is about 4 kDa to about 6 kDa. In some embodiments, the molecular weight is about 2 kDa to about 4 kDa. In some embodiments, the molecular weight is about 800 Da to about 2 kDa.

[0405] In some embodiments, the molecular weight of one or each of the first active domain and the second active domain is independently about 16 kDa or greater than 16 kDa. In some embodiments, the molecular weight is about 16 kDa to about 18 kDa. In some embodiments, the molecular weight is about 18 kDa to about 20 kDa. In some embodiments, the molecular weight is about 20 kDa to about 22 kDa. In some embodiments, the molecular weight is about 22 kDa to about 24 kDa. In some embodiments, the molecular weight is about 24 kDa to about 26 kDa. In some embodiments, the molecular weight is about 26 kDa to about 28 kDa. In some embodiments, the molecular weight is about 28 kDa to about 30 kDa. In some embodiments, the molecular weight is about 30 kDa to about 50 kDa. In some embodiments, the molecular weight is about 50 kDa to about 100 kDa. In some embodiments, the molecular weight is about 100 kDa to about 150 kDa. In some embodiments, the molecular weight is about 150 kDa to about 200 kDa. In some embodiments, the molecular weight is about 200 kDa to about 250 kDa. In some embodiments, the molecular weight is about 250 kDa to about 300 kDa.

[0406] B. Inhibitory Polypeptide Sequences In some embodiments, the linker polypeptide comprises an inhibitory polypeptide sequence capable of blocking the activity of an active domain, such as a receptor binding domain, hi some embodiments, the linker polypeptide further comprises a second linker between the receptor binding domain and the inhibitory polypeptide sequence, the second linker comprising a protease-cleavable polypeptide sequence.

[0407] Various types of inhibitory polypeptide sequences may be used within the linker polypeptide according to the present disclosure. In some embodiments, the inhibitory polypeptide sequence is a sequence that binds to an active domain, such as a ligand-binding domain from a receptor, or an immunoglobulin domain. In some embodiments, the inhibitory polypeptide sequence is a steric blocker, i.e., a sequence that sterically blocks the active domain. For example, the steric blocker may be an immunoglobulin Fc region, an albumin domain, or other relatively inactive domain that may be placed proximal to the active domain to reduce the accessibility of the active domain until the active domain is released from the inhibitory polypeptide sequence by cleavage. In some embodiments, the inhibitory polypeptide sequence interferes with the binding of a first active domain to the receptor of the first active domain and / or the binding of a second active domain to the receptor of the second active domain. In some embodiments, the inhibitory polypeptide sequence and the pharmacokinetic modulator are different elements of the linker polypeptide. In some embodiments, the inhibitory polypeptide sequence includes at least a portion of the pharmacokinetic modulator.

[0408] In some embodiments, the inhibitory polypeptide sequence comprises a cytokine binding domain. The cytokine binding domain can be a cytokine binding domain of a cytokine receptor. The cytokine binding domain of a cytokine receptor can be incorporated as an extracellular portion of the cytokine receptor or a portion thereof sufficient to bind to the cytokine polypeptide sequence of a linker polypeptide. In some embodiments, the inhibitory polypeptide sequence has at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the wild-type cytokine binding domain of a cytokine receptor, for example, the wild-type cytokine binding domain of a human cytokine receptor.

[0409] The cytokine binding domain can be a fibronectin cytokine binding domain. In some embodiments, the inhibitory polypeptide sequence has at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the cytokine binding domain of wild-type fibronectin of the cytokine receptor, e.g., the cytokine binding domain of wild-type human fibronectin.

[0410] In some embodiments, the inhibitory polypeptide sequence is selected from the group consisting of SEQ ID NOs: 10-29, 40-51, 747, 748 and 749, 850-856, 939, 940, 941 and 945, 950 and 952, 953, 954 and 955, 956, 957 and 958, 959, 960 and 961, 962, 963 and 964, 965, 966 and 967, 968, 969 and 970, 971, 972 and 973, 974, 975 and 976, 977, 978 and 979, 980, 981 and 982, 983, 984 and 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 998, 999, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1 6, 987 and 988, 989, 990, 991 and 992, 999 and 1000, 1001, 1002, 1003 and 1004, 1005, 1006, 1008 and 1010 (wherein the pair of SEQ ID NOs joined by "and" refer to VH / VL pairs that may together form an inhibitory polypeptide sequence, e.g., as separate chains or as a single chain connected by a linker). In some embodiments, the inhibitory polypeptide sequence comprises an amino acid sequence that has at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of any one of SEQ ID NOs: 1011 or 1012. In some embodiments, the inhibitory polypeptide sequence comprises an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to any one of SEQ ID NOs: 1016-1019. In some embodiments, the inhibitory polypeptide sequence comprises an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to SEQ ID NOs: 1020, 1021, or 1023. In any of the foregoing embodiments, the VH and VL domains may comprise CDRs that are identical to the CDRs of the referenced SEQ ID NO(s). The CDRs may be selected from the group consisting of CDRs from Kabat et al., (5 available at Kabat [books.google.co.uk / books?id=3jMvZYW2ZtwC&lpg=PA1137-IA1&pg=PP1#v=onepage&q&f=false].th In some embodiments, inhibitory polypeptide sequences may be identified by any suitable method, such as the method of Chothia [described in Al-Lazikani et al., (1997) JMB 273, 927-948], or the method of SEQ ID NOs: 747, 748 and 749, 939, 940, 941 and 945, 950 and 952, 953, 954 and 955, 956, 957 and 958, 959, 960 and 961, 962, 963 and 964, 965, 966 and 967, 968, 969 and 970, 971, 972 and 973, 974, The VH domain and the VL domain include any of the CDRs of 975 and 976, 977, 978 and 979, 980, 981 and 982, 983, 984 and 985, 986, 987 and 988, 989, 990, 991 and 992, 999 and 1000, 1001, 1002, 1003 and 1004, 1005, 1006, 1008 and 1010. In some embodiments, the inhibitory polypeptide sequence is selected from the group consisting of SEQ ID NOs: 747, 748 and 749, 939, 940, 941 and 945, 950 and 952, 953, 954 and 955, 956, 957 and 958, 959, 960 and 961, 962, 963 and 964, 965, 966 and 967, 968, 969 and 970, 971, 972, and and any of the sequences of 973, 974, 975 and 976, 977, 978 and 979, 980, 981 and 982, 983, 984 and 985, 986, 987 and 988, 989, 990, 991 and 992, 999 and 1000, 1001, 1002, 1003 and 1004, 1005, 1006, 1008 and 1010.

[0411] In some embodiments, the inhibitory polypeptide sequence comprises an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to any one of SEQ ID NOs: 850-856 and 863-870. In any of the foregoing embodiments, the VHH domain may comprise a CDR identical to a CDR of any one of SEQ ID NOs: 850-856 and 863-870. In some embodiments, the inhibitory polypeptide sequence comprises a VHH comprising a CDR of any one of SEQ ID NOs: 850-856 and 863-870. In some embodiments, the inhibitory polypeptide sequence comprises a sequence of any one of SEQ ID NOs: 850-856 and 863-870.

[0412] In some embodiments, the cytokine binding domain can be an immunoglobulin cytokine binding domain. In some embodiments, the immunoglobulin cytokine binding domain comprises a VH region and a VL region that binds to a cytokine. In some embodiments, the immunoglobulin cytokine binding domain can be an Fv, scFv, Fab, VHH, or other immunoglobulin sequence that has antigen binding activity for a cytokine polypeptide sequence. A VHH antibody (or nanobody) is an antigen-binding fragment of an antibody that is solely comprised of a heavy chain.

[0413] Further examples of inhibitory polypeptide sequences that can be incorporated to inhibit the cytokine polypeptide sequences of the linker polypeptide are anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, finomers, Kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as the scaffolds SpA, GroEL, lipocalin, and CTLA4.

[0414] Within a linker polypeptide that includes an IL-2 polypeptide sequence, the inhibitory polypeptide sequence can be any of the types of IL-2 inhibitory polypeptide sequences described above. In some embodiments, the IL-2 inhibitory polypeptide sequence is an immunoglobulin IL-2 inhibitory polypeptide sequence.

[0415] In some embodiments, the IL-2 inhibitory polypeptide sequence comprises an anti-IL-2 antibody or a functional fragment thereof. In some embodiments, the inhibitory polypeptide sequence comprises an IL-2 binding immunoglobulin domain. In some embodiments, the IL-2 binding immunoglobulin domain is a human IL-2 binding immunoglobulin domain.

[0416] In some embodiments, the IL-2 binding immunoglobulin domain is an scFv. In some embodiments, the IL-2 binding immunoglobulin domain comprises a set of six anti-IL-2 hypervariable regions (HVRs) as set forth in Table 1 (e.g., SEQ ID NOs: 34-39 or 750-755). In some embodiments, the IL-2 binding immunoglobulin domain comprises a set of anti-IL-2 VH and anti-IL-2 VL regions that comprise sequences that have at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequences of the set of anti-IL-2 VH and anti-IL-2 VL regions that comprise sequences set forth in Table 1, either individually or as part of an scFv. In some embodiments, the IL-2 binding immunoglobulin domain comprises a set of anti-IL-2 VH and anti-IL-2 VL regions that have sequences that are set forth in Table 1, either individually or as part of an scFv.

[0417] Exemplary IL-2 inhibitory polypeptide sequences include SEQ ID NOs: 10-31, 40-51, 747, and 850-856, as well as a combination of SEQ ID NOs: 32 and 33, or a combination of SEQ ID NOs: 748 and 749. In some embodiments, the IL-2 inhibitory polypeptide sequence comprises an IL-2-binding immunoglobulin domain comprising a VH region comprising an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 33, and a VL region comprising an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 32. In some embodiments, the IL-2-binding immunoglobulin domain comprises a VH region comprising the sequence of SEQ ID NO: 33, and a VL region comprising the sequence of SEQ ID NO: 32.

[0418] In some embodiments, the IL-2 binding immunoglobulin domain comprises a VH region comprising hypervariable regions HVR-1, HVR-2, and HVR-3 having the sequences of SEQ ID NOs: 37, 38, and 39, respectively; and a VL region comprising HVR-1, HVR-2, and HVR-3 having the sequences of SEQ ID NOs: 34, 35, and 36, respectively. In some embodiments, the IL-2 binding immunoglobulin domain comprises an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 30 or 31. In some embodiments, the IL-2 binding immunoglobulin domain comprises the sequence of SEQ ID NO: 30 or 31.

[0419] In some embodiments, the inhibitory polypeptide sequence comprises the IL-2 binding domain of the IL-2 receptor (IL-2R). In some embodiments, the IL-2R is human IL-2R.

[0420] Within a linker polypeptide that includes an IL-10 polypeptide sequence, the inhibitory polypeptide sequence can be any of the types of IL-10 inhibitory polypeptide sequences described above. In some embodiments, the IL-10 inhibitory polypeptide sequence is an immunoglobulin IL-10 inhibitory polypeptide sequence.

[0421] In some embodiments, the IL-10 inhibitory polypeptide sequence comprises an anti-IL-10 antibody or a functional fragment thereof. In some embodiments, the inhibitory polypeptide sequence comprises an IL-10 binding immunoglobulin domain. In some embodiments, the IL-10 binding immunoglobulin domain is a human IL-10 binding immunoglobulin domain.

[0422] In some embodiments, the IL-10 binding immunoglobulin domain is an scFv. In some embodiments, the IL-10 binding immunoglobulin domain comprises a set of six anti-IL-10 hypervariable regions (HVRs) set forth in Table 1 (e.g., SEQ ID NOs: 942-944 or 946-948). In some embodiments, the IL-10 binding immunoglobulin domain comprises a set of anti-IL-10 VH and VL regions that comprise sequences that have at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequences of the set of anti-IL-10 VH and VL regions that comprise the sequences set forth in Table 1, either individually or as part of an scFv. In some embodiments, the IL-10 binding immunoglobulin domain comprises a set of anti-IL-10 VH and anti-IL-10 VL regions, either individually or as part of an scFv, having the sequences of the set of anti-IL-10 VH and anti-IL-10 VL, the sequences of which are set forth in Table 1.

[0423] Exemplary IL-10 inhibitory polypeptide sequences include SEQ ID NOs: 939-948, 1011, and 1012. In some embodiments, the IL-10 inhibitory polypeptide sequence comprises an IL-10-binding immunoglobulin domain comprising a VH region comprising an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 945, and a VL region comprising an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 941. In some embodiments, the IL-10-binding immunoglobulin domain comprises a VH region comprising the sequence of SEQ ID NO: 945, and a VL region comprising the sequence of SEQ ID NO: 941.

[0424] In some embodiments, the IL-10 binding immunoglobulin domain comprises a VH region comprising hypervariable regions HVR-1, HVR-2, and HVR-3 having the sequences of SEQ ID NOs: 946, 947, and 948, respectively; and a VL region comprising HVR-1, HVR-2, and HVR-3 having the sequences of SEQ ID NOs: 942, 943, and 944, respectively. In some embodiments, the IL-10 binding immunoglobulin domain comprises an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 939 or 940. In some embodiments, the IL-10 binding immunoglobulin domain comprises the sequence of SEQ ID NO: 939 or 940.

[0425] In some embodiments, the inhibitory polypeptide sequence comprises the IL-10 binding domain of the IL-10 receptor (IL-10R). In some embodiments, the IL-10R is human IL-10R.

[0426] Within a linker polypeptide that includes an IL-15 polypeptide sequence, the inhibitory polypeptide sequence can be any of the types of IL-15 inhibitory polypeptide sequences described above. In some embodiments, the IL-15 inhibitory polypeptide sequence is an immunoglobulin IL-15 inhibitory polypeptide sequence.

[0427] In some embodiments, the IL-15 inhibitory polypeptide sequence comprises an anti-IL-15 antibody or a functional fragment thereof. In some embodiments, the inhibitory polypeptide sequence comprises an IL-15 binding immunoglobulin domain. In some embodiments, the IL-15 binding immunoglobulin domain is a human IL-15 binding immunoglobulin domain.

[0428] In some embodiments, the IL-15 binding immunoglobulin domain is an scFv. In some embodiments, the IL-15 binding immunoglobulin domain comprises a VH region comprising HVR-1, HVR-2, and HVR-3 of the VH region comprising any one of the amino acid sequences of SEQ ID NOs: 950, 955, 957, 960, 963, 966, 969, 972, 975, 978, 981, 985, and 988; and a VL region comprising HVR-1, HVR-2, and HVR-3 of the VL region comprising any one of the amino acid sequences of SEQ ID NOs: 952, 954, 958, 961, 964, 967, 970, 973, 976, 979, 982, 984, and 987. In general, those skilled in the art can easily determine the sequence of the IL-15 binding immunoglobulin domain by, for example, referring to Kabat et al. in Sequences of Proteins of Immunological Interest, 5 thHVRs within the VH and VL sequences may be identified by assigning amino acids to framework and HVR domains within the VH and VL sequences according to the definitions in US Pat. No. 6,333,623, Ed., US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991. Other numbering systems for amino acids within immunoglobulin chains include IMGT™ (international ImMunoGeneTics information system; Lefranc et al, Dev. Comp. Immunol. 29:185-203; 2005), and AHo [Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001]. In some embodiments, the IL-15 binding immunoglobulin domain comprises a set of anti-IL-15 VH and anti-IL-15 VL regions that comprise sequences that have at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequences of the set of anti-IL-15 VH and anti-IL-15 VL regions that comprise the sequences set forth in Table 1, either as individual sequences or as part of an scFv. In some embodiments, the IL-15 binding immunoglobulin domain comprises a set of anti-IL-15 VH and anti-IL-15 VL regions that have the sequences of the set of anti-IL-15 VH and anti-IL-15 VL regions that are the sequences set forth in Table 1, either as individual sequences or as part of an scFv.

[0429] Exemplary IL-15 inhibitory polypeptide sequences include SEQ ID NOs:953, 956, 959, 962, 965, 968, 971, 974, 977, 980, 983, and 986. In some embodiments, the IL-15 inhibitory polypeptide sequence comprises an IL-15-binding immunoglobulin domain comprising a VH region comprising an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to any one of SEQ ID NOs: 950, 955, 957, 960, 963, 966, 969, 972, 975, 978, 981, 985, and 988; and a VL region comprising an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to any one of SEQ ID NOs: 952, 954, 958, 961, 964, 967, 970, 973, 976, 979, 982, 984, and 987. In some embodiments, the IL-15 binding immunoglobulin domain comprises a VH region comprising the sequence of any one of SEQ ID NOs: 950, 955, 957, 960, 963, 966, 969, 972, 975, 978, 981, 985, and 988; and a VL region comprising the sequence of any one of SEQ ID NOs: 952, 954, 958, 961, 964, 967, 970, 973, 976, 979, 982, 984, and 987.

