Activatable cytokine constructs, related compositions, and methods

Abstract

We provide activatable cytokine constructs (ACCs). [Solution] (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 positioned between CP1 and DD1; and (b) An ACC comprising 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 positioned between CP2 and DD2, wherein CM1 and CM2 function as substrates for a protease, DD1 and DD2 bind to each other, and the ACC is characterized by a decrease in the activity of at least one of CP1 and / or CP2 compared to a control level of the activity of at least one of CP1 and / or CP2.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 008,542 filed April 10, 2020, U.S. Provisional Patent Application No. 63 / 161,889 filed March 16, 2021, and U.S. Provisional Patent Application No. 63 / 164,849 filed March 23, 2021. The entire contents of these provisional applications are incorporated herein by reference.

[0002] This application includes a sequence listing submitted electronically. The sequence listing was created on April 7, 2021, under the name "CYTX-071-PCT_ST25.txt" and is 379,000 bytes in size. The information in the electronic data of the sequence listing is part of this application and is incorporated herein by reference in its entirety.

[0003] Field of Invention This disclosure relates to the field of biotechnology, and more specifically to activatable cytokine constructs. [Background technology]

[0004] Cytokines are a family of small, naturally occurring proteins and glycoproteins produced and secreted by most nucleated cells in response to viral infection and / or other antigenic stimuli. Interferons are a subclass of cytokines. Interferons are currently classified into three main classes: interferon type I, interferon type II, and interferon type III. Interferons exert their cellular activity by binding to specific membrane receptors on the cell surface.

[0005] Interferon therapy has many clinical benefits. For example, interferon is known to upregulate the immune system and possess antiviral and antiproliferative properties. These biological properties have led to the clinical use of interferon as a therapeutic agent for the treatment of viral infections and malignancies. Furthermore, interferon is useful in mobilizing the patient's innate immune system to identify and attack cancer cells. Therefore, interferon therapy has been widely used in cancer and antiviral therapy, including for the treatment of hepatitis, Kaposi's sarcoma, pilocytic cell leukemia, chronic myeloid leukemia (CML), follicular lymphoma, renal cell carcinoma (RCC), melanoma, and other medical conditions. However, systemic administration of interferon comes with dose-dependent toxicities, particularly severe flu-like symptoms, neurological symptoms, hepatotoxicity, myelosuppression, and arrhythmias. In a trial of melanoma patients, the combination of pembrolizumab and pegylated IFNα resulted in an ORR of 60.5%. This combination therapy was also associated with 49% of G3 / G4 adverse events requiring a reduction in the dose of pegylated IFNα (Davar et al., J. Clin. Oncol., 2018). These undesirable side effects limit the dose of interferon therapy and sometimes lead to interruption or delay of interferon treatment.

[0006] Interleukins are another subclass of cytokines. Interleukins regulate cell proliferation, differentiation, and motility. They are particularly important in stimulating immune responses such as inflammation. Interleukins have been used to treat cancer, autoimmune diseases, and other disorders. For example, interleukin-2 (IL-2) is indicated for the treatment of melanoma, graft-versus-host disease (GVHD), neuroblastoma, and renal cell carcinoma (RCC), and is also considered useful for conditions including acute coronary syndrome, acute myelolytic syndrome, atopic dermatitis, autoimmune liver disease, basal cell carcinoma, bladder cancer, breast cancer, candidiasis, colorectal cancer, cutaneous T-cell lymphoma, endometrioma, HIV infection, ischemic heart disease, rheumatoid arthritis, nasopharyngeal adenocarcinoma, non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic cancer, systemic lupus erythematosus, tuberculosis, and other disorders. In particular, other interleukins such as IL-6, IL-7, IL-12, and IL-21 are potential treatments for cancer and other disorders. Interleukin therapy is often accompanied by undesirable side effects, including flu-like symptoms, nausea, vomiting, diarrhea, hypotension, and arrhythmias.

[0007] Therefore, the need for and desire for cytokine therapy with improved specificity and selectivity to desired targets is of great interest. Enhanced targeting of cytokine therapeutics to disease sites would reduce toxicity based on systemic mechanisms and lead to broader therapeutic benefits. [Overview of the Initiative]

[0008] This disclosure provides an activatable cytokine construct (ACC) comprising (a) a first monomer comprising a first mature cytokine protein (CP1), a first cleavable moiety (CM1), and a first dimerization domain (DD1), wherein CM1 is positioned between CP1 and DD1; and (b) a second monomer comprising a second mature cytokine protein (CP2), a second cleavable moiety (CM2), and a second dimerization domain (DD2), wherein CM2 is positioned between CP2 and DD2, wherein CM1 and CM2 function as substrates for a protease, DD1 and DD2 bind to each other, and the ACC is characterized by a decrease in the activity of at least one of CP1 and / or CP2 compared to a control level of the activity of at least one of CP1 and / or CP2. The proteases that cleave CM1 and CM2 may be overexpressed in affected tissue (e.g., tumor tissue) compared to healthy tissue. ACC is activated upon cleavage of CM1 and / or CM2, thereby allowing cytokines to exert their activity in affected tissue (e.g., in the tumor microenvironment), while cytokine activity is attenuated in healthy tissue. Therefore, the ACC provided herein may result in reduced toxicity compared to conventional cytokine therapies, enable higher effective doses of cytokines, and / or increase the therapeutic concentration range of cytokines.

[0009] Provided herein are activatable cytokine constructs (ACCs) 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 moiety (CM1), and a first dimerization domain (DD1), wherein CM1 is positioned between CP1 and DD1; and (b) the second monomer construct comprises a second mature cytokine protein (CP2), a second cleavable moiety (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 the first monomer construct and the second monomer construct, the ACC having at least one reduced level of CP1 and / or CP2 activity compared to at least one control level of CP1 and / or CP2 activity.

[0010] This 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, and the CM functions as a substrate for a protease, and the DD1 and DD2 bind to each other, and the ACC is characterized by a decrease in the activity of at least one of CP1 and / or CP2 compared to a control level of the activity of at least one of CP1 and / or CP2.

[0011] This 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 positioned 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 the DD1 and DD2 bind to each other, and the ACC is characterized by a decrease in the activity of at least one of CP1 and / or CP2 compared to a control level of the activity of at least one of CP1 and / or CP2.

[0012] This 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 comprises an amino acid sequence that functions as a substrate for a protease; DD1 and DD2 bind to each other, and the ACC is characterized by a decrease in the activity of at least one of CP1 and / or CP2 compared to a control level of the activity of at least one of CP1 and / or CP2.

[0013] The ACC of this disclosure is characterized in that CP1 and CP2 are not connected to a peptide mask, for example, an affinity masking portion.

[0014] 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 pairs selected from the group consisting of a pair of Fc domains; a sucrose domain and soluble IL-15 derived from the alpha chain of the human IL-15 receptor (IL15Rα); barnase and barnster; protein kinase A (PKA) and A-kinase anchor protein (AKAP); an adapter / docking tag module based on a mutant RNase I fragment; an epitope and a single-domain antibody (sdAb); an epitope and a single-chain variable fragment (scFv); and a soluble N-ethylmaleimide-sensitive factor adherent protein receptor (SNARE) module based on the interaction of protein syntaxin, synaptotagmin, synaptobrevinn, and SNAP25, an antigen-binding domain and an epitope.

[0015] In some embodiments, DD1 and DD2 are a pair of Fc domains. In some embodiments, the pair of Fc domains are 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 contains a sequence that is at least 80% identical to SEQ ID NO: 3. In some embodiments, the human Fc domain contains 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 contain SEQ ID NO: 3. In some embodiments, DD1 and DD2 are the same. In some embodiments, DD1 and DD2 are a pair of identical human IgG4 Fc domains. In some embodiments, the dimerized domains have the amino acid sequences of SEQ ID NOs: 315 and 316, respectively. In some embodiments, the human Fc domain includes mutations to eliminate glycosylation and / or reduce Fc-gamma receptor binding. In some embodiments, the human Fc domain includes mutations N297Q, N297A, or N297G; in some embodiments, the human Fc domain includes mutations at positions 234 and / or 235, e.g., 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 including mutations V234A, G237A, P238S, H268Q / A, V309L, A330S, or P331S, or combinations thereof (all according to EU numbering).

[0016] Examples of the addition of modified human Fc domains are known to those skilled in the art. Examples of Ig heavy chain constant region amino acids in which a mutation in at least one amino acid results in a decrease in Fc function include, but are not limited to, mutations in heavy chain constant region amino acids 228, 233, 234, 235, 236, 237, 239, 252, 254, 256, 265, 270, 297, 318, 320, 322, 327, 329, 330, and 331 (according to EU numbering). Examples of combinations of mutant amino acids are also known in the art and include, but are not limited to, combinations of mutations in amino acids 234, 235, and 331, e.g., L234F, L235E, and P331S or combinations of amino acid mutations in 318, 320, and 322, e.g., E318A, K320A, and K322A.

