Activatable cytokine constructs and related compositions and methods - Patents.com

Abstract

Provided herein is an activatable cytokine construct comprising: (a) a first monomer construct comprising a first mature cytokine protein (CP1), a first cleavable moiety (CM1) and a first dimerization domain (DD1), wherein CM1 is disposed between CP1 and DD1; and (b) a second monomer construct comprising a second mature cytokine protein (CP2), a second cleavable moiety (CM2) and a second dimerization domain (DD2), wherein CM2 is disposed between CP2 and DD2, wherein CM1 and CM2 function as substrates for a protease, DD1 and DD2 bind to each other, and wherein ACC is characterized by a reduction in at least one activity of CP1 and / or CP2 compared to a control level of at least one activity of CP1 and / or CP2.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 253,939, filed October 8, 2021, and U.S. Provisional Application No. 63 / 311,397, filed February 17, 2022, the entire contents of which are hereby incorporated by reference in their entireties.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (CYTX087.xml, size: 360,448 bytes, created on: September 29, 2022) are incorporated by reference in their entirety into this specification.

[0003] The present disclosure relates to the field of biotechnology, and more specifically to activatable cytokine constructs, such as activatable interleukin 15 (IL-15) cytokine constructs. [Background technology]

[0004] Cytokines are a family of naturally occurring small proteins and glycoproteins produced and secreted by most nucleated cells in response to viral infection and / or other antigenic stimuli. Interleukins are a subclass of cytokines. Interleukins control cell growth, differentiation and motility. Interleukins are particularly important in stimulating immune responses, such as inflammation. Interleukins have been used to treat cancer, autoimmune diseases and other diseases. For example, interleukin-2 (IL2) is indicated for the treatment of melanoma, graft-versus-host disease (GVHD), neuroblastoma, renal cell carcinoma (RCC), and is also considered useful in conditions including acute coronary syndrome, acute myeloid syndrome, atopic dermatitis, autoimmune liver disease, basal cell carcinoma, bladder cancer, breast cancer, candidiasis, colon cancer, malignant cutaneous T-cell lymphoma, endometrioma, HIV invention, ischemic heart disease, rheumatoid arthritis, nasopharyngeal adenocarcinoma, non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic cancer, systemic lupus erythematosus, tuberculosis, and other diseases.

[0005] Interleukin-15 (IL-15) is known to promote the differentiation and expansion of T cells, B cells and natural killer (NK) cells, leading to enhanced antitumor responses. IL-15 has been identified as a promising candidate for anticancer therapy and has been tested in numerous clinical trials. Despite this promise, IL-15 is known to exhibit undesirable proinflammatory effects and has been implicated in the pathogenesis of several autoimmune diseases. The maximum tolerated dose of recombinant IL-15 is 2 micrograms / kg. Recombinant soluble IL-15 also has a short half-life in vivo, which has prevented its therapeutic use. Other interleukins, such as IL-6, IL-7, IL-12 and IL-21, among others, are also potential therapeutic agents for cancer and other diseases. However, interleukin therapy is often associated with undesirable side effects, such as flu-like symptoms, nausea, vomiting, diarrhea, hypotension and arrhythmias, among others.

[0006] Interferons are another subclass of cytokines. Interferons are currently classified into three major classes: type I interferons, type II interferons, and type III interferons. Interferons exert their cellular activity by binding to specific membrane receptors on the cell surface.

[0007] Interferon therapy has many clinical benefits. For example, interferon is known to upregulate the immune system and to have antiviral and antiproliferative properties. Due to these biological properties, interferon has been used clinically as a therapeutic agent for the treatment of viral infections and malignancies. In addition, interferon is useful for recruiting the patient's innate immune system to identify and attack cancer cells. Thus, interferon therapy has been widely used in cancer and antiviral treatment, such as the treatment of hepatitis, Kaposi's sarcoma, hairy cell leukemia, chronic myeloid leukemia (CML), follicular lymphoma, renal cell carcinoma (RCC), melanoma, and other disease states. However, systemic administration of interferon is associated with dose-dependent toxicities, particularly strong flu-like symptoms, neurological symptoms, liver toxicity, bone marrow suppression, and arrhythmias. In a study of melanoma patients, the combination of pembrolizumab with pegylated IFNa resulted in an ORR of 60.5%. This combination treatment was also associated with 49% G3 / G4 adverse events, requiring dose reduction of pegylated IFNa (Davar et al., J. Clin. Oncol., 2018). These undesirable side effects limit the dosage of interferon therapy and may lead to interruption or delay of interferon therapy.

[0008] Thus, there is a great need and desire for improved specificity and selectivity of cytokine therapy towards desired targets. Enhanced targeting of cytokine therapy to disease sites may reduce toxicity based on systemic mechanisms and lead to broader therapeutic utility. Summary of the Invention

[0009] The present disclosure provides an activatable cytokine construct (ACC) comprising: (a) a first monomer comprising a first mature cytokine protein (CP1), a first cleavable portion (CM1) and a first dimerization domain (DD1), wherein CM1 is interposed between CP1 and DD1; and (b) a second monomer comprising a second mature cytokine protein (CP2), a second cleavable portion (CM2) and a second dimerization domain (DD2), wherein CM2 is interposed between CP2 and DD2, wherein CM1 and CM2 function as substrates for a protease, and DD1 and DD2 bind to each other, characterized by a reduction in at least one activity of CP1 and / or CP2 compared to a control level of at least one activity of CP1 and / or CP2. The protease(s) that cleave CM1 and CM2 may be overexpressed in diseased tissue (e.g., tumor tissue) compared to healthy tissue. ACC may be activated by cleavage of CM1 and / or CM2 in diseased tissue (e.g., tumor microenvironment) to allow cytokines to exert their activity, while in healthy tissue, cytokine activity is reduced. Thus, the ACC provided herein may result in reduced toxicity, allow higher effective dosages of cytokines, and / or widen the therapeutic window of cytokines compared to conventional cytokine therapy.

[0010] Provided herein is an activatable cytokine construct (ACC) comprising a first monomer construct and a second monomer construct, characterized in that (a) the first monomer construct comprises a first mature cytokine protein (CP1), a first cleavable portion (CM1) and a first dimerization domain (DD1), wherein CM1 is disposed between CP1 and DD1, and (b) the second monomer construct comprises a second mature cytokine protein (CP2), a second cleavable portion (CM2) and a second dimerization domain (DD2), wherein CM2 is disposed between CP2 and DD2, and DD1 and DD2 bind to each other, whereby the first monomer construct and the second monomer construct form a dimer, and the activatable cytokine construct (ACC) has a reduced activity level of at least one of CP1 and / or CP2 compared to a control level of at least one activity of CP1 and / or CP2.

[0011] The present disclosure provides an activatable cytokine construct (ACC), comprising: (a) a first monomer comprising a first mature cytokine protein (CP1) and a first dimerization domain (DD1); and (b) a second monomer comprising a second mature cytokine protein (CP2), a cleavable moiety (CM), and a second dimerization domain (DD2), wherein the CM is positioned between CP2 and DD2, wherein the CM functions as a substrate for a protease, and DD1 and DD2 bind to each other, characterized by a reduction in at least one activity of CP1 and / or CP2 compared to a control level of at least one activity of CP1 and / or CP2.

[0012] The present disclosure provides an activatable cytokine construct (ACC), comprising: (a) a first monomer comprising a first mature cytokine protein (CP1), a cleavable moiety (CM), and a first dimerization domain (DD1), wherein the CM is located between CP1 and DD1; and (b) a second monomer comprising a second mature cytokine protein (CP2) and a second dimerization domain (DD2), wherein the CM functions as a substrate for a protease, and wherein the DD1 and DD2 bind to each other, and wherein the ACC is characterized by a reduction in at least one activity of CP1 and / or CP2 compared to a control level of at least one activity of CP1 and / or CP2.

[0013] The present disclosure provides an activatable cytokine construct (ACC) comprising: (a) a first monomer comprising a first mature cytokine protein (CP1) and a first dimerization domain (DD1); and (b) a second monomer comprising a second mature cytokine protein (CP2) and a second dimerization domain (DD2), wherein CP1, CP2, or both CP1 and CP2 contain an amino acid sequence that functions as a substrate for a protease, and DD1 and DD2 bind to each other, and wherein the ACC is characterized by a reduction in at least one activity of CP1 and / or CP2 compared to a control level of at least one activity of CP1 and / or CP2.

[0014] In some embodiments, CP1 comprises an interleukin polypeptide and / or CP2 comprises an interleukin polypeptide. In some embodiments, the ACC is characterized as having a reduced level of interleukin activity compared to a corresponding control interleukin. For example, in some embodiments, the control interleukin may comprise a recombinant interleukin protein or a pegylated interleukin protein. In some embodiments, the interleukin polypeptide is a protein selected from the group consisting of IL-1α, IL-1β, IL-1RA, IL-18, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-3, IL-5, IL-6, IL-11, IL-12, IL-10, IL-20, IL-21, IL-14, IL-16, and IL-17. In some embodiments, CP1 and / or CP2 comprise IL-15.

[0015] In some embodiments, the first monomer comprising the first mature cytokine protein (CP1) and / or the second monomer comprising the second mature cytokine protein (CP2) further comprises a peptide mask (PM). In some embodiments, the ACC further comprises a CM between the PM and the CP.

[0016] In some embodiments, an activatable cytokine construct (ACC) is provided that contains a first monomer construct and a second monomer construct, where (a) the first monomer construct comprises a first peptide mask (PM1), a first mature cytokine protein (CP1), first and third cleavable portions (CM1 and CM3), and a first dimerization domain (DD1), where CM1 is disposed between CP1 and DD1, and CM3 is disposed between PM1 and CP1, and (b) the second monomer construct comprises a second mature cytokine protein (CP1), a first peptide mask (PM1), a first mature cytokine protein (CP1), a first and third cleavable portion (CM1 and CM3), and a first dimerization domain (DD1), where CM1 is disposed between CP1 and DD1, and CM3 is disposed between PM1 and CP1, and (c) the second monomer construct comprises a second mature cytokine protein (CP1), a first peptide mask (PM1), a first mature cytokine protein (CP1), a first and third cleavable portion (CM1 and CM3), and a first dimerization domain (DD1), where CM1 is disposed between CP1 and DD1, and CM3 is disposed between PM1 and CP1, and (d) the second monomer construct comprises a second mature cytokine protein (CP1), a first peptide mask (PM1), a first mature cytokine protein (CP1), a first and third cleavable portion (CM1 and CM3), and a first dimerization domain (DD1), where CM1 is disposed between CP1 and DD1, and CM3 is disposed between PM1 and CP1, and An activatable cytokine construct (ACC) is provided, comprising an intrinsic protein (CP2), a second cleavable portion (CM2), and a second dimerization domain (DD2), wherein CM2 is positioned between CP2 and DD2, and DD1 and DD2 bind to each other, thereby causing the first monomer construct and the second monomer construct to form a dimer, and wherein the activity level of at least one of CP1 and / or CP2 is reduced compared to a control level of at least one of the activities of CP1 and / or CP2.

[0017] In some embodiments, the second monomer construct further comprises a second peptide mask (PM2) and a fourth cleavable portion (CM4), where CM4 is located between PM2 and CP2. In some embodiments, the first monomer construct comprises a first polypeptide comprising PM1, CM3, CP1, CM1 and DD1. In some embodiments, the second monomer construct comprises a second polypeptide comprising CP2, CM2 and DD2. In some embodiments, the second monomer construct comprises a second polypeptide comprising PM2, CM4, CP2, CM2 and DD2.

[0018] In some embodiments, the first monomer construct comprises a first polypeptide comprising CP1, CM1 and DD1. In some embodiments, the second monomer construct comprises a second polypeptide comprising CP2, CM2 and DD2. In some embodiments, DD1 and DD2 are a pair selected from the group consisting of a pair of Fc domains; the alpha chain of the human IL-15 receptor (IL15Rα) and the sushi domain from soluble IL-15; barnase and barstar; protein kinase A (PKA) and A kinase anchor protein (AKAP); a mutant RNase I fragment-based adaptor / docking tag module; an epitope and a single domain antibody (sdAb); an epitope and a single chain variable region fragment (scFv); a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) module based on the interaction of the proteins syntaxin, synaptotagmin, synaptobrevin and SNAP25; an antigen binding domain and an epitope.

[0019] In some embodiments, DD1 and DD2 are a pair of Fc domains. In some embodiments, the pair of Fc domains is a pair of human Fc domains. In some embodiments, the human Fc domain is a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, or a human IgG4 Fc domain. In some embodiments, the human Fc domain is a human IgG4 Fc domain. In some embodiments, the human Fc domain comprises a sequence that is at least 80% identical to SEQ ID NO:3. In some embodiments, the human Fc domains each comprise a sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:3. In some embodiments, the human Fc domains each comprise SEQ ID NO:3. In some embodiments, DD1 and DD2 are identical. For example, DD1 and DD2 may be a pair of identical human IgG4 Fc domains. In some embodiments, the dimerization domains have the amino acid sequences of SEQ ID NO:315 and SEQ ID NO:316, respectively. In some embodiments, the human Fc domain contains mutations to eliminate glycosylation and / or reduce Fc-gamma receptor binding. In some embodiments, the human Fc domain contains the mutations N297Q, N297A or N297G; in some embodiments, the human Fc domain contains a mutation at position 234 and / or 235, such as L235E, or L234A and L235A (in IgG1), or F234A and L235A (in IgG4); in some embodiments, the human Fc domain is an IgG2 Fc domain containing the mutations V234A, G237A, P238S, H268Q / A, V309L, A330S, or P331S, or a combination thereof (all according to EU numbering).

[0020] Additional examples of engineered human Fc domains are known to those skilled in the art. Examples of Ig heavy chain constant region amino acids in which at least one amino acid mutation results in reduced Fc function include, but are not limited to, mutations at amino acids 228, 233, 234, 235, 236, 237, 239, 252, 254, 256, 265, 270, 297, 318, 320, 322, 327, 329, 330 and 331 of the heavy chain constant region (according to EU numbering). Examples of combinations of mutated amino acids are also known in the art, including, but are not limited to, combinations of mutations at amino acids 234, 235 and 331, such as L234F, L235E and P331S, or combinations of mutations at amino acids 318, 320 and 322, such as E318A, K320A and K322A.

[0021] Further examples of engineered Fc domains include F243L / R292P / Y300L / V305I / P396 IgG1; S239D / I332E IgG1; S239D / I332E / A330L IgG1; S298A / E333A / K334A; L234Y / L235Q / G236W / S239M / H268D / D270E / S298A IgG1 in one heavy chain, and D270E / K326D, A330M / K334E IgG1; G236A / S239D / I332E IgG1; K326W / E333S IgG1; S267E / H268F / S324T in the opposite heavy chain. These include IgG1; E345R / E430G / S440Y IgG1; N297A or N297Q or N297G IgG1; L235E IgG1; L234A / L235A IgG1; F234A / L235A IgG4; H268Q / V309L / A330S / P331S IgG2; V234A / G237A / P238S / H268A / V309L / A330S / P331S IgG2; M252Y / S254T / T256E IgG1; M428L / N434S IgG1; S267E / L328F IgG1; N325S / L328F IgG1, etc. In some embodiments, the engineered Fc domain comprises one or more substitutions selected from the group consisting of N297A IgG1, N297Q IgG1, and S228P IgG4.

[0022] In some embodiments, DD1 comprises an antigen binding domain and DD2 comprises a corresponding epitope. In some embodiments, the antigen binding domain is an anti-His tag antigen binding domain and DD2 comprises a His tag. In some embodiments, the antigen binding domain is a single chain variable region fragment (scFv). In some embodiments, the antigen binding domain is a single domain antibody (sdAb). In some embodiments, at least one of DD1 and DD2 comprises a dimerization domain substituent selected from the group consisting of a non-polypeptide polymer and a small molecule. In some embodiments, DD1 and DD2 comprise a non-polypeptide polymer covalently linked to each other. In some embodiments, the non-polypeptide polymer is a sulfur-containing polyethylene glycol and DD1 and DD2 are covalently linked to each other via one or more disulfide bonds. In some embodiments, at least one of DD1 and DD2 comprises a small molecule. In some embodiments, the small molecule is biotin. In some embodiments, DD1 comprises biotin and DD2 comprises avidin.

[0023] In some embodiments, CP1 and CP2 are mature cytokines. In some embodiments, CP1 and CP2 each comprise a mature cytokine sequence and further comprise a signal peptide (also referred to herein as a "signal sequence"). In some embodiments, CP1 and / or CP2 are each independently selected from the group consisting of interferon, interleukin, GM-CSF, G-CSF, LIF, OSM, CD154, LT-β, ​​TNF-α, TNF-β, 4-1BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE, TGF-β1, TGF-β1, TGF-β3, Epo, Tpo, Flt-3L, SCF, M-CSF, and MSP.

[0024] CP1 and / or CP2 may be wild-type human or non-human animal sequences, mutant sequences, truncated sequences, hybrid sequences, or sequences containing insertions. In some embodiments, CP1 and CP2 are the same. In some embodiments, CP1 and CP2 are different, and the disclosure encompasses any two selections and combinations of cytokine proteins listed herein. In some embodiments, CP1 and / or CP2 are interleukins. In some embodiments, CP1 and CP2 are both interleukins. In some embodiments, CP1 and CP2 are different interleukins. In some embodiments, CP1 and CP2 are the same interleukin. In some embodiments, CP1 or CP2 are interleukins. In some embodiments, one of CP1 and CP2 is an interleukin and the other CP1 or CP2 is a cytokine other than an interleukin. In some aspects, one or both cytokines are monomeric cytokines. In some aspects, one or both interleukins are monomeric interleukins. In some aspects, one of CP1 or CP2 is a monomeric interleukin and the other CP1 or CP2 is a different cytokine. In some embodiments, CP1 and / or CP2 are each independently selected from the group consisting of IL-1α, IL-1β, IL-1RA, IL-18, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-3, IL-5, IL-6, IL-11, IL-12, IL-10, IL-20, IL-21, IL-14, IL-16, and IL-17. In some embodiments, CP1 and / or CP2 comprises IL-15. In some aspects, CP1 and / or CP2 contain a variant cytokine sequence. In some aspects, CP1 and / or CP2 contain a universal cytokine sequence. In some aspects, CP1 and / or CP2 contain a truncated sequence that retains cytokine activity.

[0025] In some embodiments, the interleukin(s) is a human wild type mature interleukin. In some embodiments, the interleukin(s) may be IL-15. In some embodiments, CP1 and CP2 are both IL-15. In some embodiments, CP1 and CP2 are both human mature IL-15. In some embodiments, CP1 and CP2 both comprise an amino acid sequence derived from human mature IL-15. In some embodiments, IL-15 may be truncated. In some embodiments, IL-15 comprises amino acids 49-161 (SEQ ID NO: 347) of human IL-15. In some embodiments, IL-15 comprises amino acids 49-162 (SEQ ID NO: 348) of human IL-15. In some embodiments, the interleukin(s) is a mutant interleukin. In some embodiments, the interleukin(s) is a mutant interleukin in which an endogenous protease cleavage site has been rendered dysfunctional by substitution, deletion, or insertion of one or more amino acids. In some embodiments, the interleukin(s) is a universal cytokine molecule, e.g., having a hybrid sequence of different cytokine subtypes, or a chimeric cytokine sequence, or a humanized cytokine sequence. In some embodiments, CP1 and / or CP2 comprises a sequence that is at least 80% identical to SEQ ID NO: 347. In some embodiments, CP1 and / or CP2 comprises a sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 347. In some embodiments, CP1 and / or CP2 comprises the sequence of SEQ ID NO: 347. In some embodiments, CP1 and / or CP2 comprises an interleukin. In some embodiments, the interleukin is selected from the group consisting of IL-1α, IL-1β, IL-1RA, IL-18, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-3, IL-5, IL-6, IL-11, IL-12, IL-10, IL-20, IL-14, IL-16, and IL-17. In some embodiments, the interleukin is selected from the group consisting of IL-2 and IL-15.

[0026] In some embodiments, CM1 and / or CM2 each comprise a total of about 3 amino acids to about 15 amino acids. In some embodiments, CM1 and CM2 comprise substrates for different proteases. In some embodiments, CM1 and CM2 are the same length and comprise identical amino acid sequences. In some embodiments, CM1 and CM2 comprise substrates for the same protease. In some embodiments, the protease(s) are ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, BACE, renin, cathepsin D, cathepsin E, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, caspase 14, caspase 15, caspase 16, caspase 17, caspase 18, caspase 19, caspase 20, caspase 21, caspase 22, caspase 23, caspase 24, caspase 25, caspase 26, caspase 27, caspase 28, caspase 29, caspase 30, caspase 31, caspase 32, caspase 33, caspase 34, caspase 35, caspase 36, caspase 37, caspase 38, caspase 39, caspase 31, caspase 31, caspase 32, caspase 33, caspase 34, caspase 35, caspase 36, caspase 37, caspase 38, caspase 39, ca caspase-10, caspase-14, cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P, cruzipain, legumain, otubain-2, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, meprin, neprilysin, PSMA, BMP-1, matrix metalloproteinase (MMP) For example, MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-19, MMP-20, MMP-23, MMP-24, MMP-26, MMP-27), activated protein C, cathepsin A, cathepsin G, chymase, FVIIa, FIXa, FXa, FXIa, FXIIa, elastase, The protein is selected from the group consisting of: tPA, granzyme B, guanidinobenzoatase, HtrA1, human neutrophil lyase, lactoferrin, marapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, tryptase, uPA, DESC1, DPP-4, FAP, hepsin, matriptase-2, MT-SP1 / matriptase, TMPRSS2, TMPRSS3 and TMPRSS4.In some embodiments, the protease(s) is selected from the group consisting of uPA, legumain, MT-SP1, ADAM17, BMP-1, TMPRSS3, TMPRSS4, MMP-2, MMP-9, MMP-12, MMP-13, and MMP-14.

[0027] Suitable cleavable moieties are disclosed in WO2010 / 081173, WO2015 / 048329, WO2015 / 116933, WO2016 / 118629 and WO2020 / 118109, the disclosures of which are incorporated herein by reference in their entireties.

[0028] In some embodiments, CM1 and / or CM2 are selected from the group consisting of LSGRSDNH (SEQ ID NO:5), TGRGPSWV (SEQ ID NO:6), PLTGRSGG (SEQ ID NO:7), TARGPSFK (SEQ ID NO:8), NTLSGRSENHSG (SEQ ID NO:9), NTLSGRSGNHGS (SEQ ID NO:10), TSTSGRSANPRG (SEQ ID NO:11), TSGRSANP (SEQ ID NO:12), VHMPLGFLGP (SEQ ID NO:13), AVGLLAPP (SEQ ID NO:14), AQNLLGMV (SEQ ID NO:15), QNQALRMA (SEQ ID NO:16), LAAPLGLL( SEQ ID NO:17), STFPFGMF (SEQ ID NO:18), ISSGLLSS (SEQ ID NO:19), PAGLWLDP (SEQ ID NO:20), VAGRSMRP (SEQ ID NO:21), VVPEGRRS (SEQ ID NO:22), ILPRSPAF (SEQ ID NO:23), MVLGRSLL (SEQ ID NO:24), QGRAITFI (SEQ ID NO:25), SPRSIMLA (SEQ ID NO:26), SMLRSMPL (SEQ ID NO:27), ISSGLLLSGRSDNH (SEQ ID NO:28), AVGLLAPPGGLSGRSDNH (SEQ ID NO:29), ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 30), LSGRSGNH (SEQ ID NO: 31), SGRSANPRG (SEQ ID NO: 32), LSGRSDDH (SEQ ID NO: 33), LSGRSDIH (SEQ ID NO: 34), LSGRSDQH (SEQ ID NO: 35), LSGRSDTH (SEQ ID NO: 36), LSGRSDYH (SEQ ID NO: 37), LSGRSDNP (SEQ ID NO: 38), LSGRSANP (SEQ ID NO: 39), LSGRSANI (SEQ ID NO: 40), LSGRSDNI (SEQ ID NO: 41), MIAPVAYR (SEQ ID NO: 42), RPSPMWAY (SEQ ID NO: 43), WATPRPMR (SEQ ID NO: 44), FRLLDWQW (SEQ ID NO: 45), ISSGL (SEQ ID NO: 46), ISSGLLS (SEQ ID NO: 47), ISSGLL (SEQ ID NO: 48), ISSGLLSGRSAMPRG (SEQ ID NO: 49), AVGLLAPPTSGRSANPRG (SEQ ID NO: 50), AVGLLAPPSGRSANPRG (SEQ ID NO: 51), ISSGLLSGRSDDH (SEQ ID NO: 52), ISSGLLSGRSDIH (SEQ ID NO: 53), ISSGLLSGRSDQH (SEQ ID NO: 54), ISSGLLSGRSDTH (SEQ ID NO: 55), ISSGLLSGRSDYH (SEQ ID NO: 56),ISSGLLSGRSDNP (SEQ ID NO: 57), ISSGLLSGRSANP (SEQ ID NO: 58), ISSGLLSGRSANI (SEQ ID NO: 59), AVGLLAPPGGLSGRSDDH (SEQ ID NO: 60), AVGLLAPPGGLSGRSDIH (SEQ ID NO: 61), AVGLLAPPGGLSGRSDQH (SEQ ID NO: 62), AVGLLAPPGGLSGRSDTH (SEQ ID NO: 63), AVGLLAPPGGLSGRSDYH (SEQ ID NO: 64), AVGLLAPPGGLSGRSDNP (SEQ ID NO: 65), AV GLLAPPGGLSGRSANP (SEQ ID NO: 66), AVGLLAPPGGLSGRSANI (SEQ ID NO: 67), ISSGLLSGRSDNI (SEQ ID NO: 68), AVGLLAPPGGLSGRSDNI (SEQ ID NO: 69), GLSGRSDNHGGAVGLLAPP (SEQ ID NO: 70), GLSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 71), LSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 72), ISSGLSS (SEQ ID NO: 73), PVGYTSSL (SEQ ID NO: 74), DWLYWPGI (SEQ ID NO: 75), Row number 75), LKAAPRWA (SEQ ID NO: 76), GPSHLVLT (SEQ ID NO: 77), LPGGLSPW (SEQ ID NO: 78), MGLFSEAG (SEQ ID NO: 79), SPLPLRVP (SEQ ID NO: 80), RMHLRSLG (SEQ ID NO: 81), LLAPSHRA (SEQ ID NO: 82), GPRSFGL (SEQ ID NO: 83), GPRSFG (SEQ ID NO: 84), SARGPSRW (SEQ ID NO: 85), GGWHTGRN (SEQ ID NO: 86), HTGRSGAL (SEQ ID NO: 87), AARGPAIH (SEQ ID NO: 88), RGPAFN PM (SEQ ID NO: 89), SSRGPAYL (SEQ ID NO: 90), RGPATPIM (SEQ ID NO: 91), RGPA (SEQ ID NO: 92), GGQPSGMWGW (SEQ ID NO: 93), FPRPLGITGL (SEQ ID NO: 94), SPLTGRSG (SEQ ID NO: 95), SAGFSLPA (SEQ ID NO: 96), LAPLGLQRR (SEQ ID NO: 97), SGGPLGVR (SEQ ID NO: 98), PLGL (SEQ ID NO: 99), SGRSDNI (SEQ ID NO: 100), and LSGRSNI (SEQ ID NO: 349). In some embodiments, the CM comprises a sequence selected from the group consisting of ISSGLLSGRSDNH (SEQ ID NO: 28), LSGRSDDH (SEQ ID NO: 33), ISSGLLSGRSDQH (SEQ ID NO: 54),In some embodiments, the CM comprises a sequence selected from the group consisting of SGRSDNI (SEQ ID NO: 100), ISSGLLSGRSDNI (SEQ ID NO: 68), LSGRSDNI (SEQ ID NO: 41), and LSGRSNI (SEQ ID NO: 349). In some embodiments, the CM comprises a sequence selected from the group consisting of SGRSDNI (SEQ ID NO: 100), LSGRSDNI (SEQ ID NO: 41), and LSGRSNI (SEQ ID NO: 349). In some embodiments, the protease(s) are produced by a tumor in the subject, e.g., the protease is produced in greater amounts in the tumor than in healthy tissue of the subject. In some embodiments, the subject has been diagnosed or identified as having cancer.

[0029] In some embodiments, in the first monomer construct, CP1 and CM1 are directly adjacent to each other. In some embodiments, in the first monomer construct, CM1 and DD1 are directly adjacent to each other. In some embodiments, in the second monomer construct, CP2 and CM2 are directly adjacent to each other. In some embodiments, in the second monomer construct, CM2 and DD2 are directly adjacent to each other. In some embodiments, the first monomer construct comprises CP1 directly adjacent to CM1, and CM1 directly adjacent to DD1, wherein CM1 comprises a sequence selected from the group consisting of SEQ ID NO:5-100 and SEQ ID NO:349. In some embodiments, the second monomer construct comprises CP2 directly adjacent to CM2, and CM2 directly adjacent to DD2, wherein CM2 comprises a sequence selected from the group consisting of SEQ ID NO:5-100 and SEQ ID NO:349. In some embodiments, the first monomer construct comprises CP1 directly adjacent to CM1, and CM1 directly adjacent to DD1, where CM1 comprises a sequence of no more than 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 amino acids in length. In some embodiments, the second monomer construct comprises CP2 directly adjacent to CM2, and CM2 directly adjacent to DD2, where CM2 comprises a sequence of no more than 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 amino acids in length. In some embodiments, the first and second monomer constructs are each configured such that the cytokine (CM1 and CM2, respectively) is immediately adjacent to a cleavable portion (CM1 and CM2, respectively) that is 10, 9, 8, 7, 6, 5, or 4 amino acids or less in length, which is immediately adjacent to a dimerization domain (DD1 and DD2, respectively) that is an Fc region of human IgG, with the N-terminus of the Fc region being the first cysteine ​​residue in the hinge region reading from the N-terminus to the C-terminus (e.g., cysteine ​​226 for human IgG1, using EU numbering). In some aspects, the dimerization domain is an IgG Fc region with the upper hinge residues deleted.For example, the Fc is mutant in which the N-terminal sequence EPKSCDKTHT (SEQ ID NO: 387), ERK, ELKTPLGDTTHT (SEQ ID NO: 388) or ESKYGPP (SEQ ID NO: 389) is deleted.

[0030] In some embodiments, the first monomer construct comprises at least one linker. In some embodiments, the at least one linker is a linker L1 disposed between CP1 and CM1 and / or a linker L2 disposed between CM1 and DD1. In some embodiments, the second monomer construct comprises at least one linker. In some embodiments, the at least one linker is a linker L3 disposed between CP2 and CM2 and / or a linker L4 disposed between CM2 and DD2. In some embodiments, the first monomer construct comprises a linker L1 and the second monomer construct comprises a linker L3. In some embodiments, L1 and L3 are the same. In some embodiments, the first monomer construct comprises a linker L2 and the second monomer construct comprises a linker L4. In some embodiments, L2 and L4 are the same. In some embodiments, each linker has a total length of 1 amino acid to about 15 amino acids. In some embodiments, each linker has a total length of at least 5 amino acids. As used herein, the term "linker" means a peptide whose amino acid sequence is not a substrate for proteases.

[0031] In some embodiments, the first monomeric construct comprises at least one linker, each linker being independently selected from a single glycine (G); two glycine residues (GG); GSSGGSGGSGG (SEQ ID NO:210); GGGS (SEQ ID NO:2); GGGSGGGS (SEQ ID NO:211); GGGSGGGSGGGS (SEQ ID NO:212); GGGGSGGGGSGGGGGS (SEQ ID NO:213); GGGGSGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO:214); GGGGSGGGGS (SEQ ID NO:215); GGGGS (SEQ ID NO:216); GS; GGGGSGS (SEQ ID NO:217); GGGGSGGGGSGGGGSGS (SEQ ID NO:218); GGSLDPKGGGGS (SEQ ID NO:219); PKSCDKTHTCPPCPAPELLG (SEQ ID NO:220); SKYGPPCPPCPAPEFLG (SEQ ID NO:221); GKSSGSGSESKS (SEQ ID NO:222); GSTSGSGK SSEGKG (SEQ ID NO:223); GSTGSSGKSSEGSGSTKG (SEQ ID NO:224); GSTGSSGKPGSGEGSTKG (SEQ ID NO:225); GSTGSSGKPGSSEGST (SEQ ID NO:226); (GS)n, (GGS)n, (GSGGS)n (SEQ ID NO:227), (GGGS)n (SEQ ID NO:228), (GGGGS)n (SEQ ID NO:216), where each n is an integer that is at least 1; GGSG (SEQ ID NO:229); GGSGG (SEQ ID NO:230); GSGSG (SEQ ID NO:231; GSGGG (SEQ ID NO:232); GGGSG (SEQ ID NO:233); GSSSG (SEQ ID NO:234); GGGGSGGGGSGGGGGS (SEQ ID NO:213); GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:235); and GSTGSSGKPGSSEGST (SEQ ID NO:226). In some embodiments, the linker comprises the sequence of GGGS (SEQ ID NO:2).