[0430] In some embodiments, the inhibitory polypeptide sequence comprises the IL-15 binding domain of the IL-15 receptor (IL-15R). In some embodiments, the IL-15R is human IL-15R.

[0431] Within a linker polypeptide that includes a CXCL9 polypeptide sequence, the inhibitory polypeptide sequence can be any of the types of CXCL9 inhibitory polypeptide sequences described above, hi some embodiments, the CXCL9 inhibitory polypeptide sequence is an immunoglobulin CXCL9 inhibitory polypeptide sequence.

[0432] In some embodiments, the CXCL9 inhibitory polypeptide sequence comprises an anti-CXCL9 antibody or a functional fragment thereof. In some embodiments, the inhibitory polypeptide sequence comprises a CXCL9-binding immunoglobulin domain. In some embodiments, the CXCL9-binding immunoglobulin domain is a human CXCL9-binding immunoglobulin domain.

[0433] Exemplary CXCL9 inhibitory polypeptide sequences include SEQ ID NOs: 1020-1021. In some embodiments, the inhibitory polypeptide sequence comprises the CXCL9-binding domain of the CXCL9 receptor (CXCR3). In some embodiments, the CXCR3 is human CXCR3.

[0434] Within a linker polypeptide that includes a CXCL10 polypeptide sequence, the inhibitory polypeptide sequence can be any of the types of CXCL10 inhibitory polypeptide sequences described above. In some embodiments, the CXCL10 inhibitory polypeptide sequence is an immunoglobulin CXCL10 inhibitory polypeptide sequence.

[0435] In some embodiments, the CXCL10 inhibitory polypeptide sequence comprises an anti-CXCL10 antibody or a functional fragment thereof. In some embodiments, the inhibitory polypeptide sequence comprises a CXCL10-binding immunoglobulin domain. In some embodiments, the CXCL10-binding immunoglobulin domain is a human CXCL10-binding immunoglobulin domain.

[0436] In some embodiments, the CXCL10-binding immunoglobulin domain is an scFv. In some embodiments, the CXCL10-binding immunoglobulin domain comprises a set of six anti-CXCL10 hypervariable regions (HVRs) as set forth in Table 1 (e.g., SEQ ID NOs: 993-998). In some embodiments, the CXCL10-binding immunoglobulin domain comprises a set of anti-CXCL10 VH and VL regions that comprise sequences that have at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequences of the set of anti-CXCL10 VH and VL regions that comprise the sequences set forth in Table 1, either individually or as part of an scFv. In some embodiments, the CXCL10-binding immunoglobulin domain comprises a set of anti-CXCL10 VH and anti-CXCL10 VL regions that have the sequences of the set of anti-CXCL10 VH and anti-CXCL10 VL regions that are sequences set forth in Table 1, either individually or as part of an scFv.

[0437] Exemplary CXCL10 inhibitory polypeptide sequences include SEQ ID NOs: 989 and 990. In some embodiments, the CXCL10 inhibitory polypeptide sequence comprises a CXCL10-binding immunoglobulin domain comprising a VH region comprising an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 991, and a VL region comprising an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 992. In some embodiments, the CXCL10-binding immunoglobulin domain comprises a VH region comprising the sequence of SEQ ID NO: 991, and a VL region comprising the sequence of SEQ ID NO: 992.

[0438] In some embodiments, the CXCL10-binding immunoglobulin domain comprises a VH region comprising hypervariable regions HVR-1, HVR-2, and HVR-3 having the sequences of SEQ ID NOs: 993, 994, and 995, respectively; and a VL region comprising HVR-1, HVR-2, and HVR-3 having the sequences of SEQ ID NOs: 996, 997, and 998, respectively. In some embodiments, the CXCL10-binding immunoglobulin domain comprises an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 989 or 990. In some embodiments, the CXCL10-binding immunoglobulin domain comprises the sequence of SEQ ID NO: 989 or 990.

[0439] In some embodiments, the inhibitory polypeptide sequence comprises the CXCL10-binding domain of the CXCL10 receptor (CXCR3). In some embodiments, the CXCR3 is human CXCR3.

[0440] C. Linker A variety of linkers may be used in accordance with the present disclosure. In many embodiments, a linker may be used to connect any two domains within a linker polypeptide. In some embodiments, the linker polypeptide comprises one linker. In other embodiments, the linker polypeptide may comprise two or more linkers. In some embodiments, a first linker is present between the pharmacokinetic modulator and the first active domain. In some embodiments, a second linker is present between the receptor binding domain and the inhibitory polypeptide sequence. In some embodiments, the first linker and / or the second linker comprises a protease-cleaved polypeptide sequence. In some embodiments, after the protease-cleaved polypeptide sequence is cleaved, the first active domain and / or the second active domain are released from the remainder of the linker polypeptide. In some embodiments, the linker polypeptide comprises multiple protease-cleaved polypeptide sequences.

[0441] In these embodiments, different linkers can be used to provide different release characteristics for the different linked domains. For example, a linker for releasing a target binding domain, such as an immunoglobulin antigen binding domain, can be different from a linker for releasing a receptor binding domain, such as a cytokine polypeptide sequence. The linker can include any of the exemplary linker sequences disclosed herein, for example, in Table 1.

[0442] 1. Protease-cleavable sequence The protease cleavage sequence can be used to inhibit a wide variety of proteases, including metalloproteases, serine proteases, cysteine ​​proteases, aspartic acid proteases, threonine proteases, glutamic acid proteases, gelatinases, aspartic peptide lyases, cathepsins, kallikreins, plasmins, collagenases, hKl, hK10, hK15, stromelysins, factor Xa, chymotrypsin-like proteases, trypsin-like proteases, elastase-like proteases, subtilisin-like proteases, actinidains, bromelain, calpains, caspases, Mir1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepa, The polypeptide may include a sequence cleavable and / or recognized by psin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidase, metalloendopeptidase, ADAM10, ADAM17, ADAM12, urokinase plasminogen activator (uPA), enterokinase, prostate-specific target (PSA, hK3), interleukin 1b converting enzyme, thrombin, FAP (FAP-a), dipeptidyl peptidase or dipeptidyl peptidase IV (DPPIV / CD26), type II transmembrane serine protease (TTSP), neutrophil elastase, proteinase 3, mast cell chymase, mast cell tryptase, or dipeptidyl peptidase. In some embodiments, the protease cleavage sequence comprises any one of the protease cleavage sequences in Table 1 (e.g., SEQ ID NOs: 80-94 and 701-742), or a variant having one or two mismatches compared to any one of the protease cleavage sequences in Table 1 (e.g., SEQ ID NOs: 80-90 and 701-742). Proteases generally do not require an exact copy of a recognition sequence, and thus the exemplary sequences may vary at one or more of their amino acid positions. In some embodiments, the protease cleavage sequence comprises a sequence that matches an MMP consensus sequence, such as any one of SEQ ID NOs: 91-94.

[0443] Those of skill in the art will be familiar with additional sequences recognized by these types of proteases.

[0444] i. Matrix metalloproteinase truncated sequence In some embodiments, the protease cleavage sequence is a matrix metalloprotease (MMP) cleavage sequence and is recognized by a matrix metalloprotease. Exemplary MMP cleavage sequences are provided in Table 1. In some embodiments, the MMP cleavage sequence is cleavable and / or recognized by multiple MMPs, and / or one or more of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP12, MMP13, and / or MMP14. In some embodiments, the protease cleavage polypeptide sequence is cleavable and / or recognized by two, three, four, five, six, or seven of MMP2, MMP7, MMP8, MMP9, MMP12, MMP13, and MMP14. Table 1, e.g., SEQ ID NOs: 80-90, provides exemplary MMP cleavage sequences.

[0445] In some embodiments, the protease-cleaved polypeptide sequence comprises any one of SEQ ID NOs: 80-90. In some embodiments, the protease-cleaved polypeptide sequence comprises the sequence of SEQ ID NO: 80, or a variant sequence having one or two mismatches compared thereto. In some embodiments, the protease-cleaved polypeptide sequence comprises the sequence of SEQ ID NO: 81, or a variant sequence having one or two mismatches compared thereto. In some embodiments, the protease-cleaved polypeptide sequence comprises the sequence of SEQ ID NO: 82, or a variant sequence having one or two mismatches compared thereto. In some embodiments, the protease-cleaved polypeptide sequence comprises the sequence of SEQ ID NO: 83, or a variant sequence having one or two mismatches compared thereto. In some embodiments, the protease-cleaved polypeptide sequence comprises the sequence of SEQ ID NO: 84, or a variant sequence having one or two mismatches compared thereto. In some embodiments, the protease-cleaved polypeptide sequence comprises the sequence of SEQ ID NO: 85, or a variant sequence having one or two mismatches compared thereto. In some embodiments, the protease truncated polypeptide sequence comprises the sequence of SEQ ID NO: 86, or a variant sequence having one or two mismatches compared thereto. In some embodiments, the protease truncated polypeptide sequence comprises the sequence of SEQ ID NO: 87, or a variant sequence having one or two mismatches compared thereto. In some embodiments, the protease truncated polypeptide sequence comprises the sequence of SEQ ID NO: 88, or a variant sequence having one or two mismatches compared thereto. In some embodiments, the protease truncated polypeptide sequence comprises the sequence of SEQ ID NO: 89, or a variant sequence having one or two mismatches compared thereto. In some embodiments, the protease truncated polypeptide sequence comprises the sequence of SEQ ID NO: 90, or a variant sequence having one or two mismatches compared thereto. In some embodiments, the protease truncated polypeptide sequence comprises the sequence of SEQ ID NO: 91, or a variant sequence having one or two mismatches compared thereto.In some embodiments, the protease-cleaved polypeptide sequence comprises the sequence of SEQ ID NO: 92, or a variant sequence having one or two mismatches compared thereto. In some embodiments, the protease-cleaved polypeptide sequence comprises the sequence of SEQ ID NO: 93, or a variant sequence having one or two mismatches compared thereto. In some embodiments, the protease-cleaved polypeptide sequence comprises the sequence of SEQ ID NO: 94, or a variant sequence having one or two mismatches compared thereto.

[0446] D. Targeting Sequence In some embodiments, the linker polypeptide comprises a first targeting sequence and / or a second targeting sequence.In some embodiments, the first targeting sequence and / or the second targeting sequence is between the receptor binding domain and the protease cleavable polypeptide sequence or one of a plurality of protease cleavable polypeptide sequences.In some embodiments, at least one of the first linker and the second linker comprises a targeting sequence, for example, one of the first targeting sequence and the second targeting sequence, at least one targeting sequence, one of the first plurality of targeting sequences, one of the second plurality of targeting sequences, or one of a plurality of targeting sequences. In some embodiments, the protease-cleaved polypeptide sequence comprises a targeting sequence, e.g., one of a first targeting sequence and a second targeting sequence, at least one targeting sequence, one of a first plurality of targeting sequences, one of a second plurality of targeting sequences, or one of a plurality of targeting sequences.

[0447] In some embodiments, one or each of the first targeting sequence and the second targeting sequence, one or each of at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences increases the serum half-life of the linker polypeptide.Generally, the increase in serum half-life can be, for example, compared to the serum half-life of the linker polypeptide that lacks one or each of the first targeting sequence and the second targeting sequence, one or each of at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences. In some embodiments, one or each of the first targeting sequence and the second targeting sequence, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the multiple targeting sequences, in combination with another one of the first targeting sequence and the second targeting sequence, another one of the at least one targeting sequence, another one of the first plurality of targeting sequences, another one of the second plurality of targeting sequences, or another one of the multiple targeting sequences, synergistically increases the serum half-life of the linker polypeptide. In some embodiments, one or each of the first targeting sequence and the second targeting sequence, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences synergistically increases the serum half-life of the linker polypeptide in combination with a pharmacokinetic modulator.In some embodiments, one or each of the first targeting sequence and the second targeting sequence, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences independently increases the serum half-life of the linker polypeptide.

[0448] Serum half-life can be measured, for example, by measuring the serum level of the linker polypeptide over time after administration of the linker polypeptide. In some embodiments, any one of the above targeting sequences can independently increase the serum half-life of the linker polypeptide, when the serum half-life exceeds that of a linker polypeptide that lacks one targeting sequence but is otherwise identical to the linker polypeptide, and the increase does not depend on the other increase that comes from another targeting sequence. In some embodiments, any one of the above targeting sequences can synergistically increase the serum half-life of the linker polypeptide in combination with another one of the targeting sequences or a pharmacokinetic modulator, when the increase in serum half-life exceeds the sum of the increase that comes from one targeting sequence and the increase that comes from another one of the targeting sequences, or the increase that comes from one targeting sequence and the increase that comes from a pharmacokinetic modulator.

[0449] The targeting sequence may promote the localization, accumulation, and / or retention (e.g., after proteolysis of the protease-cleavable sequence) of the linker polypeptide and / or the first active domain and / or the second active domain in the region of interest, e.g., in the tumor microenvironment (TME). The targeting sequence may be a sequence that binds to an extracellular matrix component. Exemplary extracellular matrix components may include, for example, collagen or denatured collagen (in each case, collagen may be type I, type II, type III, or type IV collagen), poly(I), von Willebrand factor, IgB (CD79b), heparin, heparan sulfate, sulfated glycoprotein, or hyaluronic acid. In some embodiments, the extracellular matrix component is hyaluronic acid, heparin, heparan sulfate, or sulfated glycoprotein.

[0450] In some embodiments, the targeting sequence binds to a target other than an extracellular matrix component. In some embodiments, the targeting sequence binds to one or more of IgB (CD79b), fibronectin, integrin, cadherin, heparan sulfate proteoglycan, and syndecan. In some embodiments, the targeting sequence binds to at least one integrin, such as one or more of α1β1 integrin, α2β1 integrin, α3β1 integrin, α4β1 integrin, α5β1 integrin, α6β1 integrin, α7β1 integrin, α9β1 integrin, α4β7 integrin, αvβ3 integrin, αvβ5 integrin, αIIbβ3 integrin, αIIIbβ3 integrin, αMβ2 integrin, or αIIbβ3 integrin. In some embodiments, the targeting sequence binds to at least one syndecan, such as one or more of syndecan-1, syndecan-4, and syndecan-2(w). Linker polypeptides comprising such targeting sequences may also include MMP-cleavable linkers as defined elsewhere herein, such as MMP-cleavable linkers comprising any one of SEQ ID NOs: 80-90, or mutants having one or two mismatches compared to any one of SEQ ID NOs: 80-90.

[0451] In some embodiments, the targeting sequence comprises a sequence set forth in Table 2 (e.g., any one of SEQ ID NOs: 179-665, such as SEQ ID NOs: 179-640), or a variant having one or two mismatches compared to such a sequence.

[0452] In some embodiments comprising a first targeting sequence and a second targeting sequence, the first targeting sequence is configured to bind to heparin and the second targeting sequence is configured to bind to heparin, or the first targeting sequence is configured to bind to type IV collagen and the second targeting sequence is configured to bind to heparin, or the first targeting sequence is configured to bind to heparin and the second targeting sequence is configured to bind to type IV collagen.

[0453] In some embodiments, one or each of the first targeting sequence and the second targeting sequence, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences is independently configured to bind to its target with an affinity of 0.1 nM to 1 nM, 1 nM to 10 nM, 10 nM to 100 nM, 100 nM to 1 μM, 1 μM to 10 μM, or 10 μM to 100 μM. In some embodiments, one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences are independently configured to bind to their target with an affinity of 0.1 nM to 1 nM. In some embodiments, one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences are independently configured to bind to their target with an affinity of 1 nM to 10 nM. In some embodiments, one or each of the first targeting sequence and the second targeting sequence, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences is independently configured to bind to its target with an affinity of between 10 nM and 100 nM.In some embodiments, one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences are independently configured to bind to their target with an affinity of 100 nM to 1 μM. In some embodiments, one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences are independently configured to bind to their target with an affinity of 1 μM to 10 μM. In some embodiments, one or each of the first and second targeting sequences, one or each of the at least one targeting sequence, one or each of the first plurality of targeting sequences, one or each of the second plurality of targeting sequences, or one or each of the plurality of targeting sequences are independently configured to bind to their target with an affinity of 10 μM to 100 μM. In some embodiments, affinity is measured by a dissociation constant (K), which may be measured, for example, via surface plasmon resonance (SPR), enzyme-linked immunosorbent assay (ELISA), or polarization-modulated oblique-incidence reflectivity difference (OI-RD). D ) may be possible.