[0017] Further examples of modified Fc domains include F243L / R292P / Y300L / V305I / P396 IgG1;S239D / I332E IgG1;S239D / I332E / A330L IgG1;S298A / E333A / K334A; in one heavy chain, L234Y / L235Q / G236W / S239M / H268D / D270E / S298A IgG1, and in opposing heavy chains, D270E / K326D, A330M / K334E IgG;G236A / S239D / I332E IgG1;K326W / E333S IgG1;S267E / H268F / S324T Examples 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, and IgG1. In some embodiments, the modified Fc domain includes one or more substitutions selected from the group consisting of N297A IgG1, N297Q IgG1, and S228P IgG4.

[0018] 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-tagged antigen-binding domain and DD2 comprises a His tag. In some embodiments, the antigen-binding domain is a single-chain variable 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 dimerized domain substituent selected from the group consisting of non-polypeptide polymers and small molecules. In some embodiments, DD1 and DD2 comprise a non-polypeptide polymer covalently bonded to each other. In some embodiments, the non-polypeptide polymer is sulfur-containing polyethylene glycol and DD1 and DD2 are covalently bonded 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.

[0019] In some embodiments, CP1 and CP2 are mature cytokines. In some embodiments, each of CP1 and CP2 comprises a mature cytokine sequence and further comprises a signal peptide (also referred to herein as the “signal sequence”). In some embodiments, CP1 and / or CP2 are individually 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. 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 this disclosure includes any two selections and combinations of the cytokine proteins described herein. In some embodiments, CP1 and / or CP2 are interferons. In some embodiments, both CP1 and CP2 are interferons. In some embodiments, CP1 and CP2 are different interferons. In some embodiments, CP1 and CP2 are the same interferon. In some embodiments, either CP1 or CP2 is an interferon. In some embodiments, one of CP1 or CP2 is an interferon, and the other of CP1 or CP2 is a cytokine other than an interferon. In some embodiments, one or both of the cytokines are monomeric cytokines. In some embodiments, one or both of the interferons are monomeric interferons. In some embodiments, either CP1 or CP2 is a monomeric interferon, and the other of CP1 or CP2 is a different cytokine. In some embodiments, CP1 and / or CP2 include a mutant cytokine sequence.In some embodiments, CP1 and / or CP2 comprise a universal cytokine sequence. In some embodiments, CP1 and / or CP2 comprise a truncated sequence that retains cytokine activity.

[0020] In some embodiments, the interferon is human wild-type mature interferon. In some embodiments, the interferon may be type I and type II interferons, and for example, but not limited to, interferon α, interferon β, interferon ω, interferon γ, and interferon τ. In some embodiments, the interferon is interferon α. ​​In some embodiments, the interferon is selected from the group consisting of interferon α2a, interferon α2b, and interferon α-n3. In some embodiments, the interferon is interferon α2b. In some embodiments, the interferon is a mutant interferon. In some embodiments, the interferon is a mutant interferon in which the endogenous protease cleavage site is rendered dysfunctional by substitution, deletion, or insertion of one or more amino acids. In some embodiments, the interferon is a universal cytokine molecule having, for example, a hybrid sequence of different cytokine subtypes, a chimeric cytokine sequence, or a humanized cytokine sequence. In some embodiments, the interferon is a universal interferon molecule. In some embodiments, the interferon is a universal interferon α, for example, a hybrid of interferon α1 and interferon α2b. In some embodiments, CP1 and / or CP2 include a sequence that is at least 80% identical to SEQ ID NO: 1. In some embodiments, CP1 and / or CP2 include a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, CP1 and / or CP2 include the sequence of SEQ ID NO: 1. In some embodiments, the interferon is interferon β. In some embodiments, interferon β is selected from the group consisting of interferon β-1a and interferon β-1b. In some embodiments, CP1 and / or CP2 include an IFab domain. In some embodiments, CP1 and / or CP2 include an interleukin.In some applications, interleukins are 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.

[0021] In some embodiments, CM1 and / or CM2 contain a total of approximately 3 to 15 amino acids. In some embodiments, CM1 and CM2 contain substrates for different proteases. In some embodiments, CM1 and CM2 are the same length and contain the same amino acid sequence. In some embodiments, CM1 and CM2 contain substrates for the same protease. In some embodiments, the protease is selected from the group consisting of: ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, BACE, renin, cathepsin D, cathepsin E, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, ca Spase 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, regmine, otsubine-2, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, meprin, neprilysin, PSMA, BMP-1, matrix meprin Taloproteases (e.g., 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, guanidinobenzoate, HtrA1, human neutrophil lyase, lactoferrin, malapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, tryptase, uPA, DESC1, DPP-4, FAP, hepsin, matryptase-2, MT-SP1 / matryptase, TMPRSS2, TMPRSS3, and TMPRSS4.In some embodiments, the protease 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.

[0022] Suitable cleavable moieties are disclosed in International Publication Nos. WO 2010 / 081173, WO 2015 / 048329, WO 2015 / 116933, WO 2016 / 118629, and WO 2020 / 118109. The contents of these patents are hereby incorporated by reference in their entirety.

[0023] In some embodiments, CM1 and / or CM2 include sequences 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), QNQAL RMA (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), ISSGLLSGRSDNH (SEQ ID NO: 28), AVGLLAPPGGLSGRSDNH (SEQ ID NO: 29), ISSGLLSSGGSGGSLSGRSDNH (Sequence ID 30), LSGRSGNH (Sequence ID 31), SGRSANPRG (Sequence ID 32), LSGRSDDH (Sequence ID 33), LSGRSDIH (Sequence ID 34), LSGRSDQH (Sequence ID 35), LSGRSDTH (Sequence ID 36), LSGRSDYH (Sequence ID 37), LSGRSDNP (Sequence ID 38), LSGRSANP (Sequence ID 39), LSGRSANI (Sequence ID 40), LSGRSDNI (Sequence ID 41), MIAPVAYR (Sequence ID 42), RPSPMWAY (Sequence ID number 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), ISSGLLSGRSANPRG (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), AVGLL APPGGLSGRSDNP (SEQ ID NO: 65), AVGLLAPPGGLSGRSANP (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: 74), Column number 87), AARGPAIH (sequence number 88), RGPAFNPM (sequence number 89), SSRGPAYL (sequence number 90), RGPATPIM (sequence number 91), RGPA (sequence number 92), GGQPSGMWGW (sequence number 93), FPRPLGITGL (sequence number 94), SPLTGRSG (sequence number 95), SAGFSLPA (sequence number 96), LAPLGLQRR (sequence number 97), SGGPLGVR (sequence number 98), PLGL (sequence number 99), and SGRSDNI (sequence number 100). In some embodiments, CM is ISSGLLSGRSDNH (sequence number 28), LSGRSDDH (sequence number 33), ISSGLLSGRSDQH (sequence number 54),The sequence comprises a sequence selected from the group consisting of SGRSDNI (SEQ ID NO: 100) and ISSGLLSGRSDNI (SEQ ID NO: 68). In some embodiments, the protease is produced by the tumor of interest, for example, in greater quantities in the tumor than in the healthy tissue of the subject. In some embodiments, the subject is diagnosed or identified as having cancer.

[0024] In some embodiments, CP1 and CM1 are directly adjacent to each other in the first monomer construct. In some embodiments, CM1 and DD1 are directly adjacent to each other in the first monomer construct. In some embodiments, CP2 and CM2 are directly adjacent to each other in the second monomer construct. In some embodiments, CM2 and DD2 are directly adjacent to each other in the second monomer construct. In some embodiments, the first monomer construct includes CP1 directly adjacent to CM1 and CM1 directly adjacent to DD1, where CM1 includes a sequence selected from the group consisting of SEQ ID NOs. 5 to 100. In some embodiments, the second monomer construct includes CP2 directly adjacent to CM2 and CM2 directly adjacent to DD2, where CM2 includes a sequence selected from the group consisting of SEQ ID NOs. 5 to 100. In some embodiments, the first monomer construct includes CP1 directly adjacent to CM1 and CM1 directly adjacent to DD1, where CM1 includes a sequence with a length of 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 amino acids or less. In some embodiments, the second monomer construct includes a CP2 directly adjacent to CM2 and a CM2 directly adjacent to DD2, where CM2 includes a sequence with a length of 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 amino acids or less. In some embodiments, the first and second monomer constructs are configured such that the cytokines (CM1 and CM2, respectively) are directly adjacent to cleavable regions (CM1 and CM2, respectively) with a length of 10, 9, 8, 7, 6, 5, or 4 amino acids or less, and are directly adjacent to dimerization domains (DD1 and DD2, respectively) where the cleavable region is the Fc region of human IgG, and the N-terminus of the Fc region is a first cysteine ​​residue in a hinge region read from N to C (e.g., cysteine ​​226 of human IgG1 using EU numbering). In some embodiments, the dimerization domain is the IgG Fc region, and the upper hinge residue is removed. For example, Fc is a variant in which the N-terminal sequence EPKSCDKTHT (sequence number 516), ERK, ELKTPLGDTTHT (sequence number 517), or ESKYGPP (sequence number 518) has been removed.