[0032] As used herein, the term "spacer" refers herein to an amino acid residue or peptide that is incorporated into the free end of a mature ACC, for example, between a signal peptide and the N-terminus of the mature ACC. In some embodiments, the spacer (or "header") may contain a glutamine (Q) residue. In some embodiments, the residues in the spacer minimize the action of aminopeptidases and / or exopeptidases to prevent cleavage of the N-terminal amino acid. Exemplary, non-limiting spacer amino acid sequences may include or consist of any of the following exemplary amino acid sequences: QGQSGS (SEQ ID NO:375); GQSGS (SEQ ID NO:376); QSGS (SEQ ID NO:377); SGS; GS; S; QGQSGQG (SEQ ID NO:378); GQSGQG (SEQ ID NO:379); QSGQG (SEQ ID NO:380); SGQG (SEQ ID NO:381); GQG; QG; G; QGQSGQ (SEQ ID NO:382); GQSGQ (SEQ ID NO:383); QSGQ (SEQ ID NO:384); QGQSG (SEQ ID NO:385); QGQS (SEQ ID NO:386); SGQ; GQ; and Q. In some embodiments, the spacer sequence may be omitted.

[0033] In some embodiments, the first monomeric construct comprises DD1 linked directly or indirectly to the C-terminus of optional PM1, optional CM3, CP1, CM1, and CM1 in the N-terminal to C-terminal direction. In some embodiments, the first polypeptide comprises DD1 linked directly or indirectly to the N-terminus of optional PM1, optional CM3, CP1, CM1, and CM1 in the C-terminal to N-terminal direction. In some embodiments, the second polypeptide comprises DD2 linked directly or indirectly to the C-terminus of optional PM2, optional CM4, CP2, CM2, and CM2 in the N-terminal to C-terminal direction. In some embodiments, the second polypeptide comprises DD2 linked directly or indirectly to the C-terminus of CP2, CM2, and CM2 in the C-terminal to N-terminal direction.

[0034] In some embodiments, the first monomer construct comprises, in the N-terminal to C-terminal direction, CP1, an optional linker, CM1, an optional linker, and DD1, where DD1 is an IgG Fc region, the N-terminus of the Fc region is the first cysteine ​​residue in the hinge region reading in the N-terminal to C-terminal direction (e.g., cysteine ​​226 of human IgG1 or IgG4, using EU numbering), and where CM1 and the optional linker(s) located between CP1 and the N-terminal cysteine ​​of DD1 together have an overall length of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or 4 amino acids or less, preferably 10 amino acids or less, particularly preferably 7 amino acids or less. In some embodiments, the second monomer construct comprises, in the N-terminal to C-terminal direction, CP2, an optional linker, CM2, an optional linker, and DD2, where DD2 is an IgG Fc region, the N-terminus of the Fc region is the first cysteine ​​residue in the hinge region reading in the N-terminal to C-terminal direction (e.g., cysteine ​​226 of human IgG1 or IgG4, using EU numbering), and where CM2 and the optional linker(s) located between CP2 and the N-terminal cysteine ​​of DD2 together have an overall length of 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or 4 amino acids or less, preferably 10 amino acids or less, preferably 8 amino acids or less, particularly preferably 7 amino acids or less.

[0035] In some embodiments, the ACC is a homodimer in which the first and second monomeric constructs are identical and comprise the amino acid sequence of SEQ ID NO: 350. In some embodiments, the ACC is a homodimer in which the first and second monomeric constructs are identical and comprise amino acids 21-359 of SEQ ID NO: 350. In some embodiments, the ACC is a homodimer in which the first and second monomeric constructs are identical and comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 350, SEQ ID NO: 351, SEQ ID NO: 352, SEQ ID NO: 353, SEQ ID NO: 354, SEQ ID NO: 355, and SEQ ID NO: 356. In some embodiments, the first and second monomeric constructs each comprise an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to amino acids 21-359 of SEQ ID NO: 350. In some embodiments, the first and second monomer constructs each comprise an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to a sequence selected from the group consisting of SEQ ID NO:350, amino acids 21-359 of SEQ ID NO:350, SEQ ID NO:351, SEQ ID NO:352, SEQ ID NO:353, SEQ ID NO:354, SEQ ID NO:355 and SEQ ID NO:356. In some embodiments, the first and second monomer constructs each comprise an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:347. In some embodiments, the first and second monomer constructs each comprise, from N-terminal to C-terminal, SEQ ID NO:347; a CM comprising an amino acid sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:68, SEQ ID NO:100 and SEQ ID NO:349; and a dimerization domain. In some embodiments, the first monomer construct and the second monomer construct each comprise, from N-terminus to C-terminus, an optional peptide mask that specifically binds human IL-15; an optional CM3; a CP1 comprising the amino acid sequence of human IL-15; a CM1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:5-100 and SEQ ID NO:349; and an Fc domain of human IgG.In some embodiments, the first monomer construct and the second monomer construct each comprise, from N-terminus to C-terminus, an optional peptide mask that specifically binds human IL-15; an optional CM3; SEQ ID NO:347; a CM comprising an amino acid sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:68, SEQ ID NO:100, and SEQ ID NO:349; and an Fc domain of human IgG. In some embodiments, CP1 is IL-15 and ACC comprises a peptide mask comprising an amino acid sequence from the group consisting of SEQ ID NOs:358-374. In some embodiments, CP1 is IL-15 and ACC comprises a peptide mask of 40 or less amino acids derived from an amino acid sequence selected from the group consisting of SEQ ID NOs:358-374.

[0036] In some embodiments, at least one CP1 and / or CP2 activity is measured using the binding affinity (K D). For example, when CP1 or CP2 is an interleukin, the cognate receptor can be an interleukin receptor, including, for example, CD25 (IL-2Rα), CD122 (IL-2Rβ) and CD132 (IL-2Rγ). In some embodiments, the at least one CP1 and / or CP2 activity is the level of proliferation of lymphoma cells. In some embodiments, the at least one CP1 and / or CP2 activity is the level of activation of the JAK / STAT / ISGF3 pathway in lymphoma cells. In some embodiments, the at least one activity is the level of production of secreted alkaline phosphatase (SEAP) in cells, such as lymphoma cells or HEK cells. In some embodiments, the ACC (before exposure to protease) is characterized by at least one CP1 and / or CP2 activity being at least 2-fold lower compared to the level of a control. In some embodiments, the ACC is characterized by at least one CP1 and / or CP2 activity being at least 5-fold lower compared to the level of a control. In some embodiments, the ACC is characterized by at least one activity of CP1 and / or CP2 being at least 10-fold lower compared to a control level. In some embodiments, the ACC is characterized by at least one activity of CP1 and / or CP2 being at least 20-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 1100-fold, 1200-fold, 1300-fold, 1400-fold, 1500-fold, 1600-fold, 1700-fold, 1800-fold, 1900-fold, 2000-fold lower compared to a control level. In some embodiments, the control level of at least one activity of CP1 and / or CP2 is the activity of CP1 and / or CP2 of ACC after exposure of the ACC to a protease(s). In some embodiments, the control level of at least one of CP1 and / or CP2 is the corresponding CP1 and / or CP2 activity of the corresponding wild-type mature cytokine.

[0037] In some embodiments, the ACC is characterized by producing a cleavage product after exposure to a protease(s), the cleavage product comprising at least one activity of CP1 and / or CP2. In some embodiments, the at least one activity of CP1 and / or CP2 is an anti-proliferative activity. In some embodiments, the control level is the EC50 value of the wild-type mature cytokine, and the ratio of EC50 (cleavage product) to EC50 (wild-type control level) is less than about 10, or less than about 9, or less than about 8, or less than about 7, or less than about 6, or less than about 5, or less than about 4, or less than about 3, or less than about 2, or less than about 1.5, or equal to about 1. In some embodiments, the EC50 of the cleavage product is approximately the same as the EC50 of the wild-type mature cytokine, indicating that after cleavage, the activity of CP1 and / or CP2 is fully or nearly fully restored. In some embodiments, the ratio of the EC50 of the cleavage product to the EC50 of the wild type control is about 1 to about 10, or about 2 to about 8, or about 3 to about 7, or about 4 to about 6, indicating good recovery of cytokine activity after protease activation. In some embodiments, CP1 and / or CP2 are IL-15 and ACC is characterized as having a cleavage product after protease activation, and the ratio of the EC50 of the cleavage product to the EC50 of recombinant IL-15 is 1 to about 10, or about 2 to about 8, or about 3 to about 7, or about 4 to about 6, or about 5 to about 7, or about 6, when measured in IL-2 / IL-15-responsive HEK293 cells.

[0038] Provided herein is a composition comprising any one of the ACCs described herein.In some embodiments, the composition is a pharmaceutical composition.Also provided herein is a kit comprising at least one dose of any one of the compositions described herein.

[0039] Provided herein is a method of treating a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of any one of the ACCs described herein or any one of the compositions described herein.In some embodiments, the subject is identified or diagnosed as having cancer.In some non-limiting embodiments, the cancer is Kaposi's sarcoma, hairy cell leukemia, chronic myelogenous leukemia (CML), follicular lymphoma, renal cell carcinoma (RCC), melanoma, neuroblastoma, basal cell carcinoma, bladder cancer, breast cancer, colorectal cancer, malignant cutaneous T-cell lymphoma, nasopharyngeal adenocarcinoma, non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic cancer.In some non-limiting embodiments, the cancer is lymphoma.In some non-limiting embodiments, the lymphoma is Burkitt's lymphoma.

[0040] Provided herein is a nucleic acid encoding a polypeptide comprising CP1 and CM1 of any one of the ACCs described herein. In some embodiments, the polypeptide further comprises any one of the DD1s described herein. Provided herein is also a nucleic acid encoding a polypeptide comprising CP2 and CM2 of any one of the ACCs described herein. When the monomers are identical, the present disclosure provides a single nucleic acid encoding the monomers that dimerize to form the ACC. In some embodiments, the polypeptide further comprises any one of the DD2s described herein. Provided herein is also a vector comprising any one of the nucleic acids described herein. In some embodiments, the vector is an expression vector. Provided herein is also a cell comprising any one of the nucleic acids described herein or any one of the vectors described herein. In some embodiments, the nucleic acid encoding the polypeptide comprises a polynucleotide according to SEQ ID NO: 357.

[0041] Provided herein is a pair of nucleic acids that together encode a polypeptide comprising CP1 and CM1 of a first monomeric component and a polypeptide comprising CP2 and CM2 of a second monomeric component of any one of the ACCs described herein.Also provided herein is a pair of vectors that together comprise any one of the nucleic acid pairs described herein.In some embodiments, the vector pair is an expression vector pair.Also provided herein is a cell that comprises any one of the nucleic acid pairs described herein or any one of the vector pairs described herein.In other embodiments, the present invention provides a vector that comprises a vector pair.

[0042] Provided herein is a method for producing ACC, comprising culturing any one of the cells described herein in liquid medium under sufficient conditions to produce ACC, and recovering ACC from the cells or liquid medium.In some embodiments, the method further comprises isolating the ACC recovered from the cells or liquid medium.In some embodiments, the method further comprises preparing the isolated ACC into a pharmaceutical composition.

[0043] Provided herein is ACC produced by any one of the methods described herein.Also provided herein is a composition comprising any one of the ACCs described herein.Also provided herein is a composition of any one of the compositions described herein, and the composition is a pharmaceutical composition.Also provided herein is a kit comprising at least one dose of any one of the compositions described herein.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. This specification describes methods and materials for use herein. Other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0045] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims.

[0046] The terms "a" and "an" refer to one or more (i.e., to at least one) of the grammatical object of the article. As an example, "a cell" includes one or more cells.

[0047] As used herein, the terms "about" and "approximately," when used to modify a quantity specified in a numerical value or range, refer not only to that numerical value but also to reasonable deviations from that value known to those of skill in the art, e.g., ±20%, ±10%, or ±5%, where appropriate, within the intended meaning of the recited value.

[0048] Concentrations, amounts, and other numerical data may be expressed or presented in a range format herein. It should be understood that such range formats are used for convenience and brevity only, and thus should be interpreted flexibly to include not only the numerical values ​​explicitly recited as range limitations, but also all individual numerical values ​​or subranges subsumed within the range, as if each numerical value and subrange were explicitly recited. As an illustration, a numerical range of "about 0.01 to 2.0" should be interpreted to include not only the explicitly recited values ​​of about 0.01 to about 2.0, but also the individual numerical values ​​and subranges within the stated range. Thus, within this numerical range, individual values, such as 0.5, 0.7, and 1.5, as well as subranges, such as 0.5 to 1.7, 0.7 to 1.5, and 1.0 to 1.5, are included. Moreover, such interpretation should be applied regardless of the breadth or characteristics of the range described. In addition, it should be noted that all percentages are by weight unless otherwise indicated.

[0049] In understanding the scope of the present disclosure, the terms "including" or "comprising" and their derivatives, as used herein, are intended to be open-ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers, and / or steps. The above also applies to words of similar meaning, such as the terms "including" and "having" and their derivatives. The term "consisting" and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers, and / or steps. The term "consisting essentially of," as used herein, is intended to prescribe the presence of the described features, elements, components, groups, integers, and / or steps, as well as the presence of those that do not substantially affect the basic and novel characteristic(s) of the features, elements, components, groups, integers, and / or steps. Reference to any one of these transitional phrases (i.e., "comprising," "consisting," or "consisting essentially") is understood to provide direct support for the replacement of any of the other transitional phrases not specifically used. For example, the modification of the term "comprising" to "consisting essentially of" or "consisting of" finds direct support for any element disclosed throughout this disclosure to be so defined. Based on this definition, any element disclosed herein or incorporated by reference may be included or excluded from the claimed invention.

[0050] As used herein, for convenience, a plurality of compounds, elements, or steps may be presented in general lists. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, the individual members of such lists should not be construed as being de facto equivalents to any other members of the same list solely based on their presentation in a general group, unless otherwise indicated.

[0051] Furthermore, certain molecules, constructs, compositions, elements, moieties, excipients, diseases, conditions, properties, steps, etc. may be discussed in the context of certain embodiments or aspects of this disclosure or in a separate paragraph or section. This is merely for convenience and brevity, and it is understood that any such disclosure is equally applicable and intended to be combined with any other embodiments or aspects found anywhere in this disclosure and claims, all of which form the application and claimed invention as of the filing date. For example, a listing of a construct, molecule, method step, kit, or composition described with respect to a construct, composition, or method is intended to and finds direct support for the relevant embodiments of the construct, composition, formulation, and method described in any other part of this disclosure, even if those method steps, active agents, kits, or compositions are not re-listed in the context or section of that embodiment or aspect.

[0052] Unless otherwise specified, a "nucleic acid sequence encoding a protein" includes all nucleotide sequences that are degenerate versions of each other and therefore encode the same amino acid sequence.

[0053] The term "located at the N-terminus," when referring to the position of a first domain or sequence in the primary amino acid sequence of a polypeptide relative to a second domain or sequence, means that the first domain or sequence is located closer to the N-terminus of the primary amino acid sequence of the polypeptide than the second domain or sequence. In some embodiments, there may be additional sequences and / or domains between the first and second domains or sequences.

[0054] The term "located at the C-terminus," when referring to the position of a first domain or sequence in the primary amino acid sequence of a polypeptide relative to a second domain or sequence, means that the first domain or sequence is located closer to the C-terminus of the primary amino acid sequence of the polypeptide than the second domain or sequence. In some embodiments, there may be additional sequences and / or domains between the first and second domains or sequences.

[0055] The term "exogenous" means any substance introduced or derived from outside a cell, tissue or organism and that is not produced by or derived from the same cell, tissue or organism into which it is introduced.

[0056] The terms "transduced," "transfected," or "transformed" refer to the process by which exogenous nucleic acid is introduced or transferred into a cell. A "transduced," "transfected," or "transformed" cell (e.g., a mammalian cell) is a cell that has been transduced, transfected, or transformed with an exogenous nucleic acid (e.g., a vector) that contains an exogenous nucleic acid encoding any of the activatable cytokine constructs described herein.

[0057] The term "nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or combinations thereof, in single-stranded or double-stranded form. Unless otherwise limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the referenced nucleotides. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses complementary sequences in addition to the sequence explicitly indicated. In some embodiments of any of the nucleic acids described herein, the nucleic acid is DNA. In some embodiments of any of the nucleic acids described herein, the nucleic acid is RNA.

[0058] Modifications to the nucleotide sequence can be introduced by standard techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR)-mediated mutagenesis. Conservative amino acid substitutions are those in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with basic side chains (e.g., lysine, arginine, and histidine), nonpolar amino acids (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), uncharged polar amino acids (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine), hydrophilic amino acids (e.g., arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine), hydrophobic amino acids (e.g., alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine). Other families of amino acids include the aliphatic hydroxyamino acids (e.g., serine and threonine), the amide family (e.g., asparagine and glutamine), the aliphatic family (e.g., alanine, valine, leucine and isoleucine), and the aromatic family (e.g., phenylalanine, tryptophan and tyrosine).

[0059] As used herein, the phrases "specifically bind" or "immunoreact with" refer to an activatable antigen-binding protein complex that reacts with one or more antigenic determinants of a desired target antigen and does not react with other polypeptides, or does so with very low affinity, e.g., about 10 -6 It means that the bond is M or more.

[0060] The term "treatment" means improving at least one symptom of a disease. In some embodiments, the disease being treated is cancer, and is for the purpose of alleviating at least one symptom of the cancer. [Brief description of the drawings]

[0061] [Figure 1A] 1 is a schematic diagram of an exemplary activatable cytokine construct comprising a first and a second monomer construct covalently or non-covalently linked to each other via a first and a second dimerization domain DD1 140 and DD2 190, respectively. The first monomer construct comprises, from N-terminus to C-terminus, a first mature cytokine protein CP1 100, a first optional linker 110, a first cleavable moiety CM1 120, a second optional linker 130, and a first dimerization domain DD1 140. The second monomer construct comprises, from N-terminus to C-terminus, a second mature cytokine protein CP2 150, a third optional linker 160, a second cleavable moiety CM2 170, a fourth optional linker 180, and a second dimerization domain DD2 190. [Figure 1B]1 is a schematic diagram of an exemplary activatable cytokine construct comprising a first and a second monomer construct covalently or non-covalently linked to each other via a first and a second dimerization domain DD1 200 and DD2 250, respectively. The first monomer construct comprises, from N-terminus to C-terminus, a first dimerization domain DD1 200, a second optional linker 210, a first cleavable moiety CM1 220, a first optional linker 230, and a first mature cytokine protein CP1 240. The second monomer construct comprises, from N-terminus to C-terminus, a second dimerization domain DD2 250, a fourth optional linker 260, a second cleavable moiety CM2 270, a third optional linker 280, and a second mature cytokine protein CP2 290. [Figure 1C] 1 is a schematic diagram of an exemplary activatable cytokine construct comprising, from N-terminus to C-terminus: (1) a first monomer construct 110, optionally having PM1 119, optionally CM3 117, CP1 115, CM1 113, and DD1 111; (2) a second monomer construct 120, optionally having PM2 129, optionally CM4 127, CP2 125, CM2 123, and DD2 121; and (3) one or more covalent or non-covalent bonds (←→) linking the first monomer construct 110 to the second monomer construct 120. The ACC may further comprise one or more optional linkers 112, 114, 116, 118, 122, 124, 126, and 128 between the components. In one example, DD1 111 and DD2 121 are identical. In another example, DD1 111 and DD2 121 are different. [Figure 2A]1 is a schematic diagram of an exemplary activatable cytokine construct comprising a first and a second monomer construct linked to each other by non-covalent means via a first and a second dimerization domain DD1 340 and DD2 390, respectively. The first monomer construct comprises, from N-terminus to C-terminus, a first mature cytokine protein CP1 300, a first optional linker 310, a first cleavable moiety CM1 320, a second optional linker 330, and a first dimerization domain DD1 340. The second monomer construct comprises, from N-terminus to C-terminus, a second mature cytokine protein CP2 350, a third optional linker 360, a second cleavable moiety CM2 370, a fourth optional linker 380, and a second dimerization domain DD2 390. [Figure 2B] 1 is a schematic diagram of an exemplary activatable cytokine construct comprising a first and a second monomer construct bound to each other by non-covalent means via a first and a second dimerization domain DD1 400 and DD2 450, respectively. The first monomer construct comprises, from N-terminus to C-terminus, a first dimerization domain DD1 400, a second optional linker 410, a first cleavable moiety CM1 420, a first optional linker 430, and a first mature cytokine protein CP1 440. The second monomer construct comprises, from N-terminus to C-terminus, a second dimerization domain DD2 450, a fourth optional linker 460, a second cleavable moiety CM2 470, a third optional linker 480, and a second mature cytokine protein CP2 490. [Diagram 3] The sequence of the masked cytokine construct, ProC1471, is shown with the optional signal sequence in italics, the mature IL-15 (amino acids 49-161) sequence in underlined, and the sequence of the cleavable moiety (CM) in bold. [Figure 4] The activity of ProC1471 compared to recombinant IL-15 was tested in vitro using IL-2 / IL-15-responsive HEK293 cells. [Diagram 5]Activation of ProC1471 by the proteases uPA and MT-SP1. [Figure 6] FIG. 1 shows the activity of protease-activated ProC1471 compared to non-activated ProC1471 and recombinant IL-15 tested in vitro using IL-2 / IL-15-responsive HEK293 cells. [Figure 7] A shows the effect of the flexible linker length of interferon-α2b-Fc fusions on EC50 as determined by HEK293 cell-based reporter assay.B shows the effect of the linking region (LR) length of interferon-α2b-Fc fusions on EC50 as determined by HEK293 cell-based reporter assay. [Figure 8A] 1 shows the effect of linker length of interferon-α2b-Fc fusion protein on EC50, as determined from the Daudi apoptosis assay. [Figure 8B] FIG. 1 shows the effect of the length of the linking region (LR) of the interferon-α2b-Fc fusion protein on the EC50, as determined from the Daudi apoptosis assay. [Figure 9] 1 shows the results of a HEK293 cell-based reporter assay to evaluate the activity of ACC (IFNa2b 1204DNIdL NhG4); protease-treated (activated) ACC (IFNα-2b 1204DNIdL NhG4+uPA); Sylatron®; and recombinant parent cytokine (IFNa2b). The results showed that after treatment of ACC with proteases, the cytokine activity of ACC could be restored to a level comparable to that of the recombinant parent cytokine. [Figure 10]The results of Daudi lymphoma cell-based assays to measure anti-proliferative activity (top) and HEK293 cell-based reporter assays to measure activity (bottom) of ACC (ProC440), protease-treated ACC (ProC440+uPA), and stem cell IFNa2b are shown. The results showed that the activity was reduced 1000-fold by making the ACC construct of the present disclosure, and that after treatment of ACC with proteases, the activity of the ACC cytokines could be restored to levels comparable to the recombinant parent cytokines. [Figure 11] A illustrates the structure of ProC440 and shows that cleavage at the predicted site in CM with uPa was confirmed by mass spectrometry. In addition to sensitivity to uPa activation, ProC440 is cleaved by MMP4. B shows that mass spectrometry analysis identifies an MMP14 cleavage site (L161) at the C-terminal end of IFNa, close to the cleavable site. Protease activation with MMP14 restored activity to levels comparable to recombinant cytokine. [Figure 12A] 1 is a schematic diagram of an exemplary activatable cytokine construct comprising a first and a second monomer construct bound to each other by non-covalent means via a first and a second dimerization domain DD1 540 and DD2 590, respectively. The first monomer construct comprises, from N-terminus to C-terminus, a first mature cytokine protein CP1 500, a first optional linker 510, a first cleavable moiety CM1 520, a second optional linker 530, and a first dimerization domain DD1 540. The second monomer construct comprises, from N-terminus to C-terminus, a second mature cytokine protein CP2 550, a third optional linker 580, and a second dimerization domain DD2 590. [Figure 12B]1 is a schematic diagram of an exemplary activatable cytokine construct comprising a first and a second monomer construct bound to each other by non-covalent means via a first and a second dimerization domain DD1 600 and DD2 650, respectively. The first monomer construct comprises, from N-terminus to C-terminus, a first dimerization domain DD1 600, a first optional linker 630, and a first mature cytokine protein CP1 640. The second monomer construct comprises, from N-terminus to C-terminus, a second dimerization domain DD2 650, a second optional linker 660, a cleavable moiety CM 670, a third optional linker 680, and a second mature cytokine protein CP2 690. [Figure 13A] 1 is a schematic diagram of an exemplary activatable cytokine construct comprising a first and a second monomer construct bound to each other by non-covalent means via a first and a second dimerization domain DD1 740 and DD2 790, respectively. The first monomer construct comprises, from N-terminus to C-terminus, a first mature cytokine protein CP 700, a first optional linker 710, a first cleavable moiety CM1 720, a second optional linker 730, and a first dimerization domain DD1 740. The second monomer construct comprises, from N-terminus to C-terminus, a polypeptide or protein lacking cytokine activity 780, and a second dimerization domain DD2 790. The polypeptide or protein 780 lacking cytokine activity can be, for example, a truncated cytokine protein lacking cytokine activity, a mutant cytokine protein lacking cytokine activity, a stub sequence, or a polypeptide sequence that binds with high affinity to CP 700 and reduces the cytokine activity of the second portion compared to a control level of the second portion. DD1 740 and DD2 790 can be the same or different. [Figure 13B]1 is a schematic diagram of an exemplary activatable cytokine construct comprising a first and a second monomer construct bound to each other by non-covalent means via a first and a second dimerization domain, DD1 800 and DD2 850, respectively. The first monomer construct comprises, from N-terminus to C-terminus, a first dimerization domain, DD1 800, and a polypeptide or protein lacking cytokine activity 830. The second monomer construct comprises, from N-terminus to C-terminus, a second dimerization domain, DD2 850, a first optional linker 860, a cleavable moiety, CM 870, a second optional linker 880, and a mature cytokine protein, CP 890. The polypeptide or protein 830 lacking cytokine activity can be, for example, a truncated cytokine protein lacking cytokine activity, a mutant cytokine protein lacking cytokine activity, a stub sequence, or a polypeptide sequence that binds with high affinity to CP700 and reduces the cytokine activity of the second portion compared to a control level of the second portion. DD1 800 and DD2 850 can be the same or different. [Figure 14] 1 shows a schematic representation of an embodiment of an ACC illustrating the linking region (LR). [Figure 15](1) ACC IFNα-2b-hIgG4 Fc with cleavable moiety 1204 (1204); (2) product of protease membrane type serine protease 1 (MT-SP1) and ACC IFNα-2b-hIgG4 Fc with cleavable moiety 1204 (1204MT-SP1); (3) product of ACC IFNα-2b-hIgG4 Fc with cleavable moiety 1204 and protease uPA (1204uPA); (4) ACC IFNα-2b-hIgG4 Fc with cleavable moiety 1204 condensed with a 5 amino acid linker (1204+1); (5) product of IFNα-2b-hIgG4 Fc 1204+1 and MT-SP1 (1204+1 MT-SP1); (6) ACC IFNα-2b-hIgG4 with cleavable moiety 1490 (7) The product of MT-SP1 and ACC IFNα-2b-hIgG4 Fc with a cleavable moiety 1490; (8) The product of uPA and ACC IFNα-2b-hIgG4 Fc with a cleavable moiety 1490 (1490 uPA). [Figure 16] Sylatron® (pegylated interferon alpha-2b), as well as various interferon alpha-2b (IFNa2b) fusions: IFNa2b fused to the N-terminus of human IgG4 (IFNa2b NhG4); IFNa2b fused to the N-terminus of human IgG4 via a 5 amino acid linker (IFNa2b 5AA NhG4); the activatable cytokine construct IFN-α2b-1204dL-hIgG4 (IFNa2b 1204DNIdL NhG4); an activatable cytokine construct that contains the same components as IFN-α2b-1204dL-hIgG4 but also has a 5 amino acid linker placed between the mature cytokine protein component and the cleavable portion (IFNa2b 5AA 1204DNIdL NhG4); and the activatable cytokine construct IFN-α2b-1490DNI-hIgG4 (IFNa2b 1490DNI 1 provides results from a HEK293 cell-based reporter assay evaluating the interferon-α2b activity of NhG4). [Figure 17A]Figure 1 shows the structure of ProC286 and its activity compared to that of Sylatron® in the Daudi apoptosis assay. ProC286 and Sylatron® showed similar levels of activity, suggesting that ProC286 could be used as a surrogate for the Sylatron® control to assess the tolerability of IFNα-2b in hamster studies. [Figure 17B] 1 shows the structure of ProC291 and its activity compared to that of Sylatron® in the Daudi apoptosis assay. ProC291 showed significantly reduced activity compared to Sylatron® and ProC286. [Figure 18A] Figure 1 shows weight loss in golden Syrian hamsters when dosed with 2mpk control hIgG4, ProC286 or ProC440 throughout the treatment period. [Figure 18B] Figure 1 shows weight loss in golden Syrian hamsters when dosed with 10mpk of control hIgG4, ProC286 or ProC440 throughout the treatment period. [Figure 18C] Figure 1 shows weight loss in golden Syrian hamsters when dosed with 15mpk control hIgG4, ProC286 or ProC440 throughout the treatment period. [Figure 19A] Figure 1 shows clinical chemistry results (alkaline phosphatase (ALP)) in golden Syrian hamsters dosed with 2mpk, 10mpk and 15mpk of control hIgG4, ProC286 or ProC440. [Figure 19B] Figure 1 shows clinical chemistry results (alanine transaminase (ALT)) in golden Syrian hamsters dosed with 2mpk, 10mpk and 15mpk of control hIgG4, ProC286 or ProC440. [Figure 19C]Figure 1 shows clinical chemistry results (aspartate transaminase (AST)) in golden Syrian hamsters dosed with 2mpk, 10mpk and 15mpk of control hIgG4, ProC286 or ProC440. [Figure 20A] FIG. 1 shows the results of hematology analysis (reticulocyte counts) in golden Syrian hamsters dosed with 2 mpk, 10 mpk and 15 mpk of control hIgG4, ProC286 or ProC440. [Figure 20B] FIG. 1 shows the results of hematology analysis (neutrophil counts) in golden Syrian hamsters dosed with 2 mpk, 10 mpk and 15 mpk of control hIgG4, ProC286 or ProC440. [Figure 20C] FIG. 1 shows the results of hematology analysis (white blood cell (WBC) counts) in golden Syrian hamsters dosed with 2 mpk, 10 mpk and 15 mpk of control hIgG4, ProC286 or ProC440. [Figure 21] Activation of IL-15-containing ACC by uPa. A shows the cleavage of various IL-15-containing ACC by uPa by electrophoresis. B shows the activity of protease-activated IL-15-containing ACC compared to non-activated IL-15-containing ACC in a HEK-Blue reporter assay. [Figure 22] FIG. 1 shows the activity of protease-activated IL-15-containing ACC compared to non-activated IL-15-containing ACC in a human PBMC proliferation assay, based on the percentage of Ki67 expression. [Figure 23] FIG. 1 shows the activity of protease-activated IL-15-containing ACC compared to non-activated IL-15-containing ACC in a human PBMC STAT5 phosphorylation assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] Provided herein are activatable cytokine constructs (ACCs) that exhibit reduced activity levels of at least one of the corresponding cytokines, but produce cytokine products with substantially restored activity after exposure to activating conditions. The activatable cytokine constructs of the present invention can be designed to selectively activate upon exposure to diseased tissues and not in normal tissues. Thus, these compounds have the potential to provide the benefits of cytokine-based therapy with potentially less toxicity associated with certain cytokine-based therapies.

[0063] Also provided herein are related intermediates, compositions, kits, nucleic acids and recombinant cells, as well as related methods, including methods of using and making any of the activatable cytokines described herein.

[0064] The inventors have surprisingly found that ACC with the specific elements and structural orientations described herein appears to be potentially effective in improving the safety and therapeutic index of cytokines, particularly in cancer therapy. While cytokines are regulators of the innate and adaptive immune systems and have broad antitumor activity in preclinical models, clinical success has been limited by systemic toxicity and poor systemic exposure to target tissues. The inventors have surprisingly found that ACC with the specific elements and structural orientations described herein appears to reduce the systemic toxicity associated with cytokine therapy and improve targeting and exposure to target tissues. Thus, the present disclosure provides a method for reducing target-mediated drug loss (TMDD) of cytokine therapy by administering to a subject ACC with the specific elements and structural orientations described herein. Thus, the present invention solves the problem that a significant proportion of the administered cytokine dose is captured by normal tissues. This capture is problematic in conventional cytokine therapy because it limits a portion of the available dose in the systemic circulation from reaching the target tissue, e.g., cancerous tissue. The cytokine constructs of the present invention localize target binding to tumor tissue, thereby maintaining potency, reducing side effects, enabling new targeting opportunities, improving therapeutic windows for approved targets, creating therapeutic windows for undruggable targets, and providing multiple binding modes. The present disclosure allows for safe and effective systemic delivery, thereby avoiding the dose-dependent toxicity of conventional systemic cytokine therapy and the need for intratumoral injection. The present disclosure provides a means to impart localized antiviral, immunomodulatory, antiproliferative, and proapoptotic activity. The inventors have surprisingly found that dimerization of the first and second monomer constructs achieves a greater reduction in cytokine activity, particularly than when a single cytokine is attached to the dimerization domain (see FIG. 4).

