Linker polypeptides

Abstract

The present disclosure relates to linker polypeptides. In some embodiments, the linker polypeptide comprises a first targeting sequence; a second targeting sequence; and a first linker located between the first targeting sequence and the second targeting sequence, the linker comprising a protease cleavable polypeptide sequence. In some embodiments, the linker polypeptide comprises a first active domain; a second active domain; a pharmacokinetic modulator; and a first linker located 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 located between the first active domain and the inhibitory polypeptide sequence, the linker comprising a protease cleavable polypeptide sequence; and a first targeting sequence.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application number 202280063746.X, application date July 20, 2022, and invention name “Linker Polypeptide”. The original application is a national phase application with international application number PCT / US2022 / 073970, which claims priority to the U.S. provisional patent application with application number 63 / 224,350 and application date July 21, 2021.

[0002] Background and Summary of the Invention

[0003] The present disclosure relates to the field of linker polypeptides comprising one or more targeting sequences. The linker polypeptides can be used, for example, to target certain types of extracellular environments.

[0004] 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.

[0005] For example, various forms of active domains (including but not limited to immunoglobulin antigen binding domains, such as Fv, scFv, Fab or VHH, and cytokines and chemokines, such as IL-2, IL-10, IL-15, TGF-β, CXCL9, CXCL10, etc.) play a significant role in targeting diseased cells and / or maintaining effective immune cell responses. However, in some cases, systemic administration of such compounds may activate systemic immune cells. Systemic activation may lead to systemic toxicity and indiscriminate activation of immune cells (including immune cells that respond to a variety of epitopes, antigens, and stimuli). The therapeutic potential of this therapy may be affected by these severe toxicities.

[0006] Peptide, immunoglobulin and cytokine therapies may also suffer from short serum half-lives, sometimes on the order of minutes. Therefore, high doses of peptides, immunoglobulins and cytokines, which may be necessary to achieve optimal effects, can lead to severe toxicity.

[0007] Additionally, in traditional antibodies, the immunoglobulin antigen binding domain is fixed to a pharmacokinetic modulator, such as an Fc region. Thus, the activity of the Fc region is correlated with the activity of the immunoglobulin antigen binding domain, and these regions and domains cannot operate independently, even when these activities are required at different locations and / or at different times or when the activities have different requirements for Fc function, such as when one region or domain is used for target destruction and another region or domain is used for immune stimulation.

[0008] Therefore, there is a need for polypeptides that overcome the obstacles of systemic or non-targeted 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 to interfere with this stimulation while also increasing the immune response against cancer cells. The present disclosure is intended to meet one or more of these needs, provide other benefits, or at least provide the public with a useful choice.

[0009] In some aspects, a linker polypeptide is provided that can target certain types of extracellular environments by using a targeting sequence. In some embodiments, the linker polypeptide may include a first targeting sequence; a second targeting sequence; and a first linker located between the first targeting sequence and the second targeting sequence, the linker including a protease cleavable polypeptide sequence. In some embodiments, the linker polypeptide may include a first active domain; a second active domain; a pharmacokinetic modulator; and a first linker located between the pharmacokinetic modulator and the first active domain or between the first active domain and the second active domain, the first linker including a protease cleavable polypeptide sequence. In some embodiments, the linker polypeptide may include a first active domain; an inhibitory polypeptide sequence capable of blocking the activity of the first active domain; a first linker located between the first active domain and the inhibitory polypeptide sequence, the linker including a protease cleavable polypeptide sequence; and a first targeting sequence.

[0010] In some embodiments, when one or more protease cleavable polypeptide sequences are cleaved by one or more protease cleavages, the different functions of the different components of the joint polypeptide can be decoupled from each other and / or activated. For example, the cleavage of the protease cleavable polypeptide can allow the inhibitory polypeptide sequence to dissociate from the cytokine polypeptide sequence, and / or can allow the active domain (e.g., it can have an immunostimulatory function) to dissociate from the remainder of the joint polypeptide (e.g., it can have a target destruction function).

[0011] Many tumors and tumor microenvironments show abnormal expression and activation of proteases. The present disclosure provides a linker polypeptide having components that can be decoupled from each other and / or activated by proteolytic cleavage, so that it becomes active when contacted with a protease in a tumor or tumor microenvironment. In some cases, for example, relative to other parts of the subject's body or healthy tissue, this may result in an increase in active domains (e.g., cytokines or immunoglobulin domains) in and around the tumor or tumor microenvironment. An exemplary advantage that can be produced is the formation of an active domain gradient. When a linker polypeptide is administered and the linker polypeptide is selectively or preferentially activated in a tumor or tumor microenvironment and then diffuses from these regions to other parts of the body, such gradients can be formed. These gradients can, for example, increase the transport of immune cells to tumors and tumor microenvironments. Immune cells transported to tumors can infiltrate tumors. Infiltrated immune cells can produce an immune response to cancer. Infiltrated immune cells can also secrete their own chemokines and cytokines. Cytokines can have one or both of autocrine and paracrine effects in tumors and tumor microenvironments. In some cases, the immune cell comprises a T cell (such as a T effector cell or a cytotoxic T cell) or a NK cell.

[0012] Also described herein are methods of treatment and methods of administering the linker polypeptides described herein. Such administration can be systemic or local. In some embodiments, the linker polypeptides described herein are administered systemically or locally to treat cancer.

[0013] The following implementations are contemplated.

[0014] Embodiment 1 is a linker polypeptide comprising:

[0015] first targeting sequence;

[0016] a second targeting sequence; and

[0017] A first linker positioned between the first targeting sequence and the second targeting sequence, the linker comprising a protease-cleavable polypeptide sequence.

[0018] Embodiment 2 is the linker polypeptide according to the immediately preceding embodiment, further comprising a first active domain, optionally wherein the first active domain is closer to the first targeting sequence than to the second targeting sequence.

[0019] Embodiment 3 is a linker polypeptide according to the immediately preceding embodiment, further comprising an additional domain, optionally wherein the additional domain comprises an inhibitory polypeptide sequence capable of blocking the activity of the first active domain, a pharmacokinetic modulator and / or a second active domain, and optionally wherein the additional domain is closer to the second targeting sequence than the first targeting sequence.

[0020] Embodiment 4 is the linker polypeptide according to the immediately preceding embodiment, which sequentially includes the first active domain, the first targeting sequence, the first linker, the second targeting sequence and the additional domain from the N-terminus to the C-terminus or from the C-terminus to the N-terminus.

[0021] Embodiment 5 is a linker polypeptide comprising:

[0022] The first active domain;

[0023] The second active domain;

[0024] Pharmacokinetic modulators; and

[0025] A first linker is positioned between the pharmacokinetic modulator and the first active domain, wherein the first linker comprises a protease-cleavable polypeptide sequence.

[0026] Embodiment 6 is a linker polypeptide according to embodiment 5, which further comprises a first targeting sequence.

[0027] Embodiment 7 is a linker polypeptide comprising:

[0028] The first active domain;

[0029] an inhibitory polypeptide sequence capable of blocking the activity of the first active domain;

[0030] a first linker positioned between the first active domain and the inhibitory polypeptide sequence, the linker comprising a protease-cleavable polypeptide sequence; and

[0031] First targeting sequence.

[0032] Embodiment 8 is the linker polypeptide according to the immediately preceding embodiment, which comprises a pharmacokinetic modulator.

[0033] Embodiment 9 is a linker polypeptide comprising:

[0034] 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 at the C-terminus of the first domain of the pharmacokinetic modulator;

[0035] a second polypeptide chain comprising a second domain of the 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;

[0036] wherein the first linker comprises a protease-cleavable polypeptide sequence; and

[0037] The first polypeptide chain or the second polypeptide chain further comprises at least one targeting sequence.

[0038] Embodiment 10 is a linker polypeptide comprising:

[0039] 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 at the N-terminus of the first domain of the pharmacokinetic modulator;

[0040] a second polypeptide chain comprising a second domain of the 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;

[0041] wherein the first linker comprises a protease-cleavable polypeptide sequence; and

[0042] The first polypeptide chain or the second polypeptide chain further comprises at least one targeting sequence.

[0043] Embodiment 11 is a linker polypeptide according to embodiment 9 or 10, wherein the inhibitory polypeptide sequence is at the C-terminus of the second domain of the pharmacokinetic modulator.

[0044] Embodiment 12 is a linker polypeptide according to embodiment 9 or 10, wherein the inhibitory polypeptide sequence is at the N-terminus of the second domain of the pharmacokinetic modulator.

[0045] Embodiment 13 is a linker polypeptide according to any one of embodiments 9 to 12, wherein the targeting sequence is located between the protease-cleavable polypeptide sequence and the first domain of the pharmacokinetic modulator.

[0046] Embodiment 14 is a linker polypeptide according to any one of embodiments 9 to 12, wherein the targeting sequence is located between the protease-cleavable polypeptide sequence and the first active domain.

[0047] Embodiment 15 is a linker polypeptide according to any one of embodiments 9 to 12, wherein the targeting sequence is at the C-terminus of the first active domain.

[0048] Embodiment 16 is a linker polypeptide according to any one of embodiments 9 to 12, wherein the targeting sequence is at the N-terminus of the first active domain.

[0049] Embodiment 17 is a linker polypeptide according to any one of embodiments 9 to 12, wherein the targeting sequence is at the C-terminus of the inhibitory polypeptide sequence.

[0050] Embodiment 18 is a linker polypeptide according to any one of embodiments 9 to 12, wherein the targeting sequence is at the N-terminus of the inhibitory polypeptide sequence.

[0051] Embodiment 19 is a linker polypeptide according to any one of embodiments 9 to 12, wherein the targeting sequence is located between the inhibitory polypeptide sequence and the second domain of the pharmacokinetic modulator.

[0052] Embodiment 20 is a linker polypeptide according to any one of embodiments 9 to 19, wherein the targeting sequence binds to heparin, optionally wherein the targeting sequence comprises SEQ ID NO:664.

[0053] Embodiment 21 is a linker polypeptide according to any one of embodiments 9 to 19, wherein the targeting sequence binds to collagen IV, optionally wherein the targeting sequence comprises SEQ ID NO:200.

[0054] Embodiment 22 is a linker polypeptide according to any one of embodiments 9 to 19, wherein the targeting sequence binds to collagen I, optionally wherein the targeting sequence comprises SEQ ID NO:188.

[0055] Embodiment 23 is a linker polypeptide according to any one of embodiments 9 to 19, wherein the targeting sequence binds to fibronectin, optionally wherein the targeting sequence comprises SEQ ID NO:653.

[0056] 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.

[0057] Embodiment 25 is a linker polypeptide according to the immediately preceding embodiment, wherein the first targeting sequence is part of the first polypeptide chain, and the second targeting sequence is part of the second polypeptide chain.

[0058] Embodiment 26 is a linker polypeptide according to the immediately preceding embodiment, wherein the first targeting sequence is at the C-terminus of the first active domain, and the second targeting sequence is at the C-terminus of the inhibitory polypeptide sequence.

[0059] Embodiment 27 is a linker polypeptide according to any one of embodiments 24 to 26, wherein the second targeting sequence binds to heparin, optionally wherein the targeting sequence comprises SEQ ID NO:664.

[0060] Embodiment 28 is a linker polypeptide according to any one of embodiments 24 to 26, wherein the second targeting sequence binds to collagen IV, optionally wherein the targeting sequence comprises SEQ ID NO:200.

[0061] Embodiment 29 is a linker polypeptide according to any one of embodiments 24 to 26, wherein the second targeting sequence binds to collagen I, optionally wherein the targeting sequence comprises SEQ ID NO:188.

[0062] Embodiment 30 is a linker polypeptide according to any one of embodiments 24 to 26, wherein the second targeting sequence binds to fibronectin, optionally wherein the targeting sequence comprises SEQ ID NO:653.

[0063] Embodiment 31 is a linker polypeptide according to any one of embodiments 9 to 30, further comprising a second active domain, optionally wherein the second active domain is part of the second polypeptide chain.

[0064] 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.

[0065] Embodiment 33 is a linker polypeptide according to the immediately preceding embodiment, wherein the second inhibitory polypeptide sequence is a part of the second polypeptide chain.

[0066] Embodiment 34 is the linker polypeptide according to the immediately preceding embodiment, wherein the second inhibitory polypeptide sequence is at the C-terminus of the first inhibitory polypeptide sequence.

[0067] 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.

[0068] Embodiment 36 is the linker polypeptide according to the immediately preceding embodiment, wherein the first inhibitory polypeptide sequence is an immunoglobulin inhibitory polypeptide sequence.

[0069] 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.

[0070] Embodiment 38 is a linker polypeptide according to any one of embodiments 8 to 37, wherein the pharmacokinetic modulator comprises a heterodimeric Fc or a heterodimeric CH3 domain.

[0071] Embodiment 39 is a linker polypeptide according to the immediately preceding embodiment, wherein the heterodimeric Fc or the heterodimeric CH3 domain comprises a knob CH3 domain and a hole CH3 domain.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] Embodiment 48 is a linker polypeptide according to any one of the preceding embodiments, wherein the first active domain comprises a first immunoglobulin antigen binding domain.

[0081] Embodiment 49 is a linker polypeptide according to any of the preceding embodiments, wherein the second active domain comprises a second immunoglobulin antigen binding domain.

[0082] Embodiment 50 is a linker polypeptide according to any one of the preceding 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.

[0083] Embodiment 51 is a linker polypeptide according to any one of the preceding embodiments, wherein one or each of the first immunoglobulin antigen binding domain and the second immunoglobulin antigen binding domain independently comprises Fv, scFv, Fab or VHH.

[0084] Embodiment 52 is a linker polypeptide according to any of the preceding embodiments, wherein one or each of the first immunoglobulin antigen binding domain and the second immunoglobulin antigen binding domain is independently humanized or fully human.

[0085] Embodiment 53 is a linker polypeptide according to any one of the preceding 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 cancer cell surface antigen sequences, growth factor sequences and growth factor receptor sequences.

[0086] Embodiment 54 is a linker polypeptide according to the immediately preceding embodiment, wherein one or each of the first immunoglobulin antigen binding domain and the second immunoglobulin antigen binding domain is independently configured to 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.

[0087] Embodiment 55 is a linker polypeptide according to any of the preceding 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.

[0088] Embodiment 56 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 hypervariable regions (HVRs) HVR-1, HVR-2 and HVR-3 in a VH region comprising an amino acid sequence of SEQ ID NO:910, and a VL region comprising HVR-1, HVR-2 and HVR-3 in a VL region comprising an amino acid sequence of SEQ ID NO:909.

[0089] Embodiment 57 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 the amino acid sequence of SEQ ID NO:910 and a VL region comprising the amino acid sequence of SEQ ID NO:909.

[0090] 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 a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:909 or 910.

[0091] Embodiment 59 is a linker polypeptide according to embodiment 55, wherein one of the first immunoglobulin antigen binding domain and the second immunoglobulin antigen binding domain is an antigen binding domain of trastuzumab.

[0092] Embodiment 60 is a linker polypeptide according to any one of the preceding 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.

[0093] Embodiment 61 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 in a VH region comprising an amino acid sequence of SEQ ID NO:914, and a VL region comprising HVR-1, HVR-2 and HVR-3 in a VL region comprising an amino acid sequence of SEQ ID NO:913.

[0094] Embodiment 62 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 the amino acid sequence of SEQ ID NO:914 and a VL region comprising the amino acid sequence of SEQ ID NO:913.

[0095] 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 a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:913 or 914.

[0096] Embodiment 64 is a linker polypeptide according to embodiment 60, wherein one of the first immunoglobulin antigen binding domain and the second immunoglobulin antigen binding domain is an antigen binding domain of cetuximab.

[0097] Embodiment 65 is a linker polypeptide according to any one of the preceding 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.

[0098] Embodiment 66 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 in a VH region comprising an amino acid sequence of SEQ ID NO:917 and a VL region comprising HVR-1, HVR-2 and HVR-3 in a VL region comprising an amino acid sequence of SEQ ID NO:918.

[0099] Embodiment 67 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 the amino acid sequence of SEQ ID NO:917 and a VL region comprising the amino acid sequence of SEQ ID NO:918.

[0100] 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 a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:917 or 918.

[0101] Embodiment 69 is a linker polypeptide according to embodiment 65, wherein one of the first immunoglobulin antigen binding domain and the second immunoglobulin antigen binding domain is an antigen binding domain of nivolumab.

[0102] Embodiment 70 is a linker polypeptide according to any one of the preceding 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.

[0103] Embodiment 71 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 in a VH region comprising an amino acid sequence of SEQID NO:921 and a VL region comprising HVR-1, HVR-2 and HVR-3 in a VL region comprising an amino acid sequence of SEQ ID NO:922.

[0104] Embodiment 72 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 the amino acid sequence of SEQ ID NO:921 and a VL region comprising the amino acid sequence of SEQ ID NO:922.

[0105] 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 a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:921 or 922.

[0106] 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 an antigen binding domain of atezolizumab.

[0107] Embodiment 75 is a linker polypeptide according to any of the preceding 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.

[0108] 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 in a VH region comprising an 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 in a VL region comprising an amino acid sequence of any one of SEQ ID NOs: 926, 930, 934 and 938.

[0109] Embodiment 77 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 any one of the amino acid sequences of SEQ ID NO:925, 929, 933 and 937 and a VL region comprising any one of the amino acid sequences of SEQ ID NO:926, 930, 934 and 938.

[0110] 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 that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 925, 926, 929, 930, 933, 934, 937 and 938.

[0111] Embodiment 79 is a linker polypeptide according to embodiment 75, wherein one of the first immunoglobulin antigen binding domain and the second immunoglobulin antigen binding domain is an antigen binding domain of teplizumab, muromonab, otelixizumab or visilizumab.

[0112] Embodiment 80 is a linker polypeptide according to any one of the preceding embodiments, wherein the first active domain comprises a receptor binding domain.

[0113] Embodiment 81 is a linker polypeptide according to the immediately preceding embodiment, wherein the receptor binding domain comprises a cytokine polypeptide sequence.

[0114] 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, and optionally otherwise comprises a wild-type sequence.

[0115] Embodiment 83 is a linker polypeptide according to any one of embodiments 80 to 82, wherein the receptor binding domain is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of a wild-type receptor binding domain or to the receptor binding domain in Table 1.

[0116] Embodiment 84 is a linker polypeptide according to the immediately preceding embodiment, wherein the receptor binding domain is a wild-type receptor binding domain.

[0117] 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 wherein the receptor binding domain is a dimeric receptor binding domain comprising monomers that are covalently (optionally via a polypeptide linker) or non-covalently associated.

[0118] Embodiment 86 is a linker polypeptide according to any one of embodiments 80 to 85, further comprising an inhibitory polypeptide sequence capable of blocking the activity of the receptor binding domain; and

[0119] A second linker is positioned between the receptor binding domain and the inhibitory polypeptide sequence, the second linker comprising a protease cleavable polypeptide sequence.

[0120] Embodiment 87 is a linker polypeptide according to any one of embodiments 80 to 86, when it is subject to any one of embodiments 9 to 24, wherein the inhibitory polypeptide sequence includes a cytokine binding domain.

[0121] 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.

[0122] 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.

[0123] Embodiment 90 is a linker polypeptide according to the immediately preceding embodiment, wherein the cytokine binding domain is an immunoglobulin cytokine binding domain.

[0124] 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 bind to the cytokine.

[0125] Embodiment 92 is a linker polypeptide according to embodiment 90 or 91, wherein the immunoglobulin cytokine binding domain is Fv, scFv, Fab or VHH.

[0126] Embodiment 93 is a linker polypeptide according to any one of embodiments 80 to 92, which comprises a targeting sequence, wherein the targeting sequence is located between the receptor binding domain and the protease-cleavable polypeptide sequence or one of the protease-cleavable polypeptide sequences.

[0127] 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.

[0128] 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.

[0129] 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 including CD122.

[0130] 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 including CD25.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] Embodiment 102 is a linker polypeptide according to the immediately preceding embodiment, wherein the IL-2 polypeptide sequence is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 1-4.

[0136] Embodiment 103 is the linker polypeptide according to the immediately preceding embodiment, wherein the IL-2 polypeptide sequence comprises any one of SEQ ID NOs: 1-4.

[0137] 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.

[0138] Embodiment 105 is the linker polypeptide according to the immediately preceding embodiment, wherein the IL-2 polypeptide sequence comprises the sequence of SEQ ID NO:1.

[0139] 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.

[0140] Embodiment 107 is a linker polypeptide according to any of the preceding embodiments, wherein the inhibitory polypeptide sequence comprises the IL-2 binding domain of the IL-2 receptor (IL-2R).

[0141] Embodiment 108 is a linker polypeptide according to the immediately preceding embodiment, wherein the inhibitory polypeptide sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 10-29 and 40-51.

[0142] Embodiment 109 is a linker polypeptide according to embodiment 107 or 108, wherein the IL-2R is human IL-2R.

[0143] Embodiment 110 is a linker polypeptide according to any of the preceding embodiments, wherein the inhibitory polypeptide sequence comprises an IL-2 binding immunoglobulin domain.

[0144] Embodiment 111 is a linker polypeptide according to the immediately preceding embodiment, wherein the IL-2 binding immunoglobulin domain is a human IL-2 binding immunoglobulin domain.

[0145] Embodiment 112 is a linker polypeptide according to embodiment 110 or 111, wherein the IL-2 binding immunoglobulin domain comprises a VH region and a VL region, wherein the VH region comprises hypervariable regions (HVRs) HVR-1, HVR-2 and HVR-3 having sequences of SEQ ID NOs: 37, 38 and 39, respectively, and the VL region comprises HVR-1, HVR-2 and HVR-3 having sequences of SEQ ID NOs: 34, 35 and 36, respectively.