[0025] In some embodiments, the first monomer construct includes at least one linker. In some embodiments, the at least one linker is linker L1 located between CP1 and CM1 and / or linker L2 located between CM1 and DD1. In some embodiments, the second monomer construct includes at least one linker. In some embodiments, the at least one linker is linker L3 located between CP2 and CM2 and / or linker L4 located between CM2 and DD2. In some embodiments, the first monomer construct includes linker L1, and the second monomer construct includes linker L3. In some embodiments, L1 and L3 are the same. In some embodiments, the first monomer construct includes linker L2, and the second monomer construct includes linker L4. In some embodiments, L2 and L4 are the same. In some embodiments, each linker has a total length of 1 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” refers to a peptide whose amino acid sequence is not that of a protease substrate.

[0026] In some embodiments, the first monomer construct includes at least one linker, each linker being GSSGGSGGSGG(SEQ ID NO: 210);GGGS(SEQ ID NO: 2);GGGSGGGS(SEQ ID NO: 211);GGGSGGGSGGGS(SEQ ID NO: 212);GGGGSGGGGSGGGGS(SEQ ID NO: 213);GGGGSGGGGSGGGGSGGGGSGGGGS(SEQ ID NO: 214);GGGGSGGGGS(SEQ ID NO: 215);GGGGGS(SEQ ID NO: 216);GS;GGGGGSGS(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);G STSGSGKSSEGSGSTKG(sequence code 224); GTSTGSGKPGSGEGSTKG(sequence code 225); GTSTGSGKPGSSEGST(sequence code 226); (GS)n, (GGS)n, (GSGGS)n(sequence code 227), (GGGS)n(sequence code 228), (GGGGS)n(sequence code 216), where n is an integer of at least 1; GGSG(sequence code 229); GGSGG(sequence code 230); GGSSG(sequence code 231; GSGGG(sequence code 232); GGGSG(sequence code 233); GSSSG(sequence code 234); GGGGSGGGGSGGGGS(sequence code 213); GGGGSGGGGSGGGGSGGGGS(sequence code 214); and GTSTGSGKPGSSEGST(sequence code 226) are independently selected from the group. In some embodiments, the linker includes an array of GGGS(sequence code 2).

[0027] As used herein, the term “spacer” refers to an amino acid residue or peptide incorporated into the free end of a mature ACC, for example, between the signal peptide and the N-terminus of the mature ACC. In some embodiments, the spacer (or “header”) may include a glutamine (Q) residue. In some embodiments, the residue in the spacer minimizes aminopeptidase and / or exopeptidase activity to prevent cleavage of the N-terminal amino acid. Exemplary non-limiting spacer amino acid sequences include, or consist of, any of the following exemplary amino acid sequences: QGQSGS (SEQ ID NO: 504); GQSGS (SEQ ID NO: 505); QSGS ( SEQ ID NO: 506); SGS; GS; S; QGQSGQG (SEQ ID NO: 507); GQSGQG (SEQ ID NO: 508); QSGQG (SEQ ID NO: 509); SGQG (SEQ ID NO: 510); GQG; QG; G; QGQSGQ (SEQ ID NO: 511); GQSGQ (SEQ ID NO: 512); QSGQ (SEQ ID NO: 513); QGQSG (SEQ ID NO: 514); QGQS (SEQ ID NO: 515); SGQ; GQ; and Q. In some embodiments, the spacer array may be removed.

[0028] In some embodiments, the first monomer construct includes CP1, CM1, and DD1, which are directly or indirectly linked to the C-terminus of CM1 from N to C-terminus. In some embodiments, the first polypeptide includes CP1, CM1, and DD1, which are directly or indirectly linked to the N-terminus of CM1 from C to N-terminus. In some embodiments, the second polypeptide includes CP2, CM2, and DD2, which are directly or indirectly linked to the C-terminus of CM2 from N to C-terminus. In some embodiments, the second polypeptide includes CP2, CM2, and DD2, which are directly or indirectly linked to CP2, CM2, and CM2 from C to N-terminus.

[0029] In some embodiments, the first monomer construct comprises CP1, an optional linker, CM1, an optional linker, and DD1, in the N-to-C direction, where DD1 is the Fc region of IgG, the N-terminus of the Fc region being a first cysteine ​​residue in the hinge region read in the N-to-C direction (e.g., cysteine ​​226 of human IgG1 or IgG4 according to EU numbering), and CM1 and the optional linker, positioned between the N-terminal cysteines of CP1 and DD1, have a combined total 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, and particularly preferably 7 amino acids or less. In some embodiments, the second monomer construct comprises CP2, an optional linker, CM2, an optional linker, and DD2, in the direction from N to C-terminus, where DD2 is the Fc region of IgG, the N-terminus of the Fc region being a first cysteine ​​residue in the hinge region read in the direction from N to C (e.g., cysteine ​​226 of human IgG1 or IgG4 according to EU numbering), and CM2 and the optional linker, positioned between the N-terminal cysteines of CP2 and DD2, have a combined total 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, and particularly preferably 7 amino acids or less.

[0030] In some embodiments, ACC is a homodimer in which the first monomer construct and the second monomer construct are identical and contain the amino acid sequence of SEQ ID NO: 313. In some embodiments, the first monomer construct and the second monomer construct each contain an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 313. In some embodiments, the first monomer construct and the second monomer construct each contain, from N to the C-terminus, a CM containing an amino acid sequence selected from the group consisting of SEQ ID NO: 1; SEQ ID NO: 41; SEQ ID NO: 68; and SEQ ID NO: 100; and a dimerized domain.

[0031] In some embodiments, the activity of at least one CP1 and / or CP2 is measured by surface plasmon resonance (K) to determine the binding affinity (K) of CP1 and / or CP2 to their homologous receptors. D ) For example, if CP1 or CP2 is an interferon, the congeneral receptor may be an interferon α / β receptor (IFNAR). In some embodiments, at least one CP1 and / or CP2 activity is the level of lymphoma cell proliferation. In some embodiments, at least one CP1 and / or CP2 activity is the level of JAK / STAT / ISGF3 pathway activation in lymphoma cells. In some embodiments, at least one activity is the level of secreted alkaline phosphatase (SEAP) production in lymphoma cells. In some embodiments, ACC (before exposure to protease) is characterized by at least a 2-fold reduction of at least one CP1 and / or CP2 activity compared to a control level. In some embodiments, ACC is characterized by at least a 5-fold reduction of at least one CP1 and / or CP2 activity compared to a control level. In some embodiments, ACC is characterized by at least a 10-fold reduction of at least one CP1 and / or CP2 activity compared to a control level. In some embodiments, the ACC is characterized by a reduction of at least 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 times the activity of at least one CP1 and / or CP2 compared to a control level. In some embodiments, the control level of the activity of at least one CP1 and / or CP2 is the activity of CP1 and / or CP2 in the ACC after exposure of the ACC to a protease. In some embodiments, the control level of the activity of at least one CP1 and / or CP2 is the corresponding CP1 and / or CP2 activity of the corresponding wild-type mature cytokine.

[0032] In some embodiments, ACC is characterized by the generation of a cleavage product after exposure to a protease, the cleavage product containing the activity of at least one of CP1 and / or CP2. In some embodiments, the activity of at least one of CP1 and / or CP2 is antiproliferative activity. In some embodiments, the control level is the EC50 value of 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 1.5, or equal to about 1. In some embodiments, the EC50 of the cleavage product is approximately the same as the EC50 of wild-type mature cytokine, indicating that the activity of CP1 and / or CP2 is fully or nearly fully restored after cleavage.

[0033] Provided herein are compositions comprising any one of the ACCs described herein. In some embodiments, the composition is a pharmaceutical composition. Also provided herein are kits comprising at least one dosage of any one of the compositions described herein.

[0034] Provided herein are methods for treating a subject in need, comprising administering a therapeutically effective amount of any one of the ACCs described herein or any one of the compositions described herein to the subject. In some embodiments, the subject is identified or diagnosed with cancer. In some non-limiting embodiments, the cancer is Kaposi's sarcoma, pilocytic cell leukemia, chronic myeloid leukemia (CML), follicular lymphoma, renal cell carcinoma (RCC), melanoma, neuroblastoma, basal cell carcinoma, bladder cancer, breast cancer, colorectal cancer, cutaneous T-cell lymphoma, nasopharyngeal adenocarcinoma, non-small cell lung cancer (NSCLC), ovarian cancer, or pancreatic cancer. In some non-limiting embodiments, the cancer is lymphoma. In some non-limiting embodiments, the lymphoma is Burkitt lymphoma.