[0065] Furthermore, the inventors have found that the degree of reduction in cytokine activity can be adjusted by varying the length of the flexible linker or the length of the linking region. The inventors have surprisingly found that by attaching the cytokine to a sterically hindered dimerization domain (such as the Fc domain of human IgG truncated at the first cysteine ​​of the hinge region, e.g., Cys226 numbered according to EU numbering) via a short protease-cleavable sequence, a reduction in cytokine activity of approximately 1,000-fold or more can be achieved. Surprisingly, protease cleavage occurs despite the steric constraint, and upon cytokine cleavage from the dimerization domain, cytokine activity is fully restored.

[0066] The inventors have discovered that by attaching IL-15 cytokine to a sterically hindered dimerization domain (such as a human IgG Fc domain truncated at the first cysteine ​​of the hinge region, e.g., Cys226 numbered according to EU numbering), via a short protease cleavable sequence, IL-15 cytokine activity can be reduced by as much as 1,000-fold, and at least 250-fold. Moreover, upon cleavage of the IL-15 cytokine from the dimerization domain, IL-15 cytokine activity can be restored to the same or nearly the same level as standard recombinant IL-15. In some embodiments, upon cleavage of the IL-15 from the dimerization domain, IL-15 cytokine activity increases by at least 50-fold. In some embodiments, upon cleavage of the IL-15 from the dimerization domain, IL-15 cytokine activity increases by at least 60-fold.

[0067] Applicant's U.S. Provisional Application No. 63 / 008,542, filed April 10, 2020, describes certain activatable cytokine constructs and is incorporated herein by reference in its entirety. Applicant's U.S. Provisional Application Nos. 63 / 161,889 and 63 / 161,913, both filed March 16, 2021, and Applicant's U.S. Provisional Application Nos. 63 / 164,827 and 63 / 164,849, both filed March 23, 2021, describe certain activatable cytokine constructs and are also incorporated herein by reference in their entireties.

[0068] Activatable cytokine constructs The activatable cytokine construct of the present invention is a dimeric complex comprising a first monomeric construct and a second monomeric construct. Dimerization of the monomeric constructs is facilitated by a pair of dimerization domains. In one aspect, each monomeric construct contains a cytokine protein, a cleavable moiety, and a dimerization domain (DD). In one aspect, one monomeric construct contains a cytokine protein, a cleavable moiety, and a DD, and the other monomeric construct contains a cytokine protein and a DD but not a cleavable moiety. In one aspect, one monomeric construct contains a cytokine protein, a cleavable moiety, and a DD, and the other monomeric construct contains a protein or peptide lacking cytokine activity and a DD but not a cleavable moiety. In a particular embodiment, the present invention provides an activatable cytokine construct (ACC) comprising a first monomeric construct and a second monomeric construct, (a) a first monomeric construct comprising a first mature cytokine protein (CP1), a first cleavable portion (CM1), and a first dimerization domain (DD1); CM1 is placed between CP1 and DD1, (b) a second monomeric construct comprising a second mature cytokine protein (CP2), a second cleavable portion (CM2), and a second dimerization domain (DD2); CM2 is placed between CP2 and DD2, DD1 and DD2 bind to each other, thereby forming a dimer of the first monomeric component and the second monomeric component; An activatable cytokine construct (ACC) is provided, characterized in that it has a reduced activity level of at least one CP1 and / or CP2 compared to a control level of activity of at least one CP1 and / or CP2.

[0069] In certain embodiments, CP1 and CP2 each comprise an interleukin polypeptide. In one embodiment, the interleukin polypeptide is selected from the group consisting of IL-1α, IL-1β, IL-1RA, IL-18, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-3, IL-5, IL-6, IL-11, IL-12, IL-10, IL-20, IL-21 IL-14, IL-15, IL-16, and IL-17. In another embodiment of the disclosure, the interleukin polypeptide is IL-15, thereby constituting an activatable IL-15 construct. In one aspect, the activatable IL-15 construct has reduced activity compared to recombinant IL-15.

[0070] The term "activatable," when used in reference to a cytokine construct, means a cytokine construct that exhibits a first level of one or more activities and, upon exposure to conditions that cause cleavage of one or both cleavable moieties, results in the production of a cytokine construct that exhibits a second level of one or more activities, where the second level of activity is greater than the first level of activity. Non-limiting examples of activities include any of the exemplary activities of cytokines described herein or known in the art.

[0071] The term "mature cytokine protein" as used herein refers to a cytokine protein lacking a signal sequence. A cytokine protein (CP) may be a mature cytokine protein or a cytokine protein having a signal peptide. Thus, in some embodiments, the ACC disclosed herein may contain a mature cytokine protein sequence. In some embodiments, the ACC disclosed herein may contain a mature cytokine protein sequence and additionally a signal sequence. In some embodiments, the ACC disclosed herein may contain a sequence disclosed herein, containing or lacking a signal sequence as recited herein.

[0072] The terms "cleavable moiety" and "CM" are used interchangeably herein to refer to a peptide whose amino acid sequence includes a substrate for a sequence-specific protease. Cleavable moieties suitable for use as CM1 and / or CM2 include any of the protease substrates known in the art. Exemplary cleavable moieties are described in more detail below.

[0073] The terms "dimerization domain" and "DD" are used interchangeably herein and refer to one member of a pair of dimerization domains, each member of the pair being capable of binding to the other via one or more covalent or non-covalent interactions. The first DD and the second DD may be the same or different. Exemplary DDs suitable for use as DD1 and / or DD2 are described in more detail herein below.

[0074] The terms "peptide mask" and "PM" are used interchangeably herein to refer to an amino acid sequence of less than 50 amino acids that reduces or inhibits one or more activities of a cytokine protein. The PM can bind to a cytokine and limit the cytokine from interacting with its receptor. In some embodiments, the PM is 40 amino acids or less in length. In preferred embodiments, the PM is 20 amino acids or less in length. In some embodiments, the PM is 19, 18, 17, 16, or 15 amino acids or less in length.

[0075] As used herein, the term "masking efficiency" refers to the activity (e.g., EC50) of uncleaved ACC separated by the activity of a control cytokine, which may be a cleavage product of ACC or a cytokine used as a CP of ACC. ACC with reduced levels of at least one CP1 and / or CP2 activity has a masking efficiency of greater than 10. In some embodiments, the ACC described herein has a masking efficiency of greater than 10, greater than 100, greater than 1000, or greater than 5000. In some embodiments, where CP1 and / or CP2 are IL-15 polypeptides, the ACC may have a masking efficiency of about 10 to about 100, or about 10 to about 200, or about 50 to about 150, or about 50 to about 80, measured as the ratio of the EC50 of uncleaved ACC to the EC50 of the cleavage product of ACC in IL-2 / IL-15-responsive HEK293 cells.

[0076] As used herein, a polypeptide such as a cytokine or Fc domain may be a wild-type polypeptide (e.g., a naturally occurring polypeptide) or a variant of a wild-type polypeptide. A variant may be a polypeptide that has been modified by substitution, insertion, deletion and / or addition of one or more amino acids of the wild-type polypeptide, provided that the variant retains the basic function or activity of the wild-type polypeptide. In some examples, a variant may have an altered (e.g., increased or decreased) function or activity compared to the wild-type polypeptide. In some embodiments, a variant may be a functional fragment of a wild-type polypeptide. The term "functional fragment" refers to a sequence of a polypeptide (e.g., a cytokine) that may contain fewer amino acids than the full-length polypeptide sequence, but contains sufficient length of the polypeptide chain to confer an activity (e.g., cytokine activity).

[0077] The first and second monomeric constructs may further include additional elements, such as, for example, one or more linkers. The additional elements are described in more detail below. The organization of the CP, CM, and DD components in each of the first and second monomeric constructs may be arranged in the same order in each monomeric construct. The CP1, CM1, and DD1 components may be the same or different in terms of, for example, the molecular weight, size, amino acid sequence, etc. of the CP and CM components (and the DD components in embodiments where the DD components are polypeptides) compared to the corresponding CP2, CM2, and DD2. Thus, the resulting dimer may have symmetric or asymmetric monomeric construct components.

[0078] In some embodiments, the first monomer construct comprises DD1 linked directly or indirectly (through a linker) to the C-terminus of CP1, CM1, and CM1 from the N-terminus to the C-terminus of the CP and CM components. In other embodiments, the first monomer construct comprises DD1 linked directly or indirectly (through a linker) to the N-terminus of CP1, CM1, and CM1 from the C-terminus to the N-terminus of the CP and CM components. In some embodiments, the second monomer construct comprises DD2 linked directly or indirectly (through a linker) to the C-terminus of CP2, CM2, and CM2 from the N-terminus to the C-terminus of the CP and CM components. In some embodiments, the second monomer construct comprises DD2 linked directly or indirectly (through a linker) to the N-terminus of CP2, CM2, and CM2 from the C-terminus to the N-terminus of the CP and CM components.

[0079] In some embodiments, the first monomer comprising the first mature cytokine protein (CP1) and / or the second monomer comprising the second mature cytokine protein (CP2) further comprises a peptide mask (PM). In some embodiments, the ACC further comprises a CM between the PM and the CP.

[0080] In some embodiments, an activatable cytokine construct (ACC) is provided that contains a first monomer construct and a second monomer construct, where (a) the first monomer construct comprises a first peptide mask (PM1), a first mature cytokine protein (CP1), first and third cleavable portions (CM1 and CM3), and a first dimerization domain (DD1), where CM1 is disposed between CP1 and DD1, and CM3 is disposed between PM1 and CP1, and (b) the second monomer construct comprises a second mature cytokine protein (CP1), a first peptide mask (PM1), a first mature cytokine protein (CP1), a first and third cleavable portion (CM1 and CM3), and a first dimerization domain (DD1), where CM1 is disposed between CP1 and DD1, and CM3 is disposed between PM1 and CP1, and (c) the second monomer construct comprises a second mature cytokine protein (CP1), a first peptide mask (PM1), a first mature cytokine protein (CP1), a first and third cleavable portion (CM1 and CM3), and a first dimerization domain (DD1), where CM1 is disposed between CP1 and DD1, and CM3 is disposed between PM1 and CP1, and (d) the second monomer construct comprises a second mature cytokine protein (CP1), a first peptide mask (PM1), a first mature cytokine protein (CP1), a first and third cleavable portion (CM1 and CM3), and a first dimerization domain (DD1), where CM1 is disposed between CP1 and DD1, and CM3 is disposed between PM1 and CP1, and An activatable cytokine construct (ACC) is provided, comprising an intrinsic protein (CP2), a second cleavable portion (CM2) and a second dimerization domain (DD2), wherein CM2 is positioned between CP2 and DD2, and DD1 and DD2 bind to each other, thereby forming a dimer of a first monomer construct and a second monomer construct, and wherein the activity level of at least one of CP1 and / or CP2 is reduced compared to a control level of the activity of at least one of CP1 and / or CP2.

[0081] In some embodiments, the second monomer construct further comprises a second peptide mask (PM2) and a fourth cleavable portion (CM4), where CM4 is located between PM2 and CP2. In some embodiments, the first monomer construct comprises a first polypeptide comprising PM1, CM3, CP1, CM1 and DD1. In some embodiments, the second monomer construct comprises a second polypeptide comprising CP2, CM2 and DD2. In some embodiments, the second monomer construct comprises a second polypeptide comprising PM2, CM4, CP2, CM2 and DD2.

[0082] It has been found that the ACC structure is extremely effective in reducing the activity of mature cytokine protein components such that cytokine activity is not substantially impaired after activation. The activity of CP in ACC may be reduced by both the structure of ACC (e.g., dimeric structure) and the peptide mask(s) in ACC. In some embodiments, the activation condition of ACC described herein is exposure to one or more proteases that can cleave the CP from both the DD and PM. For example, the one or more proteases may cleave the CM between the CP and PM, and the CM between the CP and DD. As shown in the examples, activation of ACC resulted in substantial recovery of cytokine activity. These results suggest that the conformation of cytokine components was not irreversibly altered in the context of ACC.

[0083] In some embodiments, when the CP is linked to a PM and has a natural binding partner for the CP, there is no, or substantially no, binding of the CP to its binding partner, or binding of the CP to its binding partner is 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 50% or less compared to binding of the CP when not linked to a PM, for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, as measured in a masking efficiency assay. For example, the masking efficiency assay may include measuring the affinity of ACC to bind to cell surfaces displaying the candidate peptide mask, e.g., by FACS. Another non-limiting exemplary assay includes evaluating the ability of the peptide mask to inhibit ACC from binding to its binding partner at therapeutically relevant concentrations and times. For this second method, an immunosorbent assay has been developed to measure the time-dependent binding of a proprotein to its binding partner, as described in US20200308243, which is incorporated herein by reference. In one embodiment where the CP is an IL-15 cytokine, the masking efficiency assay may include measuring the production level of secreted alkaline phosphatase (SEAP) in IL-2 / IL15-responsive HEK293 cells, as described in Example 6.

[0084] In certain embodiments, the first and second monomeric constructs are oriented such that the components of each member of the dimer are arranged in the same order, from the N-terminus to the C-terminus of the CP and CM components. A schematic diagram of an exemplary ACC is provided in FIG. 1A. 1A, ACC comprises, from the N-terminus to the C-terminus of the CP and CM components, (1) a first monomer construct having CP1 100; CM1 120 located C-terminal to CP1 100; an optional linker 110, if present, located between the C-terminus of CP1 100 and the N-terminus of CM1 120; DD1 140; and, if present, an optional linker 130, located between the C-terminus of CM1 120 and DD1 140; (2) CP2 150; CM2 170 located C-terminal to CP2 150; an optional linker 160, if present, located between the C-terminus of CP2 150 and the N-terminus of CM2 170; DD2 190; and, if present, an optional linker 170, located between the C-terminus of CM2 170 and the N-terminus of DD2 140. a second monomeric construct having an optional linker 180 interposed between said first monomeric construct and said second monomeric construct, and (3) one or more covalent or non-covalent bonds (←→).

[0085] A schematic diagram of a further exemplary ACC, in which the ACC components are arranged in the opposite orientation, is provided in Figure 1B. Referring to Figure 1B, the ACC comprises, from the N-terminus to the C-terminus of the CP and CM components, (1) a first monomeric construct having DD1 200; CM1 220; an optional linker 210, if present, between DD1 200 and the N-terminus of CM1 220; CP1 240, which is C-terminal to CM1 220; and an optional linker 230, if present, between the C-terminus of CM1 220 and the N-terminus of CP1 240; (2) DD2 250; CM2 270; an optional linker 260, if present, between DD2 250 and the N-terminus of CM2 270; CP2 290, which is C-terminal to CM2 270; and, if present, between the C-terminus of CM2 290 and the N-terminus of CP2 240; a second monomeric construct having an optional linker 280 disposed between the N-terminus of 290 and the N-terminus of 290; and (3) one or more covalent or non-covalent bonds (←→).

[0086] 2A is a schematic diagram of an exemplary activatable cytokine construct comprising first and second monomer constructs bound to each other by non-covalent means via first and second dimerization domains DD1 340 and DD2 390, respectively. The first monomer construct comprises, from the N-terminus to the C-terminus of the CP and CM components, a first mature cytokine protein CP1 300, a first optional linker 310, a first cleavable moiety CM1 320, a second optional linker 330, and a first dimerization domain DD1 340. The second monomer construct comprises, from the N-terminus to the C-terminus of the CP and CM components, a second mature cytokine protein CP2 350, a third optional linker 360, a second cleavable moiety CM2 370, a fourth optional linker 380, and a second dimerization domain DD2 390.

[0087] 2B is a schematic diagram of an exemplary activatable cytokine construct comprising a first and a second monomer construct linked to each other by non-covalent means via a first and a second dimerization domain DD1 400 and DD2 450, respectively. The first monomer construct comprises, from the N-terminus to the C-terminus of the CP and CM components, a first dimerization domain DD1 400, a second optional linker 410, a first cleavable moiety CM1 420, a first optional linker 430, and a first mature cytokine protein CP1 440. The second monomer construct comprises, from the N-terminus to the C-terminus of the CP and CM components, a second dimerization domain DD2 450, a fourth optional linker 460, a second cleavable moiety CM2 470, a third optional linker 480, and a second mature cytokine protein CP2 490. In an alternative embodiment, either of the two moieties designated as CP1 440 and CP2 490 is a truncated cytokine protein lacking cytokine activity. For example, CP1 or CP2 may be a truncated interferon alpha 2b having the first 151 amino acids of wild-type interferon alpha 2b. In an alternative embodiment, either of the two moieties designated as CP1 440 and CP2 490 is a mutant cytokine protein lacking cytokine activity. For example, CP1 or CP2 may be a truncated interferon alpha 2b having the L130P mutation. In an alternative embodiment, either of the two moieties designated as CP1 440 and CP2 490 is a polypeptide sequence lacking cytokine activity, such as a signal portion and / or a stub sequence. In an alternative embodiment, a first of the two moieties designated CP1 440 and CP2 490 is a polypeptide sequence that binds with high affinity to a second of the two moieties designated CP1 440 and CP2 490 and reduces the cytokine activity of the second moiety compared to a control level of the second moiety.

[0088] It has been found that the ACC structure containing the dimerization domain is extremely effective in reducing the activity of mature cytokine protein components such that the cytokine activity is not substantially impaired after activation. The activation condition of ACC described herein is exposure to a protease that can cleave at least one of the cleavable moieties (CM) in ACC. As shown in the examples, activation of ACC resulted in substantial recovery of cytokine activity. These results suggest that within the context of ACC, the conformation of the cytokine components was not irreversibly altered. Importantly, ACC does not need to rely on a peptide mask that has binding affinity to the cytokine protein components to achieve the masking effect. Thus, ACC may or may not include a peptide mask that has binding affinity to the cytokine protein components.

[0089] ACC may use any of a variety of mature cytokine proteins, cleavable portions, and DDs as CP1, CP2, CM1, CM2, DD1, and DD2, respectively. For example, any of a variety of mature cytokine proteins known in the art, or any of their sequences and / or truncated variants, may be suitable for use as one or both of the CP1 and CP2 components of ACC. The mature cytokine proteins, CP1 and CP2, may be the same or different. In certain embodiments, CP1 and CP2 are the same. In other embodiments, CP1 and CP2 are different. ACC may include additional amino acid residues at one or both of the N-terminus and / or C-terminus of CP1 and / or CP2.

[0090] In some embodiments, CP1 and / or CP2 are each independently selected from the group consisting of interferon (e.g., interferon alpha, interferon beta, interferon gamma, interferon tau, and interferon omega), interleukin (e.g., IL-1alpha, IL-1beta, IL-1RA, IL-18, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-3, IL-5, GM-CSF, IL-6, IL-11, IL-21, G-CSF, IL-12, LIF, OSM, IL-10, IL-2 The cytokine protein may include mature cytokine proteins selected from the group consisting of IL-0, IL-14, IL-16, IL-17, CD154, LT-β, ​​TNF-α, TNF-β, 4-1BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE, TGF-β1, TGF-β1, TGF-β3, erythropoietin (EPO), TPO, Flt-3L, SCF, M-CSF and MSP, and sequences and / or truncated variants thereof. For example, sequences of such proteins include those exemplified herein, and further sequences can be obtained from ncbi.nlm.nih.gov / protein. The truncated variants suitable for use in the ACC of the present invention include any cytokine truncated at the N-terminus or C-terminus that retains cytokine activity. Exemplary truncated variants for use in the present invention include any of the truncated cytokine polypeptides known in the art (see, e.g., Slutzki et al., J. Mol. Biol. 360:1019-1030, 2006 and US 2009 / 0025106), as well as cytokine polypeptides truncated at the N- and / or C-terminus by 1 to about 40 amino acids, 1 to about 35 amino acids, 1 to about 30 amino acids, 1 to about 25 amino acids, 1 to about 20 amino acids, 1 to about 15 amino acids, 1 to about 10 amino acids, 1 to about 8 amino acids, 1 to about 6 amino acids, or 1 to about 4 amino acids that retain cytokine activity.In some of the foregoing embodiments, the truncated CP is a CP truncated at the N-terminus. In other embodiments, the truncated CP is a CP truncated at the C-terminus. In certain embodiments, the truncated CP is a CP truncated at both the C-terminus and the N-terminus.

[0091] In some embodiments, CP1 and / or CP2 are each independently selected from SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:12, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127 , SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, Column number 159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO: and / or a cytokine reference sequence selected from the group consisting of SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:347, and SEQ ID NO:348.The amino acid sequences of the peptide sequences of the present invention include those that are at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical. The percentage of sequence identity refers to the level of amino acid sequence identity between two or more peptide sequences when aligned using a sequence alignment program, such as the BLAST suite of programs publicly available on the Internet at the NCBI website. See also Altschul et al., J. Mol. Biol. 215:403-10, 1990. In some embodiments, the ACC contains an interferon alpha 2b mutant, such as an interferon alpha 2b molecule having a mutation at the L130 position, such as an L130P mutation, as CP1 or CP2. In some embodiments, the ACC contains an interferon alpha 2b variant having a mutation at the position of I24, F64, I60, I63, F64, W76, I116, L117, F123, or L128, or a combination thereof. For example, the interferon alpha 2b variant may contain a mutation of I116 to T, N, or R; L128 to N, H, or R; I24 to P or Q; L117H; or L128T, or a combination thereof. In some embodiments, the interferon alpha 2b variant may contain a mutation of I24Q, I60T, F64A, W76H, I116R, and L128N, or a subset thereof. In some embodiments, the ACC contains a truncated interferon alpha 2b molecule lacking cytokine activity as either CP1 or CP2. For example, truncated interferon α2b may consist of 151 or less amino acids of interferon α2b, such as 1-151, 1-150, 1-149, 1-148, ... 1-10, 1-9, 1-8, 1-7, 1-6, or 2-151, 3-151, 4-151, 5-150, 6-149, 7-148, 8-147 amino acids from the N-terminus to the C-terminus of the wild-type interferon α2b sequence, or any intervening sequence of these amino acids or variants.

[0092] In certain embodiments, CP1 and / or CP2 comprise an interleukin. Interleukins suitable for use in the compositions of the invention as CP1 and / or CP2 include, for example, IL-α1, IL-1β, IL-1RA, IL-18, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-3, IL-5, GM-CSF, IL-6, IL-11, IL-21. In some embodiments, the interleukin comprises a wild-type (WT) or recombinant interleukin. In some embodiments, the WT or recombinant interleukin polypeptide comprises IL-15. Exemplary IL-15 sequences are provided in SEQ ID NO:347, SEQ ID NO:348, SEQ ID NO:129, and SEQ ID NO:130.

[0093] In other embodiments, CP1 and / or CP2 exhibit interleukin activity and contain an amino acid sequence that is at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, or at least 99% identical, or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 111-134, 137-140, 143-146, 151-160, and 347-348. In some embodiments, CP1 and / or CP2 comprise an interleukin having an amino acid sequence selected from the group consisting of SEQ ID NOs: 111-134, 137-140, 143-146, 151-160, and 347-348. In some embodiments, CP1 and / or CP2 comprise an interleukin having an amino acid sequence selected from the group consisting of SEQ ID NO: 129, SEQ ID NO: 347, and SEQ ID NO: 348. In certain embodiments, CP1 and / or CP2 are each independently an interleukin comprising the amino acid sequence of SEQ ID NO: 347. In some of the above-described embodiments, CP1 and CP2 comprise the same amino acid sequence.

[0094] In other embodiments, CP1 and / or CP2 exhibit interleukin activity and contain an amino acid sequence that is at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, or at least 99% identical, or 100% identical to an interleukin reference sequence selected from the group consisting of SEQ ID NO: 129, SEQ ID NO: 347, and SEQ ID NO: 348. In certain embodiments, the interleukin reference sequence is a human interleukin reference sequence selected from the group consisting of SEQ ID NO: 129, SEQ ID NO: 347, and SEQ ID NO: 348. In some embodiments, CP1 and / or CP2 comprise a mature interleukin having an amino acid sequence selected from the group consisting of SEQ ID NO: 129, SEQ ID NO: 347, and SEQ ID NO: 348. In some of the above-mentioned embodiments, CP1 and CP2 comprise identical amino acid sequences.

[0095] In some embodiments, CP1 and / or CP2 exhibit interleukin activity and contain an amino acid sequence that is at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, or at least 99% identical, or 100% identical to an interleukin reference sequence that corresponds to an amino acid sequence comprising SEQ ID NO: 347. In certain embodiments, CP1 and / or CP2 comprise an interleukin polypeptide comprising the amino acid sequence of SEQ ID NO: 347. In some of the above-described embodiments, CP1 and CP2 comprise identical amino acid sequences.

[0096] In some embodiments, CP1 and / or CP2 exhibit interleukin activity and are selected from the group consisting of SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:16 4, contains an amino acid sequence that is at least 80% identical, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or 100% identical to an interleukin reference sequence selected from the group consisting of SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:347, and SEQ ID NO:348.In some embodiments, CP1 and / or CP2 are selected from the group consisting of SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:12, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:1 4, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:347, and SEQ ID NO:348. In some of the above-described embodiments, CP1 and CP2 comprise the same amino acid sequence.

[0097] In some embodiments, CP1 and / or CP2 exhibit interleukin-15 activity and contain an amino acid sequence that is at least 80% identical, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an IL-15 reference sequence selected from the group consisting of SEQ ID NO: 129 (human IL-15), SEQ ID NO: 347 (amino acids 49-161 of human IL-15), and SEQ ID NO: 348 (amino acids 49-162 of human IL-15). In some embodiments, CP1 and CP2 comprise identical amino acid sequences, where such sequences are at least 80% identical, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from the group consisting of SEQ ID NO: 129 (human IL-15), SEQ ID NO: 347 (amino acids 49-161 of human IL-15), and SEQ ID NO: 348 (amino acids 49-162 of human IL-15).

[0098] The number of amino acids in the sequence of the cytokine protein used may vary depending on the particular cytokine protein used. In some embodiments, CP1 and / or CP2 may be a total of about 10 amino acids to about 700 amino acids, about 10 amino acids to about 650 amino acids, about 10 amino acids to about 600 amino acids, about 10 amino acids to about 550 amino acids, about 10 amino acids to about 500 amino acids, about 10 amino acids to about 450 amino acids, about 10 amino acids to about 400 amino acids, about 10 amino acids to about 350 amino acids, about 10 amino acids to about 300 amino acids, about 10 amino acids to about 25 ... 0 amino acids, about 10 amino acids to about 200 amino acids, about 10 amino acids to about 150 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 80 amino acids, about 10 amino acids to about 60 amino acids, about 10 amino acids to about 40 amino acids, about 10 amino acids to about 20 amino acids, about 20 amino acids to about 700 amino acids, about 20 amino acids to about 650 amino acids, about 20 amino acids to about 600 amino acids, about 20 amino acids to about 550 amino acids amino acids, about 20 amino acids to about 500 amino acids, about 20 amino acids to about 450 amino acids, about 20 amino acids to about 400 amino acids, about 20 amino acids to about 350 amino acids, about 20 amino acids to about 300 amino acids, about 20 amino acids to about 250 amino acids, about 20 amino acids to about 200 amino acids, about 20 amino acids to about 150 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 60 amino acids acid, about 20 amino acids to about 40 amino acids, about 40 amino acids to about 700 amino acids, about 40 amino acids to about 650 amino acids, about 40 amino acids to about 600 amino acids, about 40 amino acids to about 550 amino acids, about 40 amino acids to about 500 amino acids, about 40 amino acids to about 450 amino acids, about 40 amino acids to about 400 amino acids, about 40 amino acids to about 350 amino acids, about 40 amino acids to about 300 amino acids, about 40 amino acids to about 250 amino acids,About 40 amino acids to about 200 amino acids, about 40 amino acids to about 150 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 60 amino acids, about 60 amino acids to about 700 amino acids, about 60 amino acids to about 650 amino acids, about 60 amino acids to about 600 amino acids, about 60 amino acids to about 550 amino acids, about 60 amino acids to about 500 amino acids, about 60 amino acids to about 450 amino acids, about 60 amino acids to about 400 amino acids , about 60 amino acids to about 350 amino acids, about 60 amino acids to about 300 amino acids, about 60 amino acids to about 250 amino acids, about 60 amino acids to about 200 amino acids, about 60 amino acids to about 150 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 80 amino acids, about 80 amino acids to about 700 amino acids, about 80 amino acids to about 650 amino acids, about 80 amino acids to about 600 amino acids, about 80 amino acids to about 550 amino acids, about 80 amino acids to about 500 amino acids 80 to 450 amino acids, about 80 to 400 amino acids, about 80 to 350 amino acids, about 80 to 300 amino acids, about 80 to 250 amino acids, about 80 to 200 amino acids, about 80 to 150 amino acids, about 80 to 100 amino acids, about 110 to 162 amino acids, about 100 to 120 amino acids, about 110 to 120 amino acids, about 110 to 11 5 amino acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about 650 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 550 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 450 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 350 amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 250 amino acids, about 100 amino acids to about 200 amino acids,About 100 amino acids to about 150 amino acids, about 150 amino acids to about 700 amino acids, about 150 amino acids to about 650 amino acids, about 150 amino acids to about 600 amino acids, about 150 amino acids to about 550 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 450 amino acids, about 150 amino acids to about 400 amino acids, about 150 amino acids to about 350 amino acids, about 150 amino acids to about 300 amino acids, about 150 amino acids to about 250 amino acids, about about 200 amino acids, about 150 amino acids to about 170 amino acids, about 160 amino acids to about 165 amino acids, about 200 amino acids to about 700 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 550 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 450 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 350 amino acids, about 200 amino acids to about About 300 amino acids, about 200 amino acids to about 250 amino acids, about 250 amino acids to about 700 amino acids, about 250 amino acids to about 650 amino acids, about 250 amino acids to about 600 amino acids, about 250 amino acids to about 550 amino acids, about 250 amino acids to about 500 amino acids, about 250 amino acids to about 450 amino acids, about 250 amino acids to about 400 amino acids, about 250 amino acids to about 350 amino acids, about 250 amino acids to about 300 amino acids, about 300 amino acids to about 700 amino acids , about 300 amino acids to about 650 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 550 amino acids, about 300 amino acids to about 500 amino acids, about 300 amino acids to about 450 amino acids, about 300 amino acids to about 400 amino acids, about 300 amino acids to about 350 amino acids, about 350 amino acids to about 700 amino acids, about 350 amino acids to about 650 amino acids, about 350 amino acids to about 600 amino acids, about 350 amino acids to about 550 amino acids,About 350 amino acids to about 500 amino acids, about 350 amino acids to about 450 amino acids, about 350 amino acids to about 400 amino acids, about 400 amino acids to about 700 amino acids, about 400 amino acids to about 650 amino acids, about 400 amino acids to about 600 amino acids, about 400 amino acids to about 550 amino acids, about 400 amino acids to about 500 amino acids, about 400 amino acids to about 450 amino acids, about 450 amino acids to about 700 amino acids, about 450 amino acids to about 650 amino acids, about 450 amino acids to about 600 amino acids, about 450 amino acids Each of the CP1 and CP2 polypeptides contains from about 550 amino acids, from about 450 amino acids to about 500 amino acids, from about 500 amino acids to about 700 amino acids, from about 500 amino acids to about 650 amino acids, from about 500 amino acids to about 600 amino acids, from about 500 amino acids to about 550 amino acids, from about 550 amino acids to about 700 amino acids, from about 550 amino acids to about 650 amino acids, from about 550 amino acids to about 600 amino acids, from about 600 amino acids to about 700 amino acids, from about 600 amino acids to about 650 amino acids, or from about 650 amino acids to about 700 amino acids. In some embodiments, CP1 and / or CP2 are mature wild-type human cytokine proteins.

[0099] Each monomeric component of ACC may use any of a variety of dimerization domains. Suitable DDs include both polymeric (e.g., synthetic polymers, polypeptides, polynucleotides, etc.) and small molecule (non-polymeric moieties with molecular weights of less than about 1 kilodalton, sometimes less than about 800 daltons) moieties. A pair of DDs may be any pair of moieties known in the art to bind to each other.