[0146] 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 and a VL region, wherein:

[0147] The VH region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:33, and the VL region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:32; or

[0148] The VH region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:749, and the VL region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:748.

[0149] Embodiment 114 is a linker polypeptide according to the immediately preceding embodiment, wherein the IL-2 binding immunoglobulin domain comprises a VH region and a VL region, wherein:

[0150] The VH region comprises the sequence of SEQ ID NO: 33, and the VL region comprises the sequence of SEQ ID NO: 32; or

[0151] The VH region comprises the sequence of SEQ ID NO:749, and the VL region comprises the sequence of SEQ ID NO:748.

[0152] Embodiment 115 is a linker polypeptide according to any one of embodiments 110 to 114, wherein the IL-2 binding immunoglobulin domain is a scFv.

[0153] Embodiment 116 is a linker polypeptide according to embodiments 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.

[0154] Embodiment 117 is a linker polypeptide according to embodiments 110, 111, 114 or 116, wherein the IL-2 binding immunoglobulin domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:30, 31, 747, 850-856 or 863-870.

[0155] Embodiment 118 is a linker polypeptide according to 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.

[0156] Embodiment 119 is a linker polypeptide according to any of the preceding embodiments, wherein the receptor binding domain is an IL-10 polypeptide sequence.

[0157] Embodiment 120 is a linker polypeptide according to the immediately preceding embodiment, wherein the IL-10 polypeptide sequence is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:900.

[0158] Embodiment 121 is the linker polypeptide according to the immediately preceding embodiment, wherein the IL-10 polypeptide sequence comprises the sequence of SEQ ID NO:900.

[0159] 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.

[0160] Embodiment 123 is a linker polypeptide according to any one of embodiments 118 to 122, wherein the inhibitory polypeptide sequence comprises an IL-10 binding domain of an IL-10 receptor (IL-10R).

[0161] Embodiment 124 is a linker polypeptide according to the immediately preceding embodiment, wherein the inhibitory polypeptide sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:1011 or 1012.

[0162] Embodiment 125 is a linker polypeptide according to embodiment 123 or 124, wherein the IL-10R is human IL-10R.

[0163] Embodiment 126 is a linker polypeptide according to any of the preceding embodiments, wherein the inhibitory polypeptide sequence comprises an IL-10 binding immunoglobulin domain.

[0164] Embodiment 127 is a linker polypeptide according to the immediately preceding embodiment, wherein the IL-10 binding immunoglobulin domain is a human IL-10 binding immunoglobulin domain.

[0165] Embodiment 128 is a linker polypeptide according to embodiment 126 or 127, wherein the IL-10 binding immunoglobulin domain comprises a VH region and a VL region, wherein the VH region comprises hypervariable regions (HVRs) HVR-1, HVR-2 and HVR-3 having sequences of SEQ ID NOs: 946, 947 and 948, respectively, and the VL region comprises HVR-1, HVR-2 and HVR-3 having sequences of SEQ ID NOs: 942, 943 and 944, respectively.

[0166] 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 and a VL region, wherein the VH region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:945, and the VL region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:941.

[0167] 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.

[0168] Embodiment 131 is a linker polypeptide according to any one of embodiments 126 to 130, wherein the IL-10 binding immunoglobulin domain is a scFv.

[0169] Embodiment 132 is a linker polypeptide according to the immediately preceding embodiment, wherein the IL-10 binding immunoglobulin domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:939 or 940.

[0170] 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.

[0171] Embodiment 134 is a linker polypeptide according to any of the preceding embodiments, wherein the receptor binding domain is an IL-15 polypeptide sequence.

[0172] Embodiment 135 is a linker polypeptide according to the immediately preceding embodiment, wherein the IL-15 polypeptide sequence is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:901.

[0173] Embodiment 136 is the linker polypeptide according to the immediately preceding embodiment, wherein the IL-15 polypeptide sequence comprises the sequence of SEQ ID NO:901.

[0174] 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.

[0175] 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).

[0176] Embodiment 139 is a linker polypeptide according to the immediately preceding embodiment, wherein the inhibitory polypeptide sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 1016-1019.

[0177] Embodiment 140 is a linker polypeptide according to embodiment 97 or 98, wherein the IL-15R is human IL-15R.

[0178] Embodiment 141 is a linker polypeptide according to any of the preceding embodiments, wherein the inhibitory polypeptide sequence comprises an IL-15 binding immunoglobulin domain.

[0179] Embodiment 142 is a linker polypeptide according to the immediately preceding embodiment, wherein the IL-15 binding immunoglobulin domain is a human IL-15 binding immunoglobulin domain.

[0180] 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 in a VH region comprising an 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 in a VL region comprising an amino acid sequence of any one of SEQ ID NOs: 952, 954, 958, 961, 964, 967, 970, 973, 976, 979, 982, 984 and 987.

[0181] 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 and a VL region, wherein the VH region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 950, 955, 957, 960, 963, 966, 969, 972, 975, 978, 981, 985 and 988, and the VL region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 952, 954, 958, 961, 964, 967, 970, 973, 976, 979, 982, 984 and 987.

[0182] Embodiment 145 is a linker polypeptide according to the immediately preceding embodiment, wherein the IL-15 binding immunoglobulin domain comprises a VH region and a VL region, wherein the VH region comprises any one of SEQ ID NOs: 950, 955, 957, 960, 963, 966, 969, 972, 975, 978, 981, 985 and 988, and the VL region comprises any one of SEQ ID NOs: 952, 954, 958, 961, 964, 967, 970, 973, 976, 979, 982, 984 and 987.

[0183] Embodiment 146 is a linker polypeptide according to any one of embodiments 141 to 145, wherein the IL-15 binding immunoglobulin domain is a scFv.

[0184] Embodiment 147 is a linker polypeptide according to the immediately preceding embodiment, wherein the IL-15 binding immunoglobulin domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 953, 956, 959, 962, 965, 968, 971, 974, 977, 980, 983 and 986.

[0185] Embodiment 148 is a linker polypeptide according to the immediately preceding embodiment, wherein the IL-15 binding immunoglobulin domain comprises any one of SEQ ID NOs: 953, 956, 959, 962, 965, 968, 971, 974, 977, 980, 983 and 986.

[0186] Embodiment 149 is a linker polypeptide according to any one of the preceding embodiments, wherein the receptor binding domain is a CXCL9 polypeptide sequence.

[0187] Embodiment 150 is a linker polypeptide according to the immediately preceding embodiment, wherein the CXCL9 polypeptide sequence is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:902.

[0188] Embodiment 151 is a linker polypeptide according to the immediately preceding embodiment, wherein the CXCL9 polypeptide sequence comprises the sequence of SEQ ID NO:902.

[0189] 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.

[0190] 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.

[0191] Embodiment 154 is a linker polypeptide according to the immediately preceding embodiment, wherein the inhibitory polypeptide sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:1020 or 1021.

[0192] Embodiment 155 is a linker polypeptide according to embodiment 153 or 154, wherein the CXCR3 is human CXCR3.

[0193] Embodiment 156 is a linker polypeptide according to any of the preceding embodiments, wherein the inhibitory polypeptide sequence comprises a CXCL9 binding immunoglobulin domain.

[0194] Embodiment 157 is a linker polypeptide according to the immediately preceding embodiment, wherein the CXCL9 binding immunoglobulin domain is a human CXCL9 binding immunoglobulin domain.

[0195] Embodiment 158 ​​is a linker polypeptide according to any one of the preceding embodiments, wherein the receptor binding domain is a CXCL10 polypeptide sequence.

[0196] Embodiment 159 is the linker polypeptide according to the immediately preceding embodiment, wherein the CXCL10 polypeptide sequence is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:903.

[0197] Embodiment 160 is the linker polypeptide according to the immediately preceding embodiment, wherein the CXCL10 polypeptide sequence comprises the sequence of SEQ ID NO:903.

[0198] 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.

[0199] 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.

[0200] Embodiment 163 is a linker polypeptide according to the immediately preceding embodiment, wherein the inhibitory polypeptide sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:1020 or 1021.

[0201] Embodiment 164 is a linker polypeptide according to embodiment 162 or 163, wherein the CXCR3 is human CXCR3.

[0202] Embodiment 165 is a linker polypeptide according to any of the preceding embodiments, wherein the inhibitory polypeptide sequence comprises a CXCL10 binding immunoglobulin domain.

[0203] Embodiment 166 is the linker polypeptide according to the immediately preceding embodiment, wherein the CXCL10 binding immunoglobulin domain is a human CXCL10 binding immunoglobulin domain.

[0204] Embodiment 167 is a linker polypeptide according to embodiment 165 or 166, wherein the CXCL10 binding immunoglobulin domain comprises a VH region and a VL region, wherein the VH region comprises hypervariable regions (HVRs) HVR-1, HVR-2 and HVR-3 having sequences of SEQ ID NOs: 993, 994 and 995, respectively, and the VL region comprises HVR-1, HVR-2 and HVR-3 having sequences of SEQ ID NOs: 996, 997 and 998, respectively.

[0205] Embodiment 168 is a linker polypeptide according to any one of embodiments 165 to 167, wherein the CXCL10 binding immunoglobulin domain comprises a VH region and a VL region, wherein the VH region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:991, and the VL region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:992.

[0206] Embodiment 169 is the 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.

[0207] Embodiment 170 is a linker polypeptide according to any one of embodiments 165 to 169, wherein the CXCL10 binding immunoglobulin domain is a scFv.

[0208] Embodiment 171 is a linker polypeptide according to the immediately preceding embodiment, wherein the CXCL10 binding immunoglobulin domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:989 or 990.

[0209] Embodiment 172 is the linker polypeptide according to the immediately preceding embodiment, wherein the CXCL10 binding immunoglobulin domain comprises the sequence of SEQ ID NO:989 or 990.

[0210] Embodiment 173 is a linker polypeptide according to any one of the preceding embodiments, wherein the inhibitory polypeptide sequence interferes with the binding between the first active domain and the receptor of the first active domain and / or interferes with the binding between the second active domain and the receptor of the second active domain.

[0211] Embodiment 174 is a linker polypeptide according to any of the preceding embodiments, wherein the inhibitory polypeptide sequence and the pharmacokinetic modulator are different elements of the linker polypeptide.

[0212] Embodiment 175 is a linker polypeptide according to any of the preceding embodiments, wherein the inhibitory polypeptide sequence comprises a steric blocker.

[0213] Embodiment 176 is a linker polypeptide according to any of the preceding embodiments, wherein the inhibitory polypeptide sequence comprises at least a portion of the pharmacokinetic modulator.

[0214] Embodiment 177 is a linker polypeptide according to any of the preceding embodiments, wherein the pharmacokinetic modulator comprises at least a portion of an immunoglobulin constant domain.

[0215] Embodiment 178 is a linker polypeptide according to the immediately preceding embodiment, wherein the pharmacokinetic modulator comprises at least a portion of an immunoglobulin Fc region.

[0216] Embodiment 179 is a linker polypeptide according to the immediately preceding embodiment, wherein the pharmacokinetic modulator comprises an immunoglobulin Fc region.

[0217] Embodiment 180 is a linker polypeptide according to any one of embodiments 177 to 179, wherein the immunoglobulin is a human immunoglobulin.

[0218] Embodiment 181 is a linker polypeptide according to any one of embodiments 177 to 180, wherein the immunoglobulin is IgG.

[0219] Embodiment 182 is a linker polypeptide according to the immediately preceding embodiment, wherein the IgG is IgG1, IgG2, IgG3 or IgG4.

[0220] Embodiment 183 is a linker polypeptide according to any one of the preceding embodiments, which further comprises a growth factor binding polypeptide sequence or a growth factor receptor binding polypeptide sequence.

[0221] 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.

[0222] Embodiment 185 is a linker polypeptide according to the immediately preceding embodiment, wherein the TGF-βR extracellular domain sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 1022 or 1023.

[0223] 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.

[0224] 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.

[0225] Embodiment 188 is a linker polypeptide according to embodiment 145 or 146, wherein the growth factor binding immunoglobulin domain comprises: a VH region comprising HVR-1, HVR-2 and HVR-3 in a VH region comprising the amino acid sequence of SEQ ID NO:1008 and a VL region comprising HVR-1, HVR-2 and HVR-3 in a VL region comprising the amino acid sequence of SEQ ID NO:1010.

[0226] 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.

[0227] Embodiment 190 is a linker polypeptide according to embodiments 185 to 189, wherein the growth factor binding immunoglobulin domain comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 1007 or 1009.

[0228] 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.

[0229] Embodiment 192 is a linker polypeptide according to the immediately preceding embodiment, wherein the TGF-β sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs. 904-906.

[0230] 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.

[0231] 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.

[0232] 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 in a VH region comprising an amino acid sequence of SEQ ID NO: 999 or 1003, and a VL region comprising HVR-1, HVR-2 and HVR-3 in a VL region comprising an amino acid sequence of SEQ ID NO: 1000 or 1004.

[0233] Embodiment 196 is a linker polypeptide according to the immediately preceding 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.

[0234] Embodiment 197 is a linker polypeptide according to embodiments 152 to 155, wherein the growth factor receptor binding immunoglobulin domain comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 1001, 1002, 1005 and 1006.

[0235] Embodiment 198 is a linker polypeptide according to any one of the preceding embodiments, comprising a plurality of protease-cleavable polypeptide sequences.

[0236] Embodiment 199 is a linker polypeptide according to any of the preceding embodiments, wherein the protease-cleavable polypeptide sequence is at the C-terminus of the VH region, at the C-terminus of at least a portion of the CH1 domain, between the CH1 domain and the CH2 domain, at the N-terminus of at least a portion of the CH2 domain, at the N-terminus of the disulfide bond between heavy chains, at the N-terminus of the disulfide bond within the CH2 domain, or at the N-terminus of the hinge region, or within the hinge region.

[0237] Embodiment 200 is a linker polypeptide according to any of the preceding embodiments, wherein the protease-cleavable polypeptide sequence is at the C-terminus of the first targeting sequence and at the C-terminus of the second targeting sequence.

[0238] Embodiment 201 is a linker polypeptide according to any of the preceding embodiments, wherein the protease-cleavable polypeptide sequence is at the N-terminus of the first targeting sequence and at the N-terminus of the second targeting sequence.

[0239] Embodiment 202 is a linker polypeptide according to any of the preceding embodiments, wherein the protease-cleavable polypeptide sequence is at the C-terminus of the first plurality of targeting sequences and at the N-terminus of the second plurality of targeting sequences.

[0240] Embodiment 203 is a linker polypeptide according to any of the preceding embodiments, wherein the protease-cleavable polypeptide sequence is at the C-terminus of multiple targeting sequences and at the N-terminus of at least one targeting sequence.

[0241] Embodiment 204 is a linker polypeptide according to any of the preceding embodiments, wherein the protease-cleavable polypeptide sequence is at the N-terminus of multiple targeting sequences and at the C-terminus of at least one targeting sequence.

[0242] Embodiment 205 is a linker polypeptide according to any of the preceding embodiments, wherein the protease-cleavable polypeptide sequence is at the C-terminus of the first targeting sequence and at the C-terminus of the second targeting sequence, and is not at the N-terminus of the targeting sequence.

[0243] Embodiment 206 is a linker polypeptide according to any of the preceding embodiments, wherein the protease-cleavable polypeptide sequence is at the N-terminus of the first targeting sequence and at the N-terminus of the second targeting sequence, and is not at the C-terminus of the targeting sequence.

[0244] Embodiment 207 is a linker polypeptide according to any of the preceding embodiments, wherein the linker polypeptide is configured to release the first active domain from the remainder of the linker polypeptide upon cleavage of the protease-cleavable polypeptide sequence.

[0245] Embodiment 208 is a linker polypeptide according to the immediately preceding embodiment, wherein the first active domain is configured to remain connected to one or more of the following upon cleavage of the protease-cleavable polypeptide sequence: 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 the pharmacokinetic modulator.

[0246] Embodiment 209 is a linker polypeptide according to any of the preceding embodiments, wherein the linker polypeptide is configured to release the second active domain from the remainder of the linker polypeptide upon cleavage of the protease-cleavable polypeptide sequence.

[0247] Embodiment 210 is a linker polypeptide according to the immediately preceding embodiment, wherein the second active domain is configured to remain connected to one or more of the following upon cleavage of the protease-cleavable polypeptide sequence: 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 the pharmacokinetic modulator.

[0248] Embodiment 211 is a linker polypeptide according to any of the preceding embodiments, wherein the protease-cleavable polypeptide sequence is recognized by metalloproteinases, serine proteases, cysteine ​​proteases, aspartic proteases, threonine proteases, glutamic proteases, gelatinases, asparagine peptide cleaving enzymes, cathepsins, kallikreins, plasmins, collagenases, hK1, hK10, hK15, matrix resolvants, factor Xa, chymotrypsin-like proteases, trypsin-like proteases, elastase-like proteases, subtilisin-like proteases, actinidin, bromelain, calpain, caspase, Mir 1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, protease, legumin, plasmepsin, Nepenthes protease, metalloexopeptidases, metalloendopeptidases, ADAM 10, ADAM 17, ADAM 12. 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.

[0249] Embodiment 212 is a linker polypeptide according to any of the preceding embodiments, wherein the protease-cleavable polypeptide sequence comprises any one of SEQ ID NOs: 701-742 or a variant having one or two mismatches relative to any one of SEQ ID NOs: 701-742.

[0250] Embodiment 213 is a linker polypeptide according to any of the preceding embodiments, wherein the protease-cleavable polypeptide sequence is recognized by a matrix metalloprotease.

[0251] Embodiment 214 is a linker polypeptide according to any of the preceding embodiments, wherein the protease-cleavable polypeptide sequence is recognized by MMP-1.

[0252] Embodiment 215 is a linker polypeptide according to any of the preceding embodiments, wherein the protease-cleavable polypeptide sequence is recognized by MMP-2.

[0253] Embodiment 216 is a linker polypeptide according to any of the preceding embodiments, wherein the protease-cleavable polypeptide sequence is recognized by MMP-3.

[0254] Embodiment 217 is a linker polypeptide according to any of the preceding embodiments, wherein the protease-cleavable polypeptide sequence is recognized by MMP-7.

[0255] Embodiment 218 is a linker polypeptide according to any of the preceding embodiments, wherein the protease-cleavable polypeptide sequence is recognized by MMP-8.

[0256] Embodiment 219 is a linker polypeptide according to any of the preceding embodiments, wherein the protease-cleavable polypeptide sequence is recognized by MMP-9.

[0257] Embodiment 220 is a linker polypeptide according to any of the preceding embodiments, wherein the protease-cleavable polypeptide sequence is recognized by MMP-12.

[0258] Embodiment 221 is a linker polypeptide according to any of the preceding embodiments, wherein the protease-cleavable polypeptide sequence is recognized by MMP-13.

[0259] Embodiment 222 is a linker polypeptide according to any of the preceding embodiments, wherein the protease-cleavable polypeptide sequence is recognized by MMP-14.

[0260] Embodiment 223 is a linker polypeptide according to any of the preceding embodiments, wherein the protease-cleavable polypeptide sequence is recognized by more than one MMP.

[0261] Embodiment 224 is a linker polypeptide according to any of the preceding embodiments, wherein the protease-cleavable polypeptide sequence is recognized by two, three, four, five, six or seven of MMP-2, MMP-7, MMP-8, MMP-9, MMP-12, MMP-13 and MMP-14.

[0262] Embodiment 225 is a linker polypeptide according to any of the preceding embodiments, wherein the protease-cleavable polypeptide sequence comprises any one of SEQ ID NOs: 80-94 or a variant sequence having one or two mismatches relative to any one of SEQ ID NOs: 80-90.

[0263] Embodiment 226 is a linker polypeptide according to any of the preceding embodiments, wherein the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 80 or a variant sequence having one or two mismatches relative to the sequence.

[0264] Embodiment 227 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: 81 or a variant sequence having one or two mismatches relative to the sequence.

[0265] Embodiment 228 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: 82 or a variant sequence having one or two mismatches relative to the sequence.

[0266] Embodiment 229 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: 83 or a variant sequence having one or two mismatches relative to the sequence.

[0267] Embodiment 230 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: 84 or a variant sequence having one or two mismatches relative to the sequence.

[0268] Embodiment 231 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: 85 or a variant sequence having one or two mismatches relative to the sequence.

[0269] Embodiment 232 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: 86 or a variant sequence having one or two mismatches relative to the sequence.

[0270] Embodiment 233 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: 87 or a variant sequence having one or two mismatches relative to the sequence.

[0271] Embodiment 234 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: 88 or a variant sequence having one or two mismatches relative to the sequence.

[0272] Embodiment 235 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: 89 or a variant sequence having one or two mismatches relative to the sequence.

[0273] Embodiment 236 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: 90 or a variant sequence having one or two mismatches relative to the sequence.

[0274] Embodiment 237 is a linker polypeptide according to any one of embodiments 1 to 225, wherein the protease-cleavable polypeptide sequence comprises any one of SEQ ID NOs: 80-90.

[0275] Embodiment 238 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:91.

[0276] Embodiment 239 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:92.

[0277] Embodiment 240 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:93.

[0278] 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.

[0279] Embodiment 242 is a linker polypeptide according to any of the preceding 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 is independently configured to bind to an extracellular matrix component, heparin, integrin, or syndecan; or is 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 the targeting sequence comprises any one of SEQ ID NOs: 179-665 or a variant having one or two mismatches relative to any one of SEQ ID NOs: 179-665.

[0280] Embodiment 243 is a linker polypeptide according to any of the preceding 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 independently comprises any one of SEQ ID NOs: 179-665 or a variant having one or two mismatches relative to any one of SEQ ID NOs: 179-665.

[0281] Embodiment 244 is a linker polypeptide according to any of the preceding 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 independently comprises any one of SEQ ID NOs: 179-665.