[0035] Provided herein are nucleic acids encoding polypeptides 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. Also provided herein are nucleic acids encoding polypeptides comprising CP2 and CM2 of any one of the ACCs described herein. Where the monomers are identical, the disclosure provides a single nucleic acid encoding a monomer that dimerizes to form an ACC. In some embodiments, the polypeptide further comprises any one of the DD2s described herein. Also provided herein are vectors comprising any one of the nucleic acids described herein. In some embodiments, the vector is an expression vector. Also provided herein are cells comprising any one of the nucleic acids described herein or any one of the vectors described herein.

[0036] Provided herein are nucleic acid pairs encoding together a polypeptide comprising CP1 and CM1 of a first monomer construct and a polypeptide comprising CP2 and CM2 of a second monomer construct, from any one of the ACCs described herein. Also provided herein are vector pairs comprising any one of the nucleic acid pairs described herein. In some embodiments, the vector pair is a pair of expression vectors. Also provided herein are cells comprising 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 vectors comprising vector pairs.

[0037] Provided herein are methods for producing ACC, comprising: culturing one of the cells described herein in a liquid medium under conditions sufficient to produce ACC; and recovering ACC from the cells or the liquid medium. In some embodiments, the method further comprises isolating the ACC recovered from the cells or the liquid medium. In some embodiments, the method further comprises formulating the isolated ACC into a pharmaceutical composition.

[0038] Provided herein are ACCs produced by any one of the methods described herein. Also provided herein are compositions comprising any one of the ACCs described herein. Also provided herein are any one of the compositions described herein, and the compositions are pharmaceutical compositions. Also provided herein are kits comprising at least one dosage of any one of the compositions described herein.

[0039] Unless otherwise specified, all technical and chemical terms have the same meanings as those commonly understood by those skilled in the art to which this invention belongs. While methods and materials for use in this invention are described herein, other suitable methods and materials known in the art may also be used. Materials, methods, and examples are provided for illustrative purposes only and are not intended to limit the scope. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein in their entirety by reference. In case of any inconsistency, including definitions, this specification shall prevail.

[0040] Further features and advantages of the present invention will become apparent from the detailed description and figures below, as well as from the claims.

[0041] The terms "a" and "an" refer to one or more (i.e., at least one) grammatical objects of the article. For example, "a cell" includes one or more cells.

[0042] As used herein, the terms “about” and “approximately” indicate, when used to modify a quantity specified by a number or range, a reasonable deviation from a value known to those skilled in the art, along with the number. For example, ±20%, ±10%, or ±5% are, where appropriate, within the range of the intended meaning of the described value.

[0043] Concentration, quantity, and other numerical data may be expressed or presented in range form as specified herein. Such range forms are used solely for convenience and conciseness and should be interpreted flexibly to include all individual numerical values ​​or subranges encompassed within their range, as if each numerical value and subrange were explicitly stated, rather than only including numerical values ​​explicitly listed as range limits. For example, the numerical range "approximately 0.01 to approximately 2.0" should be interpreted to include not only the explicitly stated value of approximately 0.01 to approximately 2.0, but also the individual values ​​and subranges within the indicated range. Thus, this numerical range includes individual values ​​such as 0.5, 0.7, and 1.5, and subranges such as 0.5 to 1.7, 0.7 to 1.5, and 1.0 to 1.5. Furthermore, such interpretation should apply regardless of the breadth or characteristics of the range being described. Additionally, note that all percentages are weight-based unless otherwise specified.

[0044] In understanding the scope of this disclosure, the terms “including” or “comprising” and their derivatives are intended, as used herein, to be open terms that specify the presence of the indicated features, elements, components, bases, integers, and / or steps, but do not exclude the presence of features, elements, components, bases, integers, and / or steps that are not indicated. The foregoing also applies to words with similar meanings, such as “including,” “having,” and their derivatives. The term “composed of” and its derivatives as used herein are intended to be closed terms that specify the presence of the indicated features, elements, components, bases, integers, and / or steps, but exclude the presence of other unspecified features, elements, components, bases, integers, and / or steps. The term “essentially composed of” is intended, as used herein, to identify the presence of the stated features, elements, components, bases, integers, and / or steps, as well as those that do not substantially affect the fundamental and novel characteristics of the features, elements, components, bases, integers, and / or steps. Any reference to any one of these transition terms (i.e., “comprising,” “consisting,” or “consisting essentially”) is understood to directly support the substitution of any of the other transition terms not specifically used. For example, modifying a term from “includes” to “essentially consists of” or “consisting of” may find direct support by this definition for any element disclosed through this disclosure. Based on this definition, any element disclosed herein or incorporated by reference may be included in or excluded from the claimed invention.

[0045] Where used herein, multiple compounds, elements, or steps may be presented in common lists for convenience. However, these lists should be interpreted as if each member of the list were individually identified as a separate, unique member. Therefore, individual members of such lists should not be interpreted, without pointing out the opposite, as being substantially equivalent to other members of the same list based solely on their representation in a common group.

[0046] Furthermore, specific molecules, constructs, compositions, elements, parts, excipients, obstacles, states, properties, steps, etc., may be considered in the context of one particular embodiment or aspect, or in another paragraph or section of this disclosure. This is solely for convenience and brevity, and any such disclosure is intended to be applicable to and combined with any other embodiment or aspect found anywhere in this disclosure and claims, and it is understood that they all form the claimed and claimed invention as of the filing date. For example, with respect to constructs, compositions, or methods, a list of constructs, molecules, method steps, kits, or compositions is intended, and indeed found, to be a direct reference to embodiments of constructs, compositions, formulations, and methods described in any other part of this disclosure, even if these method steps, activators, kits, or compositions are not described again in the context or section of that embodiment or aspect.

[0047] Unless otherwise specified, "protein-coding nucleic acid sequences" include all nucleotide sequences that are degenerate versions of each other and therefore encode the same amino acid sequence.

[0048] When referring to the position of a first domain or sequence relative to a second domain or sequence in the polypeptide primary amino acid sequence, the term “N-terminal” means that the first domain or sequence is located closer to the N-terminus of the polypeptide primary amino acid sequence than the second domain or sequence. In some embodiments, additional sequences and / or domains may exist between the first domain or sequence and the second domain or sequence.

[0049] When referring to the position of a first domain or sequence relative to a second domain or sequence in the polypeptide primary amino acid sequence, the term "C-terminally located" means that the first domain or sequence is located closer to the C-terminus of the polypeptide primary amino acid sequence than the second domain or sequence. In some embodiments, additional sequences and / or domains may exist between the first domain or sequence and the second domain or sequence.

[0050] The term "exogenous" refers to any substance introduced from or originating from outside a cell, tissue, or organism that is not produced by or derived from the same cell, tissue, or organism into which it is introduced.

[0051] The terms “transduced,” “transfected,” or “transformed” refer to the process by which exogenous nucleic acids are introduced or transferred into a cell. A “transduced,” “transfected,” or “transformed” cell (e.g., mammalian cell) is a cell that has been transduced, transfected, or transformed with an exogenous nucleic acid (e.g., a vector) containing an exogenous nucleic acid encoding any of the activatable cytokine constructs described herein.

[0052] The term “nucleic acid” refers to single-stranded or double-stranded deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a combination thereof. Unless otherwise specified, the term encompasses nucleic acids including known analogues of natural nucleotides having similar binding properties to a reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes complementary sequences, as well as explicitly indicated sequences. In some embodiments of any nucleic acid described herein, the nucleic acid is DNA. In some embodiments of any nucleic acid described herein, the nucleic acid is RNA.

[0053] Modifications can be introduced into nucleotide sequences by techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR)-mediated mutagenesis. Conserved amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are 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), non-charged amino acids (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine), hydrophilic amino acids (e.g., arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine), and hydrophobic amino acids (e.g., alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine). Other families of amino acids include: aliphatic hydroxyamino acids (e.g., serine and threonine), amide families (e.g., asparagine and glutamine), aliphatic families (e.g., alanine, valine, leucine and isoleucine), and aromatic families (e.g., phenylalanine, tryptophan and tyrosine).

[0054] As used herein, the expressions “specifically bind” or “immunely respond” mean that the activatable antigen-binding protein complex reacts with one or more antigenic determinants of the desired target antigen and not with other polypeptides, or for example, about 10 -6 This means binding with M or a much lower affinity than that.