[0100] For example, in some embodiments, DD1 and DD2 are selected from the group consisting of the alpha chain of the human IL-15 receptor (IL15Rα) and the sushi domain from soluble IL-15; barnase and barstar; PKA and AKAP; mutated RNase I fragment-based adaptor / docking tag molecules; a pair of antigen binding domains (e.g., a pair of single domain antibodies); soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs); modules based on the interaction of the proteins syntaxin, synaptotagmin, synaptobrevin and SNAP25; single domain antibodies (sdAbs) and corresponding epitopes; antigen binding domains (e.g., single a pair of single chain antibodies such as single chain fragment variable region (scFv), single domain antibodies, and the corresponding epitope; a pair of coiled-coil polypeptide structures (e.g., Fos-Jun coiled-coil structures, acid / base coiled-coil helices, Glu-Lys coiled-coil helices, leucine zipper structures), small molecule conjugated pairs such as biotin and avidin or streptavidin, amine / aldehyde, lectin / carbohydrate; a pair of polymers capable of binding to each other, for example, a pair of sulfur- or thiol-containing polymers (e.g., a pair of Fc domains, a pair of thiolated human serum albumin polypeptides, etc.); and the like.

[0101] In some embodiments, DD1 and DD2 are non-polypeptide polymers. The non-polypeptide polymers may be covalently bonded to each other. In some examples, the non-polypeptide polymer may be a sulfur-containing polymer, such as a sulfur-containing polyethylene glycol. In such cases, DD1 and DD2 may be covalently bonded to each other via one or more disulfide bonds.

[0102] When a pair of DD1 and DD2 is a member of a pair of epitope and antigen-binding domain, the epitope may be a natural or non-natural epitope. Exemplary non-natural epitopes include non-natural peptides such as poly-His peptides (e.g., His tags).

[0103] In certain embodiments, DD1 and DD2 are a pair of Fc domains. As used herein, "Fc domain" refers to a contiguous amino acid sequence of a single heavy chain of an immunoglobulin. A pair of Fc domains bind together to form the Fc region of an immunoglobulin.

[0104] In some embodiments, the pair of Fc domains is a pair of human Fc domains (e.g., a pair of wild-type human Fc domains). In some embodiments, the human Fc domain is a human IgG1 Fc domain (e.g., a wild-type human IgG1 Fc domain), a human IgG2 Fc domain (e.g., a wild-type human IgG2 Fc domain), a human IgG3 Fc domain (e.g., a wild-type human IgG3 Fc domain), or a human IgG4 Fc domain (e.g., a wild-type human IgG4 Fc domain). In some embodiments, the human Fc domain comprises a sequence at least 80% identical (e.g., at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to SEQ ID NO:3.

[0105] In some embodiments, a pair of Fc domains includes knob and hole variants of the Fc domain. The knob and hole variants may interact with each other to promote dimerization. In some embodiments, the knob and hole variants may include one or more amino acid modifications in the interface between the two Fc domains (e.g., in the CH3 domain). In one example, the modifications include amino acid substitutions T366W and optionally S354C in one of the antibody heavy chains, and amino acid substitutions T366S, L368A, Y407V and optionally Y349C in the other antibody heavy chain (numbered according to the EU index of the Kabat numbering system). Examples of knob and hole variants include the Fc variants of SEQ ID NOs: 315 and 316, and those described in U.S. Patent Nos. 5,731,168, 7,695,936, and 10,683,368, which are incorporated herein by reference in their entireties. In some embodiments, the dimerization domain comprises a sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to SEQ ID NOs: 315 and 316, respectively.

[0106] In some embodiments, DD1 and / or DD2 may further comprise a serum half-life extending moiety (e.g., a polypeptide that binds to a serum protein such as an immunoglobulin (e.g., IgG) or serum albumin (e.g., human serum albumin (HSA)). Examples of half-life extending moieties include hexa-hat GST (glutathione S-transferase) glutathione affinity, calmodulin binding peptide (CBP), strep tag, cellulose binding domain, maltose binding protein, S-peptide tag, chitin binding tag, immunoreactive epitope, epitope tag, E2Tag, HA epitope tag, Myc epitope, FLAG epitope, AU1 and AU5 epitope, Glu-Glu epitope, KT3 epitope, IRS epitope, Btag epitope, protein kinase-C epitope, and VSV epitope.

[0107] In some embodiments, DD1 and / or DD2 each contain a total of about 5 amino acids to about 250 amino acids, about 5 amino acids to about 200 amino acids, about 5 amino acids to about 180 amino acids, about 5 amino acids to about 160 amino acids, about 5 amino acids to about 140 amino acids, about 5 amino acids to about 120 amino acids, about 5 amino acids to about 100 amino acids, about 5 amino acids to about 80 amino acids, about 5 amino acids to about 60 amino acids, about 5 amino acids to about 40 amino acids, about 5 amino acids to about 20 amino acids, , about 5 amino acids to about 10 amino acids, about 10 amino acids to about 250 amino acids, about 10 amino acids to about 200 amino acids, about 10 amino acids to about 180 amino acids, about 10 amino acids to about 160 amino acids, about 10 amino acids to about 140 amino acids, about 10 amino acids to about 120 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 80 amino acids, about 10 amino acids to about 60 amino acids, about 10 amino acids to about 40 amino acids, about 10 amino acids to about 20 amino acids, about 0 amino acids to about 250 amino acids, about 20 amino acids to about 200 amino acids, about 20 amino acids to about 180 amino acids, about 20 amino acids to about 160 amino acids, about 20 amino acids to about 140 amino acids, about 20 amino acids to about 120 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 40 amino acids, about 40 amino acids to about 250 amino acids, about 40 amino acids to about 200 amino acids, about 40 amino acids to about 180 amino acids, about 40 amino acids to about 160 amino acids, about 40 amino acids to about 140 amino acids, about 40 amino acids to about 120 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 60 amino acids, about 60 amino acids to about 250 amino acids, about 60 amino acids to about 200 amino acids, about 60 amino acids to about 180 amino acids, about 60 amino acids to about 160 amino acids, about 60 amino acids to about 140 amino acids,About 60 amino acids to about 120 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 80 amino acids, about 80 amino acids to about 250 amino acids, about 80 amino acids to about 200 amino acids, about 80 amino acids to about 180 amino acids, about 80 amino acids to about 160 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 120 amino acids, about 80 amino acids to about 100 amino acids, about 100 amino acids to about 250 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 180 amino acids, about 100 amino acids to about 160 amino acids, about 100 amino acids to about 140 amino acids, about 100 amino acids to about 120 amino acids, about 120 amino acids to about 250 amino acids, about 120 amino acids to about 200 amino acids about 120 amino acids to about 180 amino acids, about 120 amino acids to about 160 amino acids, about 120 amino acids to about 140 amino acids, about 140 amino acids to about 250 amino acids, about 140 amino acids to about 200 amino acids, about 140 amino acids to about 180 amino acids, about 140 amino acids to about 160 amino acids, about 160 amino acids to about 250 amino acids, about 160 amino acids DD1 and DD2 each contain about 1 to about 200 amino acids, about 160 to about 180 amino acids, about 180 to about 250 amino acids, about 180 to about 200 amino acids, about 200 to about 250 amino acids, about 210 to about 220 amino acids, about 215 to about 225 amino acids, about 215 to about 220 amino acids, about 217 to about 200 amino acids, or about 218 to about 200 amino acids. In some embodiments, DD1 and DD2 are Fc domains that include a portion of a hinge region containing two cysteine ​​residues, a CH2 domain, and a CH3 domain. In some embodiments, DD1 and DD2 are Fc domains whose N-terminus is the first cysteine ​​residue in the hinge region reading from the N-terminus to the C-terminus (e.g., cysteine ​​226 of human IgG1 or IgG4 using EU numbering).

[0108] In some aspects, located directly or indirectly (e.g., via a linker) between the CP and DD components is a cleavable moiety that comprises a substrate for a protease. In some embodiments, CM1 and CM2 are each independently selected from ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADEMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, BACE, renin, cathepsin D, cathepsin E, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, caspase 14, caspase 15, caspase 16, caspase 17, caspase 18, caspase 19, caspase 20, caspase 21, caspase 22, caspase 23, caspase 24, caspase 25, caspase 26, caspase 27, caspase 28, caspase 29, caspase 30, caspase 31, caspase 32, caspase 33, caspase 34, caspase 35, caspase 36, caspase 37, caspase 38, caspase 39, caspase 31, caspase 39, caspase 31, caspase 32, caspase 33, caspase 34, caspase 35, caspase 36, caspase 37, caspase 38, caspase 39, caspase 39, caspase 39, caspase 39, caspase 39, caspase 39, ca Caspase 8, caspase 9, caspase 10, caspase 14, cathepsin A, cathepsin B, cathepsin C, cathepsin G, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P, chymase, cruzipain, DESC1, DPP-4, FAP, legumain, otubain-2, elastase, FVIIa, FiXA, FXa, FXIa, FXIIa, granza Immunoglobulin B, guanidinobenzoatase, hepsin, HtrA1, human neutrophil elastase, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, lactoferrin, marapsin, matriptase-2, meprin, MT-SP1 / matriptase, neprilysin, NS3 / 4A, PACE4, plasmin, PSMA, PSA, BMP-1, M The protease may comprise a substrate for a protease selected from the group consisting of MP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27, TMPRSS2, TMPRSS3, TMPRSS4, tPA, thrombin, tryptase and uPA.

[0109] In some embodiments of any of the ACCs described herein, the protease that cleaves any of the CMs described herein is selected from the group consisting of ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, BACE, renin, cathepsin D, cathepsin E, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, caspase 14, caspase 15, caspase 16, caspase 17, caspase 18, caspase 19, caspase 20, caspase 21, caspase 22, caspase 23, caspase 24, caspase 25, caspase 26, caspase 27, caspase 28, caspase 29, caspase 30, caspase 31, caspase 32, caspase 33, caspase 34, caspase 35, caspase 36, caspase 37, caspase 38, caspase 39 ...9, caspase 39, caspase 39, caspase 39, caspase 39, caspase 39, caspase 39, caspase 39, caspase 39, Caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 14, cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P, cruzipain, legumain, otubain-2, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, meprin, neprily Syn, PSMA, BMP-1, MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-19, MMP-20, MMP-23, MMP-24, MMP-26, MMP-27, Activated Protein C, Cathepsin A, Cathepsin G, Chymase, FVIIa, FIXa, FXa, FXIa, FXI Ia, elastase, granzyme B, guanidinobenzoatase, HtrA1, human neutrophil lyase, lactoferrin, marapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, tryptase, uPA, DESC1, DPP-4, FAP, hepsin, matriptase-2, MT-SP1 / matriptase, TMPRSS2, TMPRSS3, and TMPRSS4.

[0110] In some embodiments of any of the ACC described herein, the protease is selected from the group of uPA, legumain, MT-SP1, ADAM17, BMP-1, TMPRSS3, TMPRSS4, MMP-2, MMP-9, MMP-12, MMP-13, and MMP-14.

[0111] Elevated levels of proteases with known substrates have been reported in many cancers. See, e.g., La Roca et al., British J.Cancer 90(7):1414-1421, 2004. Suitable substrates for use with the CM1 and / or CM2 components used herein include substrates that are more prevalently found in cancer cells and tissues. Thus, in certain embodiments, CM1 and / or CM2 each independently comprise a substrate for a protease that is more prevalently found in diseased tissues associated with cancer. In some embodiments, the cancer is selected from the group consisting of gastric cancer, breast cancer, osteosarcoma, and esophageal cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is a HER2-positive cancer. In some embodiments, the cancer is Kaposi's sarcoma, hairy cell leukemia, chronic myelogenous leukemia (CML), follicular lymphoma, renal cell carcinoma (RCC), melanoma, neuroblastoma, basal cell carcinoma, malignant cutaneous T-cell lymphoma, nasopharyngeal adenocarcinoma, breast cancer, ovarian cancer, bladder cancer, BCG-resistant non-muscle invasive bladder cancer (NMIBC), endometrial cancer, pancreatic cancer, non-small cell lung cancer (NSCLC), colorectal cancer, esophageal cancer, gallbladder cancer, glioma, head and neck cancer, uterine cancer, cervical cancer, or testicular cancer, etc. In some of the above-described embodiments, the CM component comprises a substrate for a protease(s) that is more prevalent in the tumor tissue.

[0112] In some embodiments, CM1 and / or CM2 each independently contain a sequence selected from the group consisting of SEQ ID NO:5 to SEQ ID NO:100 and SEQ ID NO:349, and C-terminal and N-terminal truncated variants thereof.

[0113] In some embodiments, the CM contains a sequence selected from the group of ISSGLLSGRSDNH (SEQ ID NO: 28), LSGRSDDH (SEQ ID NO: 33), LSGRSDNI (SEQ ID NO: 41), ISSGLLSGRSDQH (SEQ ID NO: 54), ISSGLLSGRSDNI (SEQ ID NO: 68), SGRSDNI (SEQ ID NO: 100), and LSGRSNI (SEQ ID NO: 349).

[0114] In certain embodiments, CM1 and / or CM2 contain a sequence selected from the group of APRSALAHGLF (SEQ ID NO:263), AQNLLGMY (SEQ ID NO:264), LSGRSDNHGGAVGLLAPP (SEQ ID NO:265), VHMPLGFLGPGGLSGRSDNH (SEQ ID NO:266), LSGRSDNHGGVHMPLGFLGP (SEQ ID NO:267), LSGRSDNHGGSGGSISSGLLSS (SEQ ID NO:268), ISSGLLSSGGSGGSLSGRSGNH (SEQ ID NO:269), LSGRSDNHGGSGGSQNQALRMA (SEQ ID NO:270), QNQALRMAGGSGGSLSGRSDNH (SEQ ID NO:271), LSGRSGNHGGSGGSQNQALRMA (SEQ ID NO:272), QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO:273), ISSGLLSGRSGNH (SEQ ID NO:274), and C-terminal and N-terminal truncated variants thereof. Further examples of CMs include those described in U.S. Patent Application Publication Nos. 2016 / 0289324, 2019 / 0284283, and Publication Nos. WO2010 / 081173, WO2015 / 048329, WO2015 / 116933, WO2016 / 118629, and WO2020 / 118109, which are incorporated by reference in their entireties.

[0115] The truncated variants of the above-mentioned amino acid sequences suitable for use in CM1 and / or CM2 are any truncated variants that retain the recognition site of the corresponding protease. These include C-terminal and / or N-terminal truncated variants that contain at least 3 consecutive amino acids of the above-mentioned amino acid sequences, or at least 4, or at least 5, or at least 6, or at least 7 amino acids of the aforementioned amino acid sequences that retain the recognition site of the protease. In certain embodiments, the truncated variants of the above-mentioned amino acid sequences are amino acid sequences that correspond to any of the above, but are truncated at the C-terminus and / or N-terminus by 1 to about 10 amino acids, 1 to about 9 amino acids, 1 to about 8 amino acids, 1 to about 7 amino acids, 1 to about 6 amino acids, 1 to about 5 amino acids, 1 to about 4 amino acids, or 1 to about 3 amino acids, (1) having at least 3 amino acid residues; (2) retaining the recognition site of the protease. In some of the above embodiments, the truncated CM is a CM truncated at the N-terminus. In some embodiments, the truncated CM is a CM truncated at the C-terminus. In some embodiments, the truncated C is a CM truncated at the C-terminus and the N-terminus.

[0116] In some embodiments of any of the activatable cytokine constructs described herein, CM1 and / or CM2 comprise a total of about 3 amino acids to about 25 amino acids. In some embodiments, CM1 and / or CM2 comprise a total of about 3 amino acids to about 25 amino acids, about 3 amino acids to about 20 amino acids, about 3 amino acids to about 15 amino acids, about 3 amino acids to about 10 amino acids, about 3 amino acids to about 5 amino acids, about 5 amino acids to about 25 amino acids, about 5 amino acids to about 20 amino acids, about 5 amino acids to about 15 amino acids, about 5 amino acids to about 10 amino acids, about 10 amino acids to about 25 amino acids, about 10 amino acids to about 20 amino acids, about 10 amino acids to about 15 amino acids, about 15 amino acids to about 25 amino acids, about 15 amino acids to about 20 amino acids, or about 20 amino acids to about 25 amino acids.

[0117] In some embodiments, the ACC may comprise multiple CMs that comprise substrates for different proteases. In some embodiments, CM1 and CM2 comprise substrates for different proteases. In some embodiments, CM1 and CM2 comprise substrates for the same protease.

[0118] The first monomer construct and the second monomer construct may include one or more additional components, such as one or more linkers. In some embodiments, the first monomer can contain a linker disposed between CP1 and CM1. In some embodiments, in the first monomer, CP1 and CM1 are directly adjacent to each other. In some embodiments, the first monomer includes a linker disposed between CM1 and DD1. In some embodiments, the linker has a total length of 1 amino acid to about 15 amino acids. In some embodiments, in the first monomer, CM1 and DD1 are directly adjacent to each other. In some embodiments, the CM and optional linker disposed between CP1 and DD1 have a total length of 3 to 15 amino acids, or 3 to 10 amino acids, or 3 to 7 amino acids.

[0119] In some embodiments, the second monomer comprises a linker disposed between CP2 and CM2. In some embodiments, in the second monomer, CP2 and CM2 are directly adjacent to each other. In some embodiments, the second monomer comprises a linker disposed between CM2 and DD2. In some embodiments, the linker has a total length of 1 amino acid to about 15 amino acids. In some embodiments, the linker comprises a sequence of G, GG, or GGGS (SEQ ID NO: 2). In some embodiments, in the second monomer, CM2 (e.g., any of the cleavable moieties described herein) and DD2 (e.g., any of the DDs described herein) are directly adjacent to each other. In some embodiments, the CM and any linker disposed between CP2 and DD2 have a total length of 3 to 15 amino acids, or 3 to 10 amino acids, or 3 to 7 amino acids.

[0120] In some embodiments, the first monomer and / or the second monomer may have a total of about 50 amino acids to about 800 amino acids, about 50 amino acids to about 750 amino acids, about 50 amino acids to about 700 amino acids, about 50 amino acids to about 650 amino acids, about 50 amino acids to about 600 amino acids, about 50 amino acids to about 550 amino acids, about 50 amino acids to about 500 amino acids, about 50 amino acids to about 450 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 350 amino acids, About 50 amino acids to about 300 amino acids, about 50 amino acids to about 250 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 100 amino acids, about 100 amino acids to about 800 amino acids, about 100 amino acids to about 750 amino acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about 650 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 550 amino acids, about 100 amino acids to about About 500 amino acids, about 100 amino acids to about 450 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 350 amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 250 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 150 amino acids, about 150 amino acids to about 800 amino acids, about 150 amino acids to about 750 amino acids, about 150 amino acids to about 700 amino acids, about 150 amino acids to about 650 amino acids , about 150 amino acids to about 600 amino acids, about 150 amino acids to about 550 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 450 amino acids, about 150 amino acids to about 400 amino acids, about 150 amino acids to about 350 amino acids, about 150 amino acids to about 300 amino acids, about 150 amino acids to about 250 amino acids, about 150 amino acids to about 200 amino acids, about 200 amino acids to about 800 amino acids, about 200 amino acids to about 750 amino acids,About 200 amino acids to about 700 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 550 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 450 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 350 amino acids, about 200 amino acids to about 300 amino acids, about 200 amino acids to about 250 amino acids, about 250 amino acids to about 800 amino acids, about about 250 to about 750 amino acids, about 250 to about 700 amino acids, about 250 to about 650 amino acids, about 250 to about 600 amino acids, about 250 to about 550 amino acids, about 250 to about 500 amino acids, about 250 to about 450 amino acids, about 250 to about 400 amino acids, about 250 to about 350 amino acids, about 250 to about 300 amino acids, about 300 to about 800 amino acids, about 300 to about About 750 amino acids, about 300 amino acids to about 700 amino acids, about 300 amino acids to about 650 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 550 amino acids, about 300 amino acids to about 500 amino acids, about 300 amino acids to about 450 amino acids, about 300 amino acids to about 400 amino acids, about 300 amino acids to about 350 amino acids, about 350 amino acids to about 800 amino acids, about 350 amino acids to about 750 amino acids, about 350 amino acids to about 700 amino acids , about 350 amino acids to about 650 amino acids, about 350 amino acids to about 600 amino acids, about 350 amino acids to about 550 amino acids, about 350 amino acids to about 500 amino acids, about 350 amino acids to about 450 amino acids, about 350 amino acids to about 400 amino acids, about 400 amino acids to about 800 amino acids, about 400 amino acids to about 750 amino acids, about 400 amino acids to about 700 amino acids, about 400 amino acids to about 650 amino acids, about 400 amino acids to about 600 amino acids,About 400 amino acids to about 550 amino acids, about 400 amino acids to about 500 amino acids, about 400 amino acids to about 450 amino acids, about 450 amino acids to about 800 amino acids, about 450 amino acids to about 750 amino acids, about 450 amino acids to about 700 amino acids, about 450 amino acids to about 650 amino acids, about 450 amino acids to about 600 amino acids, about 450 amino acids to about 550 amino acids, about 450 amino acids to about 500 amino acids, about 500 amino acids to about 800 amino acids, about 500 amino acids to about 750 amino acids, about 500 amino acids to about 700 amino acids, about 500 amino acids to about 650 amino acids, about 500 amino acids to about 600 amino acids, about 500 amino acids to about 550 amino acids The amino acids may each contain about 550 to about 800 amino acids, about 550 to about 750 amino acids, about 550 to about 700 amino acids, about 550 to about 650 amino acids, about 550 to about 600 amino acids, about 600 to about 800 amino acids, about 600 to about 750 amino acids, about 600 to about 700 amino acids, about 600 to about 650 amino acids, about 650 to about 800 amino acids, about 650 to about 750 amino acids, about 650 to about 700 amino acids, about 700 to about 800 amino acids, about 700 to about 750 amino acids, or about 750 to about 800 amino acids.

[0121] In some embodiments of any of the ACCs described herein, one or more linkers (e.g., flexible linkers) can be introduced into the activatable cytokine construct to provide flexibility at one or more of the junctions between domains, between moieties, between moieties and domains, or at any other junctions where a linker would be beneficial. In some embodiments, when the ACC is provided as a conformationally constrained construct, a flexible linker can be inserted to facilitate the formation and maintenance of structure in the uncleaved activatable cytokine construct. Any of the linkers described herein can provide the desired flexibility to facilitate inhibition of target (e.g., cytokine receptor) binding or facilitate cleavage of the CM by proteases. In some embodiments, the linker contained in the ACC is fully or partially flexible, such that the linker can contain, in addition to the flexible linker, one or more moieties that impart a less flexible structure to provide the desired ACC. Some linkers may contain cysteine ​​residues, which may form disulfide bonds to reduce the flexibility of the construct. In some embodiments, shortening the length of the linker or linking region reduces the activity of the mature cytokine protein in ACC (see, e.g., Figures 7A-7B and 8A-8B). In most cases, the length of the linker is determined by counting the number of amino acids in the N-terminal to C-terminal direction from the N-terminus of the linker adjacent to the C-terminal amino acid of the previous component to the C-terminus of the linker adjacent to the N-terminal amino acid of the next component (i.e., the length of the linker does not contain either the C-terminal amino acid of the previous component or the N-terminal amino acid of the next component). In embodiments where the linker is used at the N-terminus of a DD containing an Fc domain, the length of the linker is determined by counting the number of amino acids from the N-terminus of the linker adjacent to the C-terminal amino acid of the previous component to the C-terminus of the linker adjacent to the first cysteine ​​of the Fc hinge region (i.e., the length of the linker does not contain the C-terminal amino acid of the previous component or the first cysteine ​​of the Fc hinge region).

[0122] As is evident from the present disclosure and FIG. 14, the ACC of the present disclosure contains a stretch of amino acids between the CP and the proximal point of interaction between the dimerization domains. That stretch of amino acids may be referred to as the linking region (LR). As used herein, the term "linking region" or "LR" refers to a stretch of amino acid residues between the C-terminus of the cytokine and the amino acid residues adjacent at the N-terminus to the proximal point of interaction between the dimerization domains (i.e., the linking region does not contain the C-terminal amino acid of the cytokine or the N-terminal amino acid of the DD that forms the proximal point of interaction with the DD of the corresponding second monomer). For example, if the DD is a pair of Fc domains, the linking region is a stretch of amino acid residues between the C-terminus of the cytokine and the first N-terminal cysteine ​​residue that participates in the disulfide bond of the Fc (e.g., cysteine ​​226 of the Fc domain of IgG1 or IgG4 according to EU numbering). If the dimerization domain is not a peptide, the linking region is a stretch of amino acid residues from the C-terminus of the cytokine to the last amino acid. For example, if DD is a biotin-streptavidin pair, the linking region of the biotin-containing monomer is a stretch of amino acid residues between the C-terminus of the cytokine and the biotin molecule, and the linking region of the streptavidin-containing monomer is a stretch of amino acid residues between the C-terminus of the cytokine and the streptavidin molecule. In some embodiments, the linking region may comprise up to 24, 18, 14, 12, 11, 10, 9, 8, 7, 6, 5 or 4 amino acids, for example 5-14, 7-12, 7-11 or 8-11 amino acids.

[0123] In some embodiments, additional amino acid sequences may be placed N-terminal or C-terminal to any domain of any ACC. Examples include, but are not limited to, targeting moieties (e.g., ligands for receptors on cells present in target tissues) and serum half-life extending moieties (e.g., polypeptides that bind to serum proteins such as immunoglobulins (e.g., IgG) or serum albumins (e.g., human serum albumin (HSA))).

[0124] In some embodiments of any of the activatable cytokine constructs described herein, the linker comprises a total of about 1 amino acid to about 25 amino acids (e.g., about 1 amino acid to about 24 amino acids, about 1 amino acid to about 22 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 18 amino acids, about 1 amino acid to about 16 amino acids, about 1 amino acid to about 15 amino acids, about 1 amino acid to about 14 amino acids, about 1 amino acid to about 12 amino acids, about 1 amino acid to about 10 amino acids, about 1 1 to about 8 amino acids, about 1 to about 6 amino acids, about 1 to about 5 amino acids, about 1 to about 4 amino acids, about 1 to about 3 amino acids, about 1 to about 2 amino acids, about 2 to about 25 amino acids, about 2 to about 24 amino acids, about 2 to about 22 amino acids, about 2 to about 20 amino acids, about 2 to about 18 amino acids, about 2 to about 16 amino acids, about 2 to about 15 amino acids, about 2 to about About 14 amino acids, about 2 amino acids to about 12 amino acids, about 2 amino acids to about 10 amino acids, about 2 amino acids to about 8 amino acids, about 2 amino acids to about 6 amino acids, about 2 amino acids to about 5 amino acids, about 2 amino acids to about 4 amino acids, about 2 amino acids to about 3 amino acids, about 4 amino acids to about 25 amino acids, about 4 amino acids to about 24 amino acids, about 4 amino acids to about 22 amino acids, about 4 amino acids to about 20 amino acids, about 4 amino acids to about 18 amino acids, about 4 amino acids to about 16 amino acids amino acids, about 4 amino acids to about 15 amino acids, about 4 amino acids to about 14 amino acids, about 4 amino acids to about 12 amino acids, about 4 amino acids to about 10 amino acids, about 4 amino acids to about 8 amino acids, about 4 amino acids to about 6 amino acids, about 4 amino acids to about 5 amino acids, about 5 amino acids to about 25 amino acids, about 5 amino acids to about 24 amino acids, about 5 amino acids to about 22 amino acids, about 5 amino acids to about 20 amino acids, about 5 amino acids to about 18 amino acids, about 5 amino acids to about 16 amino acids,about 5 amino acids to about 15 amino acids, about 5 amino acids to about 14 amino acids, about 5 amino acids to about 12 amino acids, about 5 amino acids to about 10 amino acids, about 5 amino acids to about 8 amino acids, about 5 amino acids to about 6 amino acids, about 6 amino acids to about 25 amino acids, about 6 amino acids to about 24 amino acids, about 6 amino acids to about 22 amino acids, about 6 amino acids to about 20 amino acids, about 6 amino acids to about 18 amino acids, about 6 amino acids to about 16 amino acids, about 6 amino acids to about 15 amino acids, about 6 1 to about 14 amino acids, about 6 to about 12 amino acids, about 6 to about 10 amino acids, about 6 to about 8 amino acids, about 8 to about 25 amino acids, about 8 to about 24 amino acids, about 8 to about 22 amino acids, about 8 to about 20 amino acids, about 8 to about 18 amino acids, about 8 to about 16 amino acids, about 8 to about 15 amino acids, about 8 to about 14 amino acids, about 8 to about 12 amino acids, about 8 From about 10 amino acids, from about 10 amino acids to about 25 amino acids, from about 10 amino acids to about 24 amino acids, from about 10 amino acids to about 22 amino acids, from about 10 amino acids to about 20 amino acids, from about 10 amino acids to about 18 amino acids, from about 10 amino acids to about 16 amino acids, from about 10 amino acids to about 15 amino acids, from about 10 amino acids to about 14 amino acids, from about 10 amino acids to about 12 amino acids, from about 12 amino acids to about 25 amino acids, from about 12 amino acids to about 24 amino acids, from about 12 amino acids to about 2 2 amino acids, about 12 amino acids to about 20 amino acids, about 12 amino acids to about 18 amino acids, about 12 amino acids to about 16 amino acids, about 12 amino acids to about 15 amino acids, about 12 amino acids to about 14 amino acids, about 14 amino acids to about 25 amino acids, about 14 amino acids to about 24 amino acids, about 14 amino acids to about 22 amino acids, about 14 amino acids to about 20 amino acids, about 14 amino acids to about 18 amino acids, about 14 amino acids to about 16 amino acids, about 14 amino acids to about 15 amino acids,About 15 amino acids to about 25 amino acids, about 15 amino acids to about 24 amino acids, about 15 amino acids to about 22 amino acids, about 15 amino acids to about 20 amino acids, about 15 amino acids to about 18 amino acids, about 15 amino acids to about 16 amino acids, about 16 amino acids to about 25 amino acids, about 16 amino acids to about 24 amino acids, about 16 amino acids to about 22 amino acids, about 16 amino acids to about 20 amino acids, about 16 amino acids to about 18 amino acids amino acids, about 18 amino acids to about 25 amino acids, about 18 amino acids to about 24 amino acids, about 18 amino acids to about 22 amino acids, about 18 amino acids to about 20 amino acids, about 20 amino acids to about 25 amino acids, about 20 amino acids to about 24 amino acids, about 20 amino acids to about 22 amino acids, about 22 amino acids to about 25 amino acids, about 22 amino acids to about 24 amino acids, or about 24 amino acids to about 25 amino acids.

[0125] In some embodiments of any of the ACCs described herein, the linker contains a total of about 1 amino acid, about 2 amino acids, about 3 amino acids, about 4 amino acids, about 5 amino acids, about 6 amino acids, about 7 amino acids, about 8 amino acids, about 9 amino acids, about 10 amino acids, about 11 amino acids, about 12 amino acids, about 13 amino acids, about 14 amino acids, about 15 amino acids, about 16 amino acids, about 17 amino acids, about 18 amino acids, about 19 amino acids, about 20 amino acids, about 21 amino acids, about 22 amino acids, about 23 amino acids, about 24 amino acids, or about 25 amino acids.

[0126] Surprisingly, the present inventors have found that ACC that does not include any linker between CP and DD shows the most significant reduction in cytokine activity compared to wild-type mature cytokine (see Figures 7A and 8A). Furthermore, the configuration without a linker between CP and DD still allows effective cleavage of the CM placed between CP and DD (see Figures 9 to 11). Thus, in some embodiments, ACC does not include any linker between CP and DD, and the CM between CP and DD includes 10, 9, 8, 7, 6, 5, 4 or 3 amino acids or less. In some embodiments, the total number of amino acids in the LR includes no more than 25 amino acids, e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 amino acids, or any range or specific number of amino acids selected from the range encompassed by 3-10 amino acids, or 5-15 amino acids, or 7-12 amino acids, or 3-25 amino acids.

[0127] In some embodiments of any of the ACCs described herein, the linker can be rich in glycine (Gly or G) residues. In some embodiments, the linker can be rich in serine (Ser or S) residues. In some embodiments, the linker can be rich in glycine and serine residues. In some embodiments, the linker has one or more glycine-serine residue pairs (GS) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS pairs). In some embodiments, the linker has one or more Gly-Gly-Gly-Ser (GGGS) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGS sequences). In some embodiments, the linker has one or more Gly-Gly-Gly-Gly-Ser (GGGGS) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGGS sequences). In some embodiments, the linker has one or more Gly-Gly-Ser-Gly (GGSG) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGSG sequences).

[0128] In some embodiments of any of the ACCs described herein, the linker is selected from the group consisting of G, GG, GSSGGSGGSGG (SEQ ID NO:210), GGGS (SEQ ID NO:2), GGGSGGGS (SEQ ID NO:211), GGGSGGGSGGGS (SEQ ID NO:212), GGGGSGGGGSGGGGGS (SEQ ID NO:213), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:214), GGGGSGGGGS (SEQ ID NO:215), GGGGS (SEQ ID NO:216), GS, GGGGSGS (SEQ ID NO:217), GGG It contains any one or a combination of one or more of GSGGGGSGGGGSGS (SEQ ID NO: 218), GGSLDPKGGGGS (SEQ ID NO: 219), PKSCDKTHTCPPCPAPELLG (SEQ ID NO: 220), SKYGPPCPPCPAPEFLG (SEQ ID NO: 221), GKSSGSGSESKS (SEQ ID NO: 222), GSTSGSGKSSEGKG (SEQ ID NO: 223), GSTSGSGKSSEGSGSTKG (SEQ ID NO: 224), and GSTSGSGKPGSGEGSTKG (SEQ ID NO: 225).