[0282] Embodiment 245 is a linker polypeptide according to any of the preceding 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 independently comprises any one of SEQ ID NOs: 200, 330, 619, 653, and 663-665 or a variant having one or two mismatches relative to any one of SEQ ID NOs: 200, 330, 619, 653, and 663-665.

[0283] Embodiment 246 is a linker polypeptide according to any of the preceding 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 independently comprises any one of SEQ ID NOs: 200, 330, 619, 653, and 663-665.

[0284] Embodiment 247 is a linker polypeptide according to any of the preceding 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 is independently configured to bind to denatured collagen.

[0285] Embodiment 248 is a linker polypeptide according to any of the preceding 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 is independently configured to bind to collagen.

[0286] Embodiment 249 is a linker polypeptide according to embodiment 247 or 248, wherein the collagen is collagen I.

[0287] Embodiment 250 is a linker polypeptide according to embodiment 247 or 248, wherein the collagen is collagen II.

[0288] Embodiment 251 is a linker polypeptide according to embodiment 247 or 248, wherein the collagen is collagen III.

[0289] Embodiment 252 is a linker polypeptide according to embodiment 247 or 248, wherein the collagen is collagen IV.

[0290] Embodiment 253 is a linker polypeptide according to any of the preceding 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 is independently configured to bind to an integrin.

[0291] Embodiment 254 is a linker polypeptide according to the immediately preceding embodiment, wherein the integrin is one or more of the following: 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.

[0292] Embodiment 255 is a linker polypeptide according to any of the preceding 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 is independently configured to bind to von Willebrand factor.

[0293] Embodiment 256 is a linker polypeptide according to any of the preceding 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 is independently configured to bind to IgB.

[0294] Embodiment 257 is a linker polypeptide according to any of the preceding 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 is independently configured to bind to heparin.

[0295] Embodiment 258 is a linker polypeptide according to any of the preceding embodiments, wherein the first targeting sequence is configured to bind to heparin and the second targeting sequence is configured to bind to heparin, wherein the first targeting sequence is configured to bind to collagen IV and the second targeting sequence is configured to bind to heparin, or wherein the first targeting sequence is configured to bind to heparin and the second targeting sequence is configured to bind to collagen IV.

[0296] Embodiment 259 is a linker polypeptide according to any of the preceding 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 is independently configured to bind to heparin and syndecan, heparan sulfate proteoglycan, or integrin, optionally wherein the integrin is one or more of the following: 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.

[0297] 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).

[0298] Embodiment 261 is a linker polypeptide according to any of the preceding 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 is independently configured to bind to a heparan sulfate proteoglycan.

[0299] Embodiment 262 is a linker polypeptide according to any of the preceding 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 is independently configured to bind to a sulfated glycoprotein.

[0300] Embodiment 263 is a linker polypeptide according to any of the preceding 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 is independently configured to bind to hyaluronic acid.

[0301] Embodiment 264 is a linker polypeptide according to any of the preceding 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 is independently configured to bind to fibronectin.

[0302] Embodiment 265 is a linker polypeptide according to any of the preceding 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 is independently configured to bind to a cadherin protein.

[0303] Embodiment 266 is a linker polypeptide according to any of the preceding 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 is independently configured to bind to its target in a pH-sensitive manner.

[0304] Embodiment 267 is a linker polypeptide according to any of the preceding 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 independently has a higher affinity for its target at a pH below normal physiological pH than at normal physiological pH, optionally wherein the pH below normal physiological pH is below 7 or below 6.

[0305] Embodiment 268 is a linker polypeptide according to any of the preceding 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 independently has a higher affinity for its target at a pH in the range of 5-7 (e.g., 5-5.5, 5.5-6, 6-6.5, or 6.5-7) than at normal physiological pH.

[0306] Embodiment 269 is a linker polypeptide according to any of the preceding 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 independently comprises one or more histidines, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 histidines.

[0307] Embodiment 270 is a linker polypeptide according to any of the preceding 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 independently comprises any one of SEQ ID NOs: 641-663 or a variant having one or two mismatches relative to any one of SEQ ID NOs: 641-663.

[0308] Embodiment 271 is a linker polypeptide according to any of the preceding 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 independently comprises any one of SEQ ID NOs: 641-665.

[0309] Embodiment 272 is a linker polypeptide according to any of the preceding 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 is independently configured to bind to an extracellular matrix component, IgB (CD79b), integrin, cadherin, heparan sulfate proteoglycan, syndecan, or fibronectin in a pH-sensitive manner.

[0310] 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.

[0311] Embodiment 274 is a linker polypeptide according to any of the preceding 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 is independently configured to bind to fibronectin in a pH-sensitive manner.

[0312] Embodiment 275 is a linker polypeptide according to any of the preceding 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 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.

[0313] Embodiment 276 is a linker polypeptide according to 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.

[0314] Embodiment 277 is a linker polypeptide according to 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 is independently configured to bind to its target with an affinity of 1 nM to 10 nM.

[0315] Embodiment 278 is a linker polypeptide according to 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 is independently configured to bind to its target with an affinity of 10 nM to 100 nM.

[0316] Embodiment 279 is a linker polypeptide according to 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 is independently configured to bind to its target with an affinity of 100 nM to 1 μM.

[0317] Embodiment 280 is a linker polypeptide according to 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 is independently configured to bind to its target with an affinity of 1 μM to 10 μM.

[0318] Embodiment 281 is a linker polypeptide according to 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 is independently configured to bind to its target with an affinity of 10 μM to 100 μM.

[0319] Embodiment 282 is a linker polypeptide according to any of the preceding embodiments, wherein at least one of the first linker and the second linker comprises 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, or one of the plurality of targeting sequences.

[0320] Embodiment 283 is a linker polypeptide according to any one of the immediately preceding embodiments, wherein the protease-cleavable polypeptide sequence comprises 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, or one of the plurality of targeting sequences.

[0321] Embodiment 284 is a linker polypeptide according to any of the preceding 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 increases the serum half-life of the linker polypeptide.

[0322] Embodiment 285 is a linker polypeptide according to any of the preceding 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 synergistically increases the serum half-life of the linker polypeptide with the pharmacokinetic modulator or another of the first targeting sequence and the second targeting sequence, another of the at least one targeting sequence, another of the first plurality of targeting sequences, another of the second plurality of targeting sequences, or another of the plurality of targeting sequences.

[0323] Embodiment 286 is a linker polypeptide according to 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 independently increases the serum half-life of the linker polypeptide.

[0324] Embodiment 287 is a linker polypeptide according to any of the preceding embodiments, further comprising a blocking agent conjugated to one or each of the first active domain and the second active domain.

[0325] Embodiment 288 is a linker polypeptide according to the immediately preceding embodiment, wherein the blocker is conjugated to one or each of the first active domain and the second active domain via a protease-cleavable polypeptide sequence.

[0326] Embodiment 289 is a linker polypeptide according to embodiment 287 or 288, wherein the blocking agent is albumin.

[0327] Embodiment 290 is a linker polypeptide according to any one of embodiments 287 to 289, wherein the blocking agent is serum albumin.

[0328] Embodiment 291 is a linker polypeptide according to any one of embodiments 287 to 290, wherein the blocking agent is human albumin.

[0329] Embodiment 292 is a linker polypeptide according to any of the preceding embodiments, further comprising a chemotherapeutic drug.

[0330] Embodiment 293 is a linker polypeptide according to the immediately preceding embodiment, wherein the chemotherapeutic drug is conjugated to the pharmacokinetic modulator.

[0331] Embodiment 294 is a linker polypeptide according to embodiment 292 or 293, wherein the chemotherapeutic agent is selected from altretamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, mechlorethamine, hamine, melphalan, oxaliplatin, temozolomide, thiotepa, trabectedin, carmustine, cyclohexylnitrosourea, streptozocin, azacitidine, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cytarabine, cytarabine, decitabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, nelarabine, pemetrexed, pentostatin, pralatrexate, thioguanine, trifluridine, tipiracil, daunorubicin, 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.

[0332] Embodiment 295 is a linker polypeptide according to any of the preceding embodiments, wherein the molecular weight of one or each of the first active domain and the second active domain is independently about or less than 14 kDa.

[0333] Embodiment 296 is a linker polypeptide according to the immediately preceding embodiment, wherein the molecular weight is about 12 kDa to about 14 kDa.

[0334] Embodiment 297 is a linker polypeptide according to embodiment 295, wherein the molecular weight is about 10 kDa to about 12 kDa.

[0335] Embodiment 298 is a linker polypeptide according to embodiment 295, wherein the molecular weight is about 8 kDa to about 10 kDa.

[0336] Embodiment 299 is a linker polypeptide according to embodiment 295, wherein the molecular weight is about 6 kDa to about 8 kDa.

[0337] Embodiment 300 is a linker polypeptide according to embodiment 295, wherein the molecular weight is about 4 kDa to about 6 kDa.

[0338] Embodiment 301 is a linker polypeptide according to embodiment 295, wherein the molecular weight is about 2 kDa to about 4 kDa.

[0339] Embodiment 302 is a linker polypeptide according to embodiment 295, wherein the molecular weight is about 800 Da to about 2 kDa.

[0340] Embodiment 303 is a linker polypeptide according to any one 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 or greater than 16 kDa.

[0341] Embodiment 304 is a linker polypeptide according to the immediately preceding embodiment, wherein the molecular weight is about 16 kDa to about 18 kDa.

[0342] Embodiment 305 is a linker polypeptide according to embodiment 303, wherein the molecular weight is about 18 kDa to about 20 kDa.

[0343] Embodiment 306 is a linker polypeptide according to embodiment 303, wherein the molecular weight is about 20 kDa to about 22 kDa.

[0344] Embodiment 307 is a linker polypeptide according to embodiment 303, wherein the molecular weight is about 22 kDa to about 24 kDa.

[0345] Embodiment 308 is a linker polypeptide according to embodiment 303, wherein the molecular weight is about 24 kDa to about 26 kDa.

[0346] Embodiment 309 is a linker polypeptide according to embodiment 303, wherein the molecular weight is about 26 kDa to about 28 kDa.

[0347] Embodiment 310 is a linker polypeptide according to embodiment 303, wherein the molecular weight is about 28 kDa to about 30 kDa.

[0348] Embodiment 311 is a linker polypeptide according to embodiment 303, wherein the molecular weight is about 30 kDa to about 50 kDa.

[0349] Embodiment 312 is a linker polypeptide according to embodiment 303, wherein the molecular weight is about 50 kDa to about 100 kDa.

[0350] Embodiment 313 is a linker polypeptide according to embodiment 303, wherein the molecular weight is about 100 kDa to about 150 kDa.

[0351] Embodiment 314 is a linker polypeptide according to embodiment 303, wherein the molecular weight is about 150 kDa to about 200 kDa.

[0352] Embodiment 315 is a linker polypeptide according to embodiment 303, wherein the molecular weight is about 200 kDa to about 250 kDa.

[0353] Embodiment 316 is a linker polypeptide according to embodiment 303, wherein the molecular weight is about 250 kDa to about 300 kDa.

[0354] Embodiment 317 is a linker polypeptide according to any one of the preceding embodiments, comprising a combined targeting sequence and a protease cleavable sequence, wherein the combined targeting sequence and protease cleavable sequence is any one of SEQ ID NOs: 667-673.

[0355] Embodiment 318 is a linker polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 800-848 or 1024-1041.

[0356] Embodiment 319 is a linker polypeptide according to the immediately preceding embodiment, comprising any one of SEQ ID NOs: 800-848 or 1024-1041.

[0357] Embodiment 320 is a pharmaceutical composition comprising a linker polypeptide according to any one of the preceding embodiments.

[0358] Embodiment 321 is a linker polypeptide or pharmaceutical composition according to any one of the preceding embodiments, for use in therapy.

[0359] Embodiment 322 is a linker polypeptide or a pharmaceutical composition according to any one of the preceding embodiments, which is used to treat cancer.

[0360] 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 preceding embodiments.

[0361] Embodiment 324 is the use of a linker polypeptide or a pharmaceutical composition according to any one of embodiments 1 to 321 for the preparation of a medicament for treating cancer.

[0362] Embodiment 325 is a linker polypeptide for use according to any one of embodiments 322 to 324, wherein the cancer is a solid tumor.

[0363] Embodiment 326 is a linker polypeptide for use according to the immediately preceding embodiment, wherein the solid tumor is metastatic and / or unresectable.

[0364] Embodiment 327 is a linker polypeptide for use according to any one of embodiments 322 to 326, wherein the cancer is a PD-L1 expressing cancer.

[0365] Embodiment 328 is a method, a use or 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.

[0366] Embodiment 329 is a method, a use or a linker polypeptide for use according to any one of embodiments 322 to 328, wherein the cancer is a microsatellite instability-high cancer.

[0367] Embodiment 330 is a linker polypeptide for use according to any one of embodiments 322 to 329, wherein the cancer is mismatch repair deficient.

[0368] Embodiment 331 is a nucleic acid encoding a linker polypeptide according to any one of embodiments 1 to 319.

[0369] Embodiment 332 is an expression vector comprising the nucleic acid according to the immediately preceding embodiment.

[0370] Embodiment 333 is a host cell comprising the nucleic acid according to embodiment 331 or the vector according to embodiment 332.

[0371] Embodiment 334 is a method of producing a linker polypeptide, the method comprising culturing the host cell according to the immediately preceding embodiment under conditions where the linker polypeptide is produced.

[0372] Embodiment 335 is a method according to the immediately preceding embodiment, further comprising isolating the linker polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0373] Figure 1A Shown are schematic representations of the structures of exemplary linker polypeptides and SDS-PAGE gels (with Coomassie stain) characterizing various purified linker polypeptides.

[0374] Figure 1B-1C Each shows an SDS-PAGE gel (with Coomassie stain) characterizing various purified linker polypeptides.

[0375] Figure 1D Shown is another exemplary linker polypeptide structure and a schematic representation of an SDS-PAGE gel (with Coomassie stain) characterizing various purified linker polypeptides.

[0376] Figures 2A-2F One or more SDS-PAGE gels and subsequent immunoblots characterizing various linker polypeptides with and without treatment with matrix metallopeptidase 9 (MMP9) are each shown.

[0377] Figure 3A-3BB Each shows the results of a HEK Blue IL-2 assay measuring IL-2 and IL-15 activity of a particular linker polypeptide with and without treatment with a MMP.

[0378] Figure 4A A schematic representation of the structure of different MMP linker peptides among linker polypeptides, in particular a linker peptide that binds to heparin, is shown.

[0379] Figure 4B Shows the measurement Figure 4A The results of the determination of the binding of the linker peptide to heparin.

[0380] Figure 4C Shown are illustrations of the structures of various MMP linker peptides among linker polypeptides, particularly linker peptides that bind to fibronectin, and also shown are assay results for measuring binding of linker peptides to fibronectin.

[0381] Figure 4D Shown are illustrations of the structures of different MMP linker peptides among linker polypeptides, particularly linker peptides that bind to collagen, and also shown are assay results for measuring the binding of linker peptides to collagen.

[0382] Figure 4E Schematic representations of the structures of different linker polypeptides are shown, and also shown are the results of an assay measuring the binding of the linker polypeptides to heparin.

[0383] Figure 4F The results of an assay measuring the binding of different linker polypeptides to heparin are shown, including Figure 4EThe asterisk (*) indicates that for construct NN, the software could not calculate the EC based on the fit. 50 ; however, the construct NN binding curve mimicked the construct CC binding curve.

[0384] Figure 4G The results of an assay measuring the binding of different linker polypeptides to heparin are shown, including Figure 4E Results of the assay in which the linker polypeptide constructs CC in share the same heparin binding motif.

[0385] Figure 4H The results of an assay measuring the binding of different linker polypeptides to heparin are shown, including Figure 4E The results of the determination that the linker polypeptide construct Y in shares the same heparin binding motif.

[0386] Fig. 4I The results of an assay measuring the binding of different linker polypeptides to heparin are shown, including Figure 4E The results of the determination that the linker polypeptide construct Y in shares the same heparin binding motif.

[0387] Figure 4J Shown are the results of an assay measuring the binding of different IL-15Rα-IL-15 linker polypeptides to heparin.

[0388] Figure 4K Shown are the results of an assay measuring the binding of various linker polypeptides to fibronectin.

[0389] Figure 4L Shown are the results of a pulldown assay measuring the binding of different linker polypeptides to collagen.

[0390] Figure 4M Shown are the results of assays measuring heparin binding of different linker polypeptides in the presence or absence of a heparin binding site.

[0391] Figure 5A Shown are the results of real-time whole-body imaging measuring in vivo levels of IL-2 fusion protein in tumors using fluorescently labeled proteins. Figure 5B Shows Figure 5A The levels of fusion proteins.

[0392] Figure 6 The results of measuring tumor volume in C57BL / 6 mice inoculated with B16F10 melanoma cells and treated with different linker polypeptides are shown, and a schematic diagram for ranking the anti-tumor activities of different linker polypeptides is also shown.

[0393] Figures 7A-7D The levels of full-length fusion protein in tumors are measured ( Fig. 7A), IL-2 levels in tumors ( Figure 7B ), IFN-γ levels in tumors ( Figure 7C ) and the levels of full-length fusion protein in serum ( Fig.7D )’s measurement results.

[0394] Figures 8A-8B The results show that the TNF-α ( Fig. 8A ) and IL-6 ( Figure 8B ) serum levels.

[0395] Figure 8C Shown are the results of AST activity assays after animals were treated with different linker polypeptides.

[0396] Figures 9A-9D Each shows a linker polypeptide according to certain embodiments of the present disclosure. (AD, active domain; PM, pharmacokinetic modulator; CL, protease cleavable polypeptide sequence and optionally targeting sequence; IBD, immunoglobulin antigen binding domain; D, chemotherapeutic drug).

[0397] Figures 10A-10B Each shows a linker polypeptide according to certain embodiments of the present disclosure. (AD, active domain; PM, pharmacokinetic modulator; CL, protease cleavable polypeptide sequence and optionally targeting sequence; IBD, immunoglobulin antigen binding domain; RBD, receptor binding domain; CY, cytokine polypeptide sequence).

[0398] Figures 11A-11B Each shows the release of the first active domain from the remainder of the linker polypeptide after cleavage of one or more protease-cleavable polypeptide sequences. (AD, active domain; PM, pharmacokinetic modulator; CL, protease-cleavable polypeptide sequence and optionally targeting sequence; IBD, immunoglobulin antigen binding domain; D, chemotherapeutic drug).

[0399] Figures 12A-12B Each shows the release of the first active domain from the remainder of the linker polypeptide after cleavage of one or more protease-cleavable polypeptide sequences. (AD, active domain; PM, pharmacokinetic modulator; CL, protease-cleavable polypeptide sequence and optionally targeting sequence; IBD, immunoglobulin antigen binding domain; RBD, receptor binding domain; CY, cytokine polypeptide sequence).

[0400] Figures 13A-13C The effect of treatment with different fusion proteins on tumor xenografts is shown. The mean tumor volume was Figures 13A-13B The inhibition of tumor volume was shown in Fig. 13C Shown in.

[0401] Fig.13D Shown are the levels of IFN-γ in mice bearing tumor xenografts and treated with different fusion proteins.

[0402] Figures 14A-14E Shown are the results of flow cytometric analysis of selected immune cell populations within harvested tumors in a mouse syngeneic model.

[0403] Fig.15A Schematic representation of asymmetric IL-2 Fc fusion proteins containing an ECM targeting sequence and either a single mask or a double mask is shown.

[0404] Fig. 15B Shown are the results of SDS-PAGE analysis of asymmetric IL-2 Fc fusion protein.

[0405] Figure 15C-15U Shown are the results of a HEK Blue IL-2 assay measuring the IL-2 activity of specific asymmetric IL-2 Fc fusion proteins with and without treatment with MMPs, respectively.

[0406] Figure 15V-15X Shown are the results of assays measuring the binding of different asymmetric IL-2 Fc fusion proteins to heparin and fibronectin in the presence or absence of heparin or fibronectin binding sites.

[0407] Figure 15Y Shown are the results of an assay measuring the binding of different asymmetric IL-2 Fc fusion proteins to collagen in the presence or absence of a collagen binding site. DETAILED DESCRIPTION

[0408] This specification describes exemplary embodiments and applications of the present disclosure. However, the present disclosure is not limited to these exemplary embodiments and applications or the modes of operation of the exemplary embodiments and applications or the modes described in this article. The term "or" is used in an open sense, that is, equivalent to "and / or", unless the context clearly indicates otherwise. Note that, as used in this specification and the appended claims, the singular forms "a", "an", and "the", as well as any singular use of any term, include plural indicators unless explicitly and exactly limited to one indicator. As used herein, the terms "include", "comprise", and their grammatical variants are intended to be non-restrictive, so that the listing of items in the list does not exclude other similar items that can be replaced or added to the listed items. The partial divisions in this specification are provided only for the convenience of the reader and do not limit any combination of the elements discussed. In the event of any contradiction or conflict between the material incorporated by reference and the content of the explicit description provided herein, the content of the explicit description shall prevail.

[0409] Overview

[0410] Provided herein is a linker polypeptide comprising a first targeting sequence; a second targeting sequence; and a first linker located between the first targeting sequence and the second targeting sequence, the linker comprising a protease cleavable polypeptide sequence. In some embodiments, the linker polypeptide comprises a first active domain; a second active domain; a pharmacokinetic modulator; and a first linker located 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 located between the first active domain and the inhibitory polypeptide sequence, the linker comprising a protease cleavable polypeptide sequence; and a first targeting sequence.

[0411] Proteolysis of the protease-cleavable polypeptide sequence can release the first binding domain and / or the second binding domain, so that it can, for example, neutralize tumor antigens and / or activate immune cells. In addition, in some embodiments, each of the active domains can bind to a growth factor to reduce the extent to which the growth factor is active in vivo, such as stimulating cancer cell growth.