[0454] 1. pH-sensitive targeting sequence In some embodiments, the targeting sequence is configured to bind to its target in a pH-sensitive manner. In some embodiments, the targeting sequence has a high affinity for its target at a relatively acidic pH compared to normal physiological pH (about 7.4). The high affinity may occur at a pH below 7, for example, at pH 5.5-7, 6-7, or 5.5-6.5, or below pH 6. The presence of histidine in the targeting sequence may confer pH-sensitive binding. Without being bound to any particular theory, it is believed that histidine is more likely to be protonated at low pH values, making binding to negatively charged targets more energetically favorable. Thus, in some embodiments, the targeting sequence includes one or more histidines, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 histidines. The incorporation of pH-sensitive targeting sequences can enhance the discrimination between tumor tissue and normal tissue by the linker polypeptide, so that the linker polypeptide is preferentially retained in the tumor microenvironment compared to the normal extracellular matrix. Thus, the pH-sensitive targeting element can further promote tumor-specific delivery of the linker polypeptide, which can further reduce or eliminate the toxicity resulting from the activity of the linker polypeptide in the normal extracellular matrix.

[0455] pH-sensitive binding to a target can be useful when it is desired to localize or retain the linker polypeptide and / or its cytokine polypeptide sequence in an area where the pH is different from normal physiological pH. For example, the tumor microenvironment can be more acidic than blood and / or healthy tissue. Thus, pH-sensitive binding to a target can improve the retention of the linker polypeptide and / or its cytokine polypeptide sequence in the desired area, which can facilitate lower doses than would otherwise be required and / or reduce systemic exposure and / or adverse effects.

[0456] In some embodiments, the targeting sequence is configured to bind to any target described herein in a pH-sensitive manner.In certain embodiments, the target is an extracellular matrix component, IgB (CD79b), integrin, cadherin, heparan sulfate proteoglycan, syndecan, or fibronectin.In some embodiments, the extracellular matrix component is hyaluronic acid, heparin, heparan sulfate, or sulfated glycoprotein.In another specific embodiment, the target is fibronectin.

[0457] Exemplary targeting sequences for conferring pH-sensitive binding to a target are provided in Table 2 (e.g., SEQ ID NOs: 641-663). In some embodiments, the targeting sequence comprises any one of SEQ ID NOs: 641-663, or a variant having one or two mismatches compared to any one of SEQ ID NOs: 641-663.

[0458] In some embodiments, the linker polypeptide comprises a targeting sequence adjacent to a protease cleavable sequence. The targeting sequence and the protease cleavable sequence can be any of the targeting sequences and protease cleavable sequences described herein. An exemplary combination of a targeting sequence and a protease cleavable sequence is SEQ ID NO: 667-673.

[0459] E. Pharmacokinetic Modulators In some embodiments, the linker polypeptide comprises a pharmacokinetic modulator. The pharmacokinetic modulator may be covalently or non-covalently associated with the linker polypeptide. The pharmacokinetic modulator may extend the half-life of the linker polypeptide, for example, so that smaller doses are required and less of the linker polypeptide needs to be administered over time to achieve the desired result. Various forms of pharmacokinetic modulators are known in the art and may be used in the linker polypeptides of the present disclosure. In some embodiments, the pharmacokinetic modulator comprises a polypeptide (see examples below). In some embodiments, the pharmacokinetic modulator comprises a non-polypeptide moiety (e.g., polyethylene glycol, polysaccharide, or hyaluronic acid). The non-polypeptide moiety may be associated with the linker polypeptide using known techniques, for example, conjugation with the linker polypeptide; for example, reactive amino acid residues may be used or added to the linker polypeptide to facilitate conjugation.

[0460] In some embodiments, the pharmacokinetic modulator alters the size, shape, and / or charge of the linker polypeptide, e.g., in a manner that reduces clearance. For example, a pharmacokinetic modulator with a negative charge may inhibit renal clearance. In some embodiments, the pharmacokinetic modulator increases the hydrodynamic volume of the linker polypeptide. In some embodiments, the pharmacokinetic modulator reduces renal clearance, e.g., by increasing the hydrodynamic volume of the linker polypeptide.

[0461] In some embodiments, a linker polypeptide comprising a pharmacokinetic modulator (eg, any of the pharmacokinetic modulators described herein) has a molecular weight of at least 70 kDa, eg, at least 75 or 80 kDa.

[0462] For further discussion of various approaches to incorporating pharmacokinetic modulators, see, e.g., Strohl, BioDrugs 29:215-19 (2015); and Podust et al., J. Controlled Release 240:52-66 (2016).

[0463] 1. Polypeptide pharmacokinetic modulators In some embodiments, the pharmacokinetic modulator comprises a polypeptide, e.g., an immunoglobulin sequence (see exemplary embodiments below), albumin, CTP (the negatively charged carboxy terminal peptide of the chorionic gonadotropin beta chain, which undergoes sialylation in vivo and in a suitable host cell), an inactive polypeptide (e.g., an unstructured polypeptide such as XTEN, which is a polypeptide comprising the residues Ala, Glu, Gly, Pro, Ser, and Thr), transferrin, a homoamino acid polypeptide, or an elastin-like polypeptide.

[0464] Exemplary polypeptide sequences suitable for use as pharmacokinetic modulators are provided in Table 1 (e.g., any one of SEQ ID NOs: 70-74). In some embodiments, the pharmacokinetic modulator has at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of a pharmacokinetic modulator in Table 1 (e.g., any one of SEQ ID NOs: 70-74).

[0465] In any embodiment in which the pharmacokinetic modulator comprises a polypeptide sequence derived from an organism, the polypeptide sequence may be a human polypeptide sequence.

[0466] 2. Pharmacokinetic Modulators of Immunoglobulins In some embodiments, the pharmacokinetic modulator comprises an immunoglobulin sequence, e.g., at least a portion of one or more immunoglobulin constant domains. In some embodiments, the pharmacokinetic modulator comprises an immunoglobulin constant domain. In some embodiments, the pharmacokinetic modulator comprises at least a portion of an immunoglobulin Fc region. In some embodiments, the pharmacokinetic modulator comprises an immunoglobulin Fc region.

[0467] The immunoglobulin sequence (e.g., at least a portion of one or more immunoglobulin constant domains or Fc region) can be a human immunoglobulin sequence. The immunoglobulin sequence (e.g., at least a portion of one or more immunoglobulin constant domains or Fc region) can have at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of a wild-type immunoglobulin sequence (e.g., at least a portion of one or more immunoglobulin constant domains or Fc region), such as a wild-type human immunoglobulin sequence. In any of such embodiments, the immunoglobulin sequence can be an IgG sequence (e.g., IgG1, IgG2, IgG3, or IgG4, e.g., one or more immunoglobulin constant domains or at least a portion of the Fc region of any of their isotypes), such as one or more immunoglobulin constant domains or at least a portion of the Fc region. Exemplary immunoglobulin pharmacokinetic modulator sequences include SEQ ID NOs: 70-74, 857, 858, 861, and 862, as well as combinations of SEQ ID NOs: 756 and 757; 75 and 77; 75 and 78; 76 and 77; 76 and 78; and 859 and 860.

[0468] In some embodiments, immunoglobulin pharmacokinetic modulator sequences (such as Fc regions) may perform certain functions and effects by interacting with certain targets, as set forth in Table 3 below.

[0469] F. Growth Factor Binding Polypeptide Sequences, and Growth Factor Receptor Binding Polypeptide Sequences In some embodiments, the linker polypeptide comprises a growth factor binding polypeptide sequence or a growth factor receptor binding polypeptide sequence. Such sequences can be used as active domains.

[0470] In some embodiments, the growth factor binding polypeptide sequence comprises a TGF-βR extracellular domain sequence. In some embodiments, the TGF-βR extracellular domain sequence comprises an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 1022 or 1023.

[0471] In some embodiments, the growth factor binding polypeptide sequence comprises a growth factor binding immunoglobulin domain. In some embodiments, the growth factor binding immunoglobulin domain is configured to bind to TGF-β. In some embodiments, the growth factor binding immunoglobulin domain comprises a VH region comprising HVR-1, HVR-2, and HVR-3 of the VH region comprising the amino acid sequence of SEQ ID NO: 1008; and a VL region comprising HVR-1, HVR-2, and HVR-3 of the VL region comprising the amino acid sequence of SEQ ID NO: 1010. In general, those skilled in the art will be familiar with the methods described in, for example, Kabat et al. in Sequences of Proteins of Immunological Interest, 5 thHVRs within the VH and VL sequences may be identified by assigning amino acids to framework and HVR domains within the VH and VL sequences according to the definitions in US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991. Other numbering systems for amino acids within immunoglobulin chains include IMGT™ (international ImMunoGeneTics information system; Lefranc et al, Dev. Comp. Immunol. 29:185-203; 2005), and AHo [Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001]. In some embodiments, the growth factor binding immunoglobulin domain comprises a VH region comprising the amino acid sequence of SEQ ID NO:1008; and a VL region comprising the amino acid sequence of SEQ ID NO:1010. In some embodiments, the growth factor binding immunoglobulin domain comprises a sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 1007 or 1009. In some embodiments, the growth factor receptor binding polypeptide sequence comprises a TGF-β sequence. In some embodiments, the TGF-β sequence comprises an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of any one of SEQ ID NOs: 904-906.

[0472] In some embodiments, the growth factor receptor binding polypeptide sequence comprises a growth factor receptor binding immunoglobulin domain. In some embodiments, the growth factor receptor binding immunoglobulin domain is configured to bind to a TGF-βR extracellular domain sequence. In some embodiments, the growth factor receptor binding immunoglobulin domain comprises a VH region comprising HVR-1, HVR-2, and HVR-3 of the VH region comprising the amino acid sequence of SEQ ID NO: 999 or 1003; and a VL region comprising HVR-1, HVR-2, and HVR-3 of the VL region comprising the amino acid sequence of SEQ ID NO: 1000 or 1004. In some embodiments, the growth factor receptor binding immunoglobulin domain comprises a VH region comprising the amino acid sequence of SEQ ID NO: 999 or 1003; and a VL region comprising the amino acid sequence of SEQ ID NO: 1000 or 1004. In some embodiments, the growth factor receptor binding immunoglobulin domain comprises a sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to any one of SEQ ID NOs:1001, 1002, 1005, and 1006.

[0473] [Table 3]

[0474] A. Blocking Agents In some embodiments, the linker polypeptide may include a blocking agent. In some embodiments, the blocking agent may be conjugated to one or each of the first and second active domains. In some embodiments, the blocking agent is conjugated to one or each of the first and second active domains via a protease-cleavable polypeptide sequence.

[0475] The blocking agent may prevent the immunoglobulin antigen-binding domain from binding to an antigen (e.g., a growth factor or growth factor receptor). In some embodiments, the blocking agent is linked to the immunoglobulin antigen-binding domain via the N-terminus of the heavy or light chain of the immunoglobulin antigen-binding domain.

[0476] In some embodiments, the blocking agent comprises albumin. In some embodiments, the blocking agent comprises serum albumin. In some embodiments, the blocking agent comprises human serum albumin (HAS) (e.g., SEQ ID NO: 72) or a fragment thereof.

[0477] B. Chemotherapy drugs In some embodiments, the linker polypeptide may include one or more chemotherapeutic agents. The agents may be, for example, conjugated to different elements of the linker polypeptide. In some embodiments, the chemotherapeutic agent is conjugated to a pharmacokinetic modulator of the linker polypeptide.

[0478] In some embodiments, the chemotherapeutic agent is altretamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, mechlorethamine, melphalan, oxaliplatin, temozolomide, thiotepa, trabectedin, carmustine, lomustine, streptozocin, azacitidine, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cytarabine, decitabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, nelarabine, pemetrexed, pentostatin, pralatrexate. indolent, thioguanine, trifluridine, tipiracil, daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, bleomycin, dactinomycin, mitomycin c, mitoxantrone, irinotecan, topotecan, etoposide, mitoxantrone, teniposide, cabazitaxel, docetaxel, paclitaxel, vinblastine, vincristine, vinorelbine, prednisone, methylprednisolone, dexamethasone, retinoic acid, arsenic trioxide, asparaginase, eribulin, hydroxyurea, ixabepilone, mitotane, omacetaxine, pegaspargase, procarbazine, romidepsin, and vorinostat.

[0479] III. Placement of components and their release The recitation of linker polypeptide components herein does not imply a particular order beyond what is explicitly stated (e.g., it may be explicitly stated that a protease cleavage sequence is between a cytokine polypeptide sequence and an inhibitory polypeptide sequence). The linker polypeptide components may be arranged in a variety of ways to provide properties suitable for a particular use. The linker polypeptide components may all be in one polypeptide chain or may be in multiple polypeptide chains that are crosslinked by covalent bonds such as disulfide bonds.

[0480] For example, in some embodiments where the pharmacokinetic modulator comprises an Fc, one or more components (e.g., chemotherapeutic drugs) may be attached to one chain, while one or more other components may be attached to the other chain. The Fc may be a heterodimeric Fc, such as a knob-into-hole Fc (one chain of the Fc includes a knob mutation and the other chain of the Fc includes a hole mutation). For an exemplary general discussion of knob and hole mutations, see, e.g., Xu et al., mAbs 7:1, 231-242 (2015). An exemplary knob mutation (e.g., for a human IgG1 Fc) is K360E / K409W. An exemplary hole mutation (e.g., for a human IgG1 Fc) is Q347R / D399V / F405T. See SEQ ID NOs: 756 and 757.

[0481] In some embodiments, part or all of the one or more protease-cleaved polypeptide sequences may be C-terminal to the VH region, C-terminal to at least a portion of the CH1 domain, between the CH1 and CH2 domains, N-terminal to at least a portion of the CH2 domain, N-terminal to the inter-heavy chain disulfide bond, N-terminal to a disulfide bond within the CH2 domain, or N-terminal to the hinge region or within the hinge region. In some embodiments, part or all of the one or more protease-cleaved polypeptide sequences may be between the pharmacokinetic modulator and the second active domain and / or between the blocking agent and one or each of the first and second active domains.

[0482] In some embodiments, a targeting sequence may be between the receptor binding domain and one or more protease-cleaved polypeptide sequences. In some embodiments, at least one of the first linker and the second linker comprises a targeting sequence and / or the protease-cleaved polypeptide sequence comprises a targeting sequence.

[0483] In some embodiments, the targeting sequence may be present on the same side of the protease-cleaved polypeptide sequence as the receptor binding domain (e.g., a cytokine polypeptide sequence), meaning that cleavage of the protease-cleaved polypeptide sequence does not separate the targeting sequence from the receptor binding domain. Such embodiments may be useful to promote localization or retention of both the linker polypeptide and the released receptor binding domain within the region of interest, e.g., the tumor microenvironment.

[0484] In some embodiments, a targeting sequence may be present on the same side of a protease-cleaved polypeptide sequence as an inhibitory polypeptide sequence, meaning that cleavage of the protease-cleaved polypeptide sequence does not separate the targeting sequence from the cytokine polypeptide sequence. Such embodiments may be useful for creating a gradient of cytokine from a region of interest, or for creating such a gradient more rapidly than would occur if the targeting sequence were present on the same side of the protease-cleaved sequence.

[0485] In some embodiments, the first active domain is closer to the first targeting sequence than the second targeting sequence.In other embodiments, the second active domain is closer to the first targeting sequence than the second targeting sequence.In some embodiments, the linker polypeptide comprises, in order from N-terminus to C-terminus or from C-terminus to N-terminus, the first active domain, the first targeting sequence, the first linker, the second targeting sequence and an additional domain.