[0055] The term "treatment" refers to improving at least one symptom of a disorder. In some embodiments, the disorder being treated is cancer, and at least one symptom of cancer is improved. [Brief explanation of the drawing]

[0056] [Figure 1A] This is a schematic diagram of exemplary activatable cytokine constructs, comprising first and second monomer constructs covalently or noncovalently linked to each other via first and second dimerization domains 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] This is a schematic diagram of exemplary activatable cytokine constructs comprising first and second monomer constructs covalently or noncovalently linked to each other via first and second dimerization domains 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 2A]This is a schematic diagram of exemplary activatable cytokine constructs, comprising first and second monomer constructs covalently or noncovalently linked to each other via first and second dimerization domains 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] This is a schematic diagram of exemplary activatable cytokine constructs comprising first and second monomer constructs linked to each other by non-covalent means via first and second dimerization domains 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. [Figure 3]The amino acid sequence of the exemplary activatable cytokine construct IFNα2b-1204dL-hIgG4 (SEQ ID NO: 309) is provided, and the first and second monomer constructs have the same amino acid sequence. From the N-terminus to the C-terminus, the amino acid sequences of the first and second monomer constructs encode: mouse signal peptide (not bold, but in italics); mature human interferon α2b (underlined); cleavable portion 1204dL (bold); linker (italic and bold); and human IgG4 Fc domain (not italic, bold, or underlined). [Figure 4] The amino acid sequence of the exemplary activatable cytokine construct IFNα2b-1490DNI-hIgG4 (SEQ ID NO: 311) is provided, and the first and second monomer constructs have the same amino acid sequence. From the N-terminus to the C-terminus, the amino acid sequence encodes: mouse signal peptide (not bold, but in italics); mature human interferon α2b (underlined); cleavable portion 1490DNI (not bold, but in italics); linker (italics and bold); and human IgG4 Fc domain (not italic, bold, or underlined). [Figure 5] The activatable cytokine construct IFNα2b-hIgG4 Fc (having a cleavable portion of 1204 dL or 1490) exhibits cleavage reactions with protease (uPA or MT-SP1), which generates monomer-mature IFNα2b. [Figure 6](1) ACC(1204) having a cleavable portion 1204; (2) Product of protease membrane serine protease 1 (MT-SP1) and ACC IFNα2b-hIgG4 Fc having a cleavable portion 1204 (1204 MT-SP1); (3) Product of ACC IFNα2b-hIgG4 Fc having a cleavable portion 1204 and protease uPA (1204 uPA); (4) ACC IFNα2b-hIgG4 Fc(1204+1) having a cleavable portion 1204 fused to a 5-amino acid linker; (5) Product of IFNα2b-hIgG4 Fc 1204+1 and MT-SP1 (1204+1 MT-SP1); (6) ACC IFNα2b-hIgG4 Fc having a cleavable portion 1490; (7) MT-SP1 and ACC having a cleavable portion 1490 This is a gel image loaded with the product of IFNα2b-hIgG4 Fc (1490 uPA), which has uPA and a cleavable portion of 1490. [Figure 7] Cilatron® (pegylated interferon α2b) and various interferon α2b (IFNα2b) fusions: Human IgG4 fused to IFNα2b at the N-terminus (IFNα2b NhG4); Human IgG4 fused to IFNα2b at the N-terminus via a 5-amino acid linker (IFNα2b 5AA NhG4); Activatable cytokine construct IFNα2b-1204dL-hIgG4 (IFNα2b 1204DNIdL NhG4); Activatable cytokine construct (IFNα2b 5AA 1204DNIdL NhG4) containing the same components as IFNα2b-1204dL-hIgG4 but also having a 5-amino acid linker positioned between the mature cytokine protein component and the cleavable portion; and Activatable cytokine construct IFNα2-1490DNI-hIgG4 (IFNα2b 1490DNI This report provides results from a HEK293 cell-based reporter assay to evaluate interferon α2b activity in NhG4 cells. [Figure 8A] This shows the effect of flexible linker length in interferon α2b-Fc fusion on EC50, as measured by a HEK293 cell-based reporter assay. [Figure 8B] This shows the effect of linking region (LR) length in interferon α2b-Fc fusions on EC50, as measured by a HEK293 cell-based reporter assay. [Figure 9] This paper provides results from the Daudi apoptosis assay to measure the antiproliferative activity of Cilatron® (pegylated interferon α2b) and various IFNα2b fusions: human IgG4 fused to IFNα2b at the N-terminus (IFNα2b NhG4); human IgG4 fused to IFNα2b at the N-terminus via a 5-amino acid linker (IFNα2b 5AA NhG4); the activatable cytokine construct IFNα2b-1204dL-hIgG4 (IFNα2b 1204DNIdL NhG4); the activatable cytokine construct (IFNα2b 5AA 1204DNIdL NhG4) which contains the same components as IFNα2b-1204dL-hIgG4 but also has a 5-amino acid linker positioned between the mature cytokine protein component and the cleavable portion; and the activatable cytokine construct IFNα2-1490DNI-hIgG4 (IFNα2b 1490DNI NhG4). [Figure 10A] This shows the effect of linker length in interferon α2b-Fc fusion on EC50 as measured by the Daudi apoptosis assay. [Figure 10B] This shows the effect of linking region (LR) length in interferon α2b-Fc fusions on EC50 as measured by the Daudi apoptosis assay. [Figure 11] This paper provides results from a Daudi lymphoma cell-based assay to measure the antiproliferative activity of ACC (IFNα2b 1204DNIdL NhG4), protease-treated ACC (IFNα2b 1204DNIdL NhG4+uPA), and recombinant parent cytokine (IFNα2b). The results indicate that, after protease treatment of ACC, the activity of cytokines in ACC could be restored to levels comparable to those of the recombinant parent cytokine. [Figure 12]The results of a HEK293 cell-based reporter assay to evaluate the activity of ACC(IFNα2b 1204DNIdL NhG4); protease-treated (activated) ACC(IFNα2b 1204DNIdL NhG4+uPA); cilatron®; and recombinant parent cytokine (IFNα2b) are shown. The results indicate that after protease treatment of ACC, the activity of cytokines in ACC could be restored to levels comparable to those of the recombinant parent cytokine. [Figure 13] The top panel shows the results of a Daudi lymphoma cell-based assay to measure the antiproliferative activity of ACC(ProC440), protease-treated ACC(ProC440+uPA), and stem cell IFNα2b, and the bottom panel shows the results of a HEK293 cell-based reporter assay. The results show that activity was reduced by 1000X by constructing the ACC structure of this disclosure, and that after protease treatment of ACC, the activity of cytokines in ACC was restored to levels equivalent to those of recombinant parent cytokines. [Figure 14A] The structure of ProC440 is shown, and cleavage by uPA at the predicted site in CM was confirmed by mass spectrometry. In addition to its sensitivity to uPA activation, ProC440 is also cleaved by MMP4. [Figure 14B] Mass spectrometry analysis identified the MMP14 cleavage site (L161) at the tip of the C-terminus of IFNα near the cleavable region. Protease activation by MMP14 restored activity to a level comparable to that of recombinant cytokines. [Figure 15] The structures of ProC440 and ProC657 (N IFNα2b 0AA 1204DNIdL 0AA IgG4 KiHSS) are shown. The activity of ACC ProC440 and ProC657, protease-treated ACC (ProC440 + uPA), and stem cell IFNα2b was tested using IFN-responsive HEK293 cells. The results showed that the activity of ProC657 was decreased compared to stem cell IFNα2b or uPA-activated ProC440, but increased compared to ProC440. [Figure 16] (Top panel) The antiproliferative effect of ACC ProC440 in vivo is demonstrated using the Daudi xenograft tumor model. ACC ProC440 induced complete tumor regression at a low dose of 0.1 mg / kg and reduced the rate of tumor growth at a dose of 0.02 mg / kg. [Figure 16] (Bottom panel) The antiproliferative effect of Cilatron® in vivo is demonstrated using the Daudi xenograft tumor model. [Figure 17A] The structure of ProC286 and its activity compared to that of Cilatron® in the Daudi apoptosis assay are shown. ProC286 and Cilatron® exhibit similar levels of activity, indicating that ProC286 can be used as a substitute for the Cilatron® control to assess IFNα2b tolerance in hamster studies. [Figure 17B] The structure of ProC291 and its activity compared to that of Cilatron® in the Daudi apoptosis assay are shown. ProC291 showed significantly reduced activity compared to Cilatron® and ProC286. [Figure 18] The activity of IFNα-con (recombinant interferon α, unnatural type I interferon), ProC440+uPA, PEG-IFNα2b (Cilatron®), and ProC440, as well as the predicted toxic doses in in vivo dose escalation studies at doses of, for example, 0.08, 0.4, 2, 10, and 15 mg / kg ("mpk"), are shown. [Figure 19] The top panel shows the structure of the ACC ProC859 universal interferon, its antiproliferative effect in a B16 mouse melanoma cell assay, and its activity in an IFN-responsive HEK293 assay. [Figure 20A]This is a schematic diagram of exemplary activatable cytokine constructs comprising first and second monomer constructs linked to each other by non-covalent means via first and second dimerization domains 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 560, and a second dimerization domain DD2 590. [Figure 20B] This is a schematic diagram of exemplary activatable cytokine constructs comprising first and second monomer constructs linked to each other by non-covalent means via first and second dimerization domains DD1 600 and DD2 650, respectively. The first monomer construct comprises, from N-terminus to C-terminus, the first dimerization domain DD1 600, a first optional linker 630, and the first mature cytokine protein CP1 640. The second monomer construct comprises, from N-terminus to C-terminus, the second dimerization domain DD2 650, a second optional linker 660, a cleavable portion CM 670, a third optional linker 680, and the second mature cytokine protein CP2 690. [Figure 21A]This is a schematic diagram of exemplary activatable cytokine constructs comprising first and second monomer constructs linked to each other by non-covalent means via first and second dimerization domains 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 portion 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 cytokine-inactive polypeptide or protein 780, and a second dimerization domain DD2 790. The cytokine-inactive polypeptide or protein 780 may be, for example, a cytokine-inactive truncated cytokine protein, a cytokine-inactive mutant cytokine protein, a stub sequence, or a polypeptide sequence that binds to CP 700 with high affinity and reduces the cytokine activity of the second portion compared to a control level of the second portion. DD1 740 and DD2 790 may be the same or different. [Figure 21B]This is a schematic diagram of exemplary activatable cytokine constructs comprising first and second monomer constructs linked to each other by non-covalent means via first and second dimerization domains DD1 800 and DD2 850, respectively. The first monomer construct comprises, from N-terminus to C-terminus, the first dimerization domain DD1 800 and a cytokine-inactive polypeptide or protein 830. The second monomer construct comprises, from N-terminus to C-terminus, the second dimerization domain DD2 850, a first optional linker 860, a cleavable portion CM 870, a second optional linker 880, and a mature cytokine protein CP 890. The cytokine-inactive polypeptide or protein 830 may be, for example, a cytokine-inactive truncated cytokine protein, a cytokine-inactive mutant cytokine protein, a stub sequence, or a polypeptide sequence that binds to CP 700 with high affinity and reduces the cytokine activity of the second portion compared to the control level of the second portion. DD1 800 and DD2 850 may be the same or different. [Figure 22] This study shows weight loss in Syrian Gold Hamsters when administered 2mpk, 10mpk, and 15mpk of control hIgG4, ProC286, or ProC440 over the treatment period. [Figure 23] The clinical chemistry results (alkaline phosphatase, alanine transaminase, and aspartate aminotransferase) in Syrian Gold Hamsters administered with 2mpk, 10mpk, and 15mpk control hIgG4, ProC286, or ProC440 are shown. [Figure 24] The results of hematological analyses (reticulocyte, neutrophil, and white blood cell (WBC) counts) in Syrian Gold Hamsters administered 2mpk, 10mpk, and 15mpk control hIgG4, ProC286, or ProC440 are shown. [Figure 25] An embodiment of the ACC showing the linking region (LR) is schematically shown. [Modes for carrying out the invention]