[0412] In some embodiments, the protease cleavable polypeptide sequence can be cleaved by a protease 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 metalloproteinase (MMP) cleavable linker, such as any one of the MMP cleavable linkers described herein. Without wishing to be bound by any particular theory, increased expression and / or activation of proteases (including but not limited to MMPs) in the tumor microenvironment (TME) can provide a mechanism for achieving selectivity or preferential activation of linker polypeptides at or near the tumor site. Certain protease cleavable polypeptide sequences described herein are considered to be particularly suitable for achieving such selectivity or preferential activation.

[0413] In other embodiments, the first targeting sequence and / or the second targeting sequence bind to an extracellular matrix component, an integrin or a syndecan, or are configured to bind to fibronectin in a pH-sensitive manner. In some embodiments, the targeting sequence is a targeting sequence as described herein, for example, a targeting sequence configured to bind to an extracellular matrix component, heparin, an integrin or a syndecan; or a targeting sequence configured to bind to an extracellular matrix component, heparin, IgB (CD79b), an integrin, a cadherin, a heparan sulfate proteoglycan, a syndecan or a fibronectin in a pH-sensitive manner; or a targeting sequence including a sequence of any one of SEQ ID NO: 179-665. The targeting sequence can 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 a higher level in TME and / or can be cleaved by MMP.

[0414] In some embodiments, a pharmacokinetic modulator can, for example, extend the half-life of a linker polypeptide.

[0415] The sequences of exemplary components 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 "(Pip)" means piperidine. "(Hof)" means homophenylalanine. "(Cit)" means citrulline. "(Et)" means ethionine. "C(me)" means methylcysteine. In some sequences, underscores are used to indicate mutation positions.

[0416] The present disclosure further provides the use of these linker polypeptides, for example, for treating cancer. In some embodiments, the linker polypeptide is selectively or preferentially cleaved in the tumor microenvironment, which can produce beneficial effects, for example, the recruitment and / or activation of immune cells near the tumor is improved, and / or the systemic exposure of certain components of the linker polypeptide is reduced.

[0417] Table 1: Sequence listing of linker polypeptides and their components

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

[0448]

[0449]

[0450]

[0451]

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469]

[0470]

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

[0496]

[0497]

[0498]

[0499]

[0500]

[0501]

[0502]

[0503]

[0504]

[0505]

[0506]

[0507]

[0508]

[0509]

[0510]

[0511]

[0512]

[0513]

[0514]

[0515]

[0516]

[0517]

[0518]

[0519]

[0520]

[0521]

[0522]

[0523]

[0524]

[0525]

[0526]

[0527]

[0528]

[0529]

[0530]

[0531]

[0532]

[0533]

[0534]

[0535]

[0536]

[0537]

[0538]

[0539]

[0540]

[0541]

[0542]

[0543]

[0544]

[0545]

[0546]

[0547]

[0548]

[0549]

[0550]

[0551]

[0552]

[0553]

[0554]

[0555]

[0556] Table 2: Targeting sequence list

[0557]

[0558]

[0559]

[0560]

[0561]

[0562]

[0563]

[0564]

[0565]

[0566]

[0567]

[0568]

[0569]

[0570]

[0571]

[0572]

[0573]

[0574]

[0575]

[0576]

[0577]

[0578]

[0579]

[0580] I. Definitions

[0581] As used herein, an "active domain" refers to a polypeptide or collection of polypeptides that has affinity for a target, which can be one or more polypeptides, nucleic acids, sugars, and / or combinations thereof. In some embodiments, the active domain is an agonist or antagonist of its target, or will cause and / or inhibit signal transduction associated with the target. The active domain does not need to have an exclusive affinity for the target, but only needs to have an affinity for the target that is significantly higher (e.g., 10 times or more) than the affinity of the domain for non-targets. The dissociation constant (K) between the active domain and the target is D ) can be in the range of pM, nM, μM or mM. The active domain can include one or more subdomains or subunits, each of which has a unique function and together have the function of the active domain. For example, an active domain comprising an IL-12 polypeptide sequence can include two subunits.

[0582] As used herein, "immunoglobulin antigen binding domain" refers to a domain that is an immunoglobulin or a fragment thereof, such as Fv, scFv, Fab or VHH. Exemplary immunoglobulin antigen binding domains are provided in Table 1.

[0583] As used herein, "receptor binding domain" refers to an active domain that is not an immunoglobulin antigen binding domain, such as a cytokine polypeptide sequence.

[0584] As used herein, a "cytokine polypeptide sequence" refers to a polypeptide sequence that has significant sequence identity to a wild-type cytokine (which may be part of a larger sequence, e.g., a fusion polypeptide), and the polypeptide sequence 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% 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 one, two, three, four, five, six, seven, eight, nine, or ten 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. By replacing "cytokine" with "IL-2," this definition applies to IL-2 polypeptide sequences.

[0585] As used herein, "inhibitory polypeptide sequence" refers to a polypeptide or a collection of polypeptides that inhibit the activity of an active domain in a linker polypeptide. The inhibitory polypeptide sequence can bind to or sterically hinder the active domain. In some embodiments, this binding is reduced or eliminated by acting an appropriate protease on the protease cleavable polypeptide sequence of the linker polypeptide. Exemplary inhibitory polypeptide sequences are provided in Table 1. The inhibitory polypeptide sequence can, for example, include a polypeptide having significant sequence identity with a portion of the wild-type target of the active domain or an immunoglobulin or a portion thereof (such as Fv, scFv, Fab or VHH).

[0586] As used herein, a "protease cleavable polypeptide sequence" is a sequence that serves as a substrate for cleavage by a protease. The protease cleavable polypeptide sequence is located in the linker polypeptide so that its cleavage releases one or more elements of the linker polypeptide from the remainder of the linker polypeptide, or reduces or eliminates the binding of an inhibitory polypeptide sequence to the active domain.

[0587] As used herein, a protease-cleavable polypeptide sequence is "recognized" by a given protease or class thereof if exposure of a polypeptide comprising the protease-cleavable polypeptide sequence to a protease under conditions permissive for cleavage by the protease results in an amount of cleavage that is significantly greater than the amount of cleavage of a control polypeptide having an unrelated sequence and / or if the protease-cleavable polypeptide sequence corresponds to a known recognition sequence for the protease (e.g., as described elsewhere herein for various exemplary proteases).

[0588] As used herein, a "pharmacokinetic modulator" is a moiety that prolongs the in vivo half-life of a linker polypeptide or an element of the linker polypeptide. A pharmacokinetic modulator can be a fusion domain in a linker polypeptide or can be a chemical entity that is linked post-translationally. The link can be, but is not necessarily, covalent. Exemplary pharmacokinetic modulator polypeptide sequences are provided in Table 1. Exemplary non-polypeptide pharmacokinetic modulators are described elsewhere herein.

[0589] As used herein, a "targeting sequence" is a sequence that localizes a greater portion of a linker polypeptide to an area of ​​interest (e.g., a tumor microenvironment). A targeting sequence can bind to an extracellular matrix component or other entity (e.g., an integrin or a syndecan) found in an area of ​​interest. Exemplary targeting sequences are provided in Table 2.

[0590] 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, cadherin, integrin, and syndecan) are not considered extracellular matrix components.

[0591] As used herein, "immunoglobulin constant domain" refers to a domain present in or having significant sequence identity with the constant region of an immunoglobulin such as IgG. Exemplary constant domains are C H 2 and C H 3 domains. Unless otherwise indicated, a linker polypeptide comprising an immunoglobulin constant domain may include more than one immunoglobulin constant domain. In some embodiments, the immunoglobulin constant domain has at least 80%, 85%, 90%, 95%, 97%, 98% or 99% 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 one, two, three, four, five, six, seven, eight, nine or ten 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 the same sequence as a wild-type immunoglobulin constant domain (e.g., a wild-type human immunoglobulin constant domain). Exemplary immunoglobulin constant domains are included in the sequences provided in Table 1. By using "C H 2" or "C H 3” replaces “immunoglobulin constant”, and this definition applies to C H 2 and C H 3 domains, provided that C H 2 domain sequence and non-C H 2 The percentage identity of the wild-type sequence of the immunoglobulin constant domain is no greater than that of C H2 domains with the wild-type sequence, and C H 3 domain sequence and non-C H 3 The percentage identity of the wild-type sequence of the immunoglobulin constant domain is no greater than that of C H These definitions also include domains with smaller truncations relative to the wild-type sequence, as long as the truncation does not substantially abolish the normal folding of the domain.

[0592] As used herein, "immunoglobulin Fc region" refers to a region comprising the C H 2 and C H The Fc region does not include the variable domain or C H 1 domain.

[0593] As used herein, a given component is "between" a first component and a second component if the first component is on one side of the given component and the second component is on the other side of the given component (e.g., in the primary sequence of a polypeptide). This term does not require immediate proximity. Thus, in the structure 1-2-3-4, 2 is between 1 and 4, and also between 1 and 3.

[0594] As used herein, "domain" may refer to a structural domain of a polypeptide or a functional assembly of at least one domain (but possibly multiple structural domains), depending on the context. H 2 Domain refers to a portion of a sequence that meets this condition. The immunoglobulin cytokine binding domain may include VH and VL structural domains.

[0595] As used herein, "denatured collagen" encompasses 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 in the native structure of full-length collagen.

[0596] As used herein, "configured to bind to in a pH-sensitive manner" means that a polypeptide sequence (e.g., a targeting sequence) exhibits different binding affinities for its binding partner depending on pH. For example, the affinity of a polypeptide sequence at a relatively acidic pH may be higher than the affinity at normal physiological pH (about 7.4). Higher affinity may occur at a pH below 7, for example, in the range of pH 5.5-7, 6-7, or 5.5-6.5 or below pH 6.

[0597] As used herein, "cytokine binding domain of a cytokine receptor" refers to the extracellular portion of a cytokine receptor or a fragment or truncation thereof that can bind 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% identity to the sequence of the cytokine binding domain of a wild-type cytokine receptor (e.g., 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. By replacing "cytokine" with "IL-2", "IL-10", "IL-15", "CXCL9", "CXCL10" and "TGF-β", respectively, 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.

[0598] As used herein, "immunoglobulin cytokine binding domain" refers to one or more immunoglobulin variable domains (e.g., VH and VL regions) that can bind to a cytokine polypeptide sequence. Exemplary sequences of cytokine binding immunoglobulin domains are provided in Table 1. By replacing "cytokine" with "IL-2," "IL-10," "IL-15," "CXCL9," "CXCL10," and "TGF-β," respectively, 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.

[0599] As used herein, a first element of a linker polypeptide being "closer to" a second element relative to a third element means that in the primary polypeptide sequence of the linker polypeptide, the first element is closer to the second element than to the third element, regardless of whether the first element is spatially closer to the second element than to the third element when the linker polypeptide is folded.

[0600] As used herein, "substantially" and its other grammatical forms mean sufficient to achieve the intended purpose. Thus, the term "substantially" allows for minor, insignificant variations from an absolute or perfect state, dimension, measurement, result, etc. as desired by one of ordinary skill in the art but does not significantly affect overall performance. When used with a numerical value or a parameter or characteristic that can be expressed as a numerical value, "substantially" means within ten percent.

[0601] As used herein, the term "plurality" may be 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0602] As used herein, a first sequence is said to "comprise a sequence having at least X% identity with a second sequence" if an alignment of the first sequence with a second sequence shows that X% or more of the positions of the second sequence match the first sequence as a whole. For example, the sequence QLYV (SEQ ID NO: 1168) includes a sequence having 100% identity with the sequence QLY, since the alignment would give 100% identity since there are matches to all three positions of the second sequence. Exemplary alignment algorithms are the Smith-Waterman algorithm and the Needleman-Wunsch algorithm, which are well known in the art. One skilled in the art will appreciate that the choice of algorithm and parameter settings is appropriate for a given pair of sequences to be aligned; for sequences that are generally similar in length and have an expected identity of >50% for amino acids or >75% for nucleotides, the Needleman-Wunsch algorithm with the default settings of the Needleman-Wunsch algorithm provided by the EBI on the www.ebi.ac.uk website server is generally appropriate.

[0603] As used herein, "subject" refers to any member of the animal kingdom. In some embodiments, "subject" refers to humans. In some embodiments, "subject" refers to non-human animals. In some embodiments, "subject" refers to primates. In some embodiments, "subject" includes but is not limited to mammals, birds, reptiles, amphibians, fish, insects and / or worms. In certain embodiments, non-human subjects are mammals (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates and / or pigs). In some embodiments, the subject can be a transgenic animal, a genetically engineered animal and / or a clone. In certain embodiments of the present invention, the subject is an adult, a teenager or an infant. In some embodiments, the term "individual" or "patient" is used and is intended to be interchangeable with "subject".

[0604] II. Linker polypeptide

[0605] The linker polypeptide may include a first targeting sequence; a second targeting sequence; and a first linker located between the first targeting sequence and the second targeting sequence, the linker including a protease cleavable polypeptide sequence. In some embodiments, the first targeting sequence and / or the second targeting sequence may each include two or more targeting subsequences that each bind to a target. In some embodiments, some or all of the two or more targeting subsequences may bind to the same target (e.g., tandem repeats). In some embodiments, the linker polypeptide includes a first active domain; a second active domain; a pharmacokinetic modulator; and a first linker located between the pharmacokinetic modulator and the first active domain, the first linker including a protease cleavable polypeptide sequence. In some embodiments, the linker polypeptide includes a first active domain; an inhibitory polypeptide sequence capable of blocking the activity of the first active domain; a first linker located between the first active domain and the inhibitory polypeptide sequence, the linker including a protease cleavable polypeptide sequence; and a first targeting sequence.

[0606] These elements of a linker polypeptide may be covalently linked to form a single polypeptide chain or may be present in multiple related polypeptide chains that may be linked non-covalently or covalently (eg, through one or more disulfide bonds).

[0607] In some embodiments, the linker polypeptide comprises a first polypeptide chain, the first polypeptide chain comprising a first active domain, a first domain of a pharmacokinetic modulator, and a first linker located between the first active domain and the first domain of the pharmacokinetic modulator, wherein the first active domain is at the C-terminus of the first domain of the pharmacokinetic modulator; a second polypeptide chain, the second polypeptide chain comprising a second domain of the pharmacokinetic modulator, an inhibitory polypeptide sequence capable of blocking the activity of the first active domain, and a second linker located 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.

[0608] In some embodiments, the linker polypeptide comprises a first polypeptide chain, the first polypeptide chain comprising a first active domain, a first domain of a pharmacokinetic modulator, and a first linker located between the first active domain and the first domain of the pharmacokinetic modulator, wherein the first active domain is at the N-terminus of the first domain of the pharmacokinetic modulator; a second polypeptide chain, the second polypeptide chain comprising a second domain of the pharmacokinetic modulator, an inhibitory polypeptide sequence capable of blocking the activity of the first active domain, and a second linker located 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.

[0609] A. Active domain

[0610] 1. Immunoglobulin antigen binding domain

[0611] 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.

[0612] 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 a Fv, scFv, Fab or VHH. The immunoglobulin antigen binding domain can be humanized or fully human.

[0613] In some embodiments, the immunoglobulin antigen binding domain binds to one or more sequences selected from the group consisting of cancer cell surface antigen sequences, growth factor sequences, and growth factor receptor sequences.

[0614] Under physiological conditions, cells receive signals from surrounding tissues in the form of growth factors. Growth factors can affect normal cell differentiation and constitutively activate growth-promoting pathways in cancer cells. The linker polypeptides disclosed herein can be combined with growth factors to promote the neutralization of the activity of growth factors at least to a certain extent, for example, near a tumor. Therefore, in some embodiments, the linker polypeptides disclosed herein can reduce the growth-promoting signaling received by cancer cells and stromal cells (including fibroblasts and endothelial cells) through immunoglobulin antigen binding domains, while also activating or recruiting immune cells to tumors. In some embodiments, immunoglobulin antigen binding domains can also promote the localization of linker polypeptides to tissues that specifically express specific growth factors or tissues that express specific growth factors in high amounts, for example, in and around tumors.

[0615] Growth factor receptors are typically transmembrane proteins that bind to specific growth factors and transmit instructions transmitted by the factor outside the cell to the intracellular space. In general, growth factor receptors include an extracellular domain, a transmembrane domain, and a cytoplasmic domain. In some embodiments, the linker polypeptides disclosed herein can inhibit the binding of growth factors to growth factor receptors through immunoglobulin antigen binding domains. This can promote a reduction in signaling of growth factors, at least to some extent, for example, near tumors.

[0616] In some embodiments, one or each immunoglobulin antigen binding domain of the first immunoglobulin antigen binding domain and the second immunoglobulin antigen binding domain of the joint polypeptide is independently configured to bind to the HER2 sequence. In some embodiments, one or each immunoglobulin antigen binding domain of the first immunoglobulin antigen binding domain and the second immunoglobulin antigen binding domain of the joint polypeptide independently includes: a hypervariable region (HVR) HVR-1, HVR-2 and HVR-3 in the VH region of the amino acid sequence of SEQ IDNO:910 and a VL region of HVR-1, HVR-2 and HVR-3 in the VL region of the amino acid sequence of SEQ IDNO:909. In general, one skilled in the art can identify HVRs in VH and VL sequences, for example, by assigning amino acids to framework and HVR domains within VH and VL sequences according to the following definitions: Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, Public Health Service (PHS), National Institutes of Health (NIH), NIH Publication No. 91-3242, 1991. Other numbering systems for amino acids in immunoglobulin chains include: IMGT TM(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 and second immunoglobulin antigen binding domains 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 and second immunoglobulin antigen binding domains independently comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 909 or 910. In some embodiments, one or each of the first and second immunoglobulin antigen binding domains independently is an antigen binding domain of trastuzumab.

[0617] In some embodiments, one or each of the first and second immunoglobulin antigen binding domains of the linker polypeptide is independently configured to bind to an EGFR extracellular domain sequence. In some embodiments, each of the first and second immunoglobulin antigen binding domains independently comprises: a VH region comprising HVR-1, HVR-2, and HVR-3 in a VH region comprising an amino acid sequence of SEQ ID NO: 914, and a VL region comprising HVR-1, HVR-2, and HVR-3 in a VL region comprising an amino acid sequence of SEQ ID NO: 913. In general, one skilled in the art can identify HVRs in VH and VL sequences, for example, by assigning amino acids to framework and HVR domains within VH and VL sequences according to the following definitions: Kabat et al., Protein Sequences of Immunological Significance, 5th edition, U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, NIH Publication No. 91-3242, 1991. Other numbering systems for amino acids in immunoglobulin chains include: IMGT TM(International Immunogenetics Information System; Lefranc et al., Developmental and Comparative Immunology 29:185-203; 2005) and AHo (Honegger and Pluckthun, Journal of Molecular Biology 309(3):657-670; 2001). In some embodiments, one or each of the first and second immunoglobulin antigen binding domains 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 and second immunoglobulin antigen binding domains independently comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% 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 an antigen binding domain of cetuximab.

[0618] In some embodiments, one or each of the first and second immunoglobulin antigen binding domains 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 and second immunoglobulin antigen binding domains independently comprises: a VH region comprising HVR-1, HVR-2, and HVR-3 in a VH region comprising an amino acid sequence of SEQ ID NO: 917, and a VL region comprising HVR-1, HVR-2, and HVR-3 in a VL region comprising an amino acid sequence of SEQ ID NO: 918. In general, one skilled in the art can identify HVRs in VH and VL sequences, for example, by assigning amino acids to framework and HVR domains within VH and VL sequences according to the following definitions: Kabat et al., Protein Sequences of Immunological Significance, 5th edition, U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, NIH Publication No. 91-3242, 1991. Other numbering systems for amino acids in immunoglobulin chains include: IMGT TM(International Immunogenetics Information System; Lefranc et al., Developmental and Comparative Immunology 29:185-203; 2005) and AHo (Honegger and Pluckthun, Journal of Molecular Biology 309(3):657-670; 2001). In some embodiments, one or each of the first and second immunoglobulin antigen binding domains 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 and second immunoglobulin antigen binding domains independently comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical 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 is independently an antigen binding domain of nivolumab.

[0619] In some embodiments, one or each of the first and second immunoglobulin antigen binding domains 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 and second immunoglobulin antigen binding domains independently comprises: a VH region comprising HVR-1, HVR-2, and HVR-3 in a VH region comprising an amino acid sequence of SEQ ID NO: 921, and a VL region comprising HVR-1, HVR-2, and HVR-3 in a VL region comprising an amino acid sequence of SEQ ID NO: 922. In general, one skilled in the art can identify HVRs in VH and VL sequences, for example, by assigning amino acids to framework and HVR domains within VH and VL sequences according to the following definitions: Kabat et al., Protein Sequences of Immunological Significance, 5th edition, U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, NIH Publication No. 91-3242, 1991. Other numbering systems for amino acids in immunoglobulin chains include: IMGT TM(International Immunogenetics Information System; Lefranc et al., Developmental and Comparative Immunology 29:185-203; 2005) and AHo (Honegger and Pluckthun, Journal of Molecular Biology 309(3):657-670; 2001). In some embodiments, one or each of the first and second immunoglobulin antigen binding domains 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 and second immunoglobulin antigen binding domains independently comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical 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 is independently an antigen binding domain of atezolizumab.