[0486] In some embodiments, the protease cleaved polypeptide sequence is C-terminal to the first targeting sequence and the second targeting sequence. In some embodiments, the protease cleaved polypeptide sequence is N-terminal to the first targeting sequence and the second targeting sequence. In some embodiments, the protease cleaved polypeptide sequence is C-terminal to the first multiple targeting sequence and N-terminal to the second multiple targeting sequence. In some embodiments, the protease cleaved polypeptide sequence is C-terminal to the multiple targeting sequences and N-terminal to at least one targeting sequence. In some embodiments, the protease cleaved polypeptide sequence is N-terminal to the multiple targeting sequences and C-terminal to at least one targeting sequence. In some embodiments, the protease cleaved polypeptide sequence is C-terminal to the first targeting sequence and the second targeting sequence and not N-terminal to the targeting sequence. In some embodiments, the protease-cleaved polypeptide sequence is N-terminal to the first targeting sequence and the second targeting sequence and is not C-terminal to the targeting sequences.

[0487] In some embodiments, the linker polypeptide comprises a first active domain, a second active domain, a pharmacokinetic modulator, and a first linker between the pharmacokinetic modulator and the first active domain. In some embodiments, the first linker comprises a protease-cleavable polypeptide sequence and may comprise a targeting sequence. In certain embodiments, the active domain comprises an immunoglobulin antigen-binding domain. In certain embodiments, the target-binding domain may comprise a heavy chain and a light chain, or only a heavy chain. In some embodiments, the linker polypeptide comprises a chemotherapeutic agent.

[0488] In some embodiments, the first active domain is released from the remainder of the linker polypeptide after one or more protease-cleaved polypeptide sequences are cleaved. In some embodiments, the linker polypeptide further comprises a blocking agent conjugated to one or each of the first active domain and the second active domain via a protease-cleaved polypeptide sequence. In some embodiments, the protease-cleaved polypeptide sequence connecting the first active domain to the remainder of the linker polypeptide and the protease-cleaved polypeptide sequence connecting the blocking agent to the active domain can be cleaved together (e.g., by the same protease). In some embodiments, the protease-cleaved polypeptide sequence connecting the first active domain to the remainder of the linker polypeptide and the protease-cleaved polypeptide sequence connecting the blocking agent to the active domain can be cleaved separately (e.g., by different proteases).

[0489] In some embodiments, the linker polypeptide comprises a first active domain, a second active domain, a pharmacokinetic modulator, and a first linker between the pharmacokinetic modulator and the first active domain, the first linker comprising a protease-cleavable polypeptide sequence, and may comprise a targeting sequence. In certain embodiments, the first active domain comprises a receptor-binding domain, and the second active domain comprises an immunoglobulin antigen-binding domain, which may comprise a cytokine polypeptide sequence. In some embodiments, the linker polypeptide comprises an inhibitory polypeptide sequence capable of blocking the activity of the receptor-binding domain, and a second linker between the receptor-binding domain and the inhibitory polypeptide sequence, the second linker comprising a protease-cleavable polypeptide sequence.

[0490] In some embodiments, the first active domain is released from the remainder of the linker polypeptide after one or more protease-cleavable polypeptide sequences are cleaved. In some embodiments, the first active domain comprises a receptor-binding domain, which may comprise a cytokine polypeptide sequence, and the second active domain comprises an immunoglobulin antigen-binding domain. In some embodiments, the linker polypeptide further comprises an inhibitory polypeptide sequence capable of blocking the activity of the receptor-binding domain, and a second linker between the receptor-binding domain and the inhibitory polypeptide sequence, the second linker comprising a protease-cleavable polypeptide sequence. In some embodiments, the protease-cleavable polypeptide sequence of the first linker and the protease-cleavable polypeptide sequence of the second linker can be cleaved together (e.g., by the same protease). In some embodiments, the protease-cleavable polypeptide sequence of the first linker and the protease-cleavable polypeptide sequence of the second linker can be cleaved separately (e.g., by different proteases).

[0491] In some embodiments, e.g., any of the embodiments in which there is a first and a second polypeptide chain comprising a first and a second domain of a pharmacokinetic modulator, respectively, the inhibitory polypeptide sequence is C-terminal to the second domain of the pharmacokinetic modulator, or the inhibitory polypeptide sequence is N-terminal to the second domain of the pharmacokinetic modulator. The targeting sequence may be between the protease-cleaved polypeptide sequence and the first domain of the pharmacokinetic modulator, between the protease-cleaved polypeptide sequence and the first active domain, C-terminal to the first active domain, N-terminal to the first active domain, C-terminal to the inhibitory polypeptide sequence, N-terminal to the inhibitory polypeptide sequence, or between the inhibitory polypeptide sequence and the second domain of the pharmacokinetic modulator.

[0492] In some embodiments, for example, in any of the embodiments in which there is a first polypeptide chain and a second polypeptide chain, each of which comprises a first domain and a second domain of a pharmacokinetic modulator, the linker polypeptide can comprise a first targeting sequence and a second targeting sequence.In some such embodiments, the first targeting sequence is a part of the first polypeptide chain, and the second targeting sequence is a part of the second polypeptide chain.In some such embodiments, the first targeting sequence is C-terminal to the first active domain, and the second targeting sequence is C-terminal to the inhibitory polypeptide sequence.

[0493] In some embodiments, e.g., any of the embodiments in which there is a first and a second polypeptide chain comprising a first and a second domain of a pharmacokinetic modulator, respectively, the linker polypeptide further comprises a second active domain, which may be part of the second polypeptide chain, and / or the linker polypeptide comprises a first inhibitory polypeptide sequence, which further comprises a second inhibitory polypeptide sequence. In some embodiments, the second inhibitory polypeptide sequence is part of the second polypeptide chain. In some embodiments, the second inhibitory polypeptide sequence is C-terminal to the first inhibitory polypeptide sequence. The first inhibitory polypeptide sequence and / or the second inhibitory polypeptide sequence may be an immunoglobulin inhibitory polypeptide sequence, such as a VHH.

[0494] In some embodiments, for example any of the embodiments in which there is a first and a second polypeptide chain comprising a first and a second domain of a pharmacokinetic modulator, respectively, the pharmacokinetic modulator comprises a heterodimeric Fc domain or a heterodimeric CH3 domain. The heterodimeric Fc domain or the heterodimeric CH3 domain may be in a separate polypeptide chain. In some embodiments, the heterodimeric Fc domain or the heterodimeric CH3 domain comprises a knob CH3 domain and a hole CH3 domain.

[0495] In some embodiments, the linker polypeptide comprises a polypeptide sequence of any one of SEQ ID NOs: 800-848 and 1024-1041. In some embodiments, the linker polypeptide comprises a polypeptide sequence of any one of SEQ ID NOs: 1042-1137.

[0496] IV. Pharmaceutical Formulations or Compositions Pharmaceutical formulations or compositions of the linker polypeptides described herein can be prepared by mixing such linker polypeptides having the desired purity with one or more suitable pharma- ceutically acceptable carriers [Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)] in the form of a lyophilized formulation or composition, or an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate buffer, citrate buffer, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues); (of the formula I); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0497] Formulations or compositions to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0498] V. Use In some embodiments, any one or more of the linker polypeptides, compositions, or pharmaceutical preparations described herein are for use in therapy, such as for use in the preparation of a medicament for treating or preventing a disease or disorder in a subject, such as cancer. In some embodiments, any one or more of the linker polypeptides, compositions, or pharmaceutical preparations described herein are for use in a method of treating cancer, for example, a method comprising administering a linker polypeptide or pharmaceutical composition to a subject in need thereof.

[0499] In some embodiments, a method for treating or preventing a disease or disorder in a subject is provided, comprising administering to the subject any of the linker polypeptides or pharmaceutical compositions described herein. In some embodiments, the disease or disorder is cancer, for example, a solid tumor. In some embodiments, the cancer is melanoma, colorectal cancer, breast cancer, pancreatic cancer, lung cancer, prostate cancer, ovarian cancer, cervical cancer, gastric or gastrointestinal cancer, lymphoma, colon or colorectal cancer, endometrial cancer, thyroid cancer, or bladder cancer. The cancer (e.g., any of the above cancers) may have one or more of the following characteristics: PD-L1 positive; metastatic; unresectable; mismatch repair deficient (MMRd); and / or microsatellite instability high (MSI-H). In some embodiments, the cancer is a cancer that expresses TGFβR. In some embodiments, the cancer is a cancer that expresses TGFβ. In some embodiments, the cancer is a TGFβ-dependent cancer. A cancer is considered to be dependent on a growth factor, such as TGFβ, when the cells of the cancer grow significantly slower in the absence of the growth factor than in its presence.

[0500] In some embodiments, methods are provided for enhancing regulatory T cells and / or reducing inflammatory or autoimmune activity, comprising administering a linker polypeptide to an area of ​​interest, e.g., an area of ​​inflammation. A linker polypeptide for use in such methods may comprise an IL-2 polypeptide sequence. In some embodiments, methods are provided for treating autoimmune and / or inflammatory diseases, comprising administering a linker polypeptide to an area of ​​interest, e.g., an area having inflammatory or autoimmune activity. A linker polypeptide for use in such methods may comprise an IL-2 polypeptide sequence. These methods take advantage of the ability of certain cytokines to stimulate regulatory T cells at relatively low levels, which can exert anti-inflammatory effects and reduce or suppress autoimmune activity.

[0501] The linker polypeptide in any of the above methods and uses can be delivered to a subject using any suitable administration route. In some embodiments, the linker polypeptide is delivered parenterally. In some embodiments, the linker polypeptide is delivered intravenously.

[0502] The linker polypeptides provided herein may be used alone or in combination with other agents in therapy. For example, the linker polypeptides provided herein may be co-administered with at least one additional therapeutic agent.

[0503] As mentioned above, such combination therapy includes combined administration (wherein two or more therapeutic agents are contained in the same or separate formulations) as well as separate administration, where administration of a linker polypeptide provided herein can occur prior to, simultaneously with, and / or after administration of an additional therapeutic agent and / or adjuvant.

[0504] The linker polypeptide is formulated, dosed, and administered in a manner consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the administration schedule, and other factors known to medical practitioners. In some embodiments, the linker polypeptide may be formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of linker polypeptide present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used at the same dosages and by the same routes of administration as described herein, or at about 1-99% of the dosages described herein, or at any dosage and by any route that is empirically / clinically determined to be appropriate.

[0505] For the prevention or treatment of disease, the appropriate dosage of the linker polypeptide (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease being treated, the type of linker polypeptide, the severity and course of the disease, whether the linker polypeptide is administered for prophylactic or therapeutic purposes, previous treatments, the clinical history of the patient, and response to therapeutic agents (e.g., antibodies, immunoconjugates, cytokines) that share common elements and / or sequences with the linker polypeptide, and the discretion of the attending physician. The linker polypeptide is suitably administered to the patient at one time or over a series of treatments.

[0506] VI. Nucleic Acids, Host Cells, and Methods of Production Linker polypeptides or precursors thereof may be produced using recombinant methods and compositions. In some embodiments, isolated nucleic acids are provided that encode the linker polypeptides described herein. Such nucleic acids may encode amino acid sequences that include active domains (e.g., immunoglobulin antigen-binding domains, receptor-binding domains, and / or cytokine polypeptide sequences), pharmacokinetic modulators, linkers, and inhibitory polypeptide sequences, as well as any other polypeptide components of the linker polypeptide that may be present. In further embodiments, one or more vectors (e.g., expression vectors) are provided that include such nucleic acids. In further embodiments, host cells are provided that include such nucleic acids. In some such embodiments, the host cell comprises (e.g., is transformed with) a vector that includes a nucleic acid encoding a linker polypeptide according to the present disclosure. In some embodiments, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell, or a lymphoid cell (e.g., a Y0 cell, an NS0 cell, an Sp20 cell). In some embodiments, methods of making a linker polypeptide disclosed herein are provided, comprising culturing a host cell comprising a nucleic acid encoding the linker polypeptide under conditions suitable for expression of the linker polypeptide, as provided above, and optionally comprising recovering the antibody from the host cell (or culture medium of the host cell).

[0507] For recombinant production of a linker polypeptide, for example as described above, a nucleic acid encoding the linker polypeptide is prepared and / or isolated (e.g., following construction using synthetic and / or molecular cloning methods) and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily prepared and / or isolated using known techniques.

[0508] Suitable host cells for cloning or expressing the vector encoding the linker polypeptide include the prokaryotic or eukaryotic cells described herein.For example, the linker polypeptide can be produced in bacteria, especially when glycosylation is not required.For the expression of polypeptide in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523.After expression, the linker polypeptide can be isolated from the bacterial cell paste in soluble fraction and further purified.

[0509] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding linker polypeptides, including fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of polypeptides with partial or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004); and Li et al., Nat. Biotech. 24:210-215 (2006).

[0510] Suitable host cells for the expression of the linker polypeptide are also derived from multicellular organisms (plants, invertebrates, and vertebrates). Examples of invertebrate cells include insect cells. In particular, a number of baculovirus strains have been identified that can be used with insect cells for transfection of Spodoptera frugiperda cells.

[0511] Plant cell cultures may also be utilized as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429.

[0512] Vertebrate cells may also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 cell lines (COS-7); human embryonic kidney cell lines [e.g., 293 cells or 293 cells, described in Graham et al., J. Gen Virol. 36:59 (1977)]; baby hamster kidney cells (BHK); mouse Sertoli cells [e.g., TM4 cells, described in Mather, Biol. Reprod. 23:243-251 (1980)]; monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT060562); e.g., Mather et al., Annals NY Acad. Sci. 383:44-68 (1982); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines are DHFR - Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0.

[0513] The present description and exemplary embodiments should not be understood as limiting. For the purposes of this specification and the appended claims, unless otherwise indicated, all numbers expressing quantities, percentages, or ratios, as well as other numerical values ​​used in this specification and the appended claims, shall be understood in all cases to be modified by the term "about" to the extent that they have not already been so modified. "About" refers to a degree of variation that does not substantially affect the properties of the described object, for example, within 10%, 5%, 2%, or 1%. Thus, unless otherwise indicated, the numerical parameters set forth in the following specification and the appended claims are approximations that may vary depending on the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed at least in light of the number of reported significant digits and by applying ordinary rounding techniques. EXAMPLES

[0514] The following examples are provided to illustrate certain disclosed embodiments and are not to be construed as limiting the scope of the disclosure in any way. [Example 1]

[0515] Construction of mammalian expression vectors encoding fusion proteins The coding sequences of all protein domains, including linker sequences, were synthesized as complete genes (Genscript, NJ). All synthetic genes were designed to contain coding sequences for an N-terminal signal peptide (facilitating secretion of the protein), a 5' Kozak sequence, and unique restriction sites at the 5' and 3' ends. These genes were then directionally cloned into the mammalian expression vector pcDNA3.1 (Invitrogen; Carlsbad, CA). Examples of fusion protein constructs are listed in Table 4.

[0516] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Example 2]

[0517] Fusion protein expression and purification Transient expression of fusion proteins Different mammalian cell expression systems were used to produce the fusion proteins [ExpiCHO-S™, Expi293F™, Freestyle CHO-S™, and Freestyle 293™, Life Technologies]. Briefly, the expression constructs were transiently transfected into cells using the reagents provided by the respective expression kits according to the manufacturer's protocol. The fusion proteins were then expressed and secreted into the cell culture supernatant. Samples were harvested daily from the production cultures and assessed for cell density and cell viability. Protein expression titers and product integrity in the cell culture supernatants were analyzed by SDS-PAGE to determine the optimal harvest time point. Cell culture supernatants were generally harvested between 4 and 12 days, when culture viability was typically >75%. On the day of harvest, the cell culture supernatants were clarified by centrifugation and vacuum filtration was performed before further use.