[0057] Provided herein are activatable cytokine constructs (ACCs) exhibiting reduced levels of activity of at least one corresponding cytokine, which result in cytokine products with substantially restored activity after exposure to activation conditions. The activatable cytokine constructs of the present invention may be designed to be selectively activated upon exposure to affected tissue and not activated in normal tissue. Thus, these compounds have the potential to provide the benefits of cytokine-based therapy and possess potentially low toxicity associated with specific cytokine-based therapeutics.

[0058] Furthermore, this specification provides related intermediates, compositions, kits, nucleic acids, and recombinant cells, as well as related methods, including methods for using and producing any of the activatable cytokine constructs described herein.

[0059] The inventors unexpectedly found that ACCs having the specific elements and structural orientations described herein appear to be particularly effective in improving the safety and therapeutic index of cytokines in therapies for treating cancer. Cytokines are regulators of the innate and adaptive immune systems and have broad antitumor activity in preclinical models, but their clinical outcomes have been limited by systemic toxicity and unfavorable systemic exposure to target tissues. The inventors unexpectedly found that ACCs having the specific elements and structural orientations described herein appear to reduce the systemic toxicity associated with cytokine therapeutics and improve targeting and exposure to target tissues. Accordingly, this disclosure provides a method for reducing the targeted-mediated pharmacokinetics (TMDD) of cytokine therapeutics by targeting and administering ACCs having 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 sequestrated by normal tissue. This problem limits the proportion of the dose available in the systemic circulation to reach target tissues, e.g., cancerous tissue, in conventional cytokine therapeutics. This cytokine construct localizes target binding to tumor tissue, thereby maintaining efficacy, reducing side effects, enabling novel targeting opportunities, improving the therapeutic concentration range for validated targets, creating a therapeutic concentration range for drug-unavailable targets, and providing multiple binding modalities. This disclosure enables safe and effective systemic delivery, thereby avoiding the dose-dependent toxicity of conventional systemic cytokine therapeutics and the need for intratumoral injection. This disclosure provides means for conferring localized antiviral activity, immunomodulatory activity, antiproliferative activity, and pro-apoptotic activity. The inventors unexpectedly found that dimerization of the first and second monomer constructs achieved a high reduction in cytokine activity, particularly higher than when a single cytokine is attached to the dimerized domain. See Figure 15.

[0060] Furthermore, the inventors discovered that the degree of reduction in cytokine activity could be regulated by changing the flexible linker length or linking region length. Surprisingly, the inventors found that a reduction of more than 1,000-fold in cytokine activity could be achieved by attaching the cytokine to a sterically constrained dimerization domain (e.g., the first cysteine ​​in the hinge region, e.g., the Fc domain of human IgG shortened by EU numbering Cys226) via a short protease-cleavable sequence. Surprisingly, protease cleavage occurred regardless of steric hindrance, and full cytokine activity was restored upon cytokine cleavage from the dimerization domain.

[0061] Applicant's U.S. Provisional Patent Application No. 63 / 008,542, filed April 10, 2020, which discloses specific activatable cytokine constructs, is incorporated herein by reference in its entirety.

[0062] Activatable cytokine constructs The activatable cytokine construct of the present invention is a dimer complex comprising a first monomer construct and a second monomer construct. Dimerization of the monomer components is facilitated by a pair of dimerization domains. In one embodiment, each monomer construct comprises a cytokine protein, a cleavable moiety, and a dimerization domain (DD). In one embodiment, one monomer construct comprises a cytokine protein, a cleavable moiety, and a DD, while another monomer construct comprises a cytokine protein and a DD, but does not contain a cleavable moiety. In one embodiment, one monomer construct comprises a cytokine protein, a cleavable moiety, and a DD, while another monomer construct comprises a protein or peptide lacking cytokine activity, and a DD, but does not contain a cleavable moiety. In certain embodiments, the present invention provides an activatable cytokine construct (ACC) comprising a first monomer construct and a second monomer construct. (a) The first monomer construct comprises a first mature cytokine protein (CP1), a first cleavable portion (CM1), and a first dimerization domain (DD1), CM1 is positioned 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), CM2 is positioned between CP2 and DD2; DD1 and DD2 bond to each other, thereby forming dimers of the first monomer construct and the second monomer construct, and The ACC is characterized by having a reduced level of at least one CP1 and / or CP2 activity compared to a control level of at least one CP1 and / or CP2 activity.

[0063] When used in relation to cytokine constructs, the term “activatable” refers to a cytokine construct in which exposure to a condition causing cleavage of one or more cleavable portions of a first level results in the formation of a cytokine construct exhibiting one or more second levels of activity, where the second level of activity is greater than the first level of activity. Non-limiting examples of activity include any of the exemplary activities of cytokines described herein or known in the art.

[0064] 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. Accordingly, the ACC of this disclosure may, in some embodiments, include a mature cytokine protein sequence. In some embodiments, the ACC of this disclosure may include a mature cytokine protein sequence and, additionally, a signal sequence. In some embodiments, the ACC of this disclosure may include sequences disclosed herein, with or without the signal sequence described herein.

[0065] The terms “cleavable moiety” and “CM” are used interchangeably herein and refer to a peptide sequence containing a substrate for a sequence-specific protease. Suitable cleavable moieties for use as CM1 and / or CM2 contain any protease substrate known in the art. Exemplary cleavable moieties are described in further detail below.

[0066] The terms “dimerized domain” and “DD” are used herein to mean the same thing, and refer to one member of a pair of dimerized domains, each member of the pair being able to bond to the remaining member 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 below herein.

[0067] As used herein, polypeptides such as cytokines or Fc domains may be wild-type polypeptides (e.g., naturally occurring polypeptides) or variants of wild-type polypeptides. A variant may be a polypeptide modified by substitution, insertion, deletion, and / or addition of one or more amino acids to a wild-type polypeptide, provided that the variant retains the basic function or activity of the wild-type polypeptide. In some embodiments, a variant may have modified (e.g., enhanced or reduced) 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” means that the sequence of a polypeptide (e.g., cytokine) may contain fewer amino acids than a full-length polypeptide sequence, but is long enough to confer activity (e.g., cytokine activity).