[0129] Non-limiting examples of linkers can contain a sequence that is at least 70% identical (e.g., at least 72%, at least 74%, at least 75%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to GGGS (SEQ ID NO:2), GSSGGSGGSGG (SEQ ID NO:210), GGGGSGGGGSGGGGS (SEQ ID NO:213), GGGGSGS (SEQ ID NO:217), GGGGSGGGGSGGGGSGS (SEQ ID NO:218), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:235), GGSLDPKGGGGS (SEQ ID NO:219), and GSTSGSGKPGSSEGST (SEQ ID NO:226).

[0130] In some embodiments, the linker contains a sequence selected from the group of GGSLDPKGGGGS (SEQ ID NO:219), GGGGSGGGGSGGGGSGS (SEQ ID NO:218), GGGGSGS (SEQ ID NO:217), GS, (GS)n, (GGS)n, (GSGGS)n (SEQ ID NO:227) and (GGGS)n (SEQ ID NO:228), GGSG (SEQ ID NO:229), GGSGG (SEQ ID NO:230), GSGSG (SEQ ID NO:231), GSGGG (SEQ ID NO:232), GGGSG (SEQ ID NO:233), GSSSG (SEQ ID NO:234), GGGGSGGGGSGGGGGS (SEQ ID NO:213), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:235), GSTSGSGKPGSSEGST (SEQ ID NO:226), (GGGGS)n (SEQ ID NO:216), where n is an integer of at least 1. In some embodiments, the linker contains a sequence selected from the group consisting of GGSLDPKGGGGS (SEQ ID NO:219), GGGGSGGGGSGGGGSGS (SEQ ID NO:218), GGGGSGS (SEQ ID NO:217), and GS. In some embodiments of any of the ACCs described herein, the linker contains a sequence selected from the group of GGGSGGGGSGGGGGS (SEQ ID NO:213), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:235), and GSTSGSGKPGSSEGST (SEQ ID NO:226). In some embodiments of any of the activatable cytokine constructs described herein, the linker contains a sequence selected from the group of GGGSGGGGSGGGGGS (SEQ ID NO:213) or GGGGS (SEQ ID NO:216). In some embodiments, the linker comprises a sequence of GGGS (SEQ ID NO:2). In some embodiments, the linker comprises a sequence of a single glycine residue (G), or two glycine residues (GG).

[0131] In some embodiments, the ACC can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linker sequences (e.g., linker sequences that are the same or different from any of the exemplary linker sequences described herein or known in the art). In some embodiments, the linkers include sulfo-SIAB, SMPB, and sulfo-SMPB, which linkers react with primary amine sulfhydryls.

[0132] In some embodiments of any of the ACC described herein, the ACC is characterized by a reduction in at least one activity of CP1 and / or CP2 compared to a control level of at least one activity of CP1 and / or CP2. In some embodiments, the control level can be the activity level of recombinant CP1 and / or CP2 (e.g., commercially available recombinant CP1 and / or CP2, recombinant wild-type CP1 and / or CP2, etc.). In some embodiments, the control level can be the activity level of a truncated (activated) form of ACC. In certain embodiments, the control level can be the activity level of pegylated CP1 and / or CP2.

[0133] In some embodiments, at least one activity is the binding affinity (K) of CP1 and / or CP2 to its cognate receptor, as determined using surface plasmon resonance (e.g., performed in phosphate buffered saline at 25° C.). D). In certain embodiments, the at least one activity is the level of proliferation of lymphoma cells. In other embodiments, the at least one activity is the level of activation of the JAK / STAT / ISGF3 pathway in lymphoma cells. In some embodiments, the at least one activity is the level of production of SEAP in lymphoma cells. In some embodiments, the at least one activity is the level of SEAP production in a cell-based assay using HEK cells. In further embodiments, the at least one activity of CP1 and / or CP2 is the level of cytokine-stimulated gene induction using, for example, RNAseq analysis (see, e.g., Zimmerer et al., Clin. Cancer Res. 14(18):5900-5906, 2008; Hilkens et al., J. Immunol. 171:5255-5263, 2003).

[0134] In some embodiments, the ACC is characterized by at least one CP1 and / or CP2 activity being at least 2-fold lower compared to a control level of at least one CP1 and / or CP2 activity. In some embodiments, the ACC is characterized by at least one CP1 and / or CP2 activity being at least 5-fold lower compared to a control level of at least one CP1 and / or CP2 activity. In some embodiments, the ACC is characterized by at least one CP1 and / or CP2 activity being at least 10-fold lower compared to a control level of at least one CP1 and / or CP2 activity. In some embodiments, the ACC is characterized by at least one CP1 and / or CP2 activity being at least 20-fold lower compared to a control level of at least one CP1 and / or CP2 activity. In some embodiments, the ACC is characterized by at least 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 500-fold, or 1000-fold lower in at least one activity of CP1 and / or CP2 compared to a control level of at least one activity of CP1 and / or CP2. In some embodiments, the ACC is characterized by at least 1-20-fold lower, 200-500-fold lower, 300-500-fold lower, 400-500-fold lower, 500-600-fold lower, 600-700-fold lower, 150-1000-fold lower, 100-1500-fold lower, 200-1500-fold lower, 300-1500-fold lower, or 400-1500-fold lower in at least one activity of CP1 and / or CP2 compared to a control level of at least one activity of CP1 and / or CP2. , 500 to 1500 times lower, 1000 to 1500 times lower, 100 to 1000 times lower, 200 to 1000 times lower, 300 to 1000 times lower, 400 to 1000 times lower, 500 to 1000 times lower, 100 to 500 times lower, 20 to 50 times lower, 30 to 50 times lower, 40 to 50 times lower, 100 to 400 times lower, 200 to 400 times lower, or 300 to 400 times lower, 100 to 300 times lower, 200 to 300 times lower, or 100 to 200 times lower.

[0135] In some embodiments, the control level of at least one activity of CP1 and / or CP2 is the activity of CP1 and / or CP2 released from ACC following cleavage of CM1 and CM2 by a protease(s) (the "cleavage products"). In some embodiments, the control level of at least one activity of CP1 and / or CP2 is the activity of the corresponding wild-type mature cytokine (e.g., a recombinant wild-type mature cytokine).

[0136] In some embodiments, incubation of ACC with a protease produces activated cytokine product(s), and the CP1 and / or CP2 activity(s) of the activated cytokine product(s) is / are greater than the CP1 and / or CP2 activity(s) of untreated ACC. In some embodiments, the CP1 and / or CP2 activity(s) of the activated cytokine product(s) is / are at least 1-fold greater than the CP1 and / or CP2 activity(s) of ACC. In some embodiments, the CP1 and / or CP2 activity(s) of the activated cytokine product(s) is / are at least 2-fold greater than the CP1 and / or CP2 activity(s) of ACC. In some embodiments, the CP1 and / or CP2 activity(s) of the activated cytokine product(s) is / are at least 5-fold greater than the CP1 and / or CP2 activity(s) of ACC. In some embodiments, the CP1 and / or CP2 activity(s) of the activated cytokine product(s) is / are at least 10-fold greater than the CP1 and / or CP2 activity(s) of ACC. In some embodiments, the activity of one or more of CP1 and / or CP2 of the activated cytokine product(s) is at least 20-fold greater than the activity of one or more of CP1 and / or CP2 of ACC. In some embodiments, the activity of one or more of CP1 and / or CP2 of the activated cytokine product(s) is at least 1-20 fold greater, 2-20 fold greater, 3-20 fold greater, 4-20 fold greater, 5-20 fold greater, 10-20 fold greater, 15-20 fold greater, 1-15 fold greater, 2-15 fold greater, 3-15 fold greater, 4-15 fold greater, 5-15 fold greater, 10-15 fold greater, 1-10 fold greater, 2-10 fold greater, 3-10 fold greater, 4-10 fold greater, 5-10 fold greater, 1-5 fold greater, 2-5 fold greater, 3-5 fold greater, 4-5 fold greater, 1-4 fold greater, 2-4 fold greater, 3-4 fold greater, 1-3 fold greater, 2-3 fold greater, or 1-2 fold greater than the activity of one or more of CP1 and / or CP2 of ACC.

[0137] In some embodiments, ACC can contain a sequence that is at least 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 347 or 348. In some embodiments, ACC can be encoded by a nucleic acid that contains a sequence that is at least 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 357. In some aspects, ACC may contain such sequences, but may or may not include signal sequences of those sequences. Signal sequences are not particularly limited. Some non-limiting examples of signal sequences include, for example, residues 1-20 of SEQ ID NO: 309 and the corresponding residues and nucleotides of other sequences, or substituted with a signal sequence from another species or cell line. Other examples of signal sequences include MRAWIFFLLCLAGRALA (SEQ ID NO:343) and MALTFALLVALLVLSCKSSCSVG (SEQ ID NO:344).

[0138] Various exemplary aspects of these activatable cytokine constructs are described below, which may be used in any combination, without limitation, in the methods provided herein. Exemplary aspects of activatable cytokine constructs and methods of making activatable cytokine constructs are described below.

[0139] In some embodiments, the CM is selected for use with a particular protease. The protease may be a protease produced by tumor cells (e.g., tumor cells may express a greater amount of a protease than healthy tissue). In some embodiments, the CM is a substrate for at least one protease selected from the group of ADAM17, BMP-1, cysteine ​​proteases such as cathepsins, HtrA1, legumain, matriptase (MT-SP1), matrix metalloproteases (MMPs), neutrophil elastase, TMPRSS such as TMPRSS3 or TMPRSS4, thrombin, and u-type plasminogen activator (uPA, also known as urokinase).

[0140] In some embodiments, the CM is a substrate for at least one matrix metalloprotease (MMP). Examples of MMPs include MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, and MMP27. In some embodiments, the CM is a substrate for MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11, and MMP19. In some embodiments, the CM is a substrate for MMP7. In some embodiments, the CM is a substrate for MMP9. In some embodiments, the CM is a substrate for MMP14. In some embodiments, the CM is a substrate for two or more MMPs. In some embodiments, the CM is a substrate for at least MMP9 and MMP14. In some embodiments, the CM contains two or more substrates of the same MMP. In some embodiments, the CM contains at least two or more MMP9 substrates. In some embodiments, the CM contains at least two or more MMP14 substrates.

[0141] In some embodiments, the CM is a substrate for MMPs and contains the sequences ISSGLLSS (SEQ ID NO: 19); QNQALRMA (SEQ ID NO: 16); AQNLLGMV (SEQ ID NO: 15); STFPFGMF (SEQ ID NO: 18); PVGYTSSL (SEQ ID NO: 74); DWLYWPGI (SEQ ID NO: 75); MIAPVAYR (SEQ ID NO: 42); RPSPMWAY (SEQ ID NO: 43); WATPRPMR (SEQ ID NO: 44); FRLLDWQW (SEQ ID NO: 45); LKAAPRWA (SEQ ID NO: 76); GPSHLVLT (SEQ ID NO: 77); LPGGLSPW (SEQ ID NO: 78); MGLFSEAG (SEQ ID NO: 79); SPLPLRVP (SEQ ID NO: 80); RMHLRSLG (SEQ ID NO: 81); LAAPLGLL (SEQ ID NO: 17); AVGLLAPP (SEQ ID NO: 14); LLAPSHRA (SEQ ID NO: 82); PAGLWLDP (SEQ ID NO: 20); and / or ISSGLSS (SEQ ID NO: 73).

[0142] In some embodiments, the CM is a substrate for thrombin. In some embodiments, the CM is a substrate for thrombin and contains the sequence GPRSFGL (SEQ ID NO: 83) or GPRSFG (SEQ ID NO: 84).

[0143] In some embodiments, the CM contains an amino acid sequence selected from the group of NTLSGRSENHSG (SEQ ID NO: 9); NTLSGRSGNHGS (SEQ ID NO: 10); TSTSGRSAMPRG (SEQ ID NO: 11); TSGRSAMP (SEQ ID NO: 12); VAGRSMRP (SEQ ID NO: 21); VVPEGRRS (SEQ ID NO: 22); ILPRSPAF (SEQ ID NO: 23); MVLGRSLL (SEQ ID NO: 24); QGRAITFI (SEQ ID NO: 25); SPRSIMLA (SEQ ID NO: 26); and SMLRSMPL (SEQ ID NO: 27).

[0144] In some embodiments, the CM is a substrate for neutrophil elastase. In some embodiments, the CM is a substrate for a serine protease. In some embodiments, the CM is a substrate for uPA. In some embodiments, the CM is a substrate for legumain. In some embodiments, the CM is a substrate for matriptase. In some embodiments, the CM is a substrate for a cysteine ​​protease. In some embodiments, the CM is a substrate for a cysteine ​​protease, such as a cathepsin.

[0145] In some embodiments, the CM is ISSGLLSGRSDNH (SEQ ID NO: 28); ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: 30); AVGLLAPPGGTSTSGRSANPRG (SEQ ID NO: 275); TSTSGRSANPRGGGAVGLLAPP (SEQ ID NO: 276); VHMPLGFLGPGGTSTSGRSANPRG (SEQ ID NO: 277); TSTSGRSANPRGGGVHMPLGFLGP (SEQ ID NO: 278); AVGLLAPPGGLSGRSDNH (SEQ ID NO: 29); LSGRSDNHGGAVGLLAPP (SEQ ID NO: 70); VHMPLGFLGPGGLSGRSDNH (SEQ ID NO: 266); LS Contains the sequence of GRSDNHGGVHMPLGFLGP (SEQ ID NO: 267); LSGRSDNHGGSGGSISSGLLSS (SEQ ID NO: 268); LSGRSGNHGGSGGSISSGLLSS (SEQ ID NO: 279); ISSGLLSSGGSGGSLSGRSGNH (SEQ ID NO: 269); LSGRSDNHGGSGGSQNQALRMA (SEQ ID NO: 270); QNQALRMAGGSGGSLSGRSDNH (SEQ ID NO: 271); LSGRSGNHGGSGGSQNQALRMA (SEQ ID NO: 272); QNQALRMAGGSGGSLSGRSGNH (SEQ ID NO: 273) and / or ISSGLLSGRSGNH (SEQ ID NO: 274).

[0146] In some embodiments, CM1 and / or CM2 comprise a sequence selected from the group consisting of SEQ ID NO:5 to SEQ ID NO: 100. In some embodiments, CM comprises a sequence selected from the group of ISSGLLSGRSDNH (SEQ ID NO:28), LSGRSDDH (SEQ ID NO:33), ISSGLLSGRSDQH (SEQ ID NO:54), SGRSDNI (SEQ ID NO:100), and ISSGLLSGRSDNI (SEQ ID NO:68), LSGRSDNI (SEQ ID NO:41), and LSGRSNI (SEQ ID NO:349).

[0147] In some embodiments, the ACC contains CP1 selected from SEQ ID NOs: 111-134, 137-140, 143-146, 151-160, and 347-348, CM2 selected from SEQ ID NOs: 5-100, and 263-308, and DD1, which are dimerized with CP2 selected from SEQ ID NOs: 111-134, 137-140, 143-146, 151-160, and 347-348, CM1 selected from SEQ ID NOs: 5-100, and 263-308, and DD1. In some embodiments, the ACC may contain a linker selected from SEQ ID NOs: 2, 210-234, 245, or 250 between CP1 and CM1 and / or between CM1 and DD1, and a linker selected from SEQ ID NOs: 2, 210-234, 245, or 250 between CP2 and CM2 and / or between CM2 and DD2. In some embodiments, the ACC contains DD1 and / or DD2 having an amino acid sequence at least 80% identical (e.g., at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the ACC contains DD1 having an amino acid sequence at least 80% identical (e.g., at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to SEQ ID NO: 315 or SEQ ID NO: 316. In some embodiments, the ACC contains a DD2 having an amino acid sequence that is at least 80% identical (e.g., at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical) to SEQ ID NO:315 or SEQ ID NO:316.

[0148] Drug conjugates The present disclosure also provides methods and materials for incorporating additional elements into any of the ACC described herein, such as a targeting moiety that facilitates delivery to a cell or tissue of interest, an agent (e.g., a therapeutic agent, an anti-tumor agent), a toxin, or a fragment thereof.

[0149] In some embodiments of any of the ACCs described herein, the ACC can be conjugated to a cytotoxic agent, such as, but not limited to, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof) or a radioisotope. In some embodiments of any of the ACCs described herein, the activatable cytokine construct can be conjugated to a cytotoxic agent, such as, but not limited to, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof) or a radioisotope.

[0150] Non-limiting exemplary cytotoxic agents that can be conjugated to any of the ACCs described herein include dolastatins and their derivatives (e.g., auristatin E, AFP, monomethylauristatin D (MMAD), monomethylauristatin F (MMAF), monomethylauristatin E (MMAE), desmethylauristatin E (DMAE), auristatin F, desmethylauristatin F (DMAF), dolastatin 16 (DmJ), dolastatin 16 (Dpv), auristatin derivatives (e.g., auristatin tyramine, auristatin quinolones), maytansinoids (e.g., DM-1, DM-4), maytansinoid derivatives, duocarmycins, alpha-amanitin, turbostatin, fenstatin, and the like). camptothecin, hydroxyphenstatin, spongistatin 5, spongistatin 7, halistatin 1, halistatin 2, halistatin 3, halocompstatin, pyrrolobenzimidazole (PBI), sibulostatin 6, doxaliform, cemadotin analog (CemCH2-SH), Pseudomonas toxin A (PES8) mutant, Pseudomonas toxin A (ZZ-PE38) mutant, ZJ-101, anthracyclines, doxorubicin, daunorubicin, bryostatin, camptothecin, 7-substituted camptothecins, 10,11-difluoromethylenedioxycamptothecin, combretastatin, debromoaplysiatoxin, KahaMide-F, discodermolide, and ecteinascidin.

[0151] Non-limiting, exemplary enzymatically active toxins that can be conjugated to any of the ACCs described herein include diphtheria toxin, exotoxin A chain from Pseudomonas aeruginosa, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuriies fordii proteins, dianfhin proteins, Phytoiaca Americana proteins (e.g., PAPI, PAPII, and PAP-8), momordica charantia inhibitor, curcin, crotirs, sapaonaria officinalis inhibitor, geoinin, mitogeliin, restrictocin, phenomycin, neomycin, and the trichothecenes.

[0152] Non-limiting, exemplary antineoplastic agents that can be conjugated to any of the ACCs described herein include adriamycin, cerbidine, bleomycin, alkeran, velban, oncovin, fluorouracil, methotrexate, thiotepa, bisantrene, novantrone, thioguanine, procarbazine, and cytarabine.

[0153] Non-limiting, exemplary antiviral drugs that can be conjugated to any of the ACCs described herein include acyclovir, virA, and symmetrel.

[0154] Non-limiting, exemplary antifungal agents that can be conjugated to any of the ACC described herein include nystatin.

[0155] Non-limiting, exemplary conjugable detection reagents that can be conjugated to any of the ACCs described herein include fluorescein and its derivative, fluorescein isothiocyanate (FITC).

[0156] Non-limiting, exemplary antibacterial agents that can be conjugated to any of the activatable cytokine constructs described herein include aminoglycosides, streptomycin, neomycin, kanamycin, amikacin, gentamicin, and tobramycin.

[0157] Non-limiting, exemplary, 3β,16β,17α-trihydroxycholest-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-β-D-xylopyranosyl)-(1-->3)-(2-O-acetyl-α-L-arabinopyranoside) (OSW-1) that can be conjugated to any of the activatable cytokine constructs described herein include s-nitrobenzyloxycarbonyl derivatives of O6-benzylguanine, topoisomerase inhibitors, hemiasterlin, cephalotaxine, homoharringionine, pyrrolobenzodiazepine dimers (PBDs), functionalized pyrrolobenzodiazepines, calicheamicins, podophyiitoxins, taxanes, and vinca alkaloids.

[0158] Non-limiting, exemplary radiopharmaceuticals that can be conjugated to any of the activatable cytokine constructs described herein include: 123 I, 89 Zr, 125 I, 131 I, 99 mTc, 201 T1, 62 Cu, 18 F, 68 Ga, 13 N, 15 O. 38 K, 82 Rb, 111 In, 133 Xe, 11 C and 99 One example is mTc (technetium).

[0159] Non-limiting exemplary heavy metals that can be conjugated to any of the ACCs described herein include barium, gold and platinum.

[0160] Non-limiting exemplary anti-mycoplasma agents that can be conjugated to any of the ACC described herein include tylosin, spectinomycin, streptomycin B, ampicillin, sulfanilamide, polymyxin, and chloramphenicol.

[0161] Those skilled in the art will appreciate that a wide variety of possible moieties can be conjugated to any of the activatable cytokine constructs described herein. Conjugation can include any chemical reaction that results in binding two molecules, so long as the ACC and other moieties retain their respective activities. Conjugation can include many chemical reaction mechanisms, such as covalent binding, affinity binding, intercalation, coordinate binding, and complexation. In some embodiments, the preferred bond is a covalent bond. Covalent binding can be achieved either by direct condensation of existing side chains or by incorporation of an external bridging molecule. Many bivalent or multivalent binding agents are useful in conjugating any of the activatable cytokine constructs described herein. For example, conjugates can contain organic compounds, such as thioesters, carbodiimides, succinimide esters, glutaraldehyde, diazobenzene, and hexamethylenediamine. In some embodiments, the activatable cytokine construct can contain or otherwise introduce one or more non-natural amino acid residues to provide suitable sites for conjugation.

[0162] In some embodiments of any of the ACCs described herein, the drug and / or conjugate is attached to the antigen-binding domain by a disulfide bond (e.g., a disulfide bond on a cysteine ​​molecule). Since many cancers naturally release high levels of glutathione, a reducing agent, glutathione present in the cancer tissue microenvironment can reduce the disulfide bond, followed by release of the drug and / or conjugate at the delivery site.

[0163] In some embodiments of any of the ACCs described herein, when the conjugate binds to the target in the presence of complement within the target site (e.g., diseased tissue (e.g., cancer tissue)), the amide or ester bond attaching the conjugate and / or agent to the linker is cleaved, resulting in release of the conjugate and / or agent in an activated state. These conjugates and / or agents, when administered to a subject, will achieve delivery and release of the conjugate and / or agent at the target site (e.g., diseased tissue (e.g., cancer tissue)). These conjugates and / or agents are particularly effective for in vivo delivery of any of the conjugates and / or agents described herein.

[0164] In some embodiments, the linker is not cleavable by the enzymes of the complement system. For example, the conjugate and / or agent is released without complement activation, which ultimately lyses the target cell. In such embodiments, the conjugate and / or agent is to be delivered to the target cell (e.g., a hormone, enzyme, corticosteroid, neurotransmitter, or gene). Furthermore, the linker is made mildly susceptible to cleavage by serum proteases, and the conjugate and / or agent is slowly released at the target site.

[0165] In some embodiments of any of the ACC described herein, the conjugate and / or agent are designed such that the conjugate and / or agent is delivered to a target site (e.g., diseased tissue (e.g., cancerous tissue)) but the conjugate and / or agent is not released.

[0166] In some embodiments of any of the ACCs described herein, the conjugate and / or agent is attached to the antigen-binding domain either directly or via a non-cleavable linker. Exemplary non-cleavable linkers include amino acids (e.g., D-amino acids), peptides, or other organic compounds that can be modified by the methods described herein to contain a functional group that is subsequently available for attachment to the antigen-binding domain.

[0167] In some embodiments of any of the ACCs described herein, the ACC contains at least one conjugation point of an agent. In some embodiments, all available conjugation points are available for conjugation to an agent. In some embodiments, the one or more conjugation points include, but are not limited to, sulfur atoms involved in disulfide bonds, sulfur atoms involved in interchain disulfide bonds, sulfur atoms involved in interchain sulfide bonds but not intrachain disulfide bonds, and / or sulfur atoms of cysteine ​​or other amino acid residues that contain sulfur atoms. In such cases, the residue may be naturally occurring in the protein construct structure or may be incorporated into the protein construct using methods such as, but not limited to, site-directed mutagenesis, chemical conversion, or misincorporation of non-natural amino acids.

[0168] The present disclosure also provides methods and materials for preparing ACC for conjugation.In some embodiments of any of the ACC described herein, ACC is modified to contain one or more interchain disulfide bonds.For example, the disulfide bond in ACC can be reduced after exposure to a reducing agent, such as, but not limited to, TCEP, DTT or β-mercaptoethanol.In some cases, the reduction of disulfide bond is only partial reduction. As used herein, the term "partially reduced" refers to a situation in which ACC is contacted with a reducing agent and a portion of all available conjugation sites are reduced (e.g., not all disulfide bonds are reduced). In some embodiments, an activatable cytokine construct is partially reduced if, following contact with a reducing agent, less than 99% (e.g., less than 98%, less than 97%, less than 96%, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5%) of all available conjugation sites are reduced. In some embodiments, ACC having reduction in one or more interchain disulfide bonds is conjugated to an agent that is reactive with free thiols.

[0169] The present disclosure also provides methods and materials for conjugating a therapeutic agent to a specific location on the ACC. In some embodiments of any of the ACCs described herein, the ACC is modified so that a therapeutic agent can be conjugated to the ACC at a specific location on the ACC. For example, the ACC may be partially reduced to facilitate conjugation to the ACC. In such cases, partial reduction of the ACC occurs such that the conjugation site of the ACC is not reduced. In some embodiments, the conjugation site(s) on the ACC are selected to facilitate conjugation of the agent at a specific location on the protein construct. Upon treatment with a reducing agent, various factors may affect the "reduction level" of the ACC. For example, to achieve partial reduction of the ACC using the methods and materials described herein, optimization of, but not limited to, the ratio of reducing agent to ACC, the length of incubation, the incubation temperature, and / or the pH of the reduction reaction solution may be required. Any suitable combination of factors (e.g., the ratio of reducing agent to ACC, the length and temperature of incubation with the reducing agent, and / or the pH of the reducing agent) can be used to achieve partial reduction of ACC (e.g., total reduction of available conjugation sites, or reduction of specific conjugation sites).

[0170] An effective ratio of reducing agent to ACC can be any ratio that at least partially reduces ACC in a manner that allows for conjugation to a drug (e.g., overall reduction of available conjugation sites, or reduction of specific conjugation sites). In some embodiments, the ratio of reducing agent to ACC will be in the range of about 20:1 to 1:1, about 10:1 to 1:1, about 9:1 to 1:1, about 8:1 to 1:1, about 7:1 to 1:1, about 6:1 to 1:1, about 5:1 to 1:1, about 4:1 to 1:1, about 3:1 to 1:1, about 2:1 to 1:1, about 20:1 to 1:1.5, about 10:1 to 1:1.5, about 9:1 to 1:1.5, about 8:1 to 1:1.5, about 7:1 to 1:1.5, about 6:1 to 1:1.5, about 5:1 to 1:1.5, about 4:1 to 1:1.5, about 3:1 to 1:1.5, about 2:1 to 1:1.5, about 1.5:1 to 1:1.5, or about 1:1 to 1:1.5. In some embodiments, the ratio is in the range of about 5:1 to 1:1. In some embodiments, the ratio is in the range of about 5:1 to 1.5:1. In some embodiments, the ratio is in the range of about 4:1 to 1:1. In some embodiments, the ratio is in the range of about 4:1 to 1.5:1. In some embodiments, the ratio is in the range of about 8:1 to about 1:1. In some embodiments, the ratio is in the range of about 2.5:1 to 1:1.

[0171] Effective incubation times and temperatures for treating ACC with a reducing agent can be any times and temperatures that at least partially reduce the ACC (e.g., total reduction of available conjugation sites, or reduction of specific conjugation sites) in a manner that allows for conjugation of the agent to the ACC. In some embodiments, incubation times and temperatures for treating ACC will range from about 1 hour at 37° C. to about 12 hours at 37° C. (or any subrange therein).

[0172] The effective pH of the reduction reaction for treating ACC with a reducing agent can be any pH that at least partially reduces ACC (e.g., total reduction of available conjugation sites, or reduction of specific conjugation sites) in a manner that allows for conjugation of ACC to an agent.

[0173] When the partially reduced ACC contacts with a thiol-containing drug, the drug can be conjugated to the interchain thiol in the ACC. The drug can be modified to contain a thiol using a thiol-containing reagent (e.g., cysteine ​​or N-acetylcysteine). For example, the ACC can be partially reduced after incubation with a reducing agent (e.g., TEPC) at a desired ratio of reducing agent to ACC for about 1 hour at about 37°C. The effective ratio of reducing agent to ACC can be any ratio that partially reduces at least two interchain disulfide bonds located in the ACC in a manner that allows conjugation of a thiol-containing drug (e.g., reduction of all available conjugation sites or reduction of specific conjugation sites).

[0174] In some embodiments of any of the ACCs described herein, the ACC is reduced by a reducing agent in a manner that avoids the reduction of any intrachain disulfide bonds. In some embodiments of any of the ACCs described herein, the ACC is reduced by a reducing agent in a manner that avoids the reduction of any intrachain disulfide bonds and reduces at least one interchain disulfide bond.

[0175] In some embodiments of any of the ACCs described herein, the ACC can also contain a drug conjugated to the ACC. In some embodiments, the conjugated drug is a therapeutic drug.

[0176] In some embodiments, the agent (e.g., the agent conjugated to the activatable cytokine construct) is a detectable moiety, such as a label or other marker. For example, the agent is or contains a radiolabeled amino acid, one or more biotinyl moieties that can be detected by labeled avidin (e.g., streptavidin that contains a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods), one or more radioisotopes or radionuclides, one or more fluorescent labels, one or more enzymatic labels, and / or one or more chemiluminescent agents. In some embodiments, the detectable moiety is attached by a spacer molecule.

[0177] In some embodiments, an agent (eg, a cytotoxic agent conjugated to an activatable cytokine construct) is linked to the ACC using a carbohydrate moiety, a sulfhydryl group, an amino group, or a carboxylate group.

[0178] In some embodiments of any of the ACCs described herein conjugated to an agent, the agent (e.g., a cytotoxic agent conjugated to an activatable cytokine construct) is conjugated to the ACC via a linker and / or CM (also referred to as a cleavable sequence). In some embodiments, the agent (e.g., a cytotoxic agent conjugated to an activatable cytokine construct) is conjugated to a cysteine ​​or lysine in the ACC. In some embodiments, the agent (e.g., a cytotoxic agent conjugated to an activatable cytokine construct) is conjugated to another residue of the ACC, such as a residue disclosed herein. In some embodiments, the linker is a thiol-containing linker. In some embodiments, the linker is a non-cleavable linker. Some non-limiting examples of cleavable moieties and / or linkers are shown in Table 1.

[0179] [Table 1]

[0180] Those skilled in the art will appreciate that a wide variety of possible moieties can be linked to the ACC of the present disclosure. (See, e.g., "Conjugate Vaccines," Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference.) In general, effective conjugation of a drug (e.g., a cytotoxic drug) to an ACC can be achieved by any chemical reaction that conjugates the drug to the ACC while also allowing the drug and ACC to retain functionality.

[0181] In some embodiments of any of the ACC conjugated to an agent, the agent can be conjugated to the ACC using a variety of bifunctional protein coupling agents, including, but not limited to, N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCL), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., triene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). In some embodiments, radionucleotides can be conjugated to ACC using carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) chelating agents. (See, e.g., WO94 / 11026).

[0182] Suitable linkers and CMs are described in the literature (see, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also U.S. Pat. No. 5,030,719 which describes the use of halogenated acetylhydrazide derivatives linked to ACC via oligopeptide linkers. In some embodiments, suitable linkers include (i) EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., Cat#21651G); (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6[3-(2-pyridyldithio)-propionamido]hexanoate (Pierce Chem. Co., Cat. #2165-G); and (v) sulfo-NHS (N-hydroxysulfosuccinimide) conjugated to EDC: Pierce Chem. Co., Cat. #2165-G). Chem. Co., Cat. #24510). Additional linkers include, but are not limited to, SMCC, sulfo-SMCC, SPDB, or sulfo-SPDB.

[0183] The above CMs and linkers contain components with different attributes, thus resulting in conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS ester-containing linkers are less soluble than sulfo-NHS esters. In addition, the linker SMPT contains a sterically hindered disulfide bond, which can form highly stable conjugates. Disulfide bonds are generally less stable than other bonds, as disulfide bonds are cleaved in vitro, resulting in less usable conjugates. Sulfo-NHS can particularly increase the stability of carbodiimide coupling. When carboimide coupling (e.g., EDC) is used in conjunction with sulfo-NHS, it forms esters that are more resistant to hydrolysis than the carboimide coupling reaction alone.