[0518] Purification of fusion proteins Fusion proteins were purified from cell culture supernatants by one-step or two-step procedures. Briefly, Fc domain-containing proteins were purified by Protein A affinity chromatography (HiTrap MabSelect SuRe, GE Healthcare). In some cases, Fc domain-containing proteins were further purified by size exclusion chromatography (HPLC SEC5 300A 7.8×300 mm, 5 μm; model number: 5190-2526, Agilent Bio or HiLoad 26 / 60 Superdex 200). His-tagged proteins were first purified on a nickel-agarose column (Ni-Penta™ Agarose 6 Fast Flow column, PROTEINDEX™) followed by size exclusion chromatography (HPLC SEC5 300A 7.8×300 mm, 5 μm; model number: 5190-2526, Agilent Bio). All purified samples were buffer exchanged and concentrated by ultrafiltration to a typical concentration of >1 mg / mL. The purity and homogeneity of the final samples (typically >90%) were assessed by SDS-PAGE under reducing and non-reducing conditions. The purified proteins were aliquoted and stored at -80°C until further use. Figures 1A-1D show an example of successful purification of a fusion protein. In Figures 1A-1D, analysis (Coomassie staining) of the fusion protein purified by Protein A column showed high purity of the target protein and minimal high molecular weight entities. [Example 3]

[0519] Cleavage of the fusion protein by MMP9 protease First, recombinant MMP9 (R&D Systems) was activated with p-aminophenylmercuric acetate, and the fusion protein was digested or mock digested overnight (18-22 h) at 37°C using an equal volume of the activation solution with or without the protease. Cleavage assays were prepared in TCNB buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35 (w / v), pH 7.5. The digested protein was aliquoted and stored at -80°C before testing. Aliquots of the digest were then analyzed by SDS-PAGE followed by Western blotting to assess the extent of cleavage. Digests were also evaluated by functional assays such as the HEK-Blue Interleukin reporter assay. As shown in Figures 2A-2F, after overnight incubation, we observed essentially complete cleavage of the fusion protein with the functional site by the MMP9 protease, whereas the protein containing the scrambled MMP cleavage site was not cleaved (Figure 2D). [Example 4]

[0520] Immunoblot analysis for IL-2 and IL-15 Untreated and digested fusion proteins were assessed for cleavage products by Western blot. The following antibodies were used: goat anti-mouse IL-2 polyclonal antibody (AF-402-NA; R&D systems), anti-human IL-2 antibody (Invitrogen; model number: MA5-17097, mouse IgG1), and rabbit anti-human IL-15 polyclonal antibody (ThermoFisher; model number: PA5-79466). Detection was performed using donkey anti-goat HRP-conjugated antibody, goat anti-rabbit HRP-conjugated antibody, or goat anti-mouse HRP-conjugated antibody (Jackson Immuno Research, West Grove, PA) and developed using SuperSignal West Femto Maximum sensitivity detection reagent (ThermoFisher) according to the manufacturer's recommendations. [Example 5]

[0521] Detection of mouse IL-2 / IL-2Ra fusion protein by ELISA An ELISA assay was developed to detect and quantify prodrug fusion proteins containing IL-2 / IL-2Ra moieties. Wells of a 96-well plate were coated overnight with 100 μL of rat anti-mouse IL-2 monoclonal antibody (JES6-1A12; ThermoFisher) at 1 mg / mL in PBS. After washing, the wells were blocked with TBS / 0.05% Tween 20 / 1% BSA, and then fusion proteins and / or unknown biological samples were added for 1 h at room temperature. After washing, anti-mouse IL-2Ra biotin-labeled detection antibody (BAF2438, R&D systems) was added, and binding was detected using Ultra Strepavidin HRP (ThermoFisher). ELISA plates were developed by adding chromogenic tetramethylbenzidine substrate (Ultra TMB; ThermoFisher). The reaction was stopped by the addition of 0.5 M H2SO4 and the absorbance was read at 450-650 nm. [Example 6]

[0522] Cell-based functional assays for IL-2 and IL-15 IL-2 and IL-15 are members of the four alpha helix bundle family of cytokines and share the same signaling receptor, IL2-Rβ, and a common gamma chain. Therefore, the activity of these cytokines was measured using the same reporter cell line, HEK Blue IL-2 (Invivogen, San Diego). HEK-Blue™ IL-2 cells were specifically designed to monitor activation of the JAK-STAT pathway induced by ligand binding to the IL2-Rβ and common gamma chain receptors. Stimulation with the appropriate cytokine triggered the JAK / STAT5 pathway and induced the production of secreted embryonic alkaline phosphatase (SEAP). SEAP was easily monitored using the SEAP detection medium, Quanti-Blue™. These cells responded to human / mouse IL-2 / IL-15. For the HEK Blue assay, untreated and digested samples were titrated and added to 50,000 HEK Blue cells per well in 200 μL of medium in a 96-well plate and incubated at 37° C. in 5% CO2 for 20-24 minutes. The next day, SEAP levels were measured by adding 20 μL of cell supernatant to QuantiBlue reagent followed by incubation at 37° C. for 1-3 hours and reading absorbance at 630 nm. Figures 3A-3V and 3W-3BB show the results obtained from IL-2 / IL-15 fusion proteins examined in the HEK Blue IL-2 cell assay, respectively. [Example 7]

[0523] Binding assays for linker peptides of next-generation targeting sequences A series of peptides containing MMP cleavage sites, with or without the addition of a targeting sequence, were synthesized and conjugated to the fluorophore EDANS [5-{(2-aminoethyl)amino}naphthalene-1-sulfonic acid] (custom synthesis; ThermoFisher). Table 5 shows the list of peptides. These peptides were then examined for their ability to bind to ECM proteins such as heparin, fibronectin, and collagen, which are abundantly found in the tumor stroma. In Table 5, bolded text indicates MMP cleavage sites, underlined text indicates retention motifs (targeting sequences) when present, and italicized asterisks ( * ) denotes Edans, a fluorophore conjugated to a peptide.

[0524] [Table 5]

[0525] All binding assays were prepared in 10 mM Tris-HCl, pH 7.5 and / or 10 mM Tris-HCl, pH 6. Peptides (20 μM) were incubated with agarose beads crosslinked to heparin or control agarose beads (Sigma and Pierce, respectively) for 2 hours at room temperature on a shaker. The beads were then washed four times and resuspended in 100 μL of binding buffer in a black 96-well plate. Peptide binding was quantified by measuring the fluorescence of the samples using the excitation / emission spectrum of EDANS (Ex340 / Em490). Figures 4A-4B show that several next-generation MMP linker peptides containing heparin-binding motifs bound to heparin-agarose beads, whereas first-generation MMP linkers lacking these targeting sequences did not bind to heparin-agarose beads. One such peptide provided exhibited enhanced binding to heparin at pH 6 (tumor pH) versus pH 7.5 (normal tissue pH) (FIG. 4B).

[0526] For fibronectin / collagen binding peptide assays, streptavidin-coupled magnetic beads (Mag Sepharose, Cytiva; and Dynabeads, ThermoFisher, respectively) were first incubated with biotin-labeled fibronectin (Cytoskeleton) or biotin-labeled collagen IV (Prospec) for 1 h with gentle shaking. After multiple washes, ECM-coated beads were then incubated with Edans peptide (20 μM) in neutral or acidic binding buffer for 2 h at room temperature with shaking. Beads were then washed and resuspended in 100 μL of binding buffer in a black 96-well plate. Peptide binding was quantified by measuring the fluorescence of the samples using the excitation / emission spectrum of EDANS (Ex340 / Em490). Figure 4C shows that peptide 13 was able to bind to fibronectin, resulting in enhanced binding at pH 6 (tumor pH) versus pH 7.5 (normal tissue pH), and Figure 4D shows that peptide 14 bound strongly to type IV collagen, whereas binding by peptide 15 was low. [Example 8]

[0527] Binding assays for next generation IL-2 / IL-15 fusion proteins A series of IL-2 / IL-15 fusion proteins containing a single targeting sequence, or multiple targeting sequences, in the linker region or elsewhere, were designed and successfully manufactured (Table 4, and Figures 1A-1D). These proteins were then examined for their ability to bind to ECM proteins such as heparin, fibronectin, and collagen, which are found in abundance in the tumor stroma.

[0528] 96-well plates were coated with 10 μg / mL heparin-BSA conjugate (kindly provided by Dr. Mueller, Boerhinger Ingelheim) or control BSA for 18-22 hours at room temperature on a shaker (350 rpm). After washing, wells were blocked for 90 minutes with 2% milk powder in PBS-0.05% Tween 20 or PBS-0.05% Tween 20 / 1% BSA. Fusion proteins were then titrated in 2% milk powder in PBS-0.05% Tween 20 or 1% BSA / PBS-0.05% Tween 20, pH 7.5 and / or pH 6, and added for 2 hours at room temperature with shaking. After washing, anti-mouse IL-2 biotin-labeled detection antibody (JES6-5H4; ThermoFisher), anti-6xHis tag HRP conjugate antibody (Invitrogen; 1 mg / mL; MA1-21315-HRP), or anti-human IgG HRP conjugate antibody (SouthernBiotech) were added, and binding was detected using Ultra Streptavidin HRP (ThermoFisher). Plates were developed by adding chromogenic tetramethylbenzidine substrate (Ultra TMB; ThermoFisher). The reaction was stopped by adding 0.5 M H2SO4, and absorbance was read at 450-650 nm. The IL-2 fusion proteins, Construct Y and Construct CC, bound heparin at acidic pH in a dose-dependent manner and with higher affinity than Construct B (Figure 4E). Remarkably, construct CC preferentially binds heparin at acidic pH and inhibits EC 50 Construct B showed the most robust binding, with an EC 50values ​​were over 100-fold higher. Furthermore, when the same pH-dependent heparin-binding motif was inserted into different positions in the IL-2 fusion protein, all the resulting proteins bound heparin with similar high affinity at pH 6 (Figures 4F and 4G). Similarly, similar binding affinities were observed when alternative heparin targeting sequences were engineered into different sites of the IL-2 fusion protein (Figures 4H-4I). Figure 4J shows that the IL-15Rα-IL-15 fusion protein, in the context of the linker polypeptide-IL-15 fusion protein (construct VVV), exhibits low intrinsic binding (EC) to heparin that is lost when the cytokine is bound by a blocking agent. 50 4M) and interacts with heparin. Binding activity to heparin is restored when a heparin-binding motif is engineered into the linker polypeptide-IL-15 fusion protein (construct WWW). Finally, the linker polypeptide-IL-2 fusion protein engineered with a heparin-binding site exhibits approximately 30-fold enhancement of binding to heparin in vitro compared to constructs lacking the heparin-binding site (constructs EEE and NNNN, respectively), as shown in FIG. 4M.

[0529] A similar plate-based assay was developed to probe the binding of IL-2 fusion variants to fibronectin. 96-well plates were coated with fibronectin (4-10 μg / mL; Sigma) or control BSA for 18-22 h at room temperature on a shaker (350 rpm). After washing, wells were blocked for 90 min with 2% milk powder in PBS-0.05% Tween 20 or protein-free blocking buffer (Pierce), and then fusion proteins were titrated in blocking buffer (0.1% Tween 20, pH 7.5 and / or pH 6) and added for 1 h at room temperature with shaking. After washing, anti-mouse IL-2 biotin-labeled detection antibody (JES6-5H4; ThermoFisher) or anti-human IgG HRP-conjugated antibody (SouthernBiotech) was added, and binding was detected using Ultra Streptavidin HRP (ThermoFisher). Plates were developed by adding chromogenic tetramethylbenzidine substrate (Ultra TMB; ThermoFisher). The reaction was stopped by adding 0.5 M H2SO4, and absorbance was read at 450–650 nm. Construct EE bound preferentially to fibronectin at acidic pH, exhibiting dose-dependent binding, whereas no binding was observed at pH 7.5 (Figure 4K). No significant binding of construct B was observed under neutral or acidic conditions.

[0530] To examine binding to collagen, pull-down assays were performed using agarose cross-linked to collagen (Sigma). IL-2 fusion proteins were incubated with collagen-agarose beads or control agarose beads for 18-22 h at 4°C in 1% BSA / PBS-0.05% Tween 20 with gentle rotation. After washing, beads were resuspended in SDS sample buffer (Life Technologies) to elute the proteins bound to the beads. Bound proteins were then separated by SDS-PAGE on 4-12% Bis / Tris gradient gels followed by immunoblotting with goat anti-mouse IL-2 polyclonal antibody (AF-402-NA; R&D systems). For detection, donkey anti-goat HRP-conjugated antibodies (Jackson Immuno Research, West Grove, PA) were used, and blots were developed using SuperSignal West Femto Maximum sensitivity detection reagent (ThermoFisher) according to the manufacturer's recommendations. Blot images are shown in Figure 4L. Constructs GG and II bound specifically to collagen-agarose beads, whereas no IL-2 fusion protein was found to bind to control agarose beads. Quantification of the blots using an iBright imaging system (Invitrogen) showed that the percentages of bound Constructs GG and II were 2.5 and 1.4-fold higher than the percentage of bound Construct B, despite being low (<1% of input). [Example 9]

[0531] IL-2 fusion proteins with next-generation retention linkers showed increased retention in tumors in vivo The levels of IL-2 fusion proteins present in tumors in vivo were assessed by utilizing fluorescently labeled proteins and real-time whole-body imaging. The non-cleavable constructs GGG and DD were conjugated to Dylight 650 probes (Dylight 650 Antibody Labeling Kit; ThermoFisher) according to the manufacturer's protocol. Conjugation did not significantly alter the binding of the proteins to heparin. BALB / c mice were inoculated subcutaneously with a syngeneic model of EMT6 breast cancer, and the mean tumor volume was 240 mm 3 Once tumor volume reached 100 mg / kg, animals were randomized into 3 groups (n=2 mice per treatment group) based on tumor volume. Table 6 below shows the study design.

[0532] [Table 6]

[0533] After administration of a single dose of labeled IL-2 fusion protein to tumor-bearing mice, fluorescence images (excitation: 640 / emission: 680, matching the ex / em spectrum of the Dylight 650 probe) were captured on an IVIS system (PerkinElmer, IVIS Lumina Series III) for 96 hours and are shown in FIG. 5A. Fluorescence intensity within the tumor area was quantified between groups, and at each time point, the average background tumor fluorescence (group 1) was subtracted from the values ​​of groups 2 and 3, and the data was normalized to the initial fluorescence intensity of the same amount of each labeled protein. FIG. 5B shows that the tumor-associated fluorescence from group 3 was nearly twice that of group 2 at each of the time points tested. This means that construct DD with the next-generation retention linker accumulated and was retained at twice the level in the tumor compared to construct GGG, an IL-2 fusion protein lacking a targeting sequence. [Example 10]

[0534] Multiple targeting sequences within the IL-2 fusion protein linker resulted in the greatest antitumor efficacy in vivo C57BL / 6 mice were inoculated subcutaneously with B16F10 melanoma cells, and the mean tumor volume was 70–90 mm 3 Once tumor volume reached 100%, animals were randomized into 6 groups (n=8 mice per treatment group) based on tumor volume. Mice were dosed intravenously once every 3 days (Q3D) for a total of 5 doses according to Table 7.

[0535] [Table 7]

[0536] Tumor volumes were measured twice weekly over the course of the study. Mean tumor volumes are shown in FIG. 6. Antitumor activity was observed in all treatment groups, but the most robust tumor growth inhibition (TGI) was observed with the multi-targeting linker construct, Construct III (83.5%), compared with TGIs of 52%-66% for the single targeting linker fusion proteins. On day 14, animals were sacrificed and tissues and blood (processed into serum) were collected 24 hours after the final dose (dose 5) and stored at −80° C. until further testing. [Example 11]

[0537] Multiple targeting sequences within the IL-2 fusion protein linker resulted in increased intratumoral levels of drug, IL-2, and IFN-γ, as well as enhanced circulating drug levels compared to single targeting linker constructs. Levels of full-length IL-2-IL-2Ra fusion protein, IL-2, and IFN-γ were quantified in tumor samples collected during a preclinical efficacy study comparing a panel of retained linker / IL-2 fusion drugs (see Example 10).

[0538] Tumors (n=3 per group) were harvested 24 hours after the last injection, flash frozen, and stored at -80°C until further processing. Tumor lysates were generated using tissue extraction reagent (ThermoFisher) supplemented with protease and phosphatase inhibitors. Standard techniques and protein concentrations were determined using the BCA assay (Pierce).

[0539] Lysates were examined by an in-house developed ELISA (see Example 5) measuring full-length IL-2 fusion protein (IL-2 capture / IL-2Ra detection). Results were normalized to 1 mg of tumor lysate, and the average values ​​are shown in FIG. 7A. The highest drug levels were detected with the multi-targeting linker drug Construct III (approximately 2-5-fold higher levels compared to other retained linker drugs tested). Similarly, intratumoral levels of IL-2 measured by the appropriate Luminex kit [IL-2 Mouse ProcartaPlex™ Simplex Kit; model number: EPX01A-20601-901; ThermoFisher] were also highest in the Construct III treatment group compared to other arms (FIG. 7B). This confirms that the multi-site targeting linker technology improved the retention of both full-length drug and active IL-2 released after cleavage in the TME. Furthermore, levels of IFN-γ, a major Th1-type cytokine, were also enhanced in Construct III animals [Figure 7C; Essential Th1 / Th2 Cytokine 6-Plex Mouse ProcartaPlex™ Panel; Cat. No.: EPX060-20831-901; ThermoFisher].