[0068] The first and second monomer constructs may further include additional elements, such as one or more linkers. These additional elements are described in more detail below. The configuration of the CP, CM, and DD components in each of the first and second monomer constructs may be arranged in the same order within each monomer construct. The CP1, CM1, and DD1 components may be the same as or different from the corresponding CP2, CM2, and DD2 components in terms of, for example, the molecular weight, size, amino acid sequence of the CP and CM components (and the DD component in some embodiments where the DD component is a polypeptide). Thus, the resulting dimer may have symmetric or asymmetric monomer construct components.

[0069] In some embodiments, the first monomer construct includes CP1, CM1, and DD1 directly or indirectly (via linkers) linked (via linkers) from the N-to-C terminal of the CP and CM components. In other embodiments, the first monomer construct includes CP1, CM1, and DD1 directly or indirectly (via linkers) linked (via linkers) from the N-to-C terminal of the CP and CM components. In some embodiments, the second monomer construct includes CP2, CM2, and DD2 directly or indirectly (via linkers) linked (via linkers) from the N-to-C terminal of the CP and CM components. In other embodiments, the second monomer construct includes CP2, CM2, and DD2 directly or indirectly (via linkers) linked (via linkers) from the N-to-C terminal of the CP and CM components.

[0070] In certain embodiments, the first and second monomer constructs are oriented such that the components in each member of the dimer are configured 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 Figure 1A. Referring to Figure 1A, ACC comprises the following 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, positioned between the C-terminus of CP1 100 and the N-terminus of CM1 120; DD1 140; and an optional linker 130, if present, positioned between the C-terminus of CM1 120 and DD1 140; (2) a second monomer construct having CP2 150; CM2 170 located C-terminal to CP2 150; an optional linker 160, if present, positioned between the C-terminus of CP2 150 and the N-terminus of CM2 170; DD2 190; and an optional linker 180, if present, positioned between the C-terminus of CM2 170 and DD2 190; and (3) one or more covalent or non-covalent bonds (←→).

[0071] Figure 1B provides a schematic diagram of a further exemplary ACC having components composed of the reverse orientation of ACC. Referring to Figure 1B, ACC comprises the following from the N-terminus to the C-terminus of the CP and CM components: (1) a first monomer construct having DD1 200; CM1 220; an optional linker 210 located between the N-terminus of DD1 200 and CM1 220, if present; CP1 240 located C-terminal to CM1 220; and an optional linker 230 located between the C-terminus of CM1 220 and the N-terminus of CP1 240, if present; (2) a second monomer construct having DD2 250; CM2 270; an optional linker 260 located between the N-terminus of DD2 250 and CM2 270, if present; CP2 290 located C-terminal to CM2 270; and an optional linker 280 located between the C-terminus of CM2 290 and the N-terminus of CP2 290, if present; and (3) one or more covalent or non-covalent bonds (←→).

[0072] Figure 2A is a schematic diagram of exemplary activatable cytokine constructs comprising first and second monomer constructs linked 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 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.

[0073] Figure 2B is a schematic diagram of exemplary activatable cytokine constructs comprising first and second monomer constructs linked to each other by non-covalent means via first and second dimerization domains DD1 400 and DD2 450, respectively. The first monomer construct comprises, from N-terminus to C-terminus, the 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, the 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 another embodiment, one of the two portions, represented as CP1 440 and CP2 490, is a truncated cytokine protein lacking cytokine activity. For example, CP1 or CP2 may be truncated interferon α2b having the first 151 amino acids of wild-type interferon α2b. In another embodiment, one of the two portions, represented as CP1 440 and CP2 490, is a mutant cytokine protein lacking cytokine activity. For example, CP1 or CP2 may be truncated interferon α2b having the L130P mutation. In another embodiment, one of the two portions, represented as CP1 440 and CP2 490, is a polypeptide sequence, signaling moiety, and / or stub sequence lacking cytokine activity. In another embodiment, the first portion of two portions, designated as CP1 440 and CP2 490, is a polypeptide sequence that binds with high affinity to the second portion of the two portions, designated as CP1 440 and CP2 490, and reduces the cytokine activity of the second portion compared to the control level of the second portion.

[0074] The ACC structure has been found to be highly effective in reducing the activity of mature cytokine protein components in a manner that substantially preserves the function of cytokine activity after activation. The activation condition for the ACC described herein is exposure to a protease capable of cleaving at least one cleavable moiety in the ACC. As shown in the examples, activation of the ACC results in a substantial recovery of cytokine activity. This result suggests that the conformation of the cytokine component was not irreversibly altered within the framework of the ACC. Importantly, the ACC does not rely on the need to identify and utilize a peptide mask having binding affinity to the cytokine protein component to achieve a masking effect. Therefore, the ACC does not contain a peptide mask having binding affinity to the cytokine protein component. The inventors unexpectedly found that the ACC structure is sufficient to avoid off-target and undesirable activity and / or adverse side effects of cytokines without using any masking moiety having binding affinity to the cytokine protein component. Therefore, the ACC described herein is characterized by the absence of an affinity masking moiety or peptide mask moiety.

[0075] ACC may employ various mature cytokine proteins, cleavable moieties, and DDs as CP1, CP2, CM1, CM2, DD1, and DD2, respectively. For example, any mature cytokine protein known in the art or its sequence and / or truncated variant 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 and / or C-terminuses of CP1 and / or CP2.

[0076] In some embodiments, CP1 and / or CP2 are interferons (e.g., interferon α, interferon β, interferon γ, interferon τ, and interferon ω, etc.), interleukins (e.g., IL-1α, 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, etc.), G-CSF, IL-12, LIF, OSM, IL-10, IL- The present invention may independently include mature cytokine proteins selected from the group consisting of 20, 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, EPOo, TPO, Flt-3L, SCF, M-CSF, and MSP, as well as their sequences and truncated variants. For example, sequences of such proteins include those exemplified herein, and additional sequences can be obtained from ncbi.nlm.nih.gov / protein. Trunctured variants suitable for use in the ACC of the present invention include any N- or C-terminal truncated cytokine that retains cytokine activity. The exemplary shortened variants employed in the present invention include any shortened cytokine polypeptide known in the art (see, for example, Slutzki et al., J.Mol.Biol.360:1019-1030, 2006, and U.S. Patent Application Publication No. 2009 / 0025106), as well as cytokine polypeptides that are shortened 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, and 1 to about 4 amino acids at the N and / or C terminus, and that retain cytokine activity. In some of the embodiments described above, the shortened CP is a CP shortened at the N terminus. In other embodiments, the shortened CP is a CP shortened at the C terminus. In certain embodiments, the shortened CP is a CP shortened at both the C and N termini.

[0077] In some embodiments, CP1 and / or CP2 are sequence numbers 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 12, 121, 122, 123, 124, 125, 126, 127, sequence number 127, sequence number 123, 124, 125, 126, Sequence No. 128, Sequence No. 129, Sequence No. 130, Sequence No. 131, Sequence No. 132, Sequence No. 133, Sequence No. 134, Sequence No. 135, Sequence No. 136, Sequence No. 137, Sequence No. 138, Sequence No. 139, Sequence No. 140, Sequence No. 141, Sequence No. 142, Sequence No. 143, Sequence No. 144, Sequence No. 145, Sequence No. 146, Sequence No. 147, Sequence No. 148, Sequence No. 149, Sequence No. 150, Sequence No. 151, Sequence No. 152, Sequence No. 153, Sequence No. 154, Sequence No. 155, Sequence No. 156, Sequence No. 157, Sequence No. 158, Sequence 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: 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: 1 A cytokine reference sequence selected from the group consisting of 90, SEQ ID NOs: 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, and 209 is at least 80% identical (for example, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%).It independently contains amino acid sequences that are 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, e.g., a series of BLAST programs publicly available on the NCBI website on the internet. See also Altschul et al., J.Mol.Biol.215:403-10, 1990. In some embodiments, ACC includes interferon α2b variants as CP1 or CP2, e.g., interferon α2b molecules having a mutation at position L130, e.g., an L130P mutation. In some embodiments, the ACC includes interferon α2b variants having mutations at position I24, F64, I60, I63, F64, W76, I116, L117, F123, or L128, or combinations thereof. For example, interferon α2b variants may include T, N, or R at I116; N, H, or R at L128; mutations to P or Q at I24; mutations L117H; or L128T, or combinations thereof. In some embodiments, interferon α2b variants may include mutations I24Q, I60T, F64A, W76H, I116R, and L128N, or subsets thereof. In some embodiments, the ACC includes a truncated interferon α2b molecule lacking cytokine activity as one of CP1 and CP2. For example, truncated interferon α2b may consist of interferon α2b with 151 or fewer amino acids, such as any of the amino acids from N to C-terminus in the wild-type interferon α2b sequence: 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, or any intervening sequence or variant of these amino acids.