[0184] In some embodiments of any of the ACC, agents can be conjugated to the ACC using modified amino acid sequences encompassed by the amino acid sequence of the ACC. Protein constructs can be designed to control the placement and / or dosage of conjugated agents (e.g., cytotoxic agents) by inserting conjugable amino acids at specific positions within the amino acid sequence of the ACC. For example, the ACC can be modified to contain cysteine ​​amino acid residues at positions on the first, second, third and / or fourth monomers that provide reactive thiol groups, do not negatively affect protein folding and / or assembly and do not alter antigen binding properties. In some embodiments, the ACC can be modified to contain one or more non-natural amino acid residues within the amino acid sequence of the ACC to provide suitable sites for conjugation. In some embodiments, the ACC can be modified to contain enzymatically activatable peptide sequences within the amino acid sequence of the ACC.

[0185] nucleic acid Provided herein is a nucleic acid containing a sequence encoding a first monomeric construct (or a protein portion of the first monomeric construct) (e.g., any of the first monomeric constructs described herein) and a second monomeric construct (or a protein portion of the second monomeric construct) (e.g., any of the second monomeric constructs described herein) of any of the ACCs described herein. In some embodiments, a pair of nucleic acids together encodes the first monomeric construct (or the protein portion of the first monomeric construct) and the second monomeric construct (or the protein portion of the second monomeric construct). In some embodiments, the nucleic acid sequence encoding the first monomeric construct (or the protein portion of the first monomeric construct) is at least 70% identical (e.g., at least 72% identical, at least 74% identical, at least 76% identical, at least 78% identical, at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to the nucleic acid sequence encoding the second monomeric construct (or the protein portion of the second monomeric construct).

[0186] In some embodiments, the nucleic acid encoding the protein portion of the first monomeric construct encodes a polypeptide comprising CP1 and CM1 moieties. In some embodiments, the nucleic acid encoding the protein portion of the second monomeric construct encodes a polypeptide comprising CP2 and CM2 moieties. In some embodiments, a pair of nucleic acids together encodes a protein portion of the first monomeric construct and a protein portion of the second monomeric construct, and the protein portions are subsequently conjugated to the DD1 and DD2 moieties, respectively (in a subsequent conjugation step).

[0187] In some embodiments, the nucleic acid encoding the first monomeric construct encodes a polypeptide comprising a DD1 portion. In some embodiments, the nucleic acid encoding the second monomeric construct encodes a polypeptide comprising a DD2 portion.

[0188] vector Provided herein are vectors and vector sets that contain any of the nucleic acids described herein.Those skilled in the art will be able to select a suitable vector or vector set (e.g., expression vector) to generate any of the ACCs described herein, and use the vector or vector set to express any of the ACCs described herein.For example, in selecting a vector or vector set, the cell must be considered, since the vector(s) may need to be integrated into and / or replicate in the chromosome of the cell.Exemplary vectors that can be used to generate ACC are also described below.

[0189] As used herein, the term "vector" refers to a polynucleotide that can induce expression of a recombinant protein (e.g., a first or second monomer) in a cell (e.g., any of the cells described herein). A "vector" can deliver nucleic acids and their fragments into a host cell and contains control sequences (e.g., promoters, enhancers, poly(A) signals). An exogenous polynucleotide may be inserted into an expression vector to be expressed. The term "vector" also encompasses artificial chromosomes, plasmids, retroviruses and baculovirus vectors.

[0190] Methods for making vectors containing any one of the nucleic acids described herein and suitable for transforming a cell (e.g., a mammalian cell) are well known in the art. See, for example, Sambrook et al., Eds. "Molecular Cloning: A Laboratory Manual," 2 ndEd., Cold Spring Harbor Press, 1989 and Ausubel et., Eds. "Current Protocols in Molecular Biology," Current Protocols, 1993.

[0191] Non-limiting examples of vectors include plasmids, transposons, cosmids, and viral vectors (e.g., any adenoviral vector (e.g., pSV or pCMV vectors), adeno-associated viral (AAV) vectors, lentiviral vectors, and retroviral vectors), as well as any Gateway® vectors. A vector can, for example, contain sufficient cis-acting elements for expression, and other elements for expression can be supplied by the host mammalian cell or in an in vitro expression system. One of skill in the art would be able to select suitable vectors and mammalian cells for making any of the ACCs described herein.

[0192] In some embodiments of any of the ACC described herein, the ACC may be made biosynthetically using recombinant DNA techniques and expression in eukaryotic or prokaryotic species.

[0193] In some embodiments, the vector contains a nucleic acid encoding a first monomer and a second monomer of any of the ACCs described herein. In some embodiments, the vector is an expression vector.

[0194] In some embodiments, the pair of vectors contains a pair of nucleic acids that together encode a first monomer and a second monomer of any of the ACCs described herein. In some embodiments, the pair of vectors is a pair of expression vectors.

[0195] cell Also provided herein is a host cell containing any of the vectors or sets of vectors described herein, which contain any of the nucleic acids described herein.

[0196] Any ACC described herein can be produced by any cell (e.g., mammalian cell). In some embodiments, the host cell is a mammalian cell (e.g., human cell), a rodent cell (e.g., mouse cell, rat cell, hamster cell or guinea pig cell), or a non-human primate cell.

[0197] Methods for introducing nucleic acids and vectors (e.g., any of the vectors or sets of vectors described herein) into cells are known in the art. Non-limiting examples of methods that can be used to introduce nucleic acids into cells include lipofection, transfection, calcium phosphate transfection, cationic polymer transfection, viral transfection (e.g., adenoviral transduction, lentiviral transduction), nanoparticle transfection, and electroporation.

[0198] In some embodiments, the introducing step includes introducing into the cell a vector (e.g., any of the vectors or sets of vectors described herein) containing nucleic acids encoding monomers that make up any of the ACCs described herein.

[0199] In some embodiments of any of the methods described herein, the cell can be a eukaryotic cell. As used herein, the term "eukaryotic cell" refers to a cell that has a distinct, membrane-bound nucleus. Such cells can include, for example, mammalian (e.g., rodent, non-human primate, or human), insect, fungal, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell, such as Saccharomyces cerevisiae. In some embodiments, the eukaryotic cell is a higher eukaryotic cell, such as a mammalian, avian, plant, or insect cell. Non-limiting examples of mammalian cells include Chinese hamster ovary (CHO) cells and human embryonic kidney cells (e.g., HEK293 cells).

[0200] In some embodiments, the cell contains a nucleic acid encoding a first monomer and a second monomer of any one of the ACCs described herein. In some embodiments, the cell contains a pair of nucleic acids that together encode a first monomer and a second monomer of any of the ACCs described herein.

[0201] Methods for producing activatable cytokine constructs Provided herein is a method of producing any of the ACC described herein, the method comprising: (a) culturing any of the recombinant host cells described herein in a liquid medium under conditions sufficient to produce the ACC; and (b) recovering the ACC from the host cells and / or the liquid medium.

[0202] Methods for culturing cells are well known in the art. Cells can be maintained in vitro under conditions that favor cell proliferation, cell differentiation, and cell growth. For example, cells can be cultured by contacting cells (e.g., any of the cells described herein) with a cell culture medium that contains the necessary growth factors and supplements sufficient to support cell viability and growth.

[0203] In some embodiments of any of the methods described herein, the method further comprises isolating the recovered ACC. Non-limiting examples of isolation methods include ammonium sulfate precipitation, polyethylene glycol precipitation, size exclusion chromatography, ligand-affinity chromatography, ion exchange chromatography (e.g., anion or cation), and hydrophobic interaction chromatography.

[0204] In some embodiments, the cells are capable of producing a protein portion of a first monomeric construct containing CP1, CM1, PM2 and CM3, and a protein portion of a second monomeric construct containing CP2 and CM2, and optionally PM2 and CM4, which protein portions are subsequently conjugated to the DD1 and DD2 portions, respectively.

[0205] The compositions and methods described herein may involve the use of non-reducing or partially reducing conditions that allow for the formation of disulfide bonds between the dimerization domains, forming and maintaining the dimerization of ACC.

[0206] In some embodiments of any of the methods described herein, the method further comprises preparing the isolated ACC into a pharmaceutical composition.Various formulations are known in the art and are described herein.Any of the isolated ACC described herein can be prepared for any route of administration (e.g., intravenous, intratumoral, subcutaneous, intradermal, oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, or intramuscular).

[0207] Also provided herein is the ACC produced by any of the methods described herein.Also provided is a composition (e.g., pharmaceutical composition) containing any of the ACC produced by any of the methods described herein.Also provided herein is a kit containing at least one dose of any of the compositions (e.g., pharmaceutical compositions) described herein.

[0208] Treatment method Provided herein is a method for treating a disease in a subject (e.g., cancer (e.g., any of the cancers described herein)), comprising administering to the subject a therapeutically effective amount of any of the ACCs described herein.

[0209] As used herein, the term "subject" refers to any mammal. In some embodiments, the subject is a feline (e.g., a cat), a canine (e.g., a dog), an equine (e.g., a horse), a rabbit, a pig, a rodent (e.g., a mouse, a rat, a hamster, or a guinea pig), a non-human primate (e.g., a simian (e.g., a monkey (e.g., a baboon, a marmoset), or an ape (e.g., a chimpanzee, a gorilla, an orangutan, or a gibbon)), or a human. In some embodiments, the subject is a human.

[0210] In some embodiments, the subject has been previously identified or diagnosed as having a disease (e.g., cancer (e.g., any of the cancers described herein)).

[0211] As used herein, the term "treating" includes reducing the severity, frequency, or number of one or more (e.g., 1, 2, 3, 4, or 5) symptoms or signs of a disease (e.g., cancer (e.g., any of the cancers described herein)) in a subject (e.g., any of the subjects described herein). In some embodiments where the disease is cancer, treating results in a reduction in cancer growth, inhibition of cancer progression, inhibition of cancer metastasis, or a reduction in the risk of cancer recurrence in a subject with cancer.

[0212] In some embodiments of any of the methods described herein, the disease is cancer.Also provided herein is a method for treating a subject in need of treatment (e.g., any of the exemplary subjects described herein or known in the art), comprising administering to the subject a therapeutically effective amount of any of the ACCs described herein or any of the compositions described herein (e.g., pharmaceutical compositions).

[0213] In some embodiments of these methods, the subject has been identified or diagnosed with cancer. Non-limiting examples of cancer include solid tumors, hematological tumors, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B cell neoplasms, multiple myeloma, lymphomas (e.g., B cell lymphoma, B cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, malignant cutaneous T cell lymphoma), leukemias (e.g., hairy cell leukemia, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloma, myeloma, leukemia ... The cancers include myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndrome (MDS), Kaposi's sarcoma, retinoblastoma, gastric cancer, urothelial cancer, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, brain cancer, colon cancer, bone cancer, lung cancer, breast cancer, colorectal cancer, ovarian cancer, nasopharyngeal adenocarcinoma, non-small cell lung cancer (NSCLC), squamous cell head and neck cancer, uterine cancer, bladder cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the cancer is lymphoma. In some embodiments, the lymphoma is Burkitt's lymphoma. In some aspects, the subject has been identified or diagnosed as having a familial cancer syndrome, such as Li-Fraumeni syndrome, familial breast and ovarian cancer (BRCA1 or BRCA2 mutation) syndrome, and the like. The disclosed methods are also useful for treating non-solid cancers. Exemplary solid tumors include malignant tumors (e.g., sarcomas, adenocarcinomas, and carcinomas) of various organ systems, such as the lung, breast, lymphatic system, gastrointestinal tract (e.g., colon), genitourinary tract (e.g., renal, urothelial, or testicular tumors), pharynx, prostate, and ovary. Exemplary adenocarcinomas include colorectal carcinoma, renal cell carcinoma, liver carcinoma, non-small cell carcinoma of the lung, and carcinoma of the small intestine.

[0214] Exemplary cancers described by the National Cancer Institute include: adult acute lymphocytic leukemia; childhood acute lymphocytic leukemia; adult acute myeloid leukemia; adrenal cortical carcinoma; childhood adrenocortical carcinoma; AIDS-related lymphoma; AIDS-related malignancies; anal cancer; astrocytoma (childhood, cerebellum); astrocytoma (cerebrum, child); extrahepatic bile duct cancer; bladder cancer; pediatric bladder cancer; bone cancer, osteosarcoma / malignant fibrous histiocytoma; pediatric brain stem glioma; brain tumor (adult); brain tumor, pediatric brain stem glioma; cerebellar astrocytoma (childhood); brain tumor, cerebral astrocytoma / pediatric malignant glioma; brain tumor, pediatric ependymoma; brain tumor, Medulloblastoma (pediatric);Brain tumors, supratentorial primitive neuroectodermal tumor (pediatric);Brain tumors, visual pathway and hypothalamic glioma (pediatric);Brain tumors, (pediatric, other);Breast cancer;Breast cancer and pregnancy;Pediatric breast cancer;Male breast cancer;Pediatric bronchial adenoma / carcinoid;Pediatric carcinoid tumors;Gastrointestinal carcinoid tumors;Adrenal cortical carcinoma;Pancreatic islet cell carcinoma;Cancer of unknown primary;Primary central nervous system lymphoma;Cerebellar astrocytoma (pediatric);Cerebral astrocytoma / malignant glioma (pediatric);Cervical cancer;Pediatric cancer;Chronic lymphocytic leukemia;Chronic myeloid leukemia;Chronic myeloproliferative disorder;Clear cell sarcoma of tendon sheath;Colon cancer;Pediatric colorectal cancer; Malignant cutaneous T-cell lymphoma;Endometrial carcinoma;Ependymoma (childhood);Epithelial ovarian cancer;Esophageal cancer;Childhood esophageal cancer;Ewing's sarcoma family of tumors;Childhood extracranial germ cell tumors;Extragonadal germ cell tumors;Extrahepatic bile duct cancer;Eye cancer, intraocular melanoma;Eye cancer, retinoblastoma;Gallbladder cancer;Gastric (stomach) cancer;Childhood gastric (stomach) cancer;Gastrointestinal carcinoid tumors;Childhood extracranial germ cell tumors;Extragonadal germ cell tumors;Ovarian germ cell tumors;Gestational trophoblastic tumors;Childhood brain stem glioma;Visual pathway and hypothalamic glioma (childhood);Hairy cell leukemia;Head and neck cancer;Adult primary hepatocellular (liver) cancer;Childhood primary hepatocellular (liver) cancer;adult Hodgkin's lymphoma;childhood Hodgkin's lymphoma;Hodgkin's lymphoma during pregnancy;hypopharyngeal cancer;hypothalamic and visual pathway glioma (childhood);intraocular melanoma;islet cell carcinoma (endocrine pancreas);Kaposi's sarcoma;kidney cancer;laryngeal cancer;childhood laryngeal cancer;adult acute lymphoblastic leukemia;childhood acute lymphoblastic leukemia;adult acute myeloid leukemia;childhood acute myeloid leukemia;chronic lymphocytic leukemia;chronic myeloid leukemia;hairy cell leukemia;lip and oral cavity cancer;adult primary liver cancer;childhood primary liver cancer;non-small cell lung cancer;small cell lung cancer;adult acute lymphocytic leukemia;childhood acute lymphocytic leukemia;chronic lymphocytic leukemia;AIDS-related lymphoma;primary central nervous system lymphoma;malignant cutaneous T-cell lymphoma;adult Hodgkin lymphoma;childhood Hodgkin lymphoma;Hodgkin lymphoma during pregnancy;adult non-Hodgkin lymphoma;childhood non-Hodgkin lymphoma;non-Hodgkin lymphoma during pregnancy;primary central nervous system lymphoma;Waldenstrom's macroglobulinemia;male breast cancer;malignant mesothelioma (adult);malignant mesothelioma (childhood);malignant thymoma;medulloblastoma (childhood);melanoma;intraocular melanoma;Merkel cell carcinoma;malignant mesothelioma;metastatic squamous cell carcinoma of the neck of unknown primary;multiple endocrine glands (childhood);multiple Myeloma / plasma cell neoplasms;mycosis fungoides;myelodysplastic syndrome;chronic myeloid leukemia;childhood acute myeloid leukemia;multiple myeloma;myeloproliferative disorders (chronic);nasal cavity and paranasal sinus cancer;nasopharyngeal cancer;pediatric nasopharyngeal cancer;neuroblastoma;adult non-Hodgkin's lymphoma;childhood non-Hodgkin's lymphoma;non-Hodgkin's lymphoma during pregnancy;non-small cell lung cancer;oral cavity cancer (childhood);cancer of the oral cavity and lip;oropharyngeal cancer;osteosarcoma / malignant fibrous histiocytoma of bone;childhood ovarian cancer;epithelial ovarian cancer;ovarian germ cell tumors;low-grade malignant tumors of the ovary;pancreatic cancer;childhood pancreatic cancer;pancreatic cancer, islet cell;sinus and nasal cancer;parathyroid cancer;penile cancer;brown Cell tumors;Pineal and supratentorial primitive neuroectodermal tumors (childhood);Pituitary tumors;Plasma cell neoplasms / multiple myeloma;Pleuropulmonary blastoma;Pregnancy and breast cancer;Pregnancy and Hodgkin's lymphoma;Pregnancy and non-Hodgkin's lymphoma;Primary central nervous system lymphoma;Adult primary liver cancer;Primary liver cancer in children;Prostate cancer;Rectal cancer;Renal cell (kidney) cancer;Pediatric renal cell carcinoma;Renal pelvis and ureter, transitional cell carcinoma;Retinoblastoma;Rhabdomyosarcoma (childhood);Salivary gland cancer;Pediatric salivary gland cancer;Sarcoma, Ewing's sarcoma family of tumors;Kaposi's sarcoma;Sarcoma (osteosarcoma) / malignant fibrous histiocytoma of bone;Sarcoma, childhood rhabdomyosarcoma;Adult soft tissue sarcoma ;Pediatric soft tissue sarcoma;Sézary syndrome;Skin cancer;Skin cancer (pediatric);Skin cancer (melanoma);Skin cancer, Merkel cell;Small cell lung cancer;Small intestine cancer;Adult soft tissue sarcoma;Pediatric soft tissue sarcoma;Metastatic cervical squamous cell carcinoma of unknown primary;Gastric (stomach) cancer;Pediatric gastric (stomach) cancer;Supratentorial primitive neuroectodermal tumor (pediatric);Malignant cutaneous T-cell lymphoma;Testicular cancer;Pediatric thymoma;Malignant thymoma;Thyroid cancer;Pediatric thyroid cancer;Renal pelvis and ureter, transitional cell carcinoma;Gestational trophoblastic tumor;Carcinoma of unknown primary (pediatric);Uncommon cancers of childhood;Ureter and renal pelvis, transitional cell carcinoma;Urethral cancer;Uterine sarcoma;Vaginal cancer;Visual pathway and hypothalamic glioma (pediatric);vulvar cancer; Waldenström's macroglobulinemia; and Wilms' tumor.

[0215] Further exemplary cancers include diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL).

[0216] Metastasis of the above cancers can also be treated or prevented according to the methods described herein.

[0217] In some embodiments, these methods can result in a reduction in the number, severity, or frequency of one or more symptoms of cancer in a subject (e.g., compared to the number, severity, or frequency of one or more symptoms of cancer in a subject before treatment).

[0218] In some embodiments of any of the methods described herein, the method further comprises administering to the subject an additional therapeutic agent (e.g., one or more of the therapeutic agents listed in Table 2).

[0219] [Table 2] TIFF2024537101000004.tif235159TIFF2024537101000005.tif222159

[0220] Compositions / kits Also provided herein are compositions (e.g., pharmaceutical compositions) comprising any of the ACC described herein and one or more (e.g., 1, 2, 3, 4 or 5) pharma- ceutically acceptable carriers (e.g., any of the pharma- ceutically acceptable carriers described herein), diluents or excipients.

[0221] In some embodiments, a composition (eg, a pharmaceutical composition) containing any of the ACC described herein can be placed into a sterile vial or pre-filled syringe.

[0222] In some embodiments, compositions (e.g., pharmaceutical compositions) containing any of the ACC described herein can be prepared for different routes of administration (e.g., intravenous, subcutaneous, intramuscular, intraperitoneal, or intratumoral).

[0223] In some embodiments, any of the pharmaceutical compositions described herein can contain one or more buffering agents (e.g., neutral buffered saline, phosphate buffered saline (PBS), an amino acid (e.g., glycine), one or more carbohydrates (e.g., glucose, mannose, sucrose, dextran, or mannitol), one or more antioxidants, one or more chelating agents (e.g., EDTA or glutathione), one or more preservatives, and / or a pharma- ceutically acceptable carrier (e.g., bacteriostatic water, PBS, or saline).

[0224] As used herein, the term "pharmaceutically acceptable carrier" refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration. Examples of such carriers include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and about 5% human serum albumin.

[0225] In some embodiments of any of the pharmaceutical compositions described herein, any of the ACC described herein is prepared with a carrier that protects against rapid elimination from the body, such as slow-release and controlled-release formulations, such as implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used.Methods for preparing such pharmaceutical compositions and formulations are clear to those skilled in the art.

[0226] Also provided herein is a kit containing any of the ACCs described herein, any of the compositions containing any of the ACCs described herein, or any of the pharmaceutical compositions containing any of the ACCs described herein. Also provided is a kit containing, in addition to the ACCs described herein, one or more second therapeutic agents selected from Table 2. The second therapeutic agent(s) may be provided in a dosage form separate from the ACC. Alternatively, the second therapeutic agent(s) may be prepared together with the ACC. In some embodiments, the kit comprises (1) an ACC comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 129 and SEQ ID NO: 347-356, and (2) a second therapeutic agent selected from Table 2.

[0227] Any of the kits described herein can also include instructions for using any of the compositions (e.g., pharmaceutical compositions) and / or any of the ACCs described herein. In some embodiments, the kits can include instructions for performing any of the methods described herein. In some embodiments, the kits can include at least one dose of any of the compositions (e.g., pharmaceutical compositions) described herein. In some embodiments, the kits can provide a syringe for administering any of the pharmaceutical compositions described herein.

[0228] The present disclosure includes the following non-limiting aspects.

[0229] 1. An activatable cytokine construct (ACC) comprising a first monomeric construct and a second monomeric construct, (a) the first monomeric construct comprises a first mature cytokine protein (CP1), a first cleavable portion (CM1), and a first dimerization domain (DD1), wherein the CM1 is disposed between the CP1 and the DD1; (b) the second monomeric construct comprises a second mature cytokine protein (CP2), a second cleavable portion (CM2), and a second dimerization domain (DD2), wherein the CM2 is located between the CP2 and the DD2; or (a) the first monomeric construct comprises a first mature cytokine protein (CP1) and a first dimerization domain (DD1); (b) the second monomeric construct comprises a second mature cytokine protein (CP2), a cleavable moiety (CM), and a second dimerization domain (DD2), wherein the CM is positioned between the CP2 and the DD2, and wherein the CM functions as a substrate for a protease; or (a) the first monomeric construct comprises a first mature cytokine protein (CP1), a cleavable moiety (CM), and a first dimerization domain (DD1), the CM being disposed between the CP1 and the DD1; (b) the second monomeric construct comprises a second mature cytokine protein (CP2) and a second dimerization domain (DD2), and the CM functions as a substrate for a protease; or (a) the first monomeric construct comprises a first mature cytokine protein (CP1) and a first dimerization domain (DD1); (b) the second monomeric construct comprises a second mature cytokine protein (CP2) and a second dimerization domain (DD2), wherein the CP1, the CP2, or both the CP1 and the CP2 contain an amino acid sequence that functions as a substrate for a protease; moreover, (c) said DD1 and said DD2 bind to each other, thereby forming a dimer of said first monomeric component and said second monomeric component; (d) the ACC is characterized by a reduced level of at least one activity of CP1 and / or CP2 compared to a control level of at least one activity of CP1 and / or CP2; The activatable cytokine construct (ACC).

[0230] 2. The ACC according to aspect 1, wherein the first monomeric construct comprises a first polypeptide comprising the CP1, the CM1 and the DD1.

[0231] 3. The ACC according to any one or combination of aspects 1 or 2, wherein said second monomeric construct comprises a second polypeptide comprising said CP2, said CM2 and said DD2.

[0232] 4. The ACC according to any one or combination of aspects 1 to 3, wherein said DD1 and said DD2 are a pair selected from the group consisting of: a pair of Fc domains; the alpha chain of the human IL-15 receptor (IL15Rα) and the sushi domain from soluble IL-15; barnase and barstar; PKA and AKAP; a mutated RNase I fragment-based adaptor / docking tag module; an epitope and an sdAb; an epitope and an scFv; a SNARE module based on the interaction of the proteins syntaxin, synaptotagmin, synaptobrevin and SNAP25; an antigen-binding domain and an epitope.

[0233] 5. The ACC according to aspect 4, wherein said DD1 and said DD2 are a pair of Fc domains.

[0234] 6. The ACC according to aspect 5, wherein said pair of Fc domains is a pair of human Fc domains.

[0235] 7. The ACC according to embodiment 6, wherein said human Fc domain is a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain or a human IgG4 Fc domain.

[0236] 8. The ACC according to aspect 7, wherein said human Fc domain is an Fc domain of human IgG4.

[0237] 9. The ACC according to embodiment 8, wherein the human Fc domain comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:3, SEQ ID NO:315 or SEQ ID NO:316.

[0238] 10. The ACC according to aspect 9, wherein the human Fc domain comprises a sequence that is at least 90% identical to SEQ ID NO:3, SEQ ID NO:315 or SEQ ID NO:316.

[0239] 11. The ACC according to aspect 10, wherein the human Fc domain comprises SEQ ID NO:3, SEQ ID NO:315 or SEQ ID NO:316.

[0240] 12. The ACC according to any one or combination of aspects 1 to 3 and 5 to 11, wherein said DD1 and said DD2 are identical.

[0241] 13. The ACC according to aspect 4, wherein said DD1 comprises an antigen-binding domain and said DD2 comprises the corresponding epitope.

[0242] 14. The ACC according to aspect 13, wherein the antigen-binding domain is an anti-His tag antigen-binding domain and the DD2 comprises a His tag.

[0243] 15. The ACC according to aspect 13, wherein the antigen-binding domain is a single-chain variable fragment (scFv).

[0244] 16. The ACC according to aspect 13, wherein the antigen-binding domain is a single domain antibody (sdAb).

[0245] 17. The ACC according to aspect 1, wherein at least one of said DD1 and said DD2 comprises a dimerization domain substituent selected from the group consisting of a non-polypeptide polymer and a small molecule.

[0246] 18. The ACC according to aspect 17, wherein said DD1 and said DD2 comprise a non-polypeptide polymer covalently bonded to each other.

[0247] 19. The ACC according to aspect 18, wherein the non-polypeptide polymer is a sulfur-containing polyethylene glycol, and the DD1 and the DD2 are covalently bonded to each other via one or more disulfide bonds.

[0248] 20. The ACC according to aspect 17, wherein at least one of said DD1 and said DD2 comprises a small molecule.

[0249] 21. The ACC according to aspect 20, wherein the small molecule is biotin.

[0250] 22. The ACC according to aspect 20, wherein said DD1 comprises biotin and said DD2 comprises avidin.

[0251] 23. ACC according to any one or combination of aspects 1-22, wherein said CP1 and / or said CP2 are each independently an interleukin.

[0252] 24. The ACC according to any one or combination of aspects 1 to 23, wherein said CP1 and said CP2 are identical.

[0253] 25. The ACC according to any one or combination of aspects 1 to 23, wherein CP1 and CP2 are different.

[0254] 26. ACC according to any one or combination of aspects 1 to 23, wherein said CP1 and / or said CP2 are each independently selected from the group consisting of IL-1α, IL-1β, IL-1RA, IL-18, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-3, IL-5, IL-6, IL-11, IL-12, IL-10, IL-20, IL-21, IL-14, IL-15, IL-16 and IL-17.

[0255] 27. The ACC according to aspect 26, wherein said CP1 and said CP2 are selected from the group consisting of IL-2, IL-10, IL-12, IL-15 and IL-21.

[0256] 28. The ACC according to aspect 26, wherein said CP1 and said CP2 are different interleukins.

[0257] 29. The ACC according to aspect 26, wherein said CP1 and said CP2 are the same interleukin.

[0258] 30. The ACC according to aspect 26, wherein said CP1 or said CP2 is an interleukin.

[0259] 31. ACC according to any one or combination of aspects 26 to 30, wherein said interleukin(s) is human wild-type mature interleukin.

[0260] 32. ACC according to any one or combination of aspects 26 to 31, wherein said interleukin is IL-2, IL-10, IL-12 or IL-15.

[0261] 33. The ACC according to aspect 32, wherein the interleukins are IL-2, IL-12 and IL-15.

[0262] 34. The ACC according to aspect 33, wherein the interleukin is at least one of IL-2 and IL-15.

[0263] 35. The ACC according to aspect 34, wherein the interleukin is IL-15.

[0264] 36. The ACC according to embodiment 35, wherein said CP1 and / or said CP2 comprises a sequence that is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 129, 347 and 348.

[0265] 37. The ACC according to embodiment 36, wherein said CP1 and / or said CP2 comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 129, 347 and 348.

[0266] 38. The ACC according to aspect 37, wherein said CP1 and / or said CP2 comprise the sequence of SEQ ID NO: 347.

[0267] 39. The ACC according to aspect 32, wherein the interleukin is IL-15.

[0268] 40. The ACC according to embodiment 38, wherein the interleukin has a sequence selected from the group consisting of SEQ ID NO: 347 and SEQ ID NO: 348.

[0269] 41. The ACC according to any one of aspects 1 to 40, wherein said CP1 and / or said CP2 comprise an interleukin domain.

[0270] 42. The ACC of aspect 41, wherein said CP1 and said CP2 each comprise an interleukin.

[0271] 43. The ACC according to aspect 42, wherein the interleukin is selected from the group consisting of IL-1α, IL-1β, IL-1RA, IL-18, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-3, IL-5, IL-6, IL-11, IL-12, IL-10, IL-20, IL-14, IL-16 and IL-17.

[0272] 44. The ACC according to any one or combination of aspects 1 to 43, wherein said CM1 and / or said CM2 comprises a total of about 3 amino acids to about 15 amino acids.

[0273] 45. The ACC according to any one or combination of aspects 1-44, wherein the CM1 and the CM2 comprise substrates for different proteases.

[0274] 46. ​​The ACC according to any one or combination of aspects 1 to 44, wherein the CM1 and the CM2 comprise a substrate for the same protease.

[0275] 47. The protease(s) is / are: ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, BACE, renin, cathepsin D, cathepsin E, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, caspase 14, caspase 15, caspase 16, caspase 17, caspase 18, caspase 19, caspase 20, caspase 21, caspase 22, caspase 23, caspase 24, caspase 25, caspase 26, caspase 27, caspase 28, caspase 29, caspase 30, caspase 31, caspase 32, caspase 33, caspase 34, caspase 35, caspase 36, caspase 37, caspase 38, caspase 39, caspase 40, caspase 41, caspase 42, caspase 43, caspase 44, caspase 45, caspase 46, caspase 47, caspase 48, caspase 4 ...1, caspase 42, caspase 43, ca Sarcoplasmic reticulum 14, cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P, cruzipain, legumain, otubain-2, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, meprin, neprilysin, PSMA, BMP-1, MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-19, MMP-20, MMP-23, MMP-24, MMP-26, MMP-27, Activated Protein C, Cathepsin A, Cathepsin G, Chymase, FVIIa, FIXa, FXa, FXIa, FXIIa, Elastase, Granzyme B, Guanidinobenzoatase, HtrA 1. The ACC according to any one or combination of aspects 1 to 46, which is selected from the group consisting of human neutrophil lyase, lactoferrin, marapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, tryptase, uPA, DESC1, DPP-4, FAP, hepsin, matriptase-2, MT-SP1 / matriptase, TMPRSS2, TMPRSS3 and TMPRSS4.

[0276] 48. The ACC according to aspect 47, wherein the protease(s) are selected from the group consisting of uPA, legumain, MT-SP1, ADAM17, BMP-1, TMPRSS3, TMPRSS4, MMP-2, MMP-9, MMP-12, MMP-13, and MMP-14.