[0540] Equivalent serum samples (n=3 per group) were also examined by in-house ELISA to quantify full-length IL-2 fusion drugs, and the results are shown in Figure 7D. 24 hours after administration, circulating drug levels of Construct III are approximately 1.5-4 times higher than the serum levels of other targeting drugs. This confirms that engineering multiple targeting sequences into IL-2 fusion drugs increased drug levels both in the tumor and in the circulation. Furthermore, multiple targeting sequences (e.g., a targeting sequence targeting heparin and a targeting sequence targeting type IV collagen) may result in increased serum half-life of the linker polypeptide. [Example 12]

[0541] Multiple targeting sequences within the IL-2 fusion protein linker were not associated with systemic toxicity Inflammatory cytokine levels were measured in serum using a multiplex Luminex assay (Essential Th1 / Th2 Cytokine 6-Plex Mouse ProcartaPlex™ Panel; model number: EPX060-20831-901; ThermoFisher). Low levels of TNF-α and IL-6 were detected (Figures 8A-8B; mean values ​​per group were set at 10 pg / mL and 27 pg / mL, respectively), whereas IL-12 was undetectable in all groups. In addition, no elevation of aspartate transaminase levels was observed in the treatment arms compared to control animals, indicating the absence of liver damage (Figure 8C; AST activity assay; Sigma). [Example 13]

[0542] A linker polypeptide with an immunoglobulin antigen-binding domain as an active domain. 9A-9D each illustrate a linker polypeptide according to certain embodiments of the present disclosure. The linker polypeptide of FIG. 9A comprises a first active domain (AD1); a second active domain (AD2); a pharmacokinetic modulator (PM); and a first linker between the pharmacokinetic modulator and the first active domain, the first linker comprising a protease-cleavable polypeptide sequence (CL). In some embodiments, the first linker further comprises a targeting sequence. In certain embodiments, the active domain comprises immunoglobulin antigen-binding domains (IBD1 and IBD2) that can be directed to different targets. In certain embodiments, the target-binding domain can comprise a heavy chain and a light chain (FIG. 9A), or only a heavy chain, such as a VHH (FIG. 9B). Compared to the linker polypeptide of FIG. 9A, the linker polypeptide of FIG. 9D further comprises a chemotherapeutic drug (D).

[0543] 11A-11B each illustrate the release of a first active domain from the remainder of the linker polypeptide after one or more protease-cleavable polypeptide sequences are cleaved. In these figures, the active domains may include immunoglobulin antigen-binding domains (IBD1 and IBD2). Compared to the linker polypeptide of FIG. 11A, the linker polypeptide of FIG. 11B further includes a blocking agent (B) conjugated to each of the first and second active domains via a protease-cleavable polypeptide sequence (CL). In some embodiments, the protease-cleavable polypeptide sequence connecting the first active domain to the remainder of the linker polypeptide and the protease-cleavable polypeptide sequence connecting the blocking agent to the active domain may be cleaved together (e.g., by the same protease). In some embodiments, the protease-cleavable polypeptide sequence connecting the first active domain to the remainder of the linker polypeptide and the protease-cleavable polypeptide sequence connecting the blocking agent to the active domain may be cleaved separately (e.g., by different proteases). [Example 14]

[0544] A linker polypeptide with an immunoglobulin antigen-binding domain as one active domain and a non-immunoglobulin polypeptide as the other active domain. 10A-10B each illustrate a linker polypeptide according to certain embodiments of the present disclosure. The linker polypeptide of FIG. 10A comprises a first active domain (AD1); a second active domain (AD2); a pharmacokinetic modulator (PM); and a first linker between the pharmacokinetic modulator and the first active domain, the first linker comprising a protease-cleavable polypeptide sequence (CL). In some embodiments, the first linker further comprises a targeting sequence. In certain embodiments, the first active domain comprises a receptor binding domain (RBD) and the second active domain comprises an immunoglobulin antigen binding domain (IBD). In some embodiments, the RBD comprises a cytokine polypeptide sequence (CY). Compared to the linker polypeptide of FIG. 10A, the linker polypeptide of FIG. 10B further comprises an inhibitory polypeptide sequence (IN) capable of blocking the activity of the first active domain; and a second linker between the receptor binding domain and the inhibitory polypeptide sequence, the second linker comprising a protease-cleavable polypeptide sequence (CL).

[0545] 12A-12B each illustrate the release of a first active domain from the remainder of the linker polypeptide after one or more protease-cleavable polypeptide sequences are cleaved. In these figures, the first active domain comprises a receptor binding domain (RBD), which may comprise a cytokine polypeptide sequence (CY), and the second active domain comprises an immunoglobulin antigen binding domain (IBD). Compared to the linker polypeptide of FIG. 12A, the linker polypeptide of FIG. 12B further comprises an inhibitory polypeptide sequence (IN) capable of blocking the activity of the receptor binding domain; and a second linker between the receptor binding domain and the inhibitory polypeptide sequence, the second linker comprising a protease-cleavable polypeptide sequence (CL). In some embodiments, the protease-cleavable polypeptide sequence of the first linker and the protease-cleavable polypeptide sequence of the second linker may be cleaved together (e.g., by the same protease). In some embodiments, the protease-cleavable polypeptide sequence of the first linker and the protease-cleavable polypeptide sequence of the second linker can be cleaved separately (eg, by different proteases). [Example 15]

[0546] Tumor stroma targeting sequences within the IL-2 fusion protein linker resulted in enhanced antitumor efficacy in vivo C57BL / 6 mice were inoculated subcutaneously with MC38 colorectal cancer cells. The mean tumor volume was 70-90 mm. 3 Once tumor volume reached 0.01, animals were randomized into groups of 10 (n=7 or 6 mice per treatment group) based on tumor volume. Mice were dosed intraperitoneally (IP) twice weekly (BIW) for a total of 5 doses according to the design shown in Table 8 below:

[0547] [Table 8]

[0548] Tumor volumes were measured twice weekly over the course of the study. Mean tumor volumes are shown in Figures 13A-13B, and inhibition of tumor volume is shown in Figure 13C. Antitumor activity was observed in all treatment groups at a dose of 5 mg / kg; however, the most robust tumor growth inhibition (TGI) was observed with the tumor stroma-targeted constructs NNNN, EEE, NNN, and OOOO (TGIs ranging from 74% to 86%). Although lower TGIs were observed in the low-dose treatment groups, the tumor stroma-targeted constructs EEE and NNN continued to show superior efficacy to the parent non-targeted constructs.

[0549] On day 16, animals were sacrificed, and tumors (n=3 per group) were harvested 24 hours after the last injection, flash frozen, and stored at -80°C until further processing. Tumor lysates were generated using tissue extraction reagents (ThermoFisher) supplemented with protease and phosphatase inhibitors and standard techniques, and protein concentrations were determined using a BCA assay (Pierce). Intratumoral levels of IFN-γ (IFNg), a major Th1-type cytokine, were most elevated in the group treated with the targeted construct compared to the parental non-targeted construct, as shown in Figure 13D. IFN-γ was measured using the Essential Th1 / Th2 Cytokine 6-Plex Mouse ProcartaPlex™ Panel (model number: EPX060-20831-901; ThermoFisher). [Example 16]

[0550] IL-2 fusion proteins with TME-binding motifs showed enhanced immune cell infiltration into tumors C57BL / 6 mice were inoculated subcutaneously with B16F10 melanoma cells. The mean tumor volume was 70-90 mm. 3Upon reaching 100% T cell mass, animals were randomized into 5 groups based on tumor volume (n=3 mice per treatment group). Mice were intraperitoneally administered two doses of select ODC-IL2 fusions on days 1 and 4. On day 6, tumors were harvested and processed into single cell suspensions using standard techniques (Miltenyi method, involving a combination of enzymatic and mechanical dissociation). Single cell samples were cryopreserved at -80°C before further processing. Upon thawing, cells were washed and stained for surface and intracellular targets using the antibodies listed in Table 9.

[0551] [Table 9]

[0552] Figures 14A-14E show flow cytometry analysis of select immune cell populations. Remarkably, the group treated with IL-2 fusion proteins engineered with tumor stroma targeting moieties shows enhanced intratumoral T cell infiltration (CD3+ cells) compared to the parental non-targeting fusion protein treated group or the vehicle group. More specifically, this T cell expansion appeared to be primarily driven by an expansion of both total T cells and activated cytotoxic T cells (CD8+ and CD8+CD25+ subsets). [Example 17]

[0553] Examples of IL-2 asymmetric Fc fusion proteins with tumor targeting sequences and single or double masking Additional asymmetric IL-2Fc fusion proteins containing ECM targeting sequences and single or double masks were produced, purified and functionally characterized as previously described. Figure 15A shows examples of such proteins: the rectangle indicates the Fc domain (Fc knob or Fc hole), the solid line indicates the protease-cleavable linker peptide, and the dashed line indicates the flexible linker sequence. The purity of the Fc fusion proteins was assessed by SDS-PAGE under non-reducing conditions (Figure 15B). The proteins were cleaved overnight with recombinant MMP9 protease at 37°C, and the digests were evaluated in the HEK-Blue IL-2 reporter assay as previously described. The results are shown in Figures 15C-15U. Selected IL-2 fusion proteins were assessed for their ability to bind ECM components such as heparin and fibronectin using previously described binding assays, and the results are shown in Figures 15V-15X. Fusion proteins with heparin-binding motifs inserted at different positions in the molecule all showed enhanced binding to heparin compared to the parent molecule without the tumor stroma targeting moiety (Figure 15V-15W). Similarly, only the IL-2 fusion protein engineered with a pH-dependent fibronectin-binding motif was able to bind to fibronectin compared to the parent molecule without the tumor stroma targeting moiety or the fusion protein engineered with a collagen type I binding motif (Figure 15X). Furthermore, binding to fibronectin was slightly enhanced under acidic conditions.

[0554] An image-based retention assay was performed to evaluate the ability of the fusion proteins to bind to collagen. The fusion proteins were labeled with DyLight 650 Maleimide (ThermoFisher; model number: 62295) at the reduced sulfhydryl groups according to the manufacturer's recommended procedure. The fluorescently labeled fusion proteins were then mixed with bovine type I collagen (Advanced Biomatrix; TeloCol-10; model number: 5226) and 10x concentrated PBS buffer, pH 7.4 (Invitrogen; REFAM9624) to neutralize the pH of the sample mix. The final concentrations of each component in the mix are shown in Table 10 below.

[0555] [Table 10]

[0556] 5 μL of the fusion protein-collagen mix was loaded into the inner well of an ibidi u-Slide Angiogenesis (uncoated; model number: 81501) that had been pretreated with gelatin solution (2% in H2O; Sigma; model number: G1393-20ML). The slide was incubated at room temperature for 30 minutes to allow the fusion protein-collagen mix to form a gel. Then, 50 μL of bovine type I collagen (1 mg / mL in 1× PBS) was loaded into the upper well of the slide. After the collagen gelled in the upper well, the slide was imaged using a BioTek Lionheart FX automated microscope. The fluorescence intensity of the inner well represented the amount of fusion protein present and retained in the collagen, and was measured with excitation / emission wavelengths of 628 / 685 nm. The LED intensity, integration time, and camera gain were adjusted to appropriate levels to avoid overexposure and saturation of pixel intensity. Fluorescence intensity was measured over a period of 66 hours, with images collected every 30 minutes at room temperature. Mean fluorescence intensity was calculated by Gen5 software and then normalized to the mean fluorescence intensity of the first image (T=0), which was set to 100%. The normalized mean fluorescence intensity over time showed that the fusion protein containing collagen type I binding site was retained in collagen gels to a greater extent than the non-targeting fusion protein (Figure 15Y).

Claims

**Claim 1** A first polypeptide chain comprising a first active domain, a first domain of a pharmacokinetic modulator, and a first linker between the first active domain and the first domain of the pharmacokinetic modulator, wherein the first active domain is on the C-terminal side or the N-terminal side with respect to the first domain of the pharmacokinetic modulator; A second polypeptide chain comprising a second domain of a pharmacokinetic modulator, an inhibitory polypeptide sequence capable of blocking the activity of the first active domain, and a second linker between the second domain of the pharmacokinetic modulator and the inhibitory polypeptide sequence comprising the first linker comprising a protease-cleavable polypeptide sequence; the first polypeptide chain or the second polypeptide chain further comprising at least one targeting sequence, a linker polypeptide. **Claim 2** the inhibitory polypeptide sequence is on the C-terminal side with respect to the second domain of the pharmacokinetic modulator, or the inhibitory polypeptide sequence is on the N-terminal side with respect to the second domain of the pharmacokinetic modulator; and / or the targeting sequence is between the protease-cleavable polypeptide sequence and the first domain of the pharmacokinetic modulator, or the targeting sequence is between the protease-cleavable polypeptide sequence and the first active domain, or the targeting sequence is on the C-terminal side with respect to the first active domain, or the targeting sequence is on the N-terminal side with respect to the first active domain, or the targeting sequence is on the C-terminal side with respect to the inhibitory polypeptide sequence, or the targeting sequence is on the N-terminal side with respect to the inhibitory polypeptide sequence, or the targeting sequence is between the inhibitory polypeptide sequence and the second domain of the pharmacokinetic modulator, the linker polypeptide according to claim 1. **Claim 3** the targeting sequence binds to heparin and the targeting sequence may comprise SEQ ID NO: 664; or the targeting sequence binds to type IV collagen and the targeting sequence may comprise SEQ ID NO: 200; or the targeting sequence binds to type I collagen and the targeting sequence may comprise SEQ ID NO: 188; or The linker polypeptide according to claim 1, wherein the targeting array binds to fibronectin and the targeting array may comprise SEQ ID NO:

653.

4. The linker polypeptide according to claim 1, wherein the targeting array is a first targeting array, the linker polypeptide further comprises a second targeting array, the first targeting array may be a portion of the first polypeptide chain, the second targeting array may be a portion of the second polypeptide chain, and the first targeting array may be on the C-terminal side with respect to the first active domain, and the second targeting array may be on the C-terminal side with respect to the inhibitory polypeptide sequence.

5. The second targeting array binds to heparin and the targeting array may comprise SEQ ID NO: 664; or The second targeting array binds to type IV collagen and the targeting array may comprise SEQ ID NO: 200; or The second targeting array binds to type I collagen and the targeting array may comprise SEQ ID NO: 188; or The second targeting array binds to fibronectin and the targeting array may comprise SEQ ID NO: 653, The linker polypeptide according to claim 4.