[0620] In some embodiments, one or each of the first and second immunoglobulin antigen binding domains of the linker polypeptide is independently configured to bind to a CD3 extracellular domain sequence. In some embodiments, one or each of the first and second immunoglobulin antigen binding domains independently comprises a VH region and a VL region, wherein the VH region comprises HVR-1, HVR-2, and HVR-3 in the VH region comprising any one of the amino acid sequences of SEQ ID NOs: 925, 929, 933, and 937, and the VL region comprises HVR-1, HVR-2, and HVR-3 in the VL region comprising any one of the amino acid sequences of SEQ ID NOs: 926, 930, 934, and 938. In general, one skilled in the art can identify HVRs in VH and VL sequences, for example, by assigning amino acids to framework and HVR domains within VH and VL sequences according to the following definitions: Kabat et al., Protein Sequences of Immunological Significance, 5th Edition, U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, NIH Publication No. 91-3242, 1991. Other numbering systems for amino acids in immunoglobulin chains include: IMGT TM(International Immunogenetics Information System; Lefranc et al., Developmental and Comparative Immunology 29:185-203; 2005) and AHo (Honegger and Pluckthun, Journal of Mol. Biol. 309(3):657-670; 2001). In some embodiments, one or each of the first and second immunoglobulin antigen binding domains independently comprises: 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. In some embodiments, one or each of the first and second immunoglobulin antigen binding domains independently comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 925, 926, 929, 930, 933, 934, 937 and 938. In some embodiments, one or each of the first and second immunoglobulin antigen binding domains independently is an antigen binding domain of teplizumab, muromonab, otexizumab or visilizumab.

[0621] 2. Receptor binding domain

[0622] In some embodiments, the first active domain comprises a receptor binding domain. The receptor binding domain may comprise, for example, a cytokine polypeptide sequence.

[0623] The receptor binding domain can be a wild-type receptor binding domain or a sequence having one or more differences with a wild-type receptor binding domain. In some embodiments, the receptor binding domain is a human receptor binding domain (which can be wild-type or can have one or more differences). In some embodiments, the receptor binding domain includes a modification that prevents the formation of a disulfide bond, and optionally includes a wild-type sequence in addition thereto. In some embodiments, the receptor binding domain has at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identity with the sequence of the wild-type receptor binding domain in Table 1 or with the receptor binding domain. In some embodiments, the receptor binding domain is a dimeric receptor binding domain, for example, a heterodimeric cytokine. In some embodiments, the receptor binding domain is a homodimeric receptor binding domain, for example, a homodimeric cytokine. The monomer can be connected as a fusion protein, for example, by a joint, or by a covalent bond (e.g., a disulfide bond) or by a non-covalent interaction. In some embodiments, the receptor binding domain is an interleukin polypeptide sequence. In some embodiments, the receptor binding domain can bind to a receptor comprising CD132. In some embodiments, the receptor binding domain can bind to a receptor comprising CD122. In some embodiments, the receptor binding domain can bind to a receptor comprising CD25.

[0624] In some embodiments, the receptor binding domain is an IL-2 polypeptide sequence. The IL-2 polypeptide sequence can be a wild-type IL-2 polypeptide sequence or a sequence having one or more differences from a wild-type IL-2 polypeptide sequence. In some embodiments, the IL-2 polypeptide sequence is a human IL-2 polypeptide sequence (which can be wild-type or can have one or more differences). In some embodiments, IL-2 includes modifications that prevent disulfide bond formation (e.g., aldesleukin (commercially known as ) sequence), and optionally in addition to the wild-type sequence. In some embodiments, the IL-2 polypeptide sequence is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of the wild-type IL-2 polypeptide sequence or the IL-2 polypeptide sequence in Table 1.

[0625] In some embodiments, the IL-2 polypeptide sequence comprises the sequence of any one of SEQ ID NOs: 1 to 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.

[0626] 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 having one or more differences from a wild-type IL-10 polypeptide sequence. In some embodiments, the IL-10 polypeptide sequence is a human IL-10 polypeptide sequence (which may be wild-type or may have one or more differences). In some embodiments, IL-10 includes modifications that prevent disulfide bond formation, and optionally includes a wild-type sequence in addition thereto. In some embodiments, the IL-10 polypeptide sequence has at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to a sequence of a wild-type IL-10 polypeptide sequence or an IL-10 polypeptide sequence in Table 1. In some embodiments, the IL-10 polypeptide sequence includes the sequence of SEQ ID NO: 900.

[0627] 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 having one or more differences from a 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 may have one or more differences). In some embodiments, IL-15 includes modifications that prevent disulfide bond formation, and optionally includes a wild-type sequence in addition thereto. In some embodiments, the IL-15 polypeptide sequence has at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the sequence of a wild-type IL-15 polypeptide sequence or an IL-15 polypeptide sequence in Table 1. In some embodiments, the IL-15 polypeptide sequence includes the sequence of SEQ ID NO: 901.

[0628] 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 having one or more differences with a wild-type CXCL9 polypeptide sequence. In some embodiments, the CXCL9 polypeptide sequence is a human CXCL9 polypeptide sequence (which may be wild-type or may have one or more differences). In some embodiments, CXCL9 includes modifications that prevent disulfide bond formation, and optionally includes a wild-type sequence in addition thereto. In some embodiments, the CXCL9 polypeptide sequence has at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the sequence of the wild-type CXCL9 polypeptide sequence or the CXCL9 polypeptide sequence in Table 1. In some embodiments, the CXCL9 polypeptide sequence includes the sequence of SEQ ID NO:902.

[0629] 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 having one or more differences with a wild-type CXCL10 polypeptide sequence. In some embodiments, the CXCL10 polypeptide sequence is a human CXCL10 polypeptide sequence (which may be wild-type or may have one or more differences). In some embodiments, CXCL10 includes modifications that prevent disulfide bond formation, and optionally includes a wild-type sequence in addition thereto. In some embodiments, the CXCL10 polypeptide sequence has at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the sequence of the wild-type CXCL10 polypeptide sequence or the CXCL10 polypeptide sequence in Table 1. In some embodiments, the CXCL10 polypeptide sequence includes the sequence of SEQ ID NO: 903.

[0630] 3. Size of the active domain

[0631] In some embodiments, the molecular weight of one or each of the first active domain and the second active domain is independently about or less than 14kDa. In some embodiments, the molecular weight is about 12kDa to about 14kDa. In some embodiments, the molecular weight is about 10kDa to about 12kDa. In some embodiments, the molecular weight is about 8kDa to about 10kDa. In some embodiments, the molecular weight is about 6kDa to about 8kDa. In some embodiments, the molecular weight is about 4kDa to about 6kDa. In some embodiments, the molecular weight is about 2kDa to about 4kDa. In some embodiments, the molecular weight is about 800Da to about 2kDa.

[0632] In some embodiments, the molecular weight of one or each of the first active domain and the second active domain is independently about or greater than 16kDa. In some embodiments, the molecular weight is about 16kDa to about 18kDa. In some embodiments, the molecular weight is about 18kDa to about 20kDa. In some embodiments, the molecular weight is about 20kDa to about 22kDa. In some embodiments, the molecular weight is about 22kDa to about 24kDa. In some embodiments, the molecular weight is about 24kDa to about 26kDa. In some embodiments, the molecular weight is about 26kDa to about 28kDa. In some embodiments, the molecular weight is about 28kDa to about 30kDa. In some embodiments, the molecular weight is about 30kDa to about 50kDa. In some embodiments, the molecular weight is about 50kDa to about 100kDa. In some embodiments, the molecular weight is about 100kDa to about 150kDa. In some embodiments, the molecular weight is about 150kDa to about 200kDa. In some embodiments, the molecular weight is about 200kDa to about 250kDa. In some embodiments, the molecular weight is from about 250 kDa to about 300 kDa.

[0633] B. Inhibitory polypeptide sequences

[0634] 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. In some embodiments, the linker polypeptide further comprises a second linker located between the receptor binding domain and the inhibitory polypeptide sequence, the second linker comprising a protease cleavable polypeptide sequence.

[0635] Various types of inhibitory polypeptide sequences can be used in the joint polypeptides according to the present disclosure. In some embodiments, the inhibitory polypeptide sequence is a sequence that binds to the active domain, such as a ligand binding domain or an immunoglobulin domain from a receptor. In some embodiments, the inhibitory polypeptide sequence is a steric blocker, that is, a sequence that sterically hinders the active domain. For example, a steric blocker can be an immunoglobulin Fc region, an albumin domain, or other relatively inert domains, which can be placed near the active domain to make the active domain less accessible until the active domain is released from the inhibitory polypeptide sequence by cleavage. In some embodiments, the inhibitory polypeptide sequence interferes with the binding between the first active domain and the receptor of the first active domain and / or the binding between the second active domain and the receptor of the second active domain. In some embodiments, the inhibitory polypeptide sequence and the pharmacokinetic modulator are different elements of the joint polypeptide. In some embodiments, the inhibitory polypeptide sequence includes at least a portion of the pharmacokinetic modulator.

[0636] In some embodiments, the inhibitory polypeptide sequence includes 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 provided as an extracellular portion of a 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% identity to the sequence of the wild-type cytokine binding domain of a cytokine receptor (e.g., the wild-type cytokine binding domain of a human cytokine receptor).

[0637] 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% identity to the sequence of the wild-type fibronectin cytokine binding domain of a cytokine receptor (e.g., a wild-type human fibronectin cytokine binding domain).

[0638] In some embodiments, the inhibitory polypeptide sequence comprises the same as SEQ ID NO: 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 and 988, 989, 990, 991 and 992, 999 and 1000, 1001, 1002, 1003 and 1004, 1005, 1006, 1008 and 1010 have at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to any one of the sequences (wherein the SEQ ID NOs are linked by "and" ID NO pairs indicate VH and VL pairs that together can 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 includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 1011 or 1012. In some embodiments, the inhibitory polypeptide sequence includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of any one of SEQ ID NO: 1016-1019. In some embodiments, the inhibitory polypeptide sequence includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 1020, 1021 or 1023. In any of the foregoing embodiments, the VH and VL domains may include the amino acid sequences of the referenced SEQ ID NO: 1020, 1021 or 1023. The CDRs may be identified by any suitable method, such as that described in Kabat (e.g., Kabat et al., (5th ed. 1991) Protein Sequences of Immunological Significance), which may be obtained from

[0639] books.google.co.uk / books? id=3jMvZYW2ZtwC&lpg=PA1137-IA1&pg=PP1#v=onepage&q&f=false) or Chothia (as described in Al-Lazikani et al., (1997) JMB 273, 927-948). In some embodiments, the inhibitory polypeptide sequence comprises SEQ ID NO: 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, 975 and 976 6, 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 comprises SEQ ID NO: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, 975 75 and 976, 977, 978 and 979, 980, 981 and 982, 983, 984 and 985, 986, 987 and 988, 989, 990, 9 The sequence of any one of 91 and 992, 999 and 1000, 1001, 1002, 1003 and 1004, 1005, 1006, 1008 and 1010.

[0640] In some embodiments, the inhibitory polypeptide sequence comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to the sequence of any one of SEQ ID NOs: 850-856 and 863-870. In any of the foregoing embodiments, the VHH domain may comprise CDRs identical to the CDRs of any one of SEQ ID NOs: 850-856 and 863-870. In some embodiments, the inhibitory polypeptide sequence comprises a VHH comprising the CDRs of any one of SEQ ID NOs: 850-856 and 863-870. In some embodiments, the inhibitory polypeptide sequence comprises the sequence of any one of SEQ ID NOs: 850-856 and 863-870.

[0641] In some embodiments, the cytokine binding domain can be an immunoglobulin cytokine binding domain. In some embodiments, the immunoglobulin cytokine binding domain includes a VH region and a VL region that bind to the cytokine. In some embodiments, the immunoglobulin cytokine binding domain can be Fv, scFv, Fab, VHH, or other immunoglobulin sequences that have antigen binding activity to the cytokine polypeptide sequence. VHH antibodies (or nanobodies) are antigen binding fragments of heavy chain antibodies only.

[0642] Additional examples of inhibitory polypeptide sequences that can be provided with a cytokine polypeptide sequence that inhibits a linker polypeptide are anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, lipocallin, and CTLA4 scaffolds.

[0643] In a linker polypeptide comprising an IL-2 polypeptide sequence, the inhibitory polypeptide sequence can be any type of IL-2 inhibitory polypeptide sequence described above. In some embodiments, the IL-2 inhibitory polypeptide sequence is an immunoglobulin IL-2 inhibitory polypeptide sequence.

[0644] 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.

[0645] In some embodiments, the IL-2 binding immunoglobulin domain is a scFv. In some embodiments, the IL-2 binding immunoglobulin domain includes a set of six anti-IL-2 hypervariable regions (HVRs) shown in Table 1 (e.g., SEQ ID NOs: 34-39 or 750-755). In some embodiments, the IL-2 binding immunoglobulin domain includes a set of anti-IL-2 VH and VL regions, the region including a sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identity with a sequence of a set of anti-IL-2 VH and VL regions including the sequences shown in Table 1, as a separate sequence or as part of an scFv. In some embodiments, the IL-2 binding immunoglobulin domain includes a set of anti-IL-2 VH and VL regions, the region having a sequence of a set of anti-IL-2 VH and VL sequences shown in Table 1, as a separate sequence or as part of an scFv.

[0646] Exemplary IL-2 inhibitory polypeptide sequences include SEQ ID NOs: 10-31, 40-51, 747, and 850-856, and 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 includes an IL-2 binding immunoglobulin domain comprising a VH region comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 33 and a VL region comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical 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.

[0647] In some embodiments, the IL-2 binding immunoglobulin domain comprises a VH region and a VL region, wherein the VH region comprises hypervariable regions HVR-1, HVR-2 and HVR-3 having sequences of SEQ ID NOs: 37, 38 and 39, respectively, and the VL region comprises HVR-1, HVR-2 and HVR-3 having 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% identity to a sequence of SEQ ID NOs: 30 or 31. In some embodiments, the IL-2 binding immunoglobulin domain comprises a sequence of SEQ ID NOs: 30 or 31.

[0648] In some embodiments, the inhibitory polypeptide sequence comprises an IL-2 binding domain of an IL-2 receptor (IL-2R). In some embodiments, the IL-2R is a human IL-2R.

[0649] In a linker polypeptide comprising an IL-10 polypeptide sequence, the inhibitory polypeptide sequence can be any type of IL-10 inhibitory polypeptide sequence described above. In some embodiments, the IL-10 inhibitory polypeptide sequence is an immunoglobulin IL-10 inhibitory polypeptide sequence.

[0650] 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.

[0651] In some embodiments, the IL-10 binding immunoglobulin domain is a scFv. In some embodiments, the IL-10 binding immunoglobulin domain includes a set of six anti-IL-10 hypervariable regions (HVRs) shown in Table 1 (e.g., SEQ ID NOs: 942-944 and 946-948). In some embodiments, the IL-10 binding immunoglobulin domain includes a set of anti-IL-10 VH and VL regions, the region including a sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identity with a sequence of a set of anti-IL-10 VH and VL regions including the sequences shown in Table 1, as a separate sequence or as part of an scFv. In some embodiments, the IL-10 binding immunoglobulin domain includes a set of anti-IL-10 VH and VL regions, the region having a sequence of a set of anti-IL-10 VH and VL sequences shown in Table 1, as a separate sequence or as part of an scFv.

[0652] Exemplary IL-10 inhibitory polypeptide sequences include SEQ ID NOs: 939-948, 1011, and 1012. In some embodiments, the IL-10 inhibitory polypeptide sequence includes an IL-10 binding immunoglobulin domain comprising a VH region comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 945 and a VL region comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical 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.

[0653] In some embodiments, the IL-10 binding immunoglobulin domain comprises a VH region and a VL region, wherein the VH region comprises hypervariable regions HVR-1, HVR-2 and HVR-3 having sequences of SEQ ID NOs: 946, 947 and 948, respectively, and the VL region comprises HVR-1, HVR-2 and HVR-3 having 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% identity to a sequence of SEQ ID NOs: 939 or 940. In some embodiments, the IL-10 binding immunoglobulin domain comprises a sequence of SEQ ID NOs: 939 or 940.

[0654] In some embodiments, the inhibitory polypeptide sequence comprises an IL-10 binding domain of an IL-10 receptor (IL-10R). In some embodiments, the IL-10R is a human IL-10R.

[0655] In the linker polypeptide comprising an IL-15 polypeptide sequence, the inhibitory polypeptide sequence can be any type of IL-15 inhibitory polypeptide sequence described above. In some embodiments, the IL-15 inhibitory polypeptide sequence is an immunoglobulin IL-15 inhibitory polypeptide sequence.

[0656] 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.

[0657] In some embodiments, the IL-15 binding immunoglobulin domain is a scFv. In some embodiments, the IL-15 binding immunoglobulin domain comprises: a VH region comprising HVR-1, HVR-2, and HVR-3 in a VH region comprising an 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 in a VL region comprising an amino acid sequence of any one of SEQ ID NOs: 952, 954, 958, 961, 964, 967, 970, 973, 976, 979, 982, 984, and 987. In general, one skilled in the art can identify HVRs in VH and VL sequences, for example, by assigning amino acids to framework and HVR domains within VH and VL sequences according to the following definitions: Kabat et al., Protein Sequences of Immunological Significance, 5th Edition, U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, NIH Publication No. 91-3242, 1991. Other numbering systems for amino acids in immunoglobulin chains include: IMGT TM (International Immunogenetics Information System; Lefranc et al., Developmental and Comparative Immunology 29:185-203; 2005) and AHo (Honegger and Pluckthun, Journal of 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 VL regions comprising a sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to a sequence of a set of anti-IL-15 VH and VL regions comprising the sequences shown in Table 1, as a separate sequence or as part of an scFv. In some embodiments, the IL-15 binding immunoglobulin domain comprises a set of anti-IL-15 VH and VL regions having a sequence of a set of anti-IL-15 VH and VL sequences shown in Table 1, as a separate sequence or as part of an scFv.

[0658] 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 at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical 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 at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical 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.

[0659] In some embodiments, the inhibitory polypeptide sequence comprises an IL-15 binding domain of an IL-15 receptor (IL-15R). In some embodiments, the IL-15R is a human IL-15R.

[0660] In the linker polypeptide comprising a CXCL9 polypeptide sequence, the inhibitory polypeptide sequence can be any type of CXCL9 inhibitory polypeptide sequence described above. In some embodiments, the CXCL9 inhibitory polypeptide sequence is an immunoglobulin CXCL9 inhibitory polypeptide sequence.

[0661] 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.

[0662] Exemplary CXCL9 inhibitory polypeptide sequences include SEQ ID NOs: 1020-1021. In some embodiments, the inhibitory polypeptide sequence includes the CXCL9 binding domain of the CXCL9 receptor (CXCR3). In some embodiments, the CXCR3 is human CXCR3.

[0663] In the linker polypeptide comprising a CXCL10 polypeptide sequence, the inhibitory polypeptide sequence may be any type of CXCL10 inhibitory polypeptide sequence described above. In some embodiments, the CXCL10 inhibitory polypeptide sequence is an immunoglobulin CXCL10 inhibitory polypeptide sequence.

[0664] 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.

[0665] In some embodiments, the CXCL10 binding immunoglobulin domain is an scFv. In some embodiments, the CXCL10 binding immunoglobulin domain includes a set of six anti-CXCL10 hypervariable regions (HVRs) shown in Table 1 (e.g., SEQ ID NOs: 993-998). In some embodiments, the CXCL10 binding immunoglobulin domain includes a set of anti-CXCL10 VH and VL regions, the regions including sequences having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identity with a set of anti-CXCL10 VH and VL regions including the sequences shown in Table 1, as a separate sequence or as part of an scFv. In some embodiments, the CXCL10 binding immunoglobulin domain includes a set of anti-CXCL10 VH and VL regions, the regions having a sequence of a set of anti-CXCL10 VH and VL sequences shown in Table 1, as a separate sequence or as part of an scFv.

[0666] Exemplary CXCL10 inhibitory polypeptide sequences include SEQ ID NOs: 989 and 990. In some embodiments, the CXCL10 inhibitory polypeptide sequence includes a CXCL10 binding immunoglobulin domain comprising a VH region comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 991 and a VL region comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical 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.

[0667] In some embodiments, the CXCL10 binding immunoglobulin domain comprises a VH region and a VL region, wherein the VH region comprises hypervariable regions HVR-1, HVR-2 and HVR-3 having sequences of SEQ ID NOs: 993, 994 and 995, respectively, and the VL region comprises HVR-1, HVR-2 and HVR-3 having 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% identity to a sequence of SEQ ID NOs: 989 or 990. In some embodiments, the CXCL10 binding immunoglobulin domain comprises a sequence of SEQ ID NOs: 989 or 990.

[0668] In some embodiments, the inhibitory polypeptide sequence comprises a CXCL10 binding domain of a CXCL10 receptor (CXCR3). In some embodiments, the CXCR3 is human CXCR3.

[0669] C. Connector

[0670] According to the present disclosure, various joints can be used. In many embodiments, the joint can be used to connect any two domains in the joint polypeptide. In some embodiments, the joint polypeptide includes a joint. In other embodiments, the joint polypeptide may include two or more joints. In some embodiments, the first joint is present between the pharmacokinetic modulator and the first active domain. In some embodiments, the second joint is present between the receptor binding domain and the inhibitory polypeptide sequence. In some embodiments, the first joint and / or the second joint include a protease cleavable polypeptide sequence. In some embodiments, after the protease cleavable polypeptide sequence is cleaved, the first active domain and / or the second active domain are released from the remainder of the joint polypeptide. In some embodiments, the joint polypeptide includes multiple protease cleavable polypeptide sequences.

[0671] In these embodiments, different linkers can be used to provide different release properties for different connection domains. For example, the linker used to release a target binding domain such as an immunoglobulin antigen binding domain can be different from the linker used to release a receptor binding domain such as a cytokine polypeptide sequence. The linker can include any exemplary linker sequence disclosed herein (e.g., in Table 1).