[0277] 49. The CM1 and / or the CM2 are LSGRSDNH (SEQ ID NO: 5), TGRGPSWV (SEQ ID NO: 6), PLTGRSGG (SEQ ID NO: 7), TARGPSFK (SEQ ID NO: 8), NTLSGRSENHSG (SEQ ID NO: 9), NTLSGRSGNHGS (SEQ ID NO: 10), TSTSGRSAMPRG (SEQ ID NO: 11), TSGRSANP (SEQ ID NO: 12), VHMPLGFLGP (SEQ ID NO: 13), AVGLLAPP (SEQ ID NO: 14), AQNLLGMV (SEQ ID NO: 15), QNQALRMA (SEQ ID NO: 16), LAAPLGLL (SEQ ID NO: 17), LAAPLGLL (SEQ ID NO: 18), LAAPLGLL (SEQ ID NO: 19), LAAPLGLL (SEQ ID NO: 20), LAAPLGLL (SEQ ID NO: 21), LAAPLGLL (SEQ ID NO: 22), LAAPLGLL (SEQ ID NO: 23), LAAPLGLL (SEQ ID NO: 24), LAAPLGLL (SEQ ID NO: 25), LAAPLGLL (SEQ ID NO: 26), LAAPLGLL (SEQ ID NO: 27), LAAPLGLL (SEQ ID NO: 28), LAAPLGLL (SEQ ID NO: 29), LAAPLGLL (SEQ ID NO: 30), LAAPLGLL (SEQ ID NO: 31), LAAPLGLL (SEQ ID NO: 32), LAAPLGLL (SEQ ID NO: 33), LAAPLGLL (SEQ ID NO: 34), LAAPLGLL (SEQ ID NO: 35), LAAPLGLL (SEQ ID NO: 36), LAAPLGLL (SEQ ID NO: 37), LA 17), STFPFGMF (SEQ ID NO: 18), ISSGLLSS (SEQ ID NO: 19), PAGLWLDP (SEQ ID NO: 20), VAGRSMRP (SEQ ID NO: 21), VVPEGRRS (SEQ ID NO: 22), ILPRSPAF (SEQ ID NO: 23), MVLGRSLL (SEQ ID NO: 24), QGRAITFI (SEQ ID NO: 25), SPRSIMLA (SEQ ID NO: 26), SMLRSMPL (SEQ ID NO: 27), ISSGLLSGRSDNH (SEQ ID NO: 28), AVGLLAPPGGLSGRSDNH (SEQ ID NO: 29), ISSGLLSSGGSGGSLSGRSDNH (SEQ ID NO: Row number 30), LSGRSGNH (SEQ ID NO: 31), SGRSANPRG (SEQ ID NO: 32), LSGRSDDH (SEQ ID NO: 33), LSGRSDIH (SEQ ID NO: 34), LSGRSDQH (SEQ ID NO: 35), LSGRSDTH (SEQ ID NO: 36), LSGRSDYH (SEQ ID NO: 37), LSGRSDNP (SEQ ID NO: 38), LSGRSANP (SEQ ID NO: 39), LSGRSANI (SEQ ID NO: 40), LSGRSDNI (SEQ ID NO: 41), MIAPVAYR (SEQ ID NO: 42), RPSPMWAY (SEQ ID NO: 43), WATPRPMR (SEQ ID NO: 44), F RLLDWQW (SEQ ID NO: 45), ISSGL (SEQ ID NO: 46), ISSGLLS (SEQ ID NO: 47), ISSGLL (SEQ ID NO: 48), ISSGLLSGRSAMPRG (SEQ ID NO: 49), AVGLLAPPTSGRSANPRG (SEQ ID NO: 50), AVGLLAPPSGRSANPRG (SEQ ID NO: 51), ISSGLLSGRSDDH (SEQ ID NO: 52), ISSGLLSGRSDIH (SEQ ID NO: 53), ISSGLLSGRSDQH (SEQ ID NO: 54), ISSGLLSGRSDTH (SEQ ID NO: 55), ISSGLLSGRSDYH (SEQ ID NO: 56),ISSGLLSGRSDNP (SEQ ID NO: 57), ISSGLLSGRSANP (SEQ ID NO: 58), ISSGLLSGRSANI (SEQ ID NO: 59), AVGLLAPPGGLSGRSDDH (SEQ ID NO: 60), AVGLLAPPGGLSGRSDIH (SEQ ID NO: 61), AVGLLAPPGGLSGRSDQH (SEQ ID NO: 62), AVGLLAPPGGLSGRSDTH (SEQ ID NO: 63), AVGLLAPPGGLSGRSDYH (SEQ ID NO: 64), AVGLLAPPGGLSGRSDNP (SEQ ID NO: 65), AVGLL APPGGLSGRSANP (SEQ ID NO: 66), AVGLLAPPGGLSGRSANI (SEQ ID NO: 67), ISSGLLSGRSDNI (SEQ ID NO: 68), AVGLLAPPGGLSGRSDNI (SEQ ID NO: 69), GLSGRSDNHGGAVGLLAPP (SEQ ID NO: 70), GLSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 71), LSGRSDNHGGVHMPLGFLGP (SEQ ID NO: 72), ISSGLSS (SEQ ID NO: 73), PVGYTSSL (SEQ ID NO: 74), DWLYWPGI (SEQ ID NO: 75) , LKAAPRWA (SEQ ID NO: 76), GPSHLVLT (SEQ ID NO: 77), LPGGLSPW (SEQ ID NO: 78), MGLFSEAG (SEQ ID NO: 79), SPLPLRVP (SEQ ID NO: 80), RMHLRSLG (SEQ ID NO: 81), LLAPSHRA (SEQ ID NO: 82), GPRSFGL (SEQ ID NO: 83), GPRSFG (SEQ ID NO: 84), SARGPSRW (SEQ ID NO: 85), GGWHTGRN (SEQ ID NO: 86), HTGRSGAL (SEQ ID NO: 87), AARGPAIH (SEQ ID NO: 88), RGPAFNPM (SEQ ID NO: 89) ), SSRGPAYL (SEQ ID NO: 90), RGPATPIM (SEQ ID NO: 91), RGPA (SEQ ID NO: 92), GGQPSGMWGW (SEQ ID NO: 93), FPRPLGITGL (SEQ ID NO: 94), SPLTGRSG (SEQ ID NO: 95), SAGFSLPA (SEQ ID NO: 96), LAPLGLQRR (SEQ ID NO: 97), SGGPLGVR (SEQ ID NO: 98), PLGL (SEQ ID NO: 99), SGRSDNI (SEQ ID NO: 100), and LSGRSNI (SEQ ID NO: 349).

[0278] 50. The ACC of aspect 47, wherein the CM1 and / or the CM2 comprise a sequence selected from the group consisting of ISSGLLSGRSDNH (sequence number 28), LSGRSDDH (sequence number 33), LSGRSDNI (sequence number 41), ISSGLLSGRSDQH (sequence number 54), SGRSDNI (sequence number 100), ISSGLLSGRSDNI (sequence number 68), and LSGRSNI (sequence number 349).

[0279] 51. The ACC according to any one or combination of aspects 1-50, wherein the protease(s) are produced by a tumor of the subject.

[0280] 52. The ACC of embodiment 51, wherein the subject has been diagnosed or identified as having cancer.

[0281] 53. The ACC according to any one or combination of aspects 1-52, wherein the CP1 and the CM1 are directly adjacent to each other in the first monomeric construct.

[0282] 54. The ACC according to any one or combination of aspects 1-53, wherein the CM1 and the DD1 are directly adjacent to each other in the first monomeric construct.

[0283] 55. The ACC according to any one or combination of aspects 1-54, wherein the CP2 and the CM2 are directly adjacent to each other in the second monomeric construct.

[0284] 56. The ACC according to any one or combination of aspects 1-55, wherein the CM2 and the DD2 are directly adjacent to each other in the second monomeric construct.

[0285] 57. The ACC according to any one or combination of embodiments 1 to 56, wherein the first monomeric construct comprises at least one linker.

[0286] 58. The ACC according to embodiment 57, wherein the at least one linker is a linker L1 disposed between the CP1 and the CM1 and / or a linker L2 disposed between the CM1 and the DD1.

[0287] 59. The ACC according to embodiment 58, wherein the second monomeric construct comprises at least one linker.

[0288] 60. The ACC according to embodiment 59, wherein the at least one linker is a linker L3 disposed between the CP2 and the CM2 and / or a linker L4 disposed between the CM2 and the DD2.

[0289] 61. The ACC according to embodiment 60, wherein the first monomeric construct comprises a linker L1 and the second monomeric construct comprises a linker L3.

[0290] 62. The ACC according to embodiment 61, wherein L1 and L3 are identical.

[0291] 63. The ACC according to embodiment 62, wherein the first monomeric construct comprises a linker L2 and the second monomeric construct comprises a linker L4.

[0292] 64. The ACC according to embodiment 63, wherein L2 and L4 are identical.

[0293] 65. An ACC according to embodiment 64, wherein each linker has a total length of between 1 amino acid and about 15 amino acids.

[0294] 66. The ACC according to embodiment 65, wherein each linker has a total length of at least 5 amino acids.

[0295] 67. The first monomer construct comprises at least one linker, each linker being independently selected from the group consisting of G;GG;GSSGGSGGSGG (SEQ ID NO:210);GGGS (SEQ ID NO:2);GGGSGGGS (SEQ ID NO:211);GGGSGGGSGGGS (SEQ ID NO:212);GGGGSGGGGSGGGGGS (SEQ ID NO:213);GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:214);GGGGSGGGGS (SEQ ID NO:215);GGGGS (SEQ ID NO:216);GS;GGGGSGS (SEQ ID NO:217);GGGGSGGGGSGGGGSGS (SEQ ID NO:218);GGSLDPKGGGGS (SEQ ID NO:219);PKSCDKTHTCPPCPAPELLG (SEQ ID NO:220);SKYGPPCPPCPAPEFLG (SEQ ID NO:221);GKSSGSGSESKS (SEQ ID NO:222);GSTSGSGKSSEGKG (SEQ ID NO:223); 67. The ACC according to any one or combination of aspects 1 to 66, wherein the ACC is selected from the group consisting of: (GS), (GGS), (GSGGS) (SEQ ID NO: 227), (GGGS) (SEQ ID NO: 228), (GGGGS) (SEQ ID NO: 216), where each n is an integer which is at least 1; GGSG (SEQ ID NO: 229); GGSGG (SEQ ID NO: 230); GSGSG (SEQ ID NO: 231; GSGGG (SEQ ID NO: 232); GGGSG (SEQ ID NO: 233); GSSSG (SEQ ID NO: 234); GGGGSGGGGSGGGGS (SEQ ID NO: 213); GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 235); and GSTSGSGKPGSSEGST (SEQ ID NO: 226).

[0296] 68. The ACC according to embodiment 67, wherein the linker comprises a sequence selected from the group consisting of G, GG and GGGS (sequence number 2).

[0297] 69. The ACC according to any one or combination of aspects 1 to 68, wherein the first monomeric construct comprises, in an N-terminal to C-terminal direction, the CP1, the CM1, and the DD1 linked directly or indirectly to the C-terminus of the CM1.

[0298] 70. The ACC according to any one or combination of aspects 1-69, wherein the first polypeptide comprises, in a C-terminal to N-terminal direction, the CP1, the CM1, and the DD1 linked directly or indirectly to the N-terminus of the CM1.

[0299] 71. The ACC according to any one or combination of aspects 1-70, wherein the second polypeptide comprises, in an N-terminal to C-terminal direction, the CP2, the CM2, and the DD2 linked directly or indirectly to the C-terminus of the CM2.

[0300] 72. The ACC according to any one or combination of aspects 1-71, wherein the second polypeptide comprises, in a C-terminal to N-terminal direction, the CP2, the CM2, and the DD2 linked directly or indirectly to the CM2.

[0301] 73. The ACC according to aspect 69, wherein the first monomer construct comprises, from the N-terminus to the C-terminus, the CP1, the CM1 and the DD1, wherein the CP1 and the CM1 are directly adjacent to each other, the CM1 and the DD1 are directly adjacent to each other, the CM1 is a peptide of 10 amino acids or less, the second monomer construct is identical to the first monomer construct, and the first monomer construct and the second monomer construct are covalently linked to each other via at least two disulfide bonds.

[0302] 74. The ACC according to aspect 73, wherein said CP1 is an interleukin.

[0303] 75. The ACC according to aspect 74, wherein said CP1 is IL-15.

[0304] 76. The at least one CP1 and / or CP2 activity is determined by the binding affinity (K D 76. The ACC according to any one or combination of embodiments 1 to 75, wherein

[0305] 77. ACC according to any one or combination of aspects 1 to 75, wherein the at least one CP1 and / or CP2 activity is a level of lymphoma cell proliferation.

[0306] 78. ACC according to any one or combination of aspects 1-75, wherein said at least one CP1 and / or CP2 activity is the activation level of the JAK / STAT / ISGF3 pathway in lymphoma cells.

[0307] 79. The ACC according to any one or combination of aspects 1-75, wherein the at least one activity is the production level of SEAP in HEK cells.

[0308] 80. ACC according to any one or combination of aspects 1 to 79, characterized in that the at least one CP1 and / or CP2 activity is at least 20-fold lower compared to the level of a control.

[0309] 81. ACC according to aspect 80, characterized in that the at least one CP1 and / or CP2 activity is at least 50-fold lower compared to the level of the control.

[0310] 82. ACC according to aspect 81, characterized in that the activity of at least one of CP1 and / or CP2 is at least 100-fold lower compared to the level of the control.

[0311] 83. ACC according to aspect 82, characterized in that the at least one CP1 and / or CP2 activity is at least 500-fold lower compared to the level of the control.

[0312] 84. The ACC according to any one or combination of aspects 1 to 83, wherein the control level of the at least one activity of the CP1 and / or the CP2 is the activity of the CP1 and / or the CP2 of the ACC after exposing the ACC to the protease(s).

[0313] 85. The ACC according to any one or combination of aspects 1 to 83, wherein said control level of said at least one CP1 and / or CP2 is the corresponding CP1 and / or CP2 activity of the corresponding wild-type mature cytokine.

[0314] 86. The ACC according to any one or combination of aspects 1 to 85, wherein the ACC generates a cleavage product after exposure to the protease(s), and the cleavage product comprises the at least one activity of the CP1 and / or the CP2.

[0315] 87. The ACC according to aspect 86, wherein the at least one activity of CP1 and / or CP2 is an antiproliferative activity.

[0316] 88. The ACC according to embodiment 87, wherein the control level is an EC50 value and the ratio of EC50 (cleavage product) to EC50 (control level) is less than about 10, or less than about 9, or less than about 8, or less than about 7, or less than about 6, or less than about 5, or less than about 4, or less than about 3, or less than about 2, or less than about 1.5.

[0317] 89. A composition comprising an ACC according to any one of embodiments 1 to 88 or any combination thereof.

[0318] 90. The composition according to embodiment 89, which is a pharmaceutical composition.

[0319] 91. A container, vial, syringe, injection pen or kit comprising at least one dose of the composition according to embodiment 89 or 90.

[0320] 92. A method for treating a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of ACC according to any one or combination of aspects 1 to 88, or a composition according to aspect 89 or 90.

[0321] 93. The method of embodiment 92, wherein the subject has been identified or diagnosed as having cancer.

[0322] 94. The method of aspect 93, wherein the cancer is lymphoma, solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B-cell neoplasm, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndrome (MDS), malignant cutaneous T-cell lymphoma, retinoblastoma, bladder cancer, gastric cancer, urothelial carcinoma, lung cancer, colon cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, squamous cell head and neck cancer, endometrial cancer, cervical cancer, hepatocellular carcinoma, or hepatocellular carcinoma.

[0323] 95. The method of aspect 94, wherein the lymphoma is Burkitt's lymphoma.

[0324] 96. A nucleic acid encoding a polypeptide comprising said CP1 and said CM1 of an ACC according to any one or combination of embodiments 1 to 88.

[0325] 97. The nucleic acid according to embodiment 96, wherein the polypeptide further comprises a DD1 according to any one or combination of embodiments 1 to 16 or embodiments 23 to 88.

[0326] 98. A nucleic acid encoding a polypeptide comprising said CP2 and said CM2 of an ACC according to any one or combination of embodiments 1 to 88.

[0327] 99. The nucleic acid according to aspect 98, wherein the polypeptide further comprises a DD2 according to any one or combination of aspects 1 to 16 or aspects 23 to 88.

[0328] 100. A vector comprising a nucleic acid according to any one or combination of embodiments 96 to 99.

[0329] 101. The vector according to embodiment 100, which is an expression vector.

[0330] 102. A cell comprising a nucleic acid according to any one or combination of embodiments 96 to 99, or a vector according to embodiment 100 or 101.

[0331] 103. A pair of nucleic acids which together encode a polypeptide comprising the CP1 and CM1 of the first monomeric construct and a polypeptide comprising the CP2 and CM2 of the second monomeric construct according to any one or combination of aspects 1 to 88.

[0332] 104. A pair of vectors, which together comprise a pair of nucleic acids according to embodiment 103.

[0333] 105. A pair of vectors according to embodiment 104, which is a pair of expression vectors.

[0334] 106. A cell comprising a pair of nucleic acids according to embodiment 103, or a pair of vectors according to embodiment 104 or 105.

[0335] 107. A method for making ACC, comprising the steps of: Culturing the cell of embodiment 102 or 106 in liquid culture under conditions sufficient to produce said ACC; recovering the ACC from the cells or the liquid medium; The method comprising:

[0336] 108. The method of embodiment 107, further comprising isolating the ACC recovered from the cells or the liquid medium.

[0337] 109. The method according to aspect 108, further comprising preparing the isolated ACC into a pharmaceutical composition.

[0338] 110. An ACC produced by the method described in embodiment 107.

[0339] 111. A composition comprising the ACC according to embodiment 110.

[0340] 112. The composition according to embodiment 111, which is a pharmaceutical composition.

[0341] 113. A container, vial, syringe, injection pen or kit comprising at least one dose of the composition according to embodiment 111 or 112.

[0342] 114. An activatable cytokine construct (ACC) comprising a first monomeric construct and a second monomeric construct, (a) the first monomeric construct comprises a first mature cytokine protein (CP1), a first cleavable portion (CM1), and a first dimerization domain (DD1); (b) the second monomeric construct comprises a second mature cytokine protein (CP2), a second cleavable portion (CM2), and a second dimerization domain (DD2); (c) the first monomer construct is a polypeptide including, from the N-terminus to the C-terminus, the CP1, the CM1, and the DD1; and (i) the first monomer and the second monomer each comprise a linking region comprising no more than 24 amino acids; (ii) the CP1 is a mature interleukin; (d) Furthermore, (i) the second monomeric construct is identical to the first monomeric construct; (ii) the first monomeric construct and the second monomeric construct are covalently bonded to each other via at least one disulfide bond; (iii) the DD1 and the DD2 are a pair of human IgG Fc domains; (e) the DD1 and the DD2 bind to each other, thereby forming a dimer of the first monomeric component and the second monomeric component; (f) the ACC is characterized by a reduced level of interleukin activity compared to a corresponding control interleukin; The activatable cytokine construct (ACC).

[0343] 115. The ACC according to aspect 114, wherein said CP1 is a mature human interleukin.

[0344] 116. ACC according to any one or combination of aspects 114-115, wherein said mature interleukin is mature IL-15.

[0345] 117. ACC according to any one or combination of embodiments 114-116, wherein said mature interleukin is a truncated form of IL-15.

[0346] 118. The ACC according to any one or combination of embodiments 114 to 116, wherein the mature interleukin comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NO: 129, SEQ ID NO: 347 and SEQ ID NO: 348.

[0347] 119. The ACC according to any one or combination of embodiments 114 to 116, wherein the mature interleukin comprises the sequence of SEQ ID NO: 347.

[0348] 120. The ACC according to any one or combination of aspects 114 to 119, wherein said CP1 and said CM1 are directly adjacent to each other, said CM1 and said DD1 are directly adjacent to each other, and said CM1 and said CM2 each comprise no more than 10 amino acids, optionally no more than 7 amino acids.

[0349] 121. The ACC according to any one or combination of aspects 114-120, wherein the CM1 and the CM2 each independently function as a substrate for urokinase (uPa) and / or matrix metalloproteinase (MMP).

[0350] 122. The ACC according to any one or combination of aspects 114-121, wherein said CM1 and said CM2 each independently function as a substrate for urokinase (uPa) and / or MMP-14.

[0351] 123. The ACC according to any one or combination of aspects 114 to 122, wherein said CM1 and said CM2 each comprise a sequence at least 85% identical to SEQ ID NO: 100.

[0352] 124. The ACC according to any one or combination of aspects 114 to 123, wherein CM1 and CM2 each comprise a sequence selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 68, SEQ ID NO: 100 and LSGRSNI (SEQ ID NO: 349).

[0353] 125. ACC according to any one or combination of aspects 114-124, wherein said DD1 and said DD2 are a pair of human IgG1 Fc domains, or a pair of human IgG4 Fc domains.

[0354] 126. The ACC according to embodiment 125, wherein said DD1 and said DD2 are a pair of human IgG1 Fc domains truncated at the N-terminal side of cysteine ​​226 when numbered according to EU numbering, or a pair of human IgG4 Fc domains truncated at the N-terminal side of cysteine ​​226 when numbered according to EU numbering.

[0355] 127. The ACC according to aspect 125 or 126, wherein said DD1 and said DD2 are a pair of Fc domains of human IgG4 comprising the S228P mutation when numbered according to EU numbering.

[0356] 128. The ACC according to any one or combination of aspects 114 to 127, wherein said DD1 and said DD2 each comprise a sequence at least 95% identical to SEQ ID NO:3.

[0357] 129. The ACC according to any one or combination of aspects 114 to 128, wherein the DD1 and the DD2 each comprise the sequence of SEQ ID NO:3.

[0358] 130. The ACC according to any one or combination of aspects 114-129, wherein the first monomeric construct and the second monomeric construct are covalently bonded to each other via at least two disulfide bonds.

[0359] 131. The ACC according to any one or combination of aspects 114 to 130, wherein the first monomeric construct and the second monomeric construct are covalently bonded to each other via at least three disulfide bonds.

[0360] 132. The ACC according to any one or combination of aspects 114-131, wherein the first monomeric construct and the second monomeric construct are covalently bonded to each other via at least four disulfide bonds.

[0361] 133. The ACC according to any one or combination of embodiments 114-132, wherein the first monomeric construct further comprises a signal sequence immediately adjacent to the N-terminus of the CM1.

[0362] 134. The ACC according to embodiment 133, wherein the signal sequence comprises a sequence that is at least 95% identical to SEQ ID NO: 345.

[0363] 135. The ACC according to embodiment 133, wherein the signal sequence comprises the sequence of SEQ ID NO: 345.

[0364] 136. An ACC according to any one or combination of embodiments 114 to 135, comprising a linking region comprising no more than 18 amino acids, or no more than 12 amino acids.

[0365] 137. The ACC according to embodiment 136, wherein the linking region comprises 7 to 12 amino acids.

[0366] 138. The ACC according to embodiment 136, wherein the linking region comprises 7 amino acids.

[0367] 139. ACC according to any one or combination of embodiments 114 to 138, characterized in that the interleukin activity is at least 500-fold lower compared to a corresponding control interleukin.

[0368] 140. ACC according to any one or combination of aspects 114-139, wherein said CP1 is an interleukin and said control interleukin is a recombinant interleukin.

[0369] 141. The ACC according to any one or combination of aspects 114 to 139, further comprising a peptide mask (PM1) and a cleavable moiety (CM3) located at the N-terminus of said CP1.

[0370] 142. ACC according to any one or combination of aspects 114 to 141, wherein said interleukin activity is anti-proliferative activity in lymphoma cells.

[0371] 143. ACC according to any one or combination of aspects 114 to 141, wherein said interleukin activity is induction of secreted embryonic alkaline phosphatase production in interleukin-responsive HEK293 cells.

[0372] 144. An ACC according to any of aspects 114 to 143, further characterized in that the CM1 generates cleavage products after exposure to the protease which serves as a substrate, wherein the ratio of interleukin activity of the control interleukin to the cleavage products is less than about 2, and the control interleukin is a corresponding recombinant wild-type interleukin.

[0373] 145. The ACC according to aspect 144, wherein the EC50 of the cleavage product is approximately the same as the EC50 of the corresponding recombinant wild-type interleukin.

[0374] 146. The ACC according to embodiment 114, wherein the first monomeric construct and the second monomeric construct each comprise a sequence that is at least 95% identical to a sequence selected from the group consisting of amino acids 21-359 of SEQ ID NO: 350 and SEQ ID NOs: 351-356.

[0375] 147. The ACC according to embodiment 146, further characterized in that the ACC is characterized by at least 200-fold lower interleukin activity compared to wild-type interleukin, the ACC generates cleavage products after exposure to uPA, the cleavage products having at least 50-fold higher interleukin activity than untreated ACC, and the interleukin activity is measured in an anti-proliferation assay in lymphoma cells or in a secreted embryonic alkaline phosphatase production induction assay in interleukin-responsive HEK293 cells.

[0376] 148. The ACC according to aspect 146 or 147, wherein said ACC exhibits lower toxicity in vivo compared to recombinant human IL-15.

[0377] 149. An activatable cytokine construct (ACC) comprising a first monomeric construct and a second monomeric construct, (a) the first monomeric construct comprises a first mature cytokine protein (CP1), a first cleavable portion (CM1), and a first dimerization domain (DD1); (b) the second monomeric construct comprises a second mature cytokine protein (CP2), a second cleavable portion (CM2), and a second dimerization domain (DD2); (c) the first monomer construct is a polypeptide including, from the N-terminus to the C-terminus, the CP1, the CM1, and the DD1; and (i) the ACC comprises a linking region (LR) of 7 to 10 amino acids; (ii) said CP1 comprises a sequence at least 85% identical to SEQ ID NO: 347; (iii) said CM1 comprises a sequence at least 85% identical to SEQ ID NO: 349; (d) Furthermore, (i) the second monomeric construct is identical to the first monomeric construct; (ii) the first monomeric construct and the second monomeric construct are covalently bonded to each other via at least one disulfide bond; (iii) the DD1 and the DD2 are a pair of human IgG Fc domains; (e) the DD1 and the DD2 bind to each other, thereby forming a dimer of the first monomeric component and the second monomeric component; (f) the ACC is characterized in that it has a reduced level of IL-15 activity compared to the IL-15 activity of recombinant human IL-15; The activatable cytokine construct (ACC).

[0378] 150. A composition comprising an ACC according to any one or combination of embodiments 114-149.

[0379] 151. The composition according to embodiment 150, which is a pharmaceutical composition.

[0380] 152. A container, vial, syringe, injection pen or kit comprising at least one dose of the composition according to embodiment 150 or 151.

[0381] 153. A method for treating a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of an ACC according to any one or combination of aspects 114 to 149, or a composition according to aspect 150 or 151.

[0382] 154. The method of embodiment 153, wherein the subject has been identified or diagnosed as having cancer.

[0383] 155. A nucleic acid encoding a polypeptide comprising the first monomer of an ACC according to any one or combination of embodiments 114 to 149.

[0384] 156. A vector comprising a nucleic acid according to embodiment 155.

[0385] 157. The vector according to embodiment 156, which is an expression vector.

[0386] 158. A mammalian cell comprising a nucleic acid according to embodiment 155, or a vector according to embodiment 156 or 157.

[0387] 159. A mammalian cell according to embodiment 158, which is a HEK293 cell or a CHO cell.

[0388] 160. A method for producing ACC, comprising the steps of: a. expressing said ACC in a mammalian cell according to embodiment 158 ​​or 159; b. purifying the expressed ACC; The method comprising:

[0389] 161. ACC according to any one or combination of aspects 114-149, wherein said CM1 serves as a substrate for a protease overexpressed in tumor tissue.

[0390] 162. The ACC according to embodiment 114, wherein the first monomeric construct and the second monomeric construct each comprise a sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:356.

[0391] 163. The ACC according to embodiment 162, wherein the first monomeric construct and the second monomeric construct are identical and each comprise SEQ ID NO: 356.

[0392] 164. A composition comprising an ACC according to embodiment 162 or 163.

[0393] 165. The composition according to embodiment 164, which is a pharmaceutical composition.

[0394] 166. A container, vial, syringe, injection pen or kit comprising at least one dose of the composition according to embodiment 165.

[0395] 167. A method for treating a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of an ACC according to embodiment 162 or a composition according to embodiment 165.

[0396] 168. The method of embodiment 167, wherein the subject has been identified or diagnosed as having cancer.

[0397] 169. A nucleic acid encoding a polypeptide comprising the first monomer of an ACC according to embodiment 162 or 163.

[0398] 170. A vector comprising a nucleic acid according to embodiment 169.

[0399] 171. The vector according to embodiment 170, which is an expression vector.

[0400] 172. A mammalian cell comprising a nucleic acid according to embodiment 169, or a vector according to embodiment 170 or 171.

[0401] 173. A mammalian cell according to embodiment 172, which is a HEK293 cell or a CHO cell.

[0402] 174. A method for producing ACC, comprising the steps of: a) expressing said ACC in a mammalian cell according to embodiment 172 or 173; b) purifying the expressed ACC; and The method comprising: EXAMPLES

[0403] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0404] Example 1: Preparation of activatable cytokine constructs The activatable cytokine construct IFN-α2b-1204DNIdl-hIgG4 was prepared by recombinant methods. The first and second monomer constructs of this ACC were identical and were each a polypeptide having an amino acid sequence according to SEQ ID NO: 309. Each of the first and second monomer constructs comprises, from N-terminus to C-terminus, a signal sequence from mouse IgG kappa signal sequence (residues 1-20 of SEQ ID NO: 309), a mature cytokine protein corresponding to human interferon α-2b (SEQ ID NO: 1), a cleavable portion having the amino acid sequence of SEQ ID NO: 99, a linker having the amino acid sequence GGGS (SEQ ID NO: 2), and DD corresponding to human IgG Fc (SEQ ID NO: 4). The polypeptide was prepared by transforming a host cell with a polynucleotide having the sequence of SEQ ID NO: 310 and then culturing the resulting recombinant host cell. The activatable cytokine construct IFN-α2b 1204DNIdl hIgG4 was obtained by dimerization of the resulting expressed polypeptide.

[0405] The activatable cytokine construct IFN-α-2b 1490DNI-hIgG4 was also prepared by recombinant methods. The first and second monomer constructs of this ACC were also identical, each being a polypeptide having an amino acid sequence according to SEQ ID NO: 311. Each of the first and second monomer constructs of this ACC comprises, from the N-terminus to the C-terminus, a signal sequence from the mouse IgG kappa signal sequence (residues 1-20 of SEQ ID NO: 309), a mature cytokine protein corresponding to human interferon α-2b (SEQ ID NO: 1), a cleavable portion having the amino acid sequence of SEQ ID NO: 68, a linker having the amino acid sequence GGGS (SEQ ID NO: 2), and DD corresponding to human IgG Fc (SEQ ID NO: 4). The polypeptide was prepared by transforming a host cell with a polynucleotide having the sequence of SEQ ID NO: 312, and then culturing the resulting recombinant host cell. The activatable cytokine construct IFN-α-2b 1490DNI-hIgG4 was obtained by dimerization of the resulting expressed polypeptide.

[0406] An additional activatable cytokine construct was prepared which contained an additional five amino acid residues in the linker.

[0407] Electrophoresis was performed on the activatable cytokine construct and the protease-treated activatable cytokine construct. FIG. 15 shows an electrophoretic gel of: (1) ACC IFN-α2b-1204DNIdl-hIgG4 ("1204"); (2) MT-SP1-treated IFN-α2b-1204DNIdl-hIgG4 ("1204MT-SP1"); (3) uPA-treated IFN-α2b-1204DNIdl-hIgG4 ("1204uPA"); (4) IFN-α2b-1204DNIdl-hIgG4 with five additional amino acid residues in the linker ("1204+1"); (5) MT-SP1-treated IFN-α2b-1204DNIdl-hIgG4 ("1204+1 MT-SP1"); (6) uPA-treated IFN-α2b-1204DNIdl-hIgG4 ("1204+1 Shown in the figures are (from left to right) results for (1) IFN-α-2b 1490DNI-hIgG4 ("1490"); (5) IFN-α-2b 1490DNI-hIgG4 ("1490 MT-SP1"); (6) IFN-α-2b 1490DNI-hIgG4 ("1490 MT-SP1"); and (7) IFN-α-2b 1490DNI-hIgG4 ("1490 uPA"). The results suggest that the protease was effective in cleaving the cleavable moiety within the activatable cytokine construct.

[0408] Example 2: IFN-α-2b activity of activatable cytokine constructs The activity of ACC described in Example 1 was tested using a cell-based reporter assay for human type I interferon.

[0409] IFN-responsive HEK293 cells were generated by stable transfection with human STAT2 and IRF9 genes, resulting in a fully active type I IFN signaling pathway. The cells also feature an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFNα / β-inducible ISG54 promoter. To maintain transgene expression, cells were cultured in DMEM GlutaMax medium supplemented with 10% FBS, Pen / Strep, 30 μg / mL blasticidin, 100 μg / ml zeocin, and 100 μg / mL normocin. Addition of type I IFN to these cells activates the JAK / STAT / ISGF3 pathway and subsequently induces SEAP production in the supernatant that can be readily assessed using Quanti-Blue solution, a colorimetric detection of alkaline phosphatase activity. Using this reporter assay, the activity of IFNα-2b-containing ACC was compared to that of Sylatron® (pegylated interferon α-2b). The data in Figure 16 show that the IFNα-2b activity of ACC was significantly reduced compared to that of Sylatron® (pegylated interferon α-2b).