6. A first polypeptide chain comprising a first active domain comprising a receptor-binding domain comprising a human IL-2 polypeptide sequence, a first domain of a pharmacokinetic modulator, the pharmacokinetic modulator comprising at least a portion of an immunoglobulin constant domain, and a first linker between the first active domain and the first domain of the pharmacokinetic modulator, the first active domain being on the C-terminal side with respect to the first domain of the pharmacokinetic modulator; A second polypeptide chain comprising a second domain of a pharmacokinetic modulator, an inhibitory polypeptide sequence comprising an immunoglobulin cytokine-binding domain and capable of blocking the activity of the first active domain, and a second linker between the second domain of the pharmacokinetic modulator and the inhibitory polypeptide sequence comprising a linker polypeptide; The first linker comprises a protease-cleavable polypeptide sequence comprising the amino acid sequence of SEQ ID NO: 80 or a variant sequence having one or two mismatches thereto; and The first polypeptide chain further comprises at least one targeting sequence that is at the C-terminus of the first active domain and comprises the amino acid sequence of SEQ ID NO: 664 or a variant sequence having one or two mismatches thereto. A linker polypeptide. **Claim 7** The linker polypeptide according to claim 1, further comprising a second active domain, wherein the second active domain may be a portion of the second polypeptide chain. **Claim 8** The inhibitory polypeptide sequence is a first inhibitory polypeptide sequence, the linker polypeptide further comprises a second inhibitory polypeptide sequence, the second inhibitory polypeptide sequence may be a portion of the second polypeptide chain, and the second inhibitory polypeptide sequence may be on the C-terminal side with respect to the first inhibitory polypeptide sequence; and The second inhibitory polypeptide sequence may be an immunoglobulin inhibitory polypeptide sequence, the first inhibitory polypeptide sequence may be an immunoglobulin inhibitory polypeptide sequence, and one or each of the immunoglobulin inhibitory polypeptide sequences may be a VHH. The linker polypeptide according to claim 1. **Claim 9** A first targeting sequence; A second targeting sequence; and A first linker between the first targeting sequence and the second targeting sequence, the first linker comprising a protease-cleavable polypeptide sequence A linker polypeptide comprising. **Claim 10** Further comprising a first active domain, wherein the first active domain may be proximal to the first targeting sequence as compared to the second targeting sequence. The linker polypeptide may further comprise an additional domain, the additional domain may comprise an inhibitory polypeptide sequence capable of blocking the activity of the first active domain, a pharmacokinetic modulator, and / or a second active domain, and the additional domain may be proximal to the second targeting sequence as compared to the first targeting sequence, and The linker polypeptide may sequentially comprise, from the N-terminus to the C-terminus or from the C-terminus to the N-terminus, the first active domain, the first targeting sequence, the first linker, the second targeting sequence, and the additional domain. The linker polypeptide according to claim 9. **Claim 11** A first active domain; a second active domain; a pharmacokinetic modulator; and a first linker between the pharmacokinetic modulator and the first active domain, the first linker comprising a protease-cleavable polypeptide sequence; and optionally, a first targeting sequence comprising a linker polypeptide. **Claim 12** A first active domain; an inhibitory polypeptide sequence capable of blocking the activity of the first active domain; a first linker between the first active domain and the inhibitory polypeptide sequence, the first linker comprising a protease-cleavable polypeptide sequence; and a first targeting sequence; and optionally, a pharmacokinetic modulator comprising a linker polypeptide. **Claim 13** The pharmacokinetic modulator comprises a heterodimeric Fc domain or a heterodimeric CH3 domain, the heterodimeric Fc domain or heterodimeric CH3 domain may comprise a knob CH3 domain and a hole CH3 domain, and the first domain of the pharmacokinetic modulator may be the knob CH3 domain, the second domain of the pharmacokinetic modulator may be the hole CH3 domain, or the first domain of the pharmacokinetic modulator may be the hole CH3 domain, the second domain of the pharmacokinetic modulator may be the knob CH3 domain; and the pharmacokinetic modulator may comprise the sequence of SEQ ID NO: 76, 75, 77, 78, 756, or 757, The linker polypeptide according to claim 1. **Claim 14** The first active domain comprises a first immunoglobulin antigen-binding domain and / or the second active domain comprises a second immunoglobulin antigen-binding domain; and / or One or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain independently comprises a VH region and a VL region; and / or One or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain independently comprises an Fv, scFv, Fab, or VHH; and / or One or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain independently is a humanized domain or a fully human domain; and / or One or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain is independently configured to bind to one or more sequences selected from a cancer cell surface antigen sequence, a growth factor sequence, and a growth factor receptor sequence, wherein one or each of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain may independently be configured to bind to an HER2 sequence, an EGFR extracellular domain sequence, a PD-1 extracellular domain sequence, a PD-L1 extracellular domain sequence, or a CD3 extracellular domain sequence; and / or One of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain is configured to bind to an HER2 sequence, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain may include a VH region having hypervariable regions (HVRs) HVR-1, HVR-2, and HVR-3 of the amino acid sequence of SEQ ID NO: 910 and a VL region having HVR-1, HVR-2, and HVR-3 of the VL region of the amino acid sequence of SEQ ID NO: 909, one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain may include a sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 909 or 910, one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain may include a VH region having the amino acid sequence of SEQ ID NO: 910 and a VL region having the amino acid sequence of SEQ ID NO: 909, and one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain may be the antigen-binding domain of trastuzumab; and / or One of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain is configured to bind to the EGFR extracellular domain sequence, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain is a VH region comprising HVR-1, HVR-2, and HVR-3 of the VH region containing the amino acid sequence of SEQ ID NO: 914, and a VL region comprising HVR-1, HVR-2, and HVR-3 of the VL region containing the amino acid sequence of SEQ ID NO: 913, and one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain may comprise a sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity with the sequence of SEQ ID NO: 913 or 914, and one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain may comprise a VH region containing the amino acid sequence of SEQ ID NO: 914 and a VL region containing the amino acid sequence of SEQ ID NO: 913, and one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain may be the antigen-binding domain of cetuximab; and / or One of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain is configured to bind to the PD-1 extracellular domain sequence, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain may include a VH region comprising HVR-1, HVR-2, and HVR-3 of the VH region having the amino acid sequence of SEQ ID NO: 917 and a VL region comprising HVR-1, HVR-2, and HVR-3 of the VL region having the amino acid sequence of SEQ ID NO: 918, one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain may include a sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity with the sequence of SEQ ID NO: 917 or 918, one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain may include a VH region comprising the amino acid sequence of SEQ ID NO: 917 and a VL region comprising the amino acid sequence of SEQ ID NO: 918, and one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain may be the antigen-binding domain of nivolumab; and / or One of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain is configured to bind to the PD-L1 extracellular domain sequence, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain may include an HVH-1, HVH-2, and HVH-3 of a VH region including the amino acid sequence of SEQ ID NO: 921, and an HVL-1, HVL-2, and HVL-3 of a VL region including the amino acid sequence of SEQ ID NO: 922, one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain may include a sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity with the sequence of SEQ ID NO: 921 or 922, one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain may include a VH region including the amino acid sequence of SEQ ID NO: 921 and a VL region including the amino acid sequence of SEQ ID NO: 922, and one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain may be the antigen-binding domain of atezolizumab, and / or One of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain is configured to bind to the CD3 extracellular domain sequence, wherein one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain is a VH region comprising HVR-1, HVR-2, and HVR-3 of any one of the amino acid sequences of SEQ ID NOs: 925, 929, 933, and 937, and a VL region comprising HVR-1, HVR-2, and HVR-3 of any one of the amino acid sequences of SEQ ID NOs: 926, 930, 934, and 938, and one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain may comprise a sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity with any one of the sequences of SEQ ID NOs: 925, 926, 929, 930, 933, 934, 937, and 938, and one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain may comprise a VH region comprising any one of the amino acid sequences of SEQ ID NOs: 925, 929, 933, and 937 and a VL region comprising any one of the amino acid sequences of SEQ ID NOs: 926, 930, 934, and 938, and one of the first immunoglobulin antigen-binding domain and the second immunoglobulin antigen-binding domain may be the antigen-binding domain of teprotumumab, muromonab, oterixizumab, or bisilizumab, The linker polypeptide according to claim 7.

15. The first active domain comprises a receptor-binding domain, The receptor-binding domain may comprise a cytokine polypeptide sequence; and / or The receptor-binding domain comprises a modification that prevents the formation of disulfide bonds and may comprise a wild-type sequence elsewhere; and / or The receptor-binding domain has at least 80, 85, 90, 95, 97, 98, or 99 percent identity with any one of the amino acid sequences of SEQ ID NOs: 2, 1, 3, 4, 900, and 902-938, and the receptor-binding domain may be a wild-type receptor-binding domain; and / or The receptor-binding domain is a monomeric cytokine, or the receptor-binding domain is a dimeric receptor-binding domain that comprises monomers that are covalently associated (which may be via a polypeptide linker) or non-covalently associated; and / or The linker polypeptide further comprises an inhibitory polypeptide sequence capable of blocking the activity of the receptor-binding domain and a second linker between the receptor-binding domain and the inhibitory polypeptide sequence, the second linker comprising a protease-cleavable polypeptide sequence; and / or The inhibitory polypeptide sequence comprises a cytokine-binding domain, the cytokine-binding domain may be the cytokine-binding domain of a cytokine receptor or the cytokine-binding domain of fibronectin, the cytokine-binding domain may be an immunoglobulin cytokine-binding domain, the immunoglobulin cytokine-binding domain may comprise a VL region and a VH region that bind to the cytokine, and the immunoglobulin cytokine-binding domain may be a VHH, Fv, scFv, or Fab; The linker polypeptide according to claim 1.

16. The inhibitory polypeptide sequence comprises a cytokine-binding domain, the cytokine-binding domain may be the cytokine-binding domain of a cytokine receptor or the cytokine-binding domain of fibronectin, the cytokine-binding domain may be an immunoglobulin cytokine-binding domain, the immunoglobulin cytokine-binding domain may comprise a VL region and a VH region that bind to the cytokine, and the immunoglobulin cytokine-binding domain may be a VHH, Fv, scFv, or Fab, the linker polypeptide according to claim 15.

17. Comprising a targeting sequence, the targeting sequence being between the receptor-binding domain and one of a protease-cleavable polypeptide sequence or a plurality of protease-cleavable polypeptide sequences; and / or The receptor-binding domain is an interleukin polypeptide sequence; and / or The receptor-binding domain is capable of binding to a receptor comprising CD132; and / or The receptor-binding domain is capable of binding to a receptor comprising CD122; and / or The receptor-binding domain is capable of binding to a receptor comprising CD25; and / or The receptor-binding domain is capable of binding to a receptor comprising IL-10R; and / or The receptor-binding domain is capable of binding to a receptor comprising CXCR3. The linker polypeptide according to claim 15.

18. The receptor-binding domain is an IL-2 polypeptide sequence, the IL-2 polypeptide sequence may have at least 80, 85, 90, 95, 97, 98, or 99 percent identity to any one of the sequences of SEQ ID NO: 2, 1, 3, and 4, the IL-2 polypeptide sequence may comprise any one of the sequences of SEQ ID NO: 2, 1, 3, and 4, the IL-2 polypeptide sequence may be a human IL-2 polypeptide sequence, and the IL-2 polypeptide sequence may comprise the sequence of SEQ ID NO: 2 or 1; and / or The inhibitory polypeptide sequence comprises the IL-2 binding domain of the IL-2 receptor (IL-2R), the inhibitory polypeptide sequence may comprise an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to any one of the sequences of SEQ ID NO: 10-29 and 40-51, and the IL-2R may be a human IL-2R; and / or The inhibitory polypeptide sequence includes an IL-2 binding immunoglobulin domain, which may be a human IL-2 binding immunoglobulin domain. The IL-2 binding immunoglobulin domain may include a VH region containing hypervariable regions (HVRs) HVR-1, HVR-2, and HVR-3 having the sequences of SEQ ID NOs: 37, 38, and 39, respectively, and a VL region containing HVR-1, HVR-2, and HVR-3 having the sequences of SEQ ID NOs: 34, 35, and 36, respectively. And / or the IL-2 binding immunoglobulin domain may include a VH region containing an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99% identity to the sequence of SEQ ID NO: 33, and a VL region containing an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99% identity to the sequence of SEQ ID NO: 32, or a VH region containing an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99% identity to the sequence of SEQ ID NO: 749, and a VL region containing an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99% identity to the sequence of SEQ ID NO:

748. Or the IL-2 binding immunoglobulin domain may include a VH region containing the sequence of SEQ ID NO: 33, and a VL region containing the sequence of SEQ ID NO: 32, or a VH region containing the sequence of SEQ ID NO: 749, and a VL region containing the sequence of SEQ ID NO:

748. And the IL-2 binding immunoglobulin domain may be a scFv; and / or The inhibitory polypeptide sequence comprises an IL-2 binding immunoglobulin domain, and the IL-2 binding immunoglobulin domain may be a human IL-2 binding immunoglobulin domain, the IL-2 binding immunoglobulin domain may be a scFv, the IL-2 binding immunoglobulin domain may comprise the CDRs of the amino acid sequences of SEQ ID NOs: 30, 31, 747, 850-856, or 863-870, the IL-2 binding immunoglobulin domain may comprise an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequences of SEQ ID NOs: 30, 31, 747, 850-856, or 863-870, and the IL-2 binding immunoglobulin domain may comprise the sequences of SEQ ID NOs: 30, 31, 747, 850-856, or 863-870. The linker polypeptide according to claim 15.

19. The receptor binding domain is an IL-10 polypeptide sequence, the IL-10 polypeptide sequence may have at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 900, and the IL-10 polypeptide sequence may comprise the sequence of SEQ ID NO: 900; and / or The IL-10 polypeptide sequence is a human IL-10 polypeptide sequence; and / or The inhibitory polypeptide sequence comprises the IL-10 binding domain of the IL-10 receptor (IL-10R), the inhibitory polypeptide sequence may comprise an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequences of SEQ ID NO: 1011 or 1012, and / or the IL-10R may be a human IL-10R; and / or The inhibitory polypeptide sequence includes an IL-10 binding immunoglobulin domain, and the IL-10 binding immunoglobulin domain may be a human IL-10 binding immunoglobulin domain. The IL-10 binding immunoglobulin domain may include a VH region containing hypervariable regions (HVRs) HVR-1, HVR-2, and HVR-3 having the sequences of SEQ ID NOs: 946, 947, and 948, respectively, and a VL region containing HVR-1, HVR-2, and HVR-3 having the sequences of SEQ ID NOs: 942, 943, and 944, respectively; the IL-10 binding immunoglobulin domain may include a VH region containing an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99% identity to the sequence of SEQ ID NO: 945, and a VL region containing an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99% identity to the sequence of SEQ ID NO:

941. The IL-10 binding immunoglobulin domain may include a VH region containing the sequence of SEQ ID NO: 945 and a VL region containing the sequence of SEQ ID NO:

941. The IL-10 binding immunoglobulin domain may be a scFv. The IL-10 binding immunoglobulin domain may include an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99% identity to the sequence of SEQ ID NO: 939 or 940, and the IL-10 binding immunoglobulin domain may include the sequence of SEQ ID NO: 939 or 940. The linker polypeptide according to claim 15.

20. The receptor binding domain is a CXCL9 polypeptide sequence, and the CXCL9 polypeptide sequence may be a human CXCL9 polypeptide sequence. The CXCL9 polypeptide sequence may have at least 80, 85, 90, 95, 97, 98, or 99% identity to the sequence of SEQ ID NO: 902, and the CXCL9 polypeptide sequence may include the sequence of SEQ ID NO: 902; and / or The inhibitory polypeptide sequence comprises the CXCL9-binding domain of CXCR3, CXCR3 may be human CXCR3, and the inhibitory polypeptide sequence may comprise an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 1020 or 1021; and / or The inhibitory polypeptide sequence comprises a CXCL9-binding immunoglobulin domain, and the CXCL9-binding immunoglobulin domain may be a human CXCL9-binding immunoglobulin domain. The linker polypeptide according to claim 15.

21. The receptor-binding domain is a CXCL10 polypeptide sequence, the CXCL10 polypeptide sequence may be a human CXCL10 polypeptide sequence, the CXCL10 polypeptide sequence may have at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 903, and the CXCL10 polypeptide sequence may comprise the sequence of SEQ ID NO: 903; and / or The inhibitory polypeptide sequence comprises the CXCL10-binding domain of CXCR3, CXCR3 may be human CXCR3, and the inhibitory polypeptide sequence may comprise an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 1020 or 1021; and / or The inhibitory polypeptide sequence comprises a CXCL10-binding immunoglobulin domain, which may be a human CXCL10-binding immunoglobulin domain. The CXCL10-binding immunoglobulin domain may comprise a VH region containing hypervariable regions (HVR) HVR-1, HVR-2, and HVR-3 having the sequences of SEQ ID NOs: 993, 994, and 995, respectively, and a VL region containing HVR-1, HVR-2, and HVR-3 having the sequences of SEQ ID NOs: 996, 997, and 998, respectively. The CXCL10-binding immunoglobulin domain may comprise a VH region containing an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 991, and a VL region containing an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO:

992. The CXCL10-binding immunoglobulin domain may comprise a VH region containing the sequence of SEQ ID NO: 991 and a VL region containing the sequence of SEQ ID NO:

992. The CXCL10-binding immunoglobulin domain may be a scFv. The CXCL10-binding immunoglobulin domain may contain an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 989 or 990, and the CXCL10-binding immunoglobulin domain may contain the sequence of SEQ ID NO: 989 or 990. The linker polypeptide according to claim 15.