[0672] 1. Protease cleavable sequence

[0673] The protease cleavable sequence can include sequences that can be cleaved and / or recognized by various types of proteases, such as metalloproteinases, serine proteases, cysteine ​​proteases, aspartic acid proteases, threonine proteases, glutamic acid proteases, gelatinases, asparagine peptide cleaving enzymes, cathepsins, kallikreins, plasmins, collagenases, hK1, hK10, hK15, matrix proteins, factor Xa, chymotrypsin-like proteases, trypsin-like proteases, elastase-like proteases, subtilisin-like proteases, actinidin, bromelain, calpain, caspase, Mir 1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matrices, legumin, Plasmodium plasma protease, Nepenthes protease, metalloexopeptidases, metalloendopeptidases, ADAM10, ADAM 17, ADAM 12, 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 cleavable sequence includes a sequence of any one of those in Table 1 (e.g., SEQ ID NOs: 80-94 and 701-742) or a variant with one or two mismatches relative to a sequence of any one of those in Table 1 (e.g., SEQ ID NOs: 80-90 and 701-742). Proteases generally do not require an exact copy of the recognition sequence, and thus the exemplary sequences may vary in one or more portions of their amino acid positions. In some embodiments, the protease cleavable sequence includes a sequence that matches a MMP consensus sequence, such as any one of SEQ ID NOs: 91-94.

[0674] Those skilled in the art will be familiar with additional sequences recognized by these types of proteases.

[0675] I. Matrix metalloproteinase cleavable sequence

[0676] In some embodiments, the protease cleavable sequence is a matrix metalloproteinase (MMP) cleavable sequence and is recognized by a matrix metalloproteinase. Exemplary MMP cleavable sequences are provided in Table 1. In some embodiments, the MMP cleavable sequence can be cleaved and / or recognized by one or more of a plurality of MMPs and / or MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MMP-13 and / or MMP-14. In some embodiments, the protease cleavable polypeptide sequence can be cleaved and / or recognized by two, three, four, five, six or seven of MMP-2, MMP-7, MMP-8, MMP-9, MMP-12, MMP-13 and MMP-14. Table 1, e.g., SEQ ID NOs: 80-90, provides exemplary MMP cleavable sequences.

[0677] In some embodiments, the protease cleavable polypeptide sequence includes the sequence of any one of SEQ ID NO:80-90. In some embodiments, the protease cleavable polypeptide sequence includes the sequence of SEQ ID NO:80 or a variant sequence with one or two mismatches relative to the sequence. In some embodiments, the protease cleavable polypeptide sequence includes the sequence of SEQ ID NO:81 or a variant sequence with one or two mismatches relative to the sequence. In some embodiments, the protease cleavable polypeptide sequence includes the sequence of SEQ ID NO:82 or a variant sequence with one or two mismatches relative to the sequence. In some embodiments, the protease cleavable polypeptide sequence includes the sequence of SEQ ID NO:83 or a variant sequence with one or two mismatches relative to the sequence. In some embodiments, the protease cleavable polypeptide sequence includes the sequence of SEQ ID NO:84 or a variant sequence with one or two mismatches relative to the sequence. In some embodiments, the protease cleavable polypeptide sequence includes the sequence of SEQ ID NO:85 or a variant sequence with one or two mismatches relative to the sequence. In some embodiments, the protease cleavable polypeptide sequence includes the sequence of SEQ ID NO:86 or a variant sequence with one or two mismatches relative to the sequence. In some embodiments, the protease cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 87 or a variant sequence with one or two mismatches relative to the sequence. In some embodiments, the protease cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 88 or a variant sequence with one or two mismatches relative to the sequence. In some embodiments, the protease cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 89 or a variant sequence with one or two mismatches relative to the sequence. In some embodiments, the protease cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 90 or a variant sequence with one or two mismatches relative to the sequence. In some embodiments, the protease cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 91 or a variant sequence with one or two mismatches relative to the sequence. In some embodiments, the protease cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 92 or a variant sequence with one or two mismatches relative to the sequence. In some embodiments, the protease cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 93 or a variant sequence with one or two mismatches relative to the sequence. In some embodiments, the protease-cleavable polypeptide sequence comprises the sequence of SEQ ID NO: 94 or a variant sequence having one or two mismatches relative to the sequence.

[0678] D. Targeting sequence

[0679] 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 located between the receptor binding domain and a protease cleavable polypeptide sequence or one of a plurality of protease cleavable polypeptide sequences. In some embodiments, at least one linker in the first linker and the second linker comprises a targeting sequence, for example, one of the first and second targeting sequences, at least one targeting sequence, one of the first multiple targeting sequences, one of the second multiple targeting sequences, or one of the multiple targeting sequences. In some embodiments, the protease cleavable polypeptide sequence comprises a targeting sequence, for example, one of the first and second targeting sequences, the at least one targeting sequence, one of the first multiple targeting sequences, one of the second multiple targeting sequences, or one of the multiple targeting sequences.

[0680] 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 increases the serum half-life of the linker polypeptide. In general, the increase in serum half-life can be relative to, for example, the serum half-life of a linker polypeptide lacking 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. 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 synergistically increase the serum half-life of the linker polypeptide with another of the first and second targeting sequences, another of the at least one targeting sequence, another of the first plurality of targeting sequences, another of the second plurality of targeting sequences, or another of the plurality of targeting sequences. 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 synergistically increase the serum half-life of the linker polypeptide with a pharmacokinetic modulator. 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 independently increase the serum half-life of the linker polypeptide.

[0681] 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, when the serum half-life is greater than the serum half-life of a linker polypeptide that lacks a targeting sequence but is otherwise identical to the linker polypeptide, and when the increase is independent of any other increase derived from another targeting sequence, any of the above targeting sequences can independently increase the serum half-life of the linker polypeptide. In some embodiments, when the increase in serum half-life is greater than the sum of the increase derived from one targeting sequence and the increase derived from another targeting sequence, or the sum of the increase derived from one targeting sequence and the increase derived from a pharmacokinetic modifier, any of the above targeting sequences can synergistically increase the serum half-life of the linker polypeptide with another one of the targeting sequences or with a pharmacokinetic modifier.

[0682] The targeting sequence can promote the positioning, accumulation and / or retention of the linker polypeptide and / or the first active domain and / or the second active domain (e.g., after proteolysis of the protease cleavable sequence) in the region of interest (e.g., tumor microenvironment (TME)). The targeting sequence can be a sequence that is combined with an extracellular matrix component. Exemplary extracellular matrix components can include, for example, collagen or denatured collagen (in either case, the collagen can be collagen I, II, III or IV), 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.

[0683] 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 the following: IgB (CD79b), fibronectin, integrin, cadherin, heparan sulfate proteoglycan, and syndecan. In some embodiments, the targeting sequence binds to at least one integrin, such as 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 a MMP cleavable linker as described elsewhere herein, such as a MMP cleavable linker comprising any one of SEQ ID NOs: 80-90 or a variant having one or two mismatches relative to the sequence of any one of SEQ ID NOs: 80-90.

[0684] In some embodiments, the targeting sequence comprises a sequence shown in Table 2 (e.g., SEQ ID NOs: 179-665, such as any one of SEQ ID NOs: 179-640) or a variant having one or two mismatches relative to such a sequence.

[0685] 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, wherein the first targeting sequence is configured to bind to collagen IV and the second targeting sequence is configured to bind to heparin, or wherein the first targeting sequence is configured to bind to heparin and the second targeting sequence is configured to bind to collagen IV.

[0686] 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 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 is independently configured to bind to its 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 is independently configured to bind to its target with an affinity of 1 nM to 10 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 is independently configured to bind to its target with an affinity of 10 nM to 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 is independently configured to bind to its 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 is independently configured to bind to its 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 is independently configured to bind to its target with an affinity of 10 μM to 100 μM. In some embodiments, the affinity can be a dissociation constant (K). D ), which can be measured, for example, by surface plasmon resonance (SPR), enzyme-linked immunosorbent assay (ELISA), or polarization-modulated oblique-incidence reflectivity difference (OI-RD).

[0687] 1. pH-sensitive targeting sequence

[0688] In some embodiments, the targeting sequence is configured to bind to its target in a pH-sensitive manner. In some embodiments, the affinity of the targeting sequence to its target at a relatively acidic pH is higher than the affinity to its target at a normal physiological pH (about 7.4). Higher affinity may occur at a pH lower than 7, for example, in the range of pH 5.5-7, 6-7 or 5.5-6.5 or lower than pH 6. The presence of histidine in the targeting sequence can confer pH-sensitive binding. Without wishing to be bound by any particular theory, histidine is considered to be more likely to be protonated at a lower pH and can make binding to a negatively charged target more energetically favorable. Therefore, 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. Including a pH-sensitive targeting sequence can enhance the distinction between tumors and normal tissues by a linker polypeptide, so that the linker polypeptide is more preferentially retained in the tumor microenvironment compared to the normal extracellular matrix. Therefore, the pH-sensitive targeting element can further promote tumor-specific delivery of the linker polypeptide and thereby further reduce or eliminate toxicity that may be caused by the activity of the linker polypeptide in the normal extracellular matrix.

[0689] When it is desired to locate or retain the linker polypeptide and / or its cytokine polypeptide sequence in an area where the pH is different from the normal physiological pH, it may be useful to bind to the target in a pH-sensitive manner. For example, the tumor microenvironment may be more acidic than blood and / or healthy tissue. Therefore, binding to the target in a pH-sensitive manner can improve the retention of the linker polypeptide and / or its cytokine polypeptide sequence in the area of ​​interest, which can promote lower doses and / or reduce systemic exposure and / or side effects compared to doses that would otherwise be required.

[0690] In some embodiments, the targeting sequence is configured to bind to any target described herein in a pH-sensitive manner. In a specific embodiment, 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.

[0691] Exemplary targeting sequences (e.g., SEQ ID NOs: 641-663) that confer target binding in a pH-sensitive manner are provided in Table 2. In some embodiments, the targeting sequence comprises the sequence of any one of SEQ ID NOs: 641-663 or a variant having one or two mismatches relative to the sequence of any one of SEQ ID NOs: 641-663.

[0692] In some embodiments, the linker polypeptide includes a targeting sequence adjacent to a protease cleavable sequence. The targeting sequence and the protease cleavable sequence can be any of those described herein. An exemplary combination of a targeting sequence and a protease cleavable sequence is SEQ ID NO: 667-673.

[0693] E. Pharmacokinetic modulators

[0694] In some embodiments, the joint polypeptide includes a pharmacokinetic modulator. The pharmacokinetic modulator can be covalently or non-covalently associated with the joint polypeptide. The pharmacokinetic modulator can extend the half-life of the joint polypeptide, for example, so that less dosage is required and less joint polypeptide needs to be applied over time to achieve the desired result. Various forms of pharmacokinetic modulators are known in the art and can be used in the joint polypeptide of the present disclosure. In some embodiments, the pharmacokinetic modulator includes a polypeptide (see the following examples). In some embodiments, the pharmacokinetic modulator includes a non-polypeptide portion (e.g., polyethylene glycol, polysaccharide or hyaluronic acid). The non-polypeptide portion can be associated with the joint polypeptide using known methods, for example, conjugated with the joint polypeptide; for example, reactive amino acid residues can be used or the reactive amino acid residues can be added to the joint polypeptide to promote conjugation.

[0695] In some embodiments, the pharmacokinetic modulator changes the size, shape and / or charge of the linker polypeptide, for example, in such a way as to reduce clearance. For example, a negatively charged pharmacokinetic modulator can 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, for example, by increasing the hydrodynamic volume of the linker polypeptide.

[0696] In some embodiments, the linker polypeptide comprising a pharmacokinetic modulator (eg, any pharmacokinetic modulator described herein) has a molecular weight of at least 70 kDa, eg, at least 75 or 80 kDa.

[0697] For further discussion of various methods for providing pharmacokinetic modulators, see, e.g., Strohl, BioDrugs 29:215-19 (2015) and Podust et al., J. Controlled Release 240:52-66 (2016).

[0698] 1. Peptide pharmacokinetic modulators

[0699] In some embodiments, the pharmacokinetic modulator comprises a polypeptide, e.g., an immunoglobulin sequence (see exemplary embodiments below), albumin, CTP (a negatively charged carboxyl-terminal peptide of the chorionic gonadotropin beta chain that undergoes sialylation in vivo and in a suitable host cell), an inert polypeptide (e.g., an unstructured polypeptide such as XTEN, a polypeptide comprising residues Ala, Glu, Gly, Pro, Ser, and Thr), transferrin, a homologous amino acid polypeptide, or an elastin-like polypeptide.

[0700] Exemplary polypeptide sequences suitable for use as pharmacokinetic modulators (e.g., any one of SEQ ID NOs: 70-74) are provided in Table 1. In some embodiments, the pharmacokinetic modulator has at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to the sequence of a pharmacokinetic modulator in Table 1 (e.g., any one of SEQ ID NOs: 70-74).

[0701] In any embodiment in which the pharmacokinetic modulator comprises a polypeptide sequence from an organism, the polypeptide sequence can be a human polypeptide sequence.

[0702] 2. Immunoglobulin pharmacokinetic modulators

[0703] In some embodiments, the pharmacokinetic modulator comprises an immunoglobulin sequence, for example, 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.

[0704] The immunoglobulin sequence (e.g., at least a portion of one or more immunoglobulin constant domains or an 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 an Fc region) can be at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to a wild-type immunoglobulin sequence (e.g., at least a portion of one or more immunoglobulin constant domains or an Fc region), such as a sequence of a wild-type human immunoglobulin sequence. In any such embodiment, the immunoglobulin sequence can be an IgG sequence, such as at least a portion of one or more immunoglobulin constant domains or an Fc region thereof (e.g., IgG1, IgG2, IgG3 or IgG4, such as at least a portion of one or more immunoglobulin constant domains or an Fc region of any of these isotypes). Exemplary immunoglobulin pharmacokinetic modulator sequences include SEQ ID NOs: 70-74, 857, 858, 861 and 862 and combinations of SEQ ID NOs: 756 and 757; 75 and 77; 75 and 78; 76 and 77; 76 and 78; and 859 and 860.

[0705] In some embodiments, immunoglobulin pharmacokinetic modulator sequences (such as Fc regions) can achieve certain functions and effects by interacting with certain targets, as described in Table 3 below.

[0706] F. Growth factor binding polypeptide sequences and growth factor receptor binding polypeptide sequences

[0707] In some embodiments, the linker polypeptide comprises a growth factor binding polypeptide sequence or a growth factor receptor binding polypeptide sequence. Such sequences can serve as active domains.

[0708] In some embodiments, the growth factor binding polypeptide sequence includes a TGF-βR extracellular domain sequence. In some embodiments, the TGF-βR extracellular domain sequence includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 1022 or 1023.

[0709] In some embodiments, the growth factor binding polypeptide sequence includes 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 includes: a VH region including HVR-1, HVR-2 and HVR-3 of a VH region including an amino acid sequence of SEQ ID NO: 1008 and a VL region including HVR-1, HVR-2 and HVR-3 of a VL region including an amino acid sequence of SEQ ID NO: 1010. In general, one skilled in the art can identify HVRs in VH and VL sequences, for example, by assigning amino acids to frameworks and HVR domains within VH and VL sequences according to the following definitions: Kabat et al., Protein Sequences of Immunological Significance, 5th edition, U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, NIH Publication No. 91-3242, 1991. Other numbering systems for amino acids in immunoglobulin chains include: IMGT TM (International Immunogenetics Information System; Lefranc et al., Developmental and Comparative Immunology 29:185-203; 2005) and AHo (Honegger and Pluckthun, Journal of 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% 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 that is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the sequence of any one of SEQ ID NOs: 904-906.

[0710] In some embodiments, the growth factor receptor binding polypeptide sequence includes 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 includes: a VH region of HVR-1, HVR-2, and HVR-3 including a VH region comprising an amino acid sequence of SEQ ID NO: 999 or 1003, and a VL region of HVR-1, HVR-2, and HVR-3 including a VL region comprising an amino acid sequence of SEQ ID NO: 1000 or 1004. In some embodiments, the growth factor receptor binding immunoglobulin domain includes: a VH region comprising an amino acid sequence of SEQ ID NO: 999 or 1003, and a VL region comprising an amino acid sequence of SEQ ID NO: 1000 or 1004. In some embodiments, the growth factor receptor binding immunoglobulin domain comprises a sequence that is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequence of any one of SEQ ID NOs: 1001, 1002, 1005 and 1006.

[0711] Table 3. Pharmacokinetic modulators functions, effects and targets

[0712] Functional Mode Target Effect Antibody-dependent cellular cytotoxicity (ADCC) FcgR binding site Killing of cells bound by Fab Antibody-dependent cellular phagocytosis (ADCP) FcgR binding site Killing of cells bound by Fab Complement-dependent cytotoxicity (CDC) C1q binding site Killing of cells bound by Fab Antibody Drug Conjugate (ADC) Fab Killing of cells bound by Fab Fc Recycling FcRn binding site Half-life extension

[0713] A. Blockers

[0714] 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 active domain and the second active domain. In some embodiments, the blocking agent is conjugated to one or each of the first active domain and the second active domain via a protease cleavable polypeptide sequence.

[0715] Blockers can hinder the binding of an immunoglobulin antigen-binding domain to an antigen (e.g., a growth factor or a growth factor receptor). In some embodiments, the blocker is connected to the immunoglobulin antigen-binding domain via the N-terminus of the heavy or light chain of the immunoglobulin antigen-binding domain.

[0716] 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.

[0717] B. Chemotherapy drugs

[0718] In some embodiments, the linker polypeptide may include a chemotherapeutic drug or multiple chemotherapeutic drugs. The drug can be conjugated, for example, to different elements of the linker polypeptide. In some embodiments, the chemotherapeutic drug is conjugated to a pharmacokinetic modulator of the linker polypeptide.

[0719] In some embodiments, the chemotherapeutic drug is selected from altretamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, cyclohexyl lomustine, nitrogen mustard, melphalan, oxaliplatin, temozolomide, thiotepa, trabectedin, carmustine, cyclohexyl lomustine, streptozotocin, azacitidine, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cytarabine, decitabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, nelarabine, pemetrexed, pentostatin, and platinum. Latrexate, 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, tretinoin, arsenic trioxide, asparaginase, eribulin, hydroxyurea, ixabepilone, mitotane, omacetin, pegaspargase, procarbazine, romidep, and vorinostat.

[0720] III. Arrangement of components and their release

[0721] The description of the linker polypeptide components herein does not imply any particular order beyond that explicitly stated (e.g., a protease cleavable sequence may be explicitly stated to be between a cytokine polypeptide sequence and an inhibitory polypeptide sequence). The components of the linker polypeptide may be arranged in various ways to provide properties suitable for a particular use. The components of the linker polypeptide may all be in one polypeptide chain, or they may be in multiple polypeptide chains bridged by covalent bonds such as disulfide bonds.

[0722] For example, in some embodiments, where the pharmacokinetic modulator includes an Fc, one or more components (e.g., a chemotherapeutic drug) can be bound to one chain, while one or more other components can be bound to the other chain. The Fc can be a heterodimeric Fc, such as a knob-hole Fc (wherein one Fc chain includes a knob mutation and the other Fc chain includes a hole mutation). For an exemplary general discussion of knob mutations and hole mutations, see, e.g., Xu et al., mAbs 7: 1, 231-242 (2015). An exemplary knob mutation (e.g., for human IgG1 Fc) is K360E / K409W. An exemplary hole mutation (e.g., for human IgG1 Fc) is Q347R / D399V / F405T. See SEQ ID NOs: 756 and 757.

[0723] In some embodiments, some or all of the one or more protease cleavable polypeptide sequences can be at the C-terminus of the VH region, at the C-terminus of at least a portion of the CH1 domain, between the CH1 domain and the CH2 domain, at the N-terminus of at least a portion of the CH2 domain, at the N-terminus of the disulfide bond between the heavy chains, at the N-terminus of the disulfide bond within the CH2 domain, or at the N-terminus of the hinge region, or within the hinge region. In some embodiments, some or all of the one or more protease cleavable polypeptide sequences can be between the pharmacokinetic modulator and the second active domain, and / or between the blocking agent and one or each of the first active domain and the second active domain.

[0724] In some embodiments, the targeting sequence can be between the receptor binding domain and the one or more protease cleavable polypeptide sequences. In some embodiments, at least one of the first linker and the second linker comprises a targeting sequence, and / or the protease cleavable polypeptide sequence comprises a targeting sequence.

[0725] In some embodiments, the targeting sequence can be present on the same side of the protease cleavable polypeptide sequence as the receptor binding domain (e.g., a cytokine polypeptide sequence), which means that cleavage of the protease cleavable polypeptide sequence will not separate the targeting sequence from the receptor binding domain. Such embodiments can be used to facilitate localization or retention of both the linker polypeptide and the released receptor binding domain in an area of ​​interest, such as a tumor microenvironment.

[0726] In some embodiments, the targeting sequence can be present on the same side of the protease cleavable polypeptide sequence as the inhibitory polypeptide sequence, which means that cleavage of the protease cleavable polypeptide sequence will not separate the targeting sequence from the cytokine polypeptide sequence. Such embodiments can be used to provide a cytokine gradient from an area of ​​interest, or to provide such a gradient faster than when the targeting sequence is on the same side of the protease cleavable sequence.

[0727] 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, sequentially 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.