[0410] Furthermore, the data in Figures 7A and 7B show that the activity of (uncleaved) ACC can be modulated by varying the length of the linker or linking region. The data in Figures 7A-7B show the results of IFNa-2b-hIgG4 Fc fusion constructs with various linker lengths or no linker between IFNa-2b and hIgG4 Fc tested in a HEK293 reporter assay. The fusion proteins tested in this experiment contain, from N- to C-terminal, the mature IFNα-2b cytokine sequence, an optional linker and / or cleavable moiety, and the Fc domain of human IgG4 of SEQ ID NO: 4 (including the complete hinge region, such that the N-terminus of the Fc sequence starts with the amino acid sequence ESKYGPPCPPC...). The first construct (linking region = 7) does not have a linker or cleavable moiety and its sequence consists, from N- to C-terminal, of SEQ ID NO: 1 to which it is fused SEQ ID NO: 4. The second construct (linking region=12) has a 5 amino acid linker SGGGG (SEQ ID NO:335) whose sequence consists, from the N-terminus to the C-terminus, of SEQ ID NO:1, to which it is fused SEQ ID NO:335, to which it is fused SEQ ID NO:4. The third construct (linking region=18) contains a 7 amino acid CM (SGRSDNI) and a 4 amino acid linker GGGS whose sequence consists, from the N-terminus to the C-terminus, of SEQ ID NO:1, to which it is fused SEQ ID NO:100, to which it is fused SEQ ID NO:2, to which it is fused SEQ ID NO:4. The fourth construct (linking region=23) contains a 5 amino acid linker, a 7 amino acid CM and a 4 amino acid linker whose sequence consists, from the N-terminus to the C-terminus, of SEQ ID NO:1, to which it is fused SEQ ID NO:335, to which it is fused SEQ ID NO:100, to which it is fused SEQ ID NO:2, to which it is fused SEQ ID NO:4. The fifth construct (linking region=24) contains a 13 amino acid CM (ISSGLLSGRSDNI) and a 4 amino acid linker, and its sequence consists of, from the N-terminus to the C-terminus, SEQ ID NO:1, to which it is fused SEQ ID NO:68, to which it is fused SEQ ID NO:2, to which it is fused SEQ ID NO:4.

[0411] Example 3: Activity of protease-treated ACC Protease-treated IFNα-2b-containing ACC was tested for antiproliferative responses in Daudi lymphocyte cells and in cell-based reporter assays to determine whether activity could be restored.

[0412] To cleave the dimerization domain, IFNα-2b-containing ACC was treated overnight at 37° C. with recombinant human proteases such as urokinase-type plasminogen activator (uPA) or matriptase (MT-SP1). Protease inhibitor cocktails were added to neutralize the proteases before testing for activity as described in Examples 2 and 3. Results from these assays indicate that treating IFNα-2b-containing ACC with proteases can restore activity to levels comparable to the recombinant cytokine. EC50 values ​​for ACC IFNα-2b-1204DNIdl-hIgG4, ACC IFNα-2b-1204DNIdl-hIgG4+uPA, and stem cell IFNα-2b (human recombinant IFN-α2b, available from StemCell Technologies, catalog #78077.1) were calculated from the results of the Daudi apoptosis assay and are provided in Table 3 below. [Table 3]

[0413] The EC50 values ​​for ACC IFNα-2b-1204DNIdl-hIgG4, ACC IFNα-2b-1204DNIdl-hIgG4+uPA, and stem cell IFNα-2b were calculated from the results of the IFNα / β assay and are provided in Table 4 below. [Table 4]

[0414] These results indicate that in the absence of activating proteases, the activity of IFNα-2b-1204DNIdl-hIgG4 is significantly less than the IFNα-2b control.

[0415] Example 4: In vivo tolerability of ACC activity Human IFNα-2b has previously been shown to cross-react with hamster IFNα receptors and to be active in hamsters (Altrock et al, Journal of Interferon Research, 1986). To assess the tolerability of IFNα-2b-containing ACC ProC440, golden Syrian hamsters were dosed at a starting dose of 0.4 mg / kg. Animals received one dose of test substance and continued on the study for up to 7 days after dosing unless unacceptable toxicity (DLT stands for dose-limiting toxicity) was identified. The starting dose (0.4 mg / kg ("mpk")) represents the equivalent dose at which an IFNα control (recombinant interferon α, a non-naturally occurring type I interferon manufactured by Amgen under the name Infergen®) would be expected to induce weight loss, reduced food consumption, and myelosuppression in hamsters (125 g). (In cynomolgus monkeys (cynos), 0.1 mg / kg / day IFNα control was associated with weight loss, decreased food consumption, and myelosuppression (equivalent to 1.25-2.5×10^7U in a 125 g hamster). If the starting dose was tolerated, the animal was transferred to a "medium dose" of 2 mg / kg and given three doses of the test substance as tolerated. If tolerated, the animal was transferred to a "high dose" of 10 mg / kg and given three doses of the test substance as tolerated. If tolerated, the animal was transferred to a "higher dose" of 15 mg / kg. If the test dose was not tolerated at each step, the animal was transferred to the next lower dose. If the starting dose was not tolerated, the animal was transferred to a "lower dose" of 0.08 mg / kg. The animals were dosed with ACC (ProC286) which has the structure DD-CM-CP dimer from the N-terminus to the C-terminus. As a negative control, animals were administered human IgG4, which, as expected, did not induce any toxicity in the animals.

[0416] ProC286 (ChIgG4 5AA 1204DNIdL IFNa2b) was also prepared by recombinant methods. The first and second monomer constructs were identical, and each was a polypeptide having the amino acid sequence of SEQ ID NO: 320 and a signal sequence at its N-terminus. Each of the first and second monomer constructs contained, from N-terminus to C-terminus, a signal sequence, a dimerization domain corresponding to human IgG Fc (SEQ ID NO: 3), a linker (SEQ ID NO: 321), a cleavable portion having the amino acid sequence of SEQ ID NO: 100, a linker (SEQ ID NO: 2), and a mature cytokine protein corresponding to human interferon alpha-2b (SEQ ID NO: 1).

[0417] ProC291 (NhIgG4 5AA 1204DNIdL IFNa2b) was also prepared by recombinant methods. The first and second monomer constructs were identical. Each of the first and second monomer constructs contains, from N-terminus to C-terminus, a mature cytokine protein corresponding to human interferon alpha-2b (SEQ ID NO: 1), a linker (SEQ ID NO: 321), CM (SEQ ID NO: 100), a linker (GGGS) (SEQ ID NO: 2), and the Fc region of human IgG4 (SEQ ID NO: 4) containing a complete hinge sequence.

[0418] The activity of ProC286 and ProC291 was compared to that of Sylatron® (PEG-IFN-α2b) in the Daudi apoptosis assay (FIGS. 17A-B). In this assay, ProC286 and Sylatron® showed similar levels of activity, as shown in FIG. 17A. This indicates that ProC286 has similar activity to commercially available pegylated IFN-α2b and can be used as a surrogate for the Sylatron® control to evaluate the tolerability of IFNα-2b in hamster studies. ProC291 showed reduced activity compared to ProC286 and Sylatron®, indicating that the structural orientation of IFN on the N-terminal side of the Fc was important for the reduced activity. That is, when the DD is a pair of Fc domains, placing the cytokine N-terminal to the DD (as in ProC291) may provide a greater reduction in cytokine activity than placing the cytokine C-terminal to the DD (as in ProC286).

[0419] On day 1, animals were administered a starting dose of 0.4 mg / kg. The study continued on animals for one week unless a non-tolerated dose (DLT) was reached. Clinical observations, weights and temperatures were measured for each animal before dosing and at 6, 24, 72 and 7 hours after dosing. Blood samples for hematology and chemistry analyses were collected for each animal at 72 hours and 7 days after dosing. Hematology and chemistry analyses were performed immediately after sampling. Hematology analyses included evaluation of blood smears, differential white blood cell count, hematocrit, hemoglobin, mean corpuscular hemoglobin, mean corpuscular volume, platelet count, red blood cell count, red blood cell distribution width, reticulocyte count and white blood cell count. The clinical chemistry panel included measurements of alanine aminotransferase, albumin, albumin / globulin ratio, alkaline phosphatase, aspartate aminotransferase, calcium, chloride, cholesterol, creatine kinase, creatine, gamma-glutamyltransferase, globulin, glucose, inorganic phosphorus, potassium, sodium, total bilirubin, total protein, triglycerides, urea, nitrogen, and C-reactive protein. Evidence of toxicity in the tolerability studies is summarized in Figures 18-20.

[0420] Overall, animals dosed with the unmasked ProC286 construct showed an average of 5% weight loss when dosed at 2mpk, and 15% weight loss when dosed at 10mpk and 15mpk (Figure 18). One animal dosed with ProC286 at 15mpk showed a 20% weight loss 7 days after dosing (end of study), which is considered an unacceptable dose. In contrast, animals dosed with ProC440 at 2mpk and 10mpk showed no weight loss.

[0421] Animals dosed with ProC440 at 15mpk showed an average weight loss of 5% (Figure 18). This indicates that the disclosed ACC with the dimerization structure of CP-CM-DD, starting from the N-terminus, unexpectedly limits the weight loss mediated by IFNα-2b. Without wishing to be bound by theory, it is believed that placing the interferon at the N-terminus of the DD and using a relatively short LR inhibits cytokine activity with respect to ProC440 and reduces the toxicity of the interferon compared to pegylated IFNα-2b (Sylatron®) or ProC286.

[0422] Regarding clinical chemistry, animals dosed with ProC286 showed a significant increase in alkaline phosphatase (ALP) at all doses (0.4mpk, 2mpk, 10mpk and 15mpk) 7 days after administration (end of study) (Figure 19). No significant increase in ALP was measured when animals were dosed with 10mpk or 15mpk ProC440 (Figure 19). Elevation of ALT is a marker of liver toxicity. IFNα-2b has been shown to cause liver toxicity. This indicates that the ACC of the present disclosure, which has a dimerization structure of CP-CM-DD starting from the N-terminus, unexpectedly limits liver toxicity mediated by IFNa-2b.

[0423] With regard to hematology, after 3 days and 7 days (end of study), animals dosed with ProC286 at 2mpk, 10mpk and 15mpk showed significant reductions in reticulocyte, neutrophil and white blood cell (WBC) counts (Figure 20). These reductions are reminiscent of IFNa-2b mediated bone marrow toxicity. After 3 days, animals dosed with ProC440 showed reductions in reticulocyte, neutrophil and white blood cell (WBC) counts (Figure 20). Overall, the level of reduction in hematopoietic cells observed in animals dosed with ProC440 was less significant than that observed in animals dosed with ProC286. After 7 days of administration (end of study), the overall levels of reticulocytes, neutrophils and white blood cells (WBCs) in animals dosed with ProC440 return to normal levels or levels similar to those observed in animals dosed with negative control IgG4 (Figure 20). In animals dosed with ProC286, the levels of reticulocytes, neutrophils and white blood cells (WBCs) remain low. This indicates that the ACC of the present disclosure, which has a dimerization structure of CP-CM-DD starting from the N-terminus, unexpectedly limits IFNa-2b-mediated bone marrow toxicity.

[0424] Example 5: In vitro characterization of additional IFNa-2b cytokine constructs Further activatable cytokine constructs, including IFNa-2b, were also prepared by recombinant methods. The first and second monomer constructs of these ACCs were identical. Each of the first and second monomer constructs includes, from N-terminus to C-terminus, a signal sequence from mouse IgG kappa signal sequence (residues 1-20 of SEQ ID NO: 309), a mature cytokine protein corresponding to human interferon alpha-2b (SEQ ID NO: 1), a cleavable portion (CM) having the amino acid sequence of SEQ ID NO: 100, and a dimerization domain corresponding to human IgG4 S228P Fc (including SEQ ID NO: 3). Furthermore, these ACCs either contain or do not contain a linker between the CP and the CM, which has the amino acid sequence SGGGG (SEQ ID NO: 335). These ACCs either contain or do not contain a linker between the CM and the DD, which has the amino acid sequence GGGS (SEQ ID NO: 2). These ACCs also either contain or do not contain a portion of the hinge of the DD, which is N-terminal to cysteine ​​226. These additional activatable cytokine constructs are set forth in Table 6 (see SEQ ID NOs:336-342 and SEQ ID NO:313). [Table 5]

[0425] The activity of ProC440, an ACC with no flexible linker and an Fc region truncated to Cys226, and the activity of additional ACCs containing various linker and Fc region sequences were tested in vitro using IFN-responsive HEK293 cells and Daudi cells as described above. In both assays, the activity (e.g., antiproliferative effect) of ProC440 was reduced compared to all other ACCs containing various additional sequences between the cytokine and the first amino acid (i.e., Cys226) that links the DD to the corresponding second monomer. The EC50 values ​​of the ACCs were calculated from the results of the IFNα / β assay and are provided in Table 7 below. [Table 6]

[0426] The EC50 values ​​for ACC were calculated from the results of the Daudi apoptosis assay and are provided in Table 8 below. [Table 7]

[0427] The data in Tables 7-8 also show that the activity of (uncleaved) ACC can be modulated by varying the length of the amino acid sequence between the cytokine and Cys226 of the DD.

[0428] Without wishing to be bound by theory, based on the results presented herein, the inventors hypothesize that placing the cytokine N-terminal to the DD and using a relatively short LR will inhibit cytokine activity of cytokines in addition to the interferon-α cytokine exemplified in the specific examples above.

[0429] Example 6: In vitro characterization of exemplary IL-15 cytokine constructs A human IL-15-containing activatable cytokine construct (ProC1471) was produced by recombinant methods. The first and second monomer constructs of ProC1471 were identical, each a polypeptide having the amino acid sequence of SEQ ID NO: 350 and a signal sequence at its N-terminus. Each of the first and second monomer constructs contains, from N-terminus to C-terminus, a signal sequence from a mouse IgG kappa signal sequence (residues 1-20 of SEQ ID NO: 309), a mature cytokine protein corresponding to human IL-15 amino acid residues 49-161 (SEQ ID NO: 347), a cleavable portion having the amino acid sequence of SEQ ID NO: 100, and a dimerization domain corresponding to the Fc of human IgG4 (SEQ ID NO: 3) truncated at Cys226 (according to EU numbering) and containing the mutation S228P (Figure 3). The complete monomer construct sequence of ProC1471, including the signal sequence, is shown in SEQ ID NO: 350. The linking region (LR) of this monomer construct is 7 amino acids in length.

[0430] The polypeptide was prepared by transforming a host cell with a polynucleotide having the sequence of SEQ ID NO: 357, and then culturing the resulting recombinant host cell. The cytokine construct ProC1471 was produced by dimerization of the resulting expressed polypeptide.

[0431] The activity of ProC1471 was tested in vitro using IL-2 / IL-15-responsive HEK293 cells (see Figures 4 and 6). IL-2 / IL15-responsive HEK293 cells were generated by stable transfection with human CD25 (IL-2Rα), CD122 (IL-2Rβ) and CD132 (IL-2Rγ) genes along with human JAK3 and STAT5 genes, resulting in a fully functional IL-2 / IL-15 signaling pathway. The cells were also characterized with a STAT5-inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene. To maintain transgene expression, cells were cultured in DMEM GlutaMax medium supplemented with 10% FBS, Pen / Strep, 10ug / ml puromycin and 100μg / mL Normocin. Addition of IL-2 and IL-15 to these cells activates STAT5 and subsequently induces SEAP production in the supernatant that can be readily assessed using QUANTI-Blue solution, a colorimetric detection of alkaline phosphatase activity.

[0432] IL-2 / IL-15-responsive HEK293 cells were prepared in DMEM medium supplemented with 10% FBS at a concentration of 280,000 cells / mL and 180 μL aliquots were pipetted into wells (50,000 cells / well) of white flat-bottom 96-well plates. Cytokines to be tested were diluted in DMEM medium supplemented with 10% FBS. Three-fold serial dilutions of duplicates were prepared from which 20 μL was added to each well. After 20-24 hours of incubation at 37°C, 20 μl of induced reporter cell supernatant was transferred to wells of a flat-bottom 96-well plate. 180 μl of resuspended QUANTI-Blue solution was added per well. After 1-3 hours of incubation of the plates in a 37°C incubator, SEAP levels were measured using a spectrophotometer at 620 nm. Dose-response curves were generated and EC50 values ​​obtained by nonlinear regression with sigmoidal fit using Graph Pad Prism software.

[0433] In reporter assays, the activity of ProC1471 was reduced by at least 250-fold compared to PeproTech IL-15 (recombinant human IL-15, available from PeproTech, catalog #200-15) (Figure 4), indicating that the fusion of a cleavable dimerization domain corresponding to the Fc of human IgG provided stereochemical masking for IL-15 in the ACC construct.

[0434] Example 7: Activity of protease-treated IL-15-containing ACC Protease-treated IL-15-containing ACC was tested in a reporter assay to determine whether interleukin activity could be restored. To cleave the dimerization domain, IL-15-containing ACC was treated with recombinant human proteases, such as urokinase-type plasminogen activator (uPA) or matriptase (MT-SP1), at 37°C overnight. Cleavage at the expected site in the cleavable moiety with uPa was confirmed by electrophoresis (Figure 5). The results suggest that uPa protease can cleave the cleavable moiety (CM) of ProC1471. Protease activation with uPa partially restored the activity of ProC1471 to a level close to, but lower than, that of recombinant IL-15 (Figure 6). The EC50 values ​​of ProC1471, ProC1471+uPA and PeproTech IL-15 were calculated from the results of the IL-15 reporter assay and are provided in Table 9 below. Thus, activation of ACC with uPa protease resulted in approximately 64-fold higher IL-15 activity than untreated ACC. The ratio of EC50 (cleavage product) of ProC1471 when activated by uPa to EC50 (wild-type control level) was approximately 6 (9.046 / 1.48=6.11), indicating good recovery of IL-15 activity after protease activation. [Table 8]

[0435] Example 8: Design of additional IL-15 cytokine constructs Further activatable cytokine constructs, ProC1874, ProC1875, ProC1876, ProC1877, ProC1878 and ProC1879, were also prepared by recombinant methods. The first and second monomer constructs of these ACCs were identical. Each of the first and second monomer constructs contains, from N-terminus to C-terminus, a signal sequence from mouse IgG kappa signal sequence (residues 1-20 of SEQ ID NO: 309), a mature cytokine protein corresponding to human IL-15 amino acid residues 49-162 (SEQ ID NO: 348), a cleavable moiety (CM), and a dimerization domain corresponding to the Fc of human IgG4 (SEQ ID NO: 3) truncated at Cys226 (according to EU numbering) and containing the mutation S228P. Furthermore, these ACCs contain or do not contain a linker between the cytokine and the CM having the amino acid sequence shown in Table 10 below. These additional activatable cytokine constructs are described in Table 10, and the complete amino acid sequences of these constructs are provided in Table 14 (see SEQ ID NOs:351-356). [Table 9]

[0436] Example 9: Further characterization of IL-15-containing ACC IL-15-containing ACC, ProC1471, ProC1876 and ProC1879, were treated with recombinant uPA overnight at 37°C. Cleavage at the expected site in the cleavable moiety with uPa was confirmed by electrophoresis (Figure 21A). HEK293 reporter assays characterize the activity of untreated and protease-treated IL-15-containing ACC (Figure 21B). Table 11 shows the average EC50 values ​​of IL-15-containing ACC from multiple experiments (n>3). Activation of ACC by uPa protease resulted in approximately 49- to 104-fold higher IL-15 activity than untreated ACC. [Table 10]

[0437] Example 10: Activity of IL-15-containing ACC on human PBMC proliferation In the cell proliferation assay, human PBMCs were incubated with recombinant IL-15 or IL-15-ACC (with or without prior protease activation) for 3 days. After incubation, PBMCs were stained with fixable viability dye eFlur™ 780, anti-CD3-FITC (UCHTI), anti-CD4-BV608 (RPA-T4), anti-CD8-BV480 (RPA-T8), anti-CD56-BV421 (HCD56), and anti-Ki67-APC (Ki67). As shown in FIG. 22, various cell populations including CD3-, CD56+ NK cells, CD3+, CD8+ T cells, and CD3+, CD4+ T cells were analyzed to determine the proliferation of various cell populations based on the percentage of Ki67 expression. Protease-treated IL-15-ACC shows higher proliferation activity than the corresponding untreated IL-15-containing ACC. Table 12 shows the EC50 of various IL-15-containing ACC in the PBMC proliferation assay. [Table 11]

[0438] Example 11: Activity of IL-15-containing ACC on human PBMC STAT5 phosphorylation IL-15 binding to IL-15R drives STAT5 phosphorylation and subsequent proliferation of NK and T cells. For the STAT5 phosphorylation assay, human PBMCs were first stained with anti-CD3-FITC (UCHTI), anti-CD4-BV608 (RPA-T4), anti-CD8-BV480 (RPA-T8), and anti-CD56-BV421 (HCD56) antibodies at room temperature for 30 min. After surface staining, cells were stimulated with various IL-15 test substances in RPMI medium containing 10% FBS at 37°C for 20 min. Cells were immediately fixed with pre-warmed fixative at 37°C for 10-12 min, washed, and incubated with pre-chilled (-20°C) 90% methanol at 4°C for 30 min. After fixation and permeabilization, cells were washed again and stained with anti-pSTAT5-Alexa647 (pY687). Different cell populations, including CD3-, CD56+ NK cells, CD3+, CD8+ T cells, and CD3+, CD4+ T cells, were analyzed, and STAT5 phosphorylation in different cell populations was determined as the percentage of pSTAT5 positive cells (Figure 23). The EC50 of STAT5 phosphorylation of IL15-ACC on different cell populations is summarized in Table 13. [Table 12]

[0439] [Table 13] TIFF2024537101000017.tif234159TIFF2024537101000018.tif237159TIFF2024537101000019.tif236159TIFF2024537101000020.tif232159TIFF2024537101000021.tif236159TIFF2024537101000022.tif237159TIFF2024537101000023.tif237159TIFF2024537101000024.tif235159TIFF2024537101000025.tif239159TIFF2024537101000026.tif236159TIFF2024537101000027.tif235159TIFF2024537101000028.tif236159TIFF2024537101000029.tif236159TIFF2024537101000030.tif236159TIFF2024537101000031.tif236159TIFF2024537101000032.tif235159TIFF2024537101000033.tif232159TIFF2024537101000034.tif240159TIFF2024537101000035.tif239159TIFF2024537101000036.tif240159TIFF2024537101000037.tif238159TIFF2024537101000038.tif236159TIFF2024537101000039.tif235159TIFF2024537101000040.tif236159TIFF2024537101000041.tif239159TIFF2024537101000042.tif229159TIFF2024537101000043.tif182159TIFF2024537101000044.tif219159TIFF2024537101000045.tif231159TIFF2024537101000046.tif237159TIFF2024537101000047.tif235159TIFF2024537101000048.tif57159As mentioned above, the invention described herein encompasses activatable cytokine constructs comprising various cytokine proteins discussed herein. As non-limiting examples, the CP used in the ACC of the present invention may be any of those listed in SEQ ID NOs: 111-140, 143-146, 151-160, and 347-348, and variants thereof. In particular, monomeric cytokines are suitable for use in the ACC described herein. Based on the results provided herein, it is believed that the ACC of the present invention will exhibit reduced cytokine activity compared to the corresponding wild-type cytokine, and that upon cleavage of the ACC by the relevant protease(s), the cleavage products will regain cytokine activity similar to that of the corresponding wild-type cytokine.

[0440] Other embodiments While the invention has been described in connection with the above detailed description, it is to be understood that the foregoing description is intended to be illustrative and not limiting of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 1. An activatable cytokine construct (ACC) comprising a first monomeric construct and a second monomeric construct, (a) the first monomer construct comprises a first interleukin polypeptide, a first cleavable portion (CM1), and a first dimerization domain (DD1); (b) the second monomer construct comprises a second interleukin polypeptide, a second cleavable portion (CM2), and a second dimerization domain (DD2); (c) the first monomer construct is a polypeptide comprising, from the N-terminus to the C-terminus, the interleukin polypeptide, the CM1, and the DD1; and (i) the first monomer construct and the second monomer construct each comprise a linking region comprising 18 or fewer amino acids; (ii) the interleukin polypeptide is IL-15; (d) further comprising: (i) the second monomeric component is the same as the first monomeric component; (ii) the DD1 and the DD2 are a pair of human IgG Fc domains; (e) the DD1 and the DD2 are covalently bonded to each other via at least one disulfide bond, thereby forming a dimer of the first monomeric construct and the second monomeric construct; (f) the ACC is characterized in that the level of IL-15 activity is reduced compared to recombinant human IL-15, as measured by the level of secreted embryonic alkaline phosphatase (SEAP) production in IL-2 / IL-15-responsive HEK293 cells; The activatable cytokine construct (ACC). (a) the interleukin polypeptide comprises a sequence at least 95% identical to SEQ ID NO:

347. (b) the CM1 and the CM2 each contain 8 or fewer amino acids; (c) each of the CM1 and the CM2 is independently cleavable by urokinase (uPa) and / or matrix metalloproteinase (MMP); (d) the CM1 and the CM2 each comprise a sequence at least 85% identical to SEQ ID NO: 349; (e) the CM1 and the CM2 each comprise a sequence selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 68, SEQ ID NO: 100, and SEQ ID NO: 349; (f) the DD1 and the DD2 are a pair of human IgG4 Fc domains, and optionally the human IgG4 Fc domain comprises a S228P mutation when numbered according to EU numbering; (g) the DD1 and the DD2 are a pair of human IgG1 or IgG4 Fc domains truncated at the N-terminal side of cysteine ​​226 when numbered according to EU numbering; (h) said DD1 and said DD2 each comprise a sequence at least 95% identical to SEQ ID NO:3; (i) the DD1 and the DD2 each comprise the sequence of SEQ ID NO: 3; (j) the first monomeric construct and the second monomeric construct are covalently bonded to each other via at least two disulfide bonds; (k) the first monomeric construct and the second monomeric construct are covalently bonded to each other via at least three disulfide bonds; (l) the first monomeric construct and the second monomeric construct are covalently bonded to each other via at least four disulfide bonds; (m) the first monomeric construct and the second monomeric construct each comprise a polypeptide sequence at least 95% identical to amino acids 21-359 of SEQ ID NO: 350; or (n) the first monomeric construct and the second monomeric construct each comprise a polypeptide sequence selected from the group consisting of SEQ ID NOs: 350-356.

3. 1. An activatable cytokine construct (ACC) comprising a first monomeric construct and a second monomeric construct, (a) the first monomer construct comprises a first interleukin polypeptide, a first cleavable portion (CM1), and a first dimerization domain (DD1); (b) the second monomer construct comprises a second interleukin polypeptide, a second cleavable portion (CM2), and a second dimerization domain (DD2); (c) the first monomer construct is a polypeptide comprising, from the N-terminus to the C-terminus, the interleukin polypeptide, the CM1, and the DD1; and (i) the interleukin polypeptide and the CM1 are directly adjacent to each other; (ii) the CM1 and the DD1 are directly adjacent to each other; (iii) the interleukin polypeptide comprises a sequence at least 85% identical to SEQ ID NO: 347; or (iv) the CM1 comprises a sequence at least 85% identical to SEQ ID NO: 349; (d) further comprising: (i) the second monomeric component is the same as the first monomeric component; (ii) the DD1 and the DD2 are a pair of human IgG1 or IgG4 Fc domains; (e) the DD1 and the DD2 are covalently bonded to each other via at least one disulfide bond, thereby forming a dimer of the first monomeric construct and the second monomeric construct; (f) the ACC is characterized by a reduced level of IL-15 activity compared to the activity of recombinant human IL-15; The activatable cytokine construct (ACC).

4. An activatable cytokine construct (ACC) comprising a first monomeric construct and a second monomeric construct, (a) the first monomer construct comprises a first interleukin polypeptide, a first cleavable portion (CM1), and a first dimerization domain (DD1); said CM1 being positioned between said interleukin polypeptide and said DD1; (b) the second monomer construct comprises a second interleukin polypeptide, a second cleavable portion (CM2), and a second dimerization domain (DD2); the CM2 is positioned between the second interleukin polypeptide and the DD2; or (a) the first monomer construct comprises a first interleukin polypeptide and a first dimerization domain (DD1); (b) the second monomer construct comprises a second interleukin polypeptide, a cleavable moiety (CM), and a second dimerization domain (DD2), wherein the CM is positioned between the second interleukin polypeptide and the DD2, and the CM functions as a substrate for a protease; or (a) the first monomer construct comprises a first interleukin polypeptide, a cleavable moiety (CM), and a first dimerization domain (DD1), wherein the CM is disposed between the interleukin polypeptide and the DD1; (b) the second monomer construct comprises a second interleukin polypeptide and a second dimerization domain (DD2); the CM serves as a substrate for a protease; or (a) the first monomer construct comprises a first interleukin polypeptide and a first dimerization domain (DD1); (b) the second monomer construct comprises a second interleukin polypeptide and a second dimerization domain (DD2), and the first interleukin polypeptide, the second interleukin polypeptide, or both the first interleukin polypeptide and the second interleukin polypeptide contain an amino acid sequence that functions as a substrate for a protease; the DD1 and the DD2 bind to each other, thereby forming a dimer of the first monomeric component and the second monomeric component; and The ACC is characterized by a reduced level of interleukin activity compared to a control level of interleukin activity. The activatable cytokine construct (ACC).

5. (a) said DD1 and said DD2 are a pair of Fc domains, and optionally (i) the pair of Fc domains is a pair of human Fc domains; (ii) the human Fc domain is a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, or a human IgG4 Fc domain; (iii) the human Fc domain is a human IgG4 Fc domain; and (iv) the human Fc domain comprises a sequence at least 90% identical to SEQ ID NO:3, SEQ ID NO:315, or SEQ ID NO:316, or the human Fc domain comprises SEQ ID NO:3, SEQ ID NO:315, or SEQ ID NO:316; (b) the first interleukin polypeptide and / or the second interleukin polypeptide comprises a sequence at least 90% identical to SEQ ID NO: 347; (c) the first monomeric construct and the second monomeric construct have the structures, from N-terminal to C-terminal, of first interleukin polypeptide-CM1-DD1 and second interleukin polypeptide-CM2-DD1, respectively; or (d) the first interleukin polypeptide and / or the second interleukin polypeptide comprises the sequence of SEQ ID NO:

347.

6. 6. The ACC according to any one of claims 1 to 5, characterized in that the level of IL-15 activity is reduced by about 100 to about 500 fold compared to recombinant human IL-15, as measured by the level of SEAP (secreted embryonic alkaline phosphatase) production in IL-2 / IL-15-responsive HEK293 cells.

7. ACC according to any one of claims 1 to 5, characterized in that the level of IL-15 activity is reduced by at least 200-fold compared to recombinant human IL-15.

8. 6. The ACC according to any one of claims 1 to 5, characterized in that the level of IL-15 activity is reduced by about 250-fold compared to recombinant human IL-15.

9. having an EC50 that is about 1 to about 10-fold that of wild-type recombinant IL-15, as measured in IL-2 / IL15-responsive HEK293 cells, following cleavage of said ACC by uPA protease; or 6. The ACC according to any one of claims 1 to 5, characterized in that it has an EC50 that is about 3 to about 7 times the EC50 of wild-type recombinant IL-15, as measured in IL-2 / IL15-responsive HEK293 cells, after cleavage of the ACC by uPA protease.

10. 6. A polynucleotide encoding a polypeptide comprising said CP1 and said CM1 of the ACC according to any one of claims 1 to 5, wherein optionally said polypeptide further comprises a DD1 having a sequence at least 95% identical to SEQ ID NO:

3.

11. A vector comprising the polynucleotide of claim 10, optionally wherein the vector is an expression vector.

12. A pair of nucleic acids which together encode a polypeptide comprising CP1 and CM1 of the first monomer structure and a polypeptide comprising CP2 and CM2 of the second monomer structure described in any one of claims 1 to 5. (a) the polynucleotide of claim 10; (b) the vector of claim 11, or (c) a pair of nucleic acids according to claim 12 Optionally, said host cell is a mammalian cell.

14. 1. A method of making an ACC, comprising: Culturing the host cell of claim 13 in a liquid medium under conditions sufficient to produce the ACC; recovering the ACC from the cells or the liquid medium; The method comprising:

15. The method of claim 1, further comprising: (a) isolating the ACC recovered from the cells or the liquid medium; and, optionally, 15. The method of claim 14, further comprising (b) preparing the isolated ACC into a pharmaceutical composition.

16. A composition comprising the ACC according to any one of claims 1 to 5, which is optionally a pharmaceutical composition.

17. 17. A container, vial, syringe, injection pen or kit comprising at least one dose of the composition of claim 16.

18. A composition for use in a method for treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the ACC or the composition, the subject has been identified or diagnosed as having cancer, and optionally 17. The composition of claim 16, wherein the cancer is leukemia, lymphoma, or a solid tumor.

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