22. The inhibitory polypeptide sequence interferes with the binding of the first active domain to the receptor of the first active domain; and / or The inhibitory polypeptide sequence and the pharmacokinetic modulator are different elements of the linker polypeptide; and / or The inhibitory polypeptide sequence comprises a steric blocker; and / or The inhibitory polypeptide sequence comprises at least a part of the pharmacokinetic modulator. The linker polypeptide according to claim 1.

23. The pharmacokinetic modulator comprises at least a part of the immunoglobulin constant domain, the pharmacokinetic modulator may comprise at least a part of the immunoglobulin Fc region, and the pharmacokinetic modulator may comprise the immunoglobulin Fc region; and / or the immunoglobulin is a human immunoglobulin; and / or the immunoglobulin is IgG, and IgG may be IgG1, IgG2, IgG3, or IgG4, The linker polypeptide according to claim 1.

24. further comprising a growth factor-binding polypeptide sequence or a growth factor receptor-binding polypeptide sequence; the growth factor-binding polypeptide sequence may comprise a TGF-βR extracellular domain sequence, and the TGF-βR extracellular domain sequence may comprise an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 1022 or 1023; and / or the growth factor-binding polypeptide sequence comprises a growth factor-binding immunoglobulin domain, the growth factor-binding immunoglobulin domain may be configured to bind TGF-β, the growth factor-binding immunoglobulin domain may comprise HV R-1, HV R-2, and HV R-3 of the VH region comprising the amino acid sequence of SEQ ID NO: 1008, and HV R-1, HV R-2, and HV R-3 of the VL region comprising the amino acid sequence of SEQ ID NO: 1010, the growth factor-binding immunoglobulin domain may comprise the VH region comprising the amino acid sequence of SEQ ID NO: 1008 and the VL region comprising the amino acid sequence of SEQ ID NO: 1010, and the growth factor-binding immunoglobulin domain may comprise a sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to the sequence of SEQ ID NO: 1007 or 1009; and / or the growth factor receptor-binding polypeptide sequence comprises a TGF-β sequence, and the TGF-β sequence may comprise an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to any one of the sequences of SEQ ID NO: 904-906; and / or The growth factor receptor-binding polypeptide sequence may include a growth factor receptor-binding immunoglobulin domain, and the growth factor receptor-binding immunoglobulin domain may be configured to bind to the TGF-βR extracellular domain sequence. The growth factor receptor-binding immunoglobulin domain may include an HV region including HV-1, HV-2, and HV-3 of the VH region containing the amino acid sequence of SEQ ID NO: 999 or 1003, and an HV region including HV-1, HV-2, and HV-3 of the VL region containing the amino acid sequence of SEQ ID NO: 1000 or 1004. The growth factor receptor-binding immunoglobulin domain may include a VH region containing the amino acid sequence of SEQ ID NO: 999 or 1003 and a VL region containing the amino acid sequence of SEQ ID NO: 1000 or 1004. And the growth factor receptor-binding immunoglobulin domain may include a sequence having at least 80, 85, 90, 95, 97, 98, or 99 percent identity to any one of the sequences of SEQ ID NO: 1001, 1002, 1005, and 1006. The linker polypeptide according to claim 1. **Claim 25** Comprising a plurality of protease cleavage-type polypeptide sequences; and / or The protease cleavage-type polypeptide sequence is on the C-terminal side with respect to the VH region, on the C-terminal side with respect to at least a part of the CH1 domain, between the CH1 domain and the CH2 domain, on the N-terminal side with respect to at least a part of the CH2 domain, on the N-terminal side with respect to the inter-heavy chain disulfide bond, on the N-terminal side with respect to the disulfide bond within the CH2 domain, or on the N-terminal side with respect to the hinge region, or within the hinge region; and / or The protease cleavage-type polypeptide sequence is on the C-terminal side with respect to the first targeting sequence and the second targeting sequence; and / or The protease cleavage-type polypeptide sequence is on the N-terminal side with respect to the first targeting sequence and the second targeting sequence; and / or The protease cleavage-type polypeptide sequence is on the C-terminal side with respect to the first plurality of targeting sequences and on the N-terminal side with respect to the second plurality of targeting sequences; and / or The protease cleavage-type polypeptide sequence is on the C-terminal side with respect to the plurality of targeting sequences and on the N-terminal side with respect to at least one targeting sequence; and / or The protease-cleavable polypeptide sequence is on the N-terminal side with respect to a plurality of targeting sequences and on the C-terminal side with respect to at least one targeting sequence; and / or The protease-cleavable polypeptide sequence is on the C-terminal side with respect to the first targeting sequence and the second targeting sequence and not on the N-terminal side with respect to the targeting sequence; and / or The protease-cleavable polypeptide sequence is on the N-terminal side with respect to the first targeting sequence and the second targeting sequence and not on the C-terminal side with respect to the targeting sequence, The linker polypeptide according to claim 1. Claim 26 Upon cleavage of the protease-cleavable polypeptide sequence, it is configured to release the first active domain from the remaining portion of the linker polypeptide, and the first active domain may be configured to maintain connection to the first targeting sequence or the pharmacokinetic modulator upon cleavage of the protease-cleavable polypeptide sequence. The linker polypeptide according to claim 1. Claim 27 The protease-cleavable polypeptide sequence is recognized by a metalloprotease, a serine protease, a cysteine protease, an aspartic protease, a threonine protease, a glutamic protease, a gelatinase, an asparaginyl peptidase, a cathepsin, a kallikrein, a plasmin, a collagenase, hK1, hK10, hK15, a stromelysin, factor Xa, a chymotrypsin-like protease, a trypsin-like protease, an elastase-like protease, a subtilisin-like protease, an actinidin, a bromelain, a calpain, a caspase, Mir1-CP, a papain, an HIV-1 protease, an HSV protease, a CMV protease, a chymosin, a renin, a pepsin, a matriptase, a legumain, a plasminopepsin, a nepenthesin, a metalloexopeptidase, a metalloendopeptidase, ADAM10, ADAM17, ADAM12, urokinase plasminogen activator (uPA), enterokinase, a prostate-specific target (PSA, hK3), an interleukin 1β converting enzyme, thrombin, FAP (FAP-α), a dipeptidyl peptidase or dipeptidyl peptidase IV (DPPIV / CD26), a type II transmembrane serine protease (TTSP), neutrophil elastase, proteinase 3, mast cell chymase, mast cell tryptase, or a dipeptidyl peptidase; and / or The protease-cleavable polypeptide sequence comprises a variant sequence having one or two mismatches as compared with any one of the sequences of SEQ ID NOs: 80 to 94, or any one of the sequences of SEQ ID NOs: 80 to 90; and / or The protease-cleavable polypeptide sequence comprises a variant sequence having one or two mismatches as compared with any one of the sequences of SEQ ID NOs: 701 to 742, or any one of the sequences of SEQ ID NOs: 701 to 742; and / or The protease-cleavable polypeptide sequence is recognized by a matrix metalloprotease; and / or The protease-cleavable polypeptide sequence is recognized by MMP1; and / or The protease-cleavable polypeptide sequence is recognized by MMP2; and / or The protease-cleavable polypeptide sequence is recognized by MMP3; and / or The protease-cleavable polypeptide sequence is recognized by MMP7; and / or The protease-cleavable polypeptide sequence is recognized by MMP8; and / or The protease-cleavable polypeptide sequence is recognized by MMP9; and / or The protease-cleavable polypeptide sequence is recognized by MMP12; and / or The protease-cleavable polypeptide sequence is recognized by MMP13; and / or The protease-cleavable polypeptide sequence is recognized by MMP14; and / or The protease-cleavable polypeptide sequence is recognized by more than one MMP; and / or The protease-cleavable polypeptide sequence is recognized by two, three, four, five, six, or seven of MMP2, MMP7, MMP8, MMP9, MMP12, MMP13, and MMP14, The linker polypeptide according to claim 1. **Claim 28** At least one targeting sequence is configured to bind to an extracellular matrix component, heparin, integrin, or syndecan; or is configured to bind pH-sensitively to an extracellular matrix component, heparin, IgB (CD79b), integrin, cadherin, heparan sulfate proteoglycan, syndecan, or fibronectin; or the targeting sequence comprises any one of the sequences of SEQ ID NOs: 179 to 665, or a variant sequence having one or two mismatches compared to any one of the sequences of SEQ ID NOs: 179 to 665; and / or At least one targeting sequence comprises any one of the sequences of SEQ ID NOs: 179 to 665, or a variant sequence having one or two mismatches compared to any one of the sequences of SEQ ID NOs: 179 to 665; and / or At least one targeting sequence comprises any one of the sequences of SEQ ID NOs: 179 to 665; and / or At least one targeting sequence comprises any one of the sequences of SEQ ID NOs: 200, 330, 619, 653, and 663 to 665, or a variant sequence having one or two mismatches compared to any one of the sequences of SEQ ID NOs: 200, 330, 619, 653, and 663 to 665; and / or At least one targeting array comprises any one of the sequences of SEQ ID NOs: 200, 330, 619, 653, and 663-665; and / or At least one targeting array is configured to bind to denatured collagen or collagen, and the collagen may be collagen I, collagen II, collagen III, or collagen IV, The linker polypeptide according to claim 1. [

29. ] At least one targeting array is configured to bind to an integrin, and the integrin may be one or more of α1β1 integrin, α2β1 integrin, α3β1 integrin, α4β1 integrin, α5β1 integrin, α6β1 integrin, α7β1 integrin, α9β1 integrin, α4β7 integrin, ανβ3 integrin, ανβ5 integrin, αIIbβ3 integrin, αIIIbβ3 integrin, αMβ2 integrin, or αIIbβ3 integrin; and / or At least one targeting array is configured to bind to von Willebrand factor; and / or At least one targeting array is configured to bind to IgB; and / or At least one targeting array is configured to bind to heparin; and / or At least one targeting array is configured to bind to heparin and syndecan, heparan sulfate proteoglycan, or integrin, the integrin may be one or more of α1β1 integrin, α2β1 integrin, α3β1 integrin, α4β1 integrin, α5β1 integrin, α6β1 integrin, α7β1 integrin, α9β1 integrin, α4β7 integrin, ανβ3 integrin, ανβ5 integrin, αIIbβ3 integrin, αIIIbβ3 integrin, αMβ2 integrin, or αIIbβ3 integrin, and the syndecan may be one or more of syndecan 1, syndecan 4, and syndecan 2(w); and / or At least one targeting array is configured to bind to heparan sulfate proteoglycan; and / or At least one targeting array is configured to bind to a sulfated glycoprotein; and / or At least one targeting array is configured to bind to hyaluronic acid; and / or At least one targeting array is configured to bind to fibronectin; and / or At least one targeting array is configured to bind to cadherin; and / or At least one targeting array is configured to bind to its target in a pH-sensitive manner; and / or At least one targeting array has a higher affinity for its target at a pH below normal physiological pH, where the pH below normal physiological pH may be below 7 or below 6; and / or At least one targeting array has a higher affinity for its target in the range of pH 5-7, such as pH 5-5.5, 5.5-6, 6-6.5 or 6.5-7; and / or At least one targeting array contains one or more histidines, for example, one, two, three, four, five, six, seven, eight, nine or ten histidines; and / or At least one targeting array contains any one of the sequences of SEQ ID NOs: 641-663, or a variant sequence having one or two mismatches compared to any one of the sequences of SEQ ID NOs: 641-663; and / or At least one targeting array contains any one of the sequences of SEQ ID NOs: 641-665; and / or At least one targeting array is configured to bind to an extracellular matrix component, IgB (CD79b), integrin, cadherin, heparan sulfate proteoglycan, syndecan, or fibronectin in a pH-sensitive manner, and the extracellular matrix component may be hyaluronic acid, heparin, heparan sulfate, or a sulfated glycoprotein; and / or At least one targeting array is configured to bind to fibronectin in a pH-sensitive manner; and / or At least one targeting array is configured to bind to its target with an affinity of 0.1 nM to 1 nM, 1 nM to 10 nM, 10 nM to 100 nM, 100 nM to 1 μM, 1 μM to 10 μM, or 10 μM to 100 μM. The linker polypeptide according to claim 1.

30. At least one of the first linker and the second linker comprises at least one targeting array; and / or The protease-cleavable polypeptide sequence comprises at least one targeting array; and / or At least one targeting array increases the serum half-life of the linker polypeptide; and / or At least one targeting array synergistically increases the serum half-life of the linker polypeptide in combination with a pharmacokinetic modulator; and / or At least one targeting array independently increases the serum half-life of the linker polypeptide. The linker polypeptide according to claim 1.

31. Further comprising a blocker conjugated to the first active domain, The blocker may be conjugated to the first active domain via a protease-cleavable polypeptide sequence, and The blocker may be albumin, serum albumin, and / or human albumin. The linker polypeptide according to claim 1.

32. Further comprising a chemotherapeutic agent, the chemotherapeutic agent may be conjugated to a pharmacokinetic modulator, and the chemotherapeutic agent is selected from altretamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, mechlorethamine, melphalan, oxaliplatin, temozolomide, thiotepa, trabectedin, carmustine, lomustine, streptozocin, azacitidine, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cytarabine, decitabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, nelarabine, pemetrexed, pentostatin, pralatrexate, thioguanine, trifluridine, tipiracil, daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, bleomycin, dactinomycin, mitomycin c, mitoxantrone, irinotecan, topotecan, etoposide, mitoxantrone, teniposide, cabazitaxel, docetaxel, paclitaxel, vinblastine, vincristine, vinorelbine, prednisone, methylprednisolone, dexamethasone, retinoic acid, arsenic trioxide, asparaginase, eribulin, hydroxyurea, ixabepilone, mitotane, omacetaxine, pegaspargase, procarbazine, romidepsin, and vorinostat, and may be selected from: The linker polypeptide according to claim 1.

33. The molecular weight of the first active domain is independently about 14 kDa or less, about 12 kDa to about 14 kDa, about 10 kDa to about 12 kDa, about 8 kDa to about 10 kDa, about 6 kDa to about 8 kDa, about 4 kDa to about 6 kDa, about 2 kDa to about 4 kDa, or about 800 Da to about 2 kDa; or The molecular weight of the first active domain is about 16 kDa, or greater than 16 kDa, about 16 kDa to about 18 kDa, about 18 kDa to about 20 kDa, about 20 kDa to about 22 kDa, about 22 kDa to about 24 kDa, about 24 kDa to about 26 kDa, about 26 kDa to about 28 kDa, about 28 kDa to about 30 kDa, about 30 kDa to about 50 kDa, about 50 kDa to about 100 kDa, about 100 kDa to about 150 kDa, about 150 kDa to about 200 kDa, about 200 kDa to about 250 kDa, or about 250 kDa to about 300 kDa. The linker polypeptide according to claim 1.

34. The linker polypeptide according to claim 1, comprising a combination of a targeting sequence and a protease cleavage-type sequence, wherein the combination of the targeting sequence and the protease cleavage-type sequence is any one of SEQ ID NOs: 667 to 673.

35. A linker polypeptide comprising an amino acid sequence having at least 80, 85, 90, 95, 97, 98, or 99% identity to any one of the sequences of SEQ ID NOs: 1119, 800 to 848, 1024 to 1118, and 1120 to 1137, which may include any one of the sequences of SEQ ID NOs: 1119, 800 to 848, 1024 to 1118, and 1120 to 1137.

36. A pharmaceutical composition for use in the treatment of cancer, comprising the linker polypeptide according to any one of claims 1 to 35.

37. The cancer is a solid tumor, and the solid tumor may be metastatic and / or inoperable; and / or The cancer is a cancer that expresses PD-L1; and / or The cancer is melanoma, colorectal cancer, breast cancer, pancreatic cancer, lung cancer, prostate cancer, ovarian cancer, cervical cancer, gastric cancer or gastrointestinal cancer, lymphoma, colon cancer or colorectal cancer, endometrial cancer, thyroid cancer, or bladder cancer; and / or The cancer is a high-frequency microsatellite instability cancer; and / or The cancer has a mismatch repair function deficiency. The pharmaceutical composition according to claim 36.

38. A nucleic acid encoding the linker polypeptide according to any one of claims 1 to 35, or an expression vector comprising the nucleic acid encoding the linker polypeptide according to any one of claims 1 to 35.

39. A host cell comprising the nucleic acid or vector according to claim 38.

40. A method for producing a linker polypeptide, the method comprising culturing the host cell according to claim 39 under conditions under which the linker polypeptide is produced, the method may further comprise isolating the linker polypeptide.