[0728] In some embodiments, the protease-cleavable polypeptide sequence is at the C-terminus of the first targeting sequence and at the C-terminus of the second targeting sequence. In some embodiments, the protease-cleavable polypeptide sequence is at the N-terminus of the first targeting sequence and at the N-terminus of the second targeting sequence. In some embodiments, the protease-cleavable polypeptide sequence is at the C-terminus of the first plurality of targeting sequences and at the N-terminus of the second plurality of targeting sequences. In some embodiments, the protease-cleavable polypeptide sequence is at the C-terminus of the plurality of targeting sequences and at the N-terminus of at least one targeting sequence. In some embodiments, the protease-cleavable polypeptide sequence is at the N-terminus of the plurality of targeting sequences and at the C-terminus of at least one targeting sequence. In some embodiments, the protease-cleavable polypeptide sequence is at the C-terminus of the first targeting sequence and at the C-terminus of the second targeting sequence, and not at the N-terminus of the targeting sequences. In some embodiments, the protease-cleavable polypeptide sequence is at the N-terminus of the first targeting sequence and at the N-terminus of the second targeting sequence, and not at the C-terminus of the targeting sequences.

[0729] In some embodiments, the joint polypeptide comprises a first active domain, a second active domain, a pharmacokinetic modulator, and a first joint between the pharmacokinetic modulator and the first active domain. In some embodiments, the first joint comprises a protease cleavable polypeptide sequence and optionally 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 joint polypeptide comprises a chemotherapeutic drug.

[0730] In some embodiments, after one or more protease cleavable polypeptide sequences are cleaved, the first active domain is released from the remainder of the joint polypeptide. In some embodiments, the joint polypeptide further includes a blocker that is conjugated with one or each of the protease cleavable polypeptide sequence and the first active domain and the second active domain. In some embodiments, the protease cleavable polypeptide sequence connected to the remainder of the first active domain and the joint polypeptide and the protease cleavable polypeptide sequence connecting the blocker to the active domain can be cleaved together (for example, by the same protease). In some embodiments, the protease cleavable polypeptide sequence connected to the remainder of the first active domain and the joint polypeptide and the protease cleavable polypeptide sequence connecting the blocker to the active domain can be cleaved separately (for example, by different proteases).

[0731] 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 optionally 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 that may include 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.

[0732] In some embodiments, after one or more protease cleavable polypeptide sequences are cleaved, the first active domain is released from the remainder of the joint polypeptide. In some embodiments, the first active domain includes a receptor binding domain that can include a cytokine polypeptide sequence, and the second active domain includes an immunoglobulin antigen binding domain. In some embodiments, the joint polypeptide further includes an inhibitory polypeptide sequence that can block the activity of the receptor binding domain; and a second joint between the receptor binding domain and the inhibitory polypeptide sequence, wherein the second joint includes a protease cleavable polypeptide sequence. In some embodiments, the protease cleavable polypeptide sequences of the first joint and the second joint can be cleaved together (e.g., by the same protease). In some embodiments, the protease cleavable polypeptide sequences of the first joint and the second joint can be cleaved separately (e.g., by different proteases).

[0733] In some embodiments, for example, in any embodiment in which there are first and second polypeptide chains comprising the first and second domains of the pharmacokinetic modulator, respectively, the inhibitory polypeptide sequence is at the C-terminus of the second domain of the pharmacokinetic modulator, or the inhibitory polypeptide sequence is at the N-terminus of the second domain of the pharmacokinetic modulator. The targeting sequence can be located between the protease cleavable polypeptide sequence and the first domain of the pharmacokinetic modulator, between the protease cleavable polypeptide sequence and the first active domain, at the C-terminus of the first active domain, at the N-terminus of the first active domain, at the C-terminus of the inhibitory polypeptide sequence, at the N-terminus of the inhibitory polypeptide sequence, or between the inhibitory polypeptide sequence and the second domain of the pharmacokinetic modulator.

[0734] In some embodiments, for example, in any embodiment in which there are a first polypeptide chain and a second polypeptide chain comprising a first domain and a second domain of a pharmacokinetic modulator, respectively, the linker polypeptide may include a first targeting sequence and a second targeting sequence. In some such embodiments, the first targeting sequence is part of the first polypeptide chain and the second targeting sequence is part of the second polypeptide chain. In some such embodiments, the first targeting sequence is at the C-terminus of the first active domain and the second targeting sequence is at the C-terminus of the inhibitory polypeptide sequence.

[0735] In some embodiments, for example, in any embodiment in which there are a first polypeptide chain and a second polypeptide chain comprising a first domain and a second domain of a pharmacokinetic modulator, respectively, the linker polypeptide further comprises a second active domain, optionally wherein the second active domain is part of the second polypeptide chain, and / or the linker polypeptide comprises a first inhibitory polypeptide sequence, and the linker polypeptide 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 at the C-terminus of the first inhibitory polypeptide sequence. The first inhibitory polypeptide sequence and / or the second inhibitory polypeptide sequence can be an immunoglobulin inhibitory polypeptide sequence, such as a VHH.

[0736] In some embodiments, for example, in any embodiment in which there are a first polypeptide chain and a second polypeptide chain comprising a first domain and a second domain of a pharmacokinetic modulator, respectively, the pharmacokinetic modulator comprises a heterodimer Fc or heterodimer CH3 domain. The heterodimer Fc or heterodimer CH3 domain can be in a separate polypeptide chain. In some embodiments, the heterodimer Fc or heterodimer CH3 domain comprises a knob CH3 domain and a hole CH3 domain.

[0737] In some embodiments, the linker polypeptide comprises the polypeptide sequence of any one of SEQ ID NOs: 800-848 and 1024- 1041. In some embodiments, the linker polypeptide comprises the polypeptide sequence of any one of SEQ ID NOs: 1042-1137.

[0738] IV. Pharmaceutical Preparations or Compositions

[0739] Pharmaceutical preparations or compositions of linker polypeptides as described herein can be prepared by mixing such linker polypeptides having the desired purity with one or more optional pharmaceutically 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 doses and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium 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 1 0 residues) polypeptides; 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 nonionic surfactants such as polyethylene glycol (PEG).

[0740] Formulations or compositions for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

[0741] V. Uses

[0742] In some embodiments, any one or more of the linker polypeptides, compositions, or pharmaceutical preparations described herein are used in therapy, e.g., for preparing a medicament for treating or preventing a disease or condition, such as cancer, in a subject. In some embodiments, any one or more of the linker polypeptides, compositions, or pharmaceutical preparations described herein are used in a method of treating cancer, the method comprising, e.g., administering a linker polypeptide or pharmaceutical composition to a subject in need thereof.

[0743] In some embodiments, a method for treating or preventing a disease or condition of a subject is provided, the method comprising administering any linker polypeptide or pharmaceutical composition described herein to the subject. In some embodiments, the disease or condition 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 cancer or gastrointestinal cancer, lymphoma, colon cancer or colorectal cancer, endometrial cancer, thyroid cancer or bladder cancer. Cancer (e.g., any of the foregoing cancers) may have one or more of the following features: PD-L1 positive; metastatic; unresectable; mismatch repair defective (MMRd); and / or high microsatellite instability (MSI-H). In some embodiments, cancer is a cancer expressing TGFβR. In some embodiments, cancer is a cancer expressing TGFβ. In some embodiments, cancer is a TGFβ-dependent cancer. If the growth rate of cancer cells in the absence of growth factors is significantly slower than the growth rate in the presence of growth factors, the cancer is considered to be dependent on growth factors such as TGFβ.

[0744] In some embodiments, a method for strengthening T regulatory cells and / or reducing inflammation or autoimmune activity is provided, the method comprising applying a linker polypeptide to an area of ​​interest, e.g., an area of ​​inflammation. The linker polypeptide used in such methods may include an IL-2 polypeptide sequence. In some embodiments, a method for treating an autoimmune disease and / or inflammatory disease is provided, the method comprising applying a linker polypeptide to an area of ​​interest, e.g., an area of ​​inflammation or autoimmune activity. The linker polypeptide used in such methods may include an IL-2 polypeptide sequence. These methods utilize the ability of certain cytokines to stimulate T regulatory cells at relatively low levels, which can exert an anti-inflammatory effect and reduce or inhibit autoimmune activity.

[0745] The linker polypeptide in any of the foregoing methods and uses can be delivered to a subject using any suitable route of administration. In some embodiments, the linker polypeptide is delivered parenterally. In some embodiments, the linker polypeptide is delivered intravenously.

[0746] The linker polypeptides provided herein can be used alone or in combination with other agents in therapy. For example, the linker polypeptides provided herein can be co-administered with at least one additional therapeutic agent.

[0747] Such combination therapies noted above encompass both combined administration (wherein two or more therapeutic agents are contained in the same or separate formulations) and separate administration, in which case administration of the linker polypeptide provided herein may occur prior to, simultaneously with, and / or after administration of the additional therapeutic agents and / or adjuvants.

[0748] The joint polypeptide will be prepared, administered and used in a manner that meets good medical practice. The factors considered in this context include the specific condition treated, the specific mammal treated, the clinical condition of individual patients, the cause of the condition, the delivery site of the medicament, the method of administration, the timetable for administration and other factors known to medical practitioners. In some embodiments, the joint polypeptide is optionally prepared together with one or more medicaments currently used to prevent or treat the condition in question. The effective amount of such other medicaments depends on the amount of the joint polypeptide present in the preparation, the type of condition or treatment and other factors discussed above. These are usually with the same dosage and with about 1% to 99% of the route of administration as described herein or the dosage described herein or with empirically / clinically determining that any dosage and any approach are appropriate.

[0749] For the prevention or treatment of a 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 to be treated, the type of linker polypeptide, the severity and progression of the disease, whether the linker polypeptide is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history, and the response to therapeutic agents (e.g., antibodies or immunoconjugates, cytokines) that share common elements and / or sequences with the linker polypeptide, and the judgment of the attending physician. The linker polypeptide is suitable for administration to the patient at one time or in a series of treatments.

[0750] VI. Nucleic Acids, Host Cells and Production Methods

[0751] Recombinant methods and compositions can be used to produce joint polypeptides or their precursors. In some embodiments, a kind of nucleic acid encoding the separation of joint polypeptides described herein is provided. Such nucleic acids can encode amino acid sequences including active domains (including, for example, immunoglobulin antigen binding domains, receptor binding domains and / or cytokine polypeptide sequences), pharmacokinetic modifiers, joints and inhibitory polypeptide sequences, and any other polypeptide components that may be present in the joint polypeptide. In other embodiments, one or more vectors (for example, expression vectors) including such nucleic acids are provided. In other embodiments, a host cell including such nucleic acids is provided. In some such embodiments, the host cell includes a vector (for example, transformed with a vector), and the vector includes a nucleic acid encoding a joint polypeptide according to the present disclosure. In some embodiments, the host cell is eukaryotic, for example, Chinese hamster ovary (CHO) cells or lymphocytes (for example, Y0, NS0, Sp20 cells). In some embodiments, a method for preparing a joint polypeptide disclosed herein is provided, wherein the method includes culturing a host cell including a nucleic acid encoding a joint polypeptide as provided above under conditions suitable for expressing the joint polypeptide, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0752] For the recombinant production of a joint polypeptide, for example, a nucleic acid encoding a joint polypeptide as described above is prepared and / or separated (for example, after using synthesis and / or molecular cloning techniques to construct) and inserted into one or more vectors to further clone and / or express in a host cell. Such nucleic acids can be easily prepared and / or separated using known techniques.

[0753] Suitable host cells for cloning or expressing the linker polypeptide encoding vector include prokaryotes or eukaryotic cells as described herein. For example, linker polypeptides can be produced in bacteria, particularly when glycosylation is not required. For expression of polypeptides in bacteria, see, for example, U.S. Patents Nos. 5,648,237, 5,789,199, and 5,840,523. After expression, the linker polypeptide can be separated from the bacterial cell paste with a soluble fraction, and the linker polypeptide can be further purified.

[0754] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for linker polypeptide encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized" to produce polypeptides with partially or completely human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006).

[0755] Suitable host cells for expressing the linker polypeptides are also derived from multicellular organisms (plants, invertebrates and vertebrates). Examples of invertebrate cells include insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfecting Spodopterafrugiperda cells.

[0756] Plant cell cultures can also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429.

[0757] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (such as 293 or 293 cells as described herein, e.g., Graham et al., J. Gen. Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (such as TM4 cells as described herein, e.g., Mather, Biol. Reprod. 23:243-251 (1980); monkey kidney cells (CV1) as described herein; African green monkey kidney cells (VERO-76); human cervical tumor cells (HELA); canine kidney cells (MDCK; Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, e.g., Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980); and myeloma cell lines such as Y0, NS0, and Sp2 / 0.

[0758] ***

[0759] This description and exemplary embodiments should not be considered restrictive. For the purpose of this specification and the appended claims, unless otherwise stated, all numbers representing amounts, percentages or ratios and other numerical values ​​used in this specification and claims should be understood in all cases to be modified by the term "about" to the extent that it has not been so modified. "About" means that the degree of change in the properties of the described subject matter will not be substantially affected, for example, within 10%, 5%, 2% or 1%. Therefore, unless otherwise indicated, the numerical parameters set forth in the following specification and the appended claims are approximate values ​​that can be changed according to the desired properties sought to be obtained. At least, and not attempting to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be interpreted according to the number of reported significant figures and by applying ordinary rounding techniques.

[0760] Example

[0761] The following examples are provided to illustrate certain disclosed embodiments and should not be construed as limiting the scope of the disclosure in any way.

[0762] Example 1: Construction of a mammalian expression vector encoding a fusion protein

[0763] The coding sequences of all protein domains containing the linker sequence were synthesized into complete genes (Genscript, NJ). All synthetic genes were designed to contain the coding sequence of the N-terminal signal peptide (to promote protein secretion), 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.

[0764] Table 4. Linker polypeptide constructs

[0765]

[0766]

[0767]

[0768]

[0769]

[0770] Example 2: Expression and purification of fusion protein

[0771] Transient expression of fusion proteins

[0772] Different mammalian cell expression systems are used to produce fusion proteins (ExpiCHO-S TM 、Expi293F TM 、Freestyle CHO-S TM and Freestyle 293 TM, Life Technologies). In brief, expression constructs were transiently transfected into cells according to the manufacturer's protocol and using reagents provided in the corresponding expression kit. The fusion protein was then expressed and secreted into the cell culture supernatant. Samples were collected from production cultures every day and cell density and viability were assessed. Protein expression titers and product integrity in cell culture supernatants were analyzed by SDS-PAGE to determine the optimal collection time. Cell culture supernatants were usually collected between 4 and 12 days when culture viability was typically >75%. On the day of collection, cell culture supernatants were removed by centrifugation and vacuum filtration before further use.

[0773] Purification of fusion proteins

[0774] Fusion proteins were purified from cell culture supernatants in a one-step or two-step procedure. Briefly, proteins containing Fc domains were purified by protein A affinity chromatography (HiTrap MabSelect SuRe, GE Healthcare). In some cases, proteins containing Fc domains were further purified by size exclusion chromatography (HPLC SEC5 300A 7.8×300mm, 5μm, part number 5190-2526, Agilent Bio or HiLoad 26 / 60 Superdex 200). First, the Fc domains were purified by HPLC on a nickel-agarose column (Ni-Penta TM Sepharose 6 Fast Flow Column, PROTEINDEX TM ) followed by size exclusion chromatography (HPLC SEC5 300A 7.8x 300mm, 5μm part 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 were assessed by SDS PAGE under reducing and non-reducing conditions (usually >90%). The purified protein was aliquoted and stored at -80°C before further use. Figures 1A-1D An example of a successfully purified fusion protein is shown. Figures 1A-1D In Figure 3, analysis of the fusion protein purified by a protein A column (by Coomassie staining) shows the high purity of the target protein and minimal high molecular weight entities.

[0775] Example 3: Cleavage of fusion protein by MMP9 protease

[0776] Recombinant MMP9 (R&D Systems) was first activated with p-aminophenylmercuric acetate, and the fusion protein was digested or mock-digested overnight (18-22 hours) at 37°C using either this activated protease or an equivalent amount of activation solution without the protease. The cleavage assay was set up 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 prior to testing. Aliquots of the digest were then analyzed by SDS-PAGE followed by Western blotting to assess the extent of cleavage. The digests were also evaluated in functional assays such as the HEK-Blue interleukin reporter assay. As in Figures 2A-2F As shown in , the fusion protein with functional sites was observed to be essentially completely cleaved by the MMP9 protease after overnight incubation. In contrast, the protein containing the scrambled MMP cleavage site was not cleaved ( Figure 2D ).

[0777] Example 4: Immunoblot analysis of IL-2 and IL-15

[0778] The cleavage products of untreated and digested fusion proteins were assessed by Western blotting. The following antibodies were used: goat anti-mouse IL-2 polyclonal antibody (AF-402-NA; R&D Systems), anti-human IL-2 antibody (Invitrogen, catalog number MA5-17097, mouse IgG1) and rabbit anti-human IL-15 polyclonal antibody (ThermoFisher, catalog number PA5-79466). 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, West Grove, PA) were used for detection, and SuperSignal West Femto maximum sensitivity detection reagent (ThermoFisher) was used for color development according to the manufacturer's recommendation.

[0779] Example 5: Detection of mouse IL-2 / IL-2Ra fusion protein by ELISA

[0780] An ELISA assay was developed to detect and quantify prodrug fusion proteins including IL-2 and 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; Thermo Fisher Scientific) at 1 mg / mL in PBS. After washing, the wells were blocked with TBS / 0.05% Tween 20 / 1% BSA, and then the fusion protein and / or unknown biological sample was added for 1 hour at room temperature. After washing, anti-mouse IL-2Ra biotinylated detection antibody (BAF2438, R&D Systems) was added and binding was detected using Ultra Strepavidin HRP (Thermo Fisher Scientific). The ELISA plate was developed by adding chromogenic tetramethylbenzidine substrate (Ultra TMB, Thermo Fisher Scientific). The reaction was stopped by adding 0.5 M H2SO4, and the absorbance was read at 450-650 nm.

[0781] Example 6: Determination of IL-2 and IL-15 functional cells

[0782] IL-2 and IL-15 are members of the four α-helical bundle family of cytokines and share the same signaling receptor IL2-Rβ and a common γ chain. Therefore, the same reporter cell line HEK Blue IL-2 (InvivoGen, San Diego) was used to measure the activity of these cytokines. TM IL-2 cells are specifically designed to monitor activation of the JAK-STAT pathway induced by ligand binding to the IL2-Rβ and common γ chain receptors. Stimulation with appropriate cytokines triggers the JAK / STAT5 pathway and induces secreted embryonic alkaline phosphatase (SEAP) production. TM (a SEAP detection medium) can easily monitor SEAP. These cells respond to human / murine IL-2 and 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 hours. The next day, the level of SEAP was measured by adding 20 μL of cell supernatant to QuantiBlue reagent, followed by incubation at 37°C for 1-3 hours and reading the absorbance at 630nm. Figure 3A-3V and Figure 3W-3BB Results obtained from IL-2 and IL-15 fusion proteins tested in the HEK Blue IL-2 cell assay are shown separately.

[0783] Example 7: Next Generation Targeting Sequence Linker Peptide Binding Assay

[0784] A series of peptides including MMP cleavable sites with or without the addition of targeting sequences were synthesized and conjugated to the fluorophore EDANS (5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid) (custom synthesis, Thermo Fisher Scientific). Table 5 shows the list of peptides. These peptides were then tested for their ability to bind to ECM proteins such as heparin, fibronectin, and collagen, which are found in large quantities in the tumor stroma. In Table 5, the bold text shows the MMP cleavage site, the underlined text shows the retention motif (targeting sequence) when present, and the italic asterisk (*) shows the Edans fluorophore conjugated to the peptide.

[0785] Table 5. Next generation MMP cleavable linkers with targeting sequences

[0786]

[0787] All binding assays were set up in 10 mM TrisHCl, pH 7.5 and / or 10 mM TrisHCl, pH 6. Peptides (20 μM) were incubated on a shaker at room temperature with agarose cross-linked with heparin or control agarose beads (Sigma and Pierce, respectively) for 2 hours. The beads were then washed 4 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 (excitation 340 / emission 490). Figure 4A-4B Several next generation MMP linker peptides containing heparin binding motifs were shown to bind to heparin-agarose beads, whereas first generation MMP linkers lacking these targeting sequences did not. One such peptide showed enhanced binding to heparin at pH 6 (the pH of tumors) relative to pH 7.5 (the pH of normal tissues) ( Figure 4B ).

[0788] For fibronectin and collagen binding peptide assays, streptavidin-coupled magnetic beads (Mag Sepharose, Cytiva and Dynabeads, Thermo Fisher Scientific, respectively) were first incubated with biotin-labeled fibronectin (Cytoskeleton) or biotin-labeled collagen IV (Prospec) for 1 hour under gentle shaking. After multiple washes, the ECM-coated beads were then incubated with Edans peptide (20 μM) in neutral or acidic binding buffer under shaking for 2 hours at room temperature. The 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 (excitation 340 / emission 490). Figure 4C It was shown that peptide 13 is able to bind to fibronectin and exhibits enhanced binding at pH 6 (the pH of tumors) relative to pH 7.5 (the pH of normal tissue). Figure 4D Peptide 14 was shown to bind strongly to collagen IV, while peptide 15 bound to a lesser extent.

[0789] Example 8: Next Generation IL-2 / IL-15 Fusion Protein Binding Assay

[0790] A series of IL-2 and IL-15 fusion proteins including single or multiple targeting sequences in the linker region or other locations were designed and successfully produced (Table 4 and Figures 1A-1D These proteins were then tested for their ability to bind to ECM proteins such as heparin, fibronectin and collagen, which are found in large quantities in the tumor stroma.