[0078] In certain embodiments, CP1 and / or CP2 include interferon. Interferons suitable for use in the constructs of the present invention as CP1 and / or CP2 include, for example, interferon α, interferon β, interferon ω, and interferon τ. In some embodiments, if the interferon is interferon α, it may be interferon α2a, interferon α2b, or interferon α-n3. Further examples of interferon α include interferon α-1, interferon α-4, interferon α-5, interferon α-6, interferon α-7, interferon α-8, interferon α-10, interferon α-13, interferon α-14, interferon α-16, interferon α-17, and interferon α-21. In some embodiments, the interferon is recombinant or purified interferon α. In certain embodiments, if the interferon is interferon β, it is selected from the group consisting of interferon β-1a and interferon β-1b. In some embodiments, CP1 and / or CP2 contain the IFab domain of interferon α or interferon β. IFab is involved in the cytokine release and antiviral functions of interferon. Exemplary IFab sequences are provided in SEQ ID NOs: 325-334.

[0079] In some embodiments, CP1 and / or CP2 include an amino acid sequence that exhibits interferon activity and 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, at least 99% identical, or at least 99% identical, or 100% identical, to an interferon α reference sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105. In some embodiments, CP1 and / or CP2 include a mature α-interferon having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105. In some embodiments, CP1 and / or CP2 include a mature human α-interferon having the amino acid sequence of SEQ ID NO: 1. In some of the embodiments described above, CP1 and CP2 contain the same amino acid sequence.

[0080] In other embodiments, CP1 and / or CP2 include an amino acid sequence that exhibits interferon activity and 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, at least 99% identical, or 100% identical to an interferon-beta reference sequence selected from the group consisting of SEQ ID NOs: 106 and SEQ ID NOs: 107. In certain embodiments, the interferon-beta reference sequence is a human interferon-beta reference sequence selected from the group consisting of SEQ ID NOs: 106 and SEQ ID NOs: 107. In some embodiments, CP1 and / or CP2 include a mature β-interferon having an amino acid sequence selected from the group consisting of SEQ ID NOs: 106, SEQ ID NOs: 107, SEQ ID NOs: 108, and SEQ ID NOs: 109. In some of the embodiments described above, CP1 and CP2 include the same amino acid sequence.

[0081] In some embodiments, CP1 and / or CP2 exhibit interferon activity and include 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, at least 99% identical, or 100% identical to the interferon ω reference sequence corresponding to SEQ ID NO: 110. In certain embodiments, CP1 and / or CP2 include a mature human ω interferon having the amino acid sequence of SEQ ID NO: 110. In some of the embodiments described above, CP1 and CP2 include the same amino acid sequence.

[0082] In some embodiments, CP1 and / or CP2 exhibit interleukin activity, as shown in SEQ ID NOs: 111, 112, 113, 114, 115, 116, 117, 118, 119, 12, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, The amino acid sequence 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 number 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, and SEQ ID NO: 160. In some embodiments, CP1 and / or CP2 include a mature interleukin having an amino acid sequence selected from the group consisting of SEQ ID NOs: 111, 112, 113, 114, 115, 116, 117, 118, 119, 12, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 143, 144, 145, 146, 151, 152, 153, 154, 155, 156, 157, 158, 159, and 160.In some of the embodiments described above, CP1 and CP2 contain the same amino acid sequence.

[0083] In some embodiments, CP1 and / or CP2 exhibit interleukin activity, such as SEQ ID NO: 111 (Human IL-1α), SEQ ID NO: 113 (Human IL-1β), SEQ ID NO: 115 (Human IL-1RA), SEQ ID NO: 117 (Human IL-18), SEQ ID NO: 119 (Human IL-2), SEQ ID NO: 121 (Human IL-4), SEQ ID NO: 123 (Human IL-7), SEQ ID NO: 125 (Human IL-9), SEQ ID NO: 127 (Human IL-13), SEQ ID NO: 129 (Human IL-15), SEQ ID NO: 131 (Human IL-3), SEQ ID NO: 133 (Human IL-5), SEQ ID NO: 137 (Human IL-6), SEQ ID NO: 139 (Human IL-11), SEQ ID NO: 1 The amino acid sequence 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 interleukin reference sequence selected from the group consisting of 43 (human IL-12α), SEQ ID NO: 144 (human IL-12β), SEQ ID NO: 151 (human IL-10), SEQ ID NO: 153 (human IL-20); SEQ ID NO: 155 (human IL-14), SEQ ID NO: 157 (human IL-16), and SEQ ID NO: 159 (human IL-17). In these specific embodiments, CP1 and / or CP2 are sequence numbers 111 (human IL-1α), 113 (human IL-1β), 115 (human IL-1RA), 117 (human IL-18), 119 (human IL-2), 121, 123 (human IL-7), 125 (human IL-9), 127 (human IL-13), 129 (human IL-15), and 131 (human IL-1). L-3), includes an amino acid sequence selected from the group consisting of SEQ ID NO: 133 (Human IL-5), SEQ ID NO: 137 (Human IL-6), SEQ ID NO: 139 (Human IL-11), SEQ ID NO: 143 (Human IL-12α), SEQ ID NO: 144 (Human IL-12β), SEQ ID NO: 151 (Human IL-10), SEQ ID NO: 153 (Human IL-20); SEQ ID NO: 155 (Human IL-14), SEQ ID NO: 157 (Human IL-16), and SEQ ID NO: 159 (Human IL-17). In some of the embodiments described above, CP1 and CP2 include the same amino acid sequence.

[0084] The number of amino acids in the sequence of the cytokine protein used may vary depending on the specific cytokine protein used. In some embodiments, CP1 and / or CP2 may have a total of approximately 10 to 700 amino acids, approximately 10 to 650 amino acids, approximately 10 to 600 amino acids, approximately 10 to 550 amino acids, approximately 10 to 500 amino acids, approximately 10 to 450 amino acids, approximately 10 to 400 amino acids, approximately 10 to 350 amino acids, approximately 10 to 300 amino acids, approximately 10 to 250 amino acids, approximately 10 to 200 amino acids, approximately 10 to 150 amino acids, and approximately 10 amino acids to approximately 100 amino acids, approximately 10 amino acids to approximately 80 amino acids, approximately 10 amino acids to approximately 60 amino acids, approximately 10 amino acids to approximately 40 amino acids, approximately 10 amino acids to approximately 20 amino acids, approximately 20 amino acids to approximately 700 amino acids, approximately 20 amino acids to approximately 650 amino acids, approximately 20 amino acids to approximately 600 amino acids, approximately 20 amino acids to approximately 550 amino acids, approximately 20 amino acids to approximately 500 amino acids, approximately 20 amino acids to approximately 450 amino acids, approximately 20 amino acids to approximately 400 amino acids, approximately 20 amino acids to approximately 350 amino acids, approximately 20 amino acids to approximately 300 amino acids, approximately 2 0 amino acids to approximately 250 amino acids, approximately 20 amino acids to approximately 200 amino acids, approximately 20 amino acids to approximately 150 amino acids, approximately 20 amino acids to approximately 100 amino acids, approximately 20 amino acids to approximately 80 amino acids, approximately 20 amino acids to approximately 60 amino acids, approximately 20 amino acids to approximately 40 amino acids, approximately 40 amino acids to approximately 700 amino acids, approximately 40 amino acids to approximately 650 amino acids, approximately 40 amino acids to approximately 600 amino acids, approximately 40 amino acids to approximately 550 amino acids, approximately 40 amino acids to approximately 500 amino acids, approximately 40 amino acids to approximately 450 amino acids, approximately 40 amino acids to approximately 400 amino acids, approximately 40 amino acids to approximately 350 amino acids, approximately 40 amino acids to approximately 300 amino acids, approximately 40 amino acids to approximately 250 amino acids, approximately 40 amino acids to approximately 200 amino acids, approximately 40 amino acids to approximately 150 amino acids, approximately 40 amino acids to approximately 100 amino acids, approximately 40 amino acids to approximately 80 amino acids, approximately 40 amino acids to approximately 60 amino acids, approximately 60 amino acids to approximately 700 amino acids, approximately 60 amino acids to approximately 650 amino acids, approximately 60 amino acids to approximately 600 amino acids, approximately 60 amino acids to approximately 550 amino acids, approximately 60 amino acids to approximately 500 amino acids, approximately 60 amino acids to approximately 450 amino acids,Approximately 60 amino acids to approximately 400 amino acids, approximately 60 amino acids to approximately 350 amino acids, approximately 60 amino acids to approximately 300 amino acids, approximately 60 amino acids to approximately 250 amino acids, approximately 60 amino acids to approximately 200 amino acids, approximately 60 amino acids to approximately 150