[0791] 96-well plates were coated with 10 μg / mL of heparin-BSA conjugate (provided by Dr. Mueller of Boerhinger Ingelheim) or control BSA at room temperature on a shaker (350 rpm) for 18-22 hours. After washing, wells were blocked with PBS-0.05% Tween 20 or PBS-0.05% Tween 20 / 1% BSA containing 2% milk powder for 90 minutes. The fusion protein was then titrated in PBS-0.05% Tween 20 or 1% BSA / PBS-0.05% Tween 20 at pH 7.5 and / or pH 6 containing 2% milk powder and added at room temperature for 2 hours with shaking. After washing, anti-mouse IL-2 biotinylated detection antibody (JES6-5H4, Thermo Fisher Scientific), anti-6x-His tag HRP conjugated antibody (Invitrogen, 1 mg / mL, catalog number MA1-21315-HRP) or anti-human IgG HRP conjugated antibody (Southern Biotech) were added, and binding was detected using ultrastreptavidin HRP (Thermo Fisher Scientific). The plate was developed by adding chromogenic tetramethylbenzidine substrate (Ultra TMB, Thermo Fisher Scientific). The reaction was terminated by adding 0.5M H2SO4, and the absorbance was read at 450-650nm. IL-2 fusion protein constructs Y and CC bind to heparin in a dose-dependent manner at acidic pH and have a higher affinity than construct B ( Figure 4E ). Strikingly, construct CC preferentially bound to heparin at acidic pH and exhibited the most robust binding, with an EC 50 10 nM, while construct B bound much weaker, with an EC 50 Furthermore, when the same pH-dependent heparin-binding motif was inserted into different positions of IL-2 fusion proteins, all resulting proteins bound heparin with similar high affinity at pH 6 ( Figure 4F and 4G Similarly, similar binding affinity was observed when another heparin targeting sequence was engineered into a different site of the IL-2 fusion protein ( Figures 4H-4I ). Figure 4J The IL-15Rα-IL-15 fusion protein has low intrinsic binding (EC 50 This interaction was abolished when the cytokine was bound by a blocker in the context of a linker polypeptide-IL-15 fusion protein (construct VVV). When a heparin binding motif was engineered into the linker polypeptide-IL-15 fusion protein (construct WWW), heparin binding activity was restored. Finally, as in Figure 4MAs shown in , linker polypeptide-IL-2 fusion proteins engineered with a heparin binding site showed approximately 30-fold enhanced binding to heparin in vitro compared to constructs lacking the heparin binding site (constructs EEE and NNNN versus constructs AAA and NNN, respectively).

[0792] A similar plate-based assay was developed to interrogate the binding of IL-2 fusion variants to fibronectin. 96-well plates were coated with fibronectin (4-10 μg / mL, Sigma) or control BSA on a shaker (350 rpm) at room temperature for 18-22 hours. After washing, the wells were blocked with PBS-0.05% Tween 20 or protein-free blocking buffer (Pierce) containing 2% milk powder for 90 minutes, then the fusion protein was titrated in blocking buffer-0.1% Tween 20 at pH 7.5 and / or pH 6, and added at room temperature under shaking for 1 hour. After washing, anti-mouse IL-2 biotin-labeled detection antibodies (JES6-5H4, Thermo Fisher Scientific) or anti-human IgG HRP conjugated antibodies (Southern Biotechnology) were added, and binding was detected using superstreptavidin HRP (Thermo Fisher Scientific). The plates were developed by adding chromogenic tetramethylbenzidine substrate (Ultra TMB, Thermo Fisher Scientific). The reaction was terminated by adding 0.5 M H2SO4 and the absorbance was read at 450-650 nm. Construct EE preferentially bound to fibronectin at acidic pH and showed dose-dependent binding, while no binding was observed at pH 7.5 ( Figure 4K ). No significant binding of construct B was observed under either neutral or acidic conditions.

[0793] To test binding to collagen, pull-down assays were performed using agarose cross-linked to collagen (Sigma). IL-2 fusion proteins were incubated with collagen-agarose or control agarose beads in 1% BSA / PBS-0.05% Tween 20 at 4°C for 18-22 hours with gentle rotation. After washing, proteins bound to the beads were eluted by resuspending the beads in SDS sample buffer (Life Technologies). The bound proteins were then separated by SDS-PAGE on a 4% to 12% BisTris gradient gel, followed by immunoblotting with a goat anti-mouse IL-2 polyclonal antibody (AF-402-NA; R&D Systems). Donkey anti-goat HRP-conjugated antibody was used for detection (Jackson ImmunoResearch, West Grove, Pennsylvania), and blots were developed using SuperSignal West Femto Maximum Sensitivity Detection Reagent (Thermo Fisher Scientific) as recommended by the manufacturer. Blot images are shown in Figure 4L. Construct GG and construct II were specifically bound by collagen-agarose beads, while no IL-2 fusion protein bound to control agarose beads. Quantification of the blot using the iBright imaging system (Invitrogen) showed that although the fractions of bound constructs GG and II were low (<1% of input), their fractions were 2.5 and 1.4 times higher than the fraction of bound construct B.

[0794] Example 9: Next Generation Retention Linker IL-2 Fusion Protein Shows Greater Retention in Tumors in Vivo.

[0795] The level of IL-2 fusion protein present in tumors in vivo was assessed by utilizing fluorescently labeled proteins and real-time whole body imaging. According to the manufacturer's protocol (Dylight 650 antibody labeling kit, Thermo Fisher Scientific), non-cleavable constructs GGG and constructs DD were conjugated with Dylight 650 probes. Conjugation did not significantly change the binding of protein to heparin. BALB / c mice were subcutaneously inoculated with EMT6 breast cancer isogenic models, and when the average tumor volume reached 240mm 3 At 4 hr, animals were randomized into 3 groups based on tumor volume (n=2 mice per treatment group). Table 6 below shows the study design.

[0796] Table 6. Study Design for Evaluation of IL-2 Fusion Proteins

[0797]

[0798] After administration of a single dose of labeled IL-2 fusion protein to tumor-bearing mice, fluorescence images (excitation 640 / emission 680, consistent with the Dylight 650 probe excitation / emission spectrum) were captured on an IVIS system (PerkinElmer, IVIS Lumina Series III) for 96 hours and Figure 5A Fluorescence intensity in tumor areas was quantified across groups, mean background tumor fluorescence (Group 1) was subtracted from Group 2 and Group 3 values ​​at each time point, and data were normalized to the initial fluorescence intensity of the same amount of each tagged protein. Figure 5B It is shown that at each time point tested, the tumor-associated fluorescence of Group 3 was approximately 2-fold higher than that of Group 2. This means that the next generation linker-retaining construct DD accumulated and was retained in the tumor at a level that was twice that of the IL-2 fusion protein construct GGG lacking any targeting sequence.

[0799] Example 10: Multiple targeting sequences in the linker of IL-2 fusion protein produce maximal anti-tumor efficacy in vivo

[0800] C57BL / 6 mice were subcutaneously inoculated with B16F10 melanoma cells and the average tumor volume reached 70-90 mm 3 At 4 pm, animals were randomized into 6 groups based on tumor volume (n=8 mice per treatment group). Mice were intravenously dosed every 3 days (Q3D) according to Table 7 for a total of 5 doses.

[0801] Table 7. Study design for evaluating IL-2 fusion proteins using multiple targeting sequences

[0802]

[0803] During the study, tumor volumes were measured twice a week. The average tumor volume was Figure 6 Antitumor activity was observed in all treatment groups, but the most robust tumor growth inhibition (TGI) was observed with multiple targeting linker constructs Construct III (83.5%), compared to 52% to 66% TGI in single targeting linker fusion proteins. On day 14, animals were sacrificed, and tissues and blood (processed into serum) were collected 24 hours after the last dose (dose #5) and stored at -80°C until further testing.

[0804] Example 11: Multiple targeting sequences in the linker of an IL-2 fusion protein result in increased levels of intratumoral drug, IL-2, and IFN-γ, as well as enhanced levels of drug in the circulation compared to a single targeting linker construct.

[0805] Levels of full-length IL-2-IL-2Ra fusion protein, IL-2, and IFN-γ were quantified in tumor samples collected during preclinical efficacy studies to compare a panel of linker-retaining IL-2 fusion drugs (see Example 10).

[0806] Tumors (n=3 per group) were collected 24 hours after the last dose injection, snap frozen and stored at -80°C until further processing. Tumor lysates were generated using tissue extraction reagent (Thermo Fisher Scientific) supplemented with protease and phosphatase inhibitors. Standard techniques and protein concentrations were determined using the BCA assay (Pierce).

[0807] The lysates were tested with an in-house developed ELISA (see Example 5) to measure the full-length IL-2 fusion protein (IL-2 capture / IL-2Ra detection). The results were normalized to 1 mg of tumor lysate, and Fig. 7A The average values ​​are shown in Figure 2. The highest levels of drug detected with multiple targeted linker drug constructs III (which were about 2- to 5-fold higher than the levels of other retained linker drugs tested) were also detected with an appropriate Luminex kit (IL-2 Mouse ProcartaPlexTM The intratumoral level of IL-2 measured by Simplex kit, catalog number EPX01A-20601-901, Thermo Fisher Scientific) was highest in the construct III treated group compared with the other groups ( Figure 7B ). This suggests that the multisite targeting linker technology improves TME retention of both full-length drugs and releases active IL-2 after cleavage. In addition, levels of the major Th1 cytokine IFN-γ were enhanced in construct III animals ( Figure 7C ; Essential Th1 / Th2 cytokine 6-Plex mouse ProcartaPlex TM Detection panel (Panel, catalog number EPX060-20831-901, Thermo Fisher Scientific).

[0808] Equivalent serum samples (n=3 per group) were tested by in-house ELISA to quantify the full-length IL-2 fusion drug and the results are presented in Fig.7D 24 hours after administration, the circulating drug level of construct III was about 1.5 times to 4 times of other targeted drug serum levels. This shows that multiple targeting sequences are engineered into IL-2 fusion drugs to increase the drug levels in both tumors and circulation. In addition, multiple targeting sequences (for example, targeting sequences targeting heparin and targeting sequences targeting collagen IV) can increase the serum half-life of linker polypeptides.

[0809] Example 12: Multiple targeting sequences in the linker of IL-2 fusion protein are not associated with any systemic toxicity

[0810] Multiplex Luminex Assay (Essential Th1 / Th2 Cytokine 6-Plex Mouse ProcartaPlex TM Detection panel, catalog number EPX060-20831-901, Thermo Fisher Scientific) measured the levels of inflammatory cytokines in serum. Low levels of TNF-α and IL-6 ( Figures 8A-8B ; the mean values ​​of each group were equal to or lower than 10pg / mL and 27pg / mL, respectively), while IL-12 was not detected in all groups. In addition, no increase in aspartate aminotransferase levels was observed in the treated groups compared with the control animals, indicating the absence of any liver damage ( Figure 8C ; AST activity assay, Sigma).

[0811] Example 13: Linker polypeptides having an immunoglobulin antigen binding domain as an active domain

[0812] Figures 9A-9D Each shows a linker polypeptide according to certain embodiments of the present disclosure. Fig. 9AThe linker polypeptide 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, wherein the first linker comprises a protease cleavable polypeptide sequence (CL). In some embodiments, the first linker further comprises a targeting sequence. In certain embodiments, the active domains comprise immunoglobulin antigen binding domains (IBD1 and IBD2) that may be involved in different targets. In certain embodiments, the target binding domain may comprise a heavy chain and a light chain ( Fig. 9A ) or only the heavy chain ( Fig. 9B ), such as VHH. Fig. 9A Compared with the linker peptide Fig.9D The linker polypeptide further comprises a chemotherapeutic drug (D).

[0813] Figures 11A-11B Each shows the release of the 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). Fig.11A Compared with the linker peptide Fig. 11B The linker polypeptide further comprises a blocking agent (B) conjugated to each of the first active domain and the second active domain 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 can 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 can be cleaved separately (e.g., by different proteases).

[0814] Example 14: Linker polypeptide having an immunoglobulin antigen binding domain as one active domain and a non-immunoglobulin polypeptide as another active domain

[0815] Figures 10A-10B Each shows a linker polypeptide according to certain embodiments of the present disclosure. Fig. 10AThe linker polypeptide includes 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, wherein the first linker includes a protease-cleavable polypeptide sequence (CL). In some embodiments, the first linker further includes a targeting sequence. In certain embodiments, the first active domain includes a receptor binding domain (RBD), and the second active domain includes an immunoglobulin antigen binding domain (IBD). In some embodiments, the RBD includes a cytokine polypeptide sequence (CY). Fig. 10A Compared with the linker peptide Fig. 10B The linker polypeptide further includes 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, wherein the second linker includes a protease cleavable polypeptide sequence (CL).

[0816] Figures 12A-12B Each shows the release of the 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 includes a receptor binding domain (RBD) that may include a cytokine polypeptide sequence (CY), and the second active domain includes an immunoglobulin antigen binding domain (IBD). Fig. 12A Compared with the linker peptide Fig. 12B The linker polypeptide further includes 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 including a protease cleavable polypeptide sequence (CL). In some embodiments, the protease cleavable polypeptide sequences of the first linker and the second linker can be cleaved together (e.g., by the same protease). In some embodiments, the protease cleavable polypeptide sequences of the first linker and the second linker can be cleaved separately (e.g., by different proteases).

[0817] Example 15: Tumor stromal targeting sequences in the linker of IL-2 fusion proteins produce enhanced anti-tumor efficacy in vivo

[0818] MC38 colorectal cancer cells were subcutaneously inoculated into C57BL / 6 mice. When the average tumor volume reached 70-90 mm 3 Animals were randomly divided into 10 groups (n=7 or 6 mice per treatment group) based on tumor volume. Mice were intraperitoneally (IP) dosed twice a week (BIW) for a total of 5 doses according to the design shown in Table 8 below:

[0819] Table 8. Dosing of C57BL / 6 mice inoculated with MC38 cells

[0820]

[0821]

[0822] During the study, tumor volumes were measured twice a week. The average tumor volume was Figures 13A-13B The inhibition of tumor volume was shown in Fig. 13C Antitumor activity was observed in all treatment groups at the 5 mg / kg dose; however, the most robust tumor growth inhibition (TGI) was observed with tumor matrix targeting constructs NNNN, EEE, NNN, and OOOO (TGI ranged from 74% to 86%). Relatively moderate TGIs were observed in the low dose treatment groups, and tumor matrix targeting constructs EEE and NNN continued to show superior efficacy to the parental non-targeting constructs.

[0823] On day 16, animals were sacrificed and tumors (n=3 per group) were collected 24 hours after the last dose injection, snap frozen and stored at -80°C until further processing. Tumor lysates were generated using tissue extraction reagent (Thermo Fisher Scientific) supplemented with protease and phosphatase inhibitors, and standard techniques and protein concentrations were determined using the BCA assay (Pierce). Fig.13D As shown in , the intratumoral levels of the major Th1 cytokine IFN-γ (IFNg) were mostly elevated in the groups treated with the targeting constructs compared to the groups treated with the parental non-targeting constructs. TM IFN-γ was measured by the assay panel (Cat. No. EPX060-20831-901, Thermo Fisher Scientific).

[0824] Example 16: IL-2 fusion protein with TME binding motif shows enhanced intratumoral immune cell infiltration

[0825] B16F10 melanoma cells were subcutaneously inoculated into C57BL / 6 mice. When the average tumor volume reached 70-90 mm 3 Animals were randomly divided into 5 groups (n=3 mice per treatment group) based on tumor volume. Mice were intraperitoneally administered twice with selected 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, which is a combination of enzymatic and mechanical dissociation). Single cell samples were cryopreserved at -80°C before further processing. After thawing, cells were washed and stained for surface and intracellular targets using the antibodies listed in Table 9.

[0826] Table 9. Antibodies for staining immune cell markers

[0827] Markers form clone Catalog Number Manufacturer CD3 AF700 17A2 100216 Biolegend CD4 AF488 GK1.5 100423 Baijin Biotechnology Co., Ltd. CD8a BV785 53-6.7 100750 Baijin Biotechnology Co., Ltd. CD25 PE-Cy7 3C7 101916 Baijin Biotechnology Co., Ltd. DX5 PCp / Cy5.5 DX5 108916 Baijin Biotechnology Co., Ltd. CD44 BV650 IM7 103049 Baijin Biotechnology Co., Ltd. PD-1 BV510 29F.1A12 135241 Baijin Biotechnology Co., Ltd. CD45 BV421 30-F11 103134 Baijin Biotechnology Co., Ltd. Ki-67 PE 11F6 151210 Baijin Biotechnology Co., Ltd. FoxP3 APC FJK-16s 17-5773-82 Thermo Fisher Scientific

[0828] Figures 14A-14E Flow cytometry analysis of selected immune cell populations is shown. Remarkably, the group treated with the IL-2 fusion protein engineered through the tumor matrix targeting site shows enhanced intratumoral T cell infiltration (CD3+ cells) compared to the group treated with the parent non-targeted fusion protein or vehicle group. More specifically, the increase in this T cell seems to be mainly driven by the increase of both total and activated cytotoxic T cells (CD8+ and CD8+CD25+ subsets).

[0829] Example 17: Examples of IL-2 asymmetric Fc fusion proteins with tumor targeting sequences and single or double masks.

[0830] Additional asymmetric IL-2 Fc fusion proteins containing an ECM targeting sequence and either a single mask or a double mask were prepared, purified, and functionally characterized as previously described. Fig.15A Examples of such proteins are shown: rectangles represent Fc domains (Fc knobs or Fc holes), solid lines represent protease-cleavable linker peptides, and dashed lines represent flexible linker sequences. The purity of Fc fusion proteins was assessed by SDS-PAGE under non-reducing conditions ( Fig. 15B ). Proteins were cleaved with recombinant MMP-9 protease overnight at 37°C, and digests were evaluated in the HEK-Blue IL-2 reporter assay as previously described. Figure 15C-15U The ability of selected IL-2 fusion proteins to bind to ECM components such as heparin and fibronectin was assessed using the binding assay described previously, and the results are presented in Figure 15V-15X Compared with the parent molecule without tumor matrix targeting site, the fusion proteins with heparin binding motif inserted at different positions of the molecule all showed enhanced binding to heparin ( Figure 15V-15W Similarly, IL-2 fusion proteins engineered with only the pH-dependent fibronectin binding motif were able to bind to fibronectin compared to the parent molecule without the tumor stroma targeting site or the fusion protein engineered with the collagen I binding motif ( Figure 15X ). In addition, the binding to fibronectin was slightly enhanced under acidic conditions.

[0831] In order to evaluate the ability of fusion protein to bind to collagen, image-based retention assay was carried out. According to the manufacturer's recommended procedure (Thermo Fisher Scientific, catalog number 62295), fusion protein was labeled at reduced sulfhydryl groups with DyLight 650 maleimide. Then fluorescently labeled fusion protein was mixed with bovine type I collagen (Advanced Biomatrix, TeloCol-10, catalog number 5226) and 10X PBS buffer, pH 7.4 (Invitrogen, REFAM9624) to make the sample mixture reach neutral pH. The final concentration of various components in the mixture is as shown in the following table 10.

[0832] Table 10. Concentrations of components in fusion protein-collagen mixtures

[0833] Components concentration Construct BBBBBB / Construct TTTTT 5.4μM (right side) Construct KKKKKKK / construct TTTTT 3.4μM (right side) Bovine Collagen Type I 4mg / ml PBS 1X

[0834] 5 μL of fusion protein-collagen mixture is loaded into the inner hole of ibidi u-Slide angiogenesis (uncoated, part 81501) pretreated with gelatin solution (2% in H2O, Sigma, catalog number G1393-20ML). The slide is incubated at room temperature for 30 minutes to form a gel of the fusion protein-collagen mixture. Then, 50 μL of bovine type I collagen (1 mg / mL in 1X PBS) is loaded into the upper hole of the slide. After collagen gels in the upper hole, the slide is imaged using a BioTek Lionheart FX automated microscope. The fluorescence intensity of the inner hole represents the amount of fusion protein present and retained in the collagen, and is measured at excitation / emission 628 / 685nm. LED intensity, integration time and camera gain are adjusted to appropriate levels to avoid overexposure and pixel intensity saturation. Fluorescence intensity is measured within 66 hours, and images are taken every 30 minutes at room temperature. The mean fluorescence intensity was calculated by Gen5 software and then normalized to the mean fluorescence intensity of the first image (T=0) set as 100%. The normalized mean fluorescence intensity over time showed that the fusion protein containing the collagen I binding site was retained in the collagen gel to a greater extent than the non-targeted fusion protein ( Figure 15Y ).

Claims

1. A linker polypeptide comprising: first targeting sequence; a second targeting sequence; as well as A first linker positioned between the first targeting sequence and the second targeting sequence, the linker comprising a protease-cleavable polypeptide sequence.

2. The linker polypeptide of the immediately preceding claim, further comprising a first active domain, optionally wherein the first active domain is closer to the first targeting sequence than to the second targeting sequence.

3. The linker polypeptide of the immediately preceding claim, further comprising an additional domain, optionally wherein the additional domain comprises an inhibitory polypeptide sequence capable of blocking the activity of the first active domain, a pharmacokinetic modulator and / or a second active domain, and optionally wherein the additional domain is closer to the second targeting sequence than the first targeting sequence.

4. The linker polypeptide according to the immediately preceding claim, which comprises, sequentially 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.

5. A linker polypeptide comprising The first active domain; The second active domain; Pharmacokinetic modulators; and A first linker is positioned between the pharmacokinetic modulator and the first active domain, wherein the first linker comprises a protease-cleavable polypeptide sequence. The linker polypeptide of claim 5 , further comprising a first targeting sequence.

7. A linker polypeptide comprising: The first active domain; an inhibitory polypeptide sequence capable of blocking the activity of the first active domain; a first linker positioned between the first active domain and the inhibitory polypeptide sequence, the linker comprising a protease-cleavable polypeptide sequence; and First targeting sequence.

8. A linker polypeptide according to the immediately preceding claim comprising a pharmacokinetic modulator.

9. A linker polypeptide comprising: 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 at the C-terminus of 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 the 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.

10. A linker polypeptide comprising: 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 at the N-terminus of 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 the 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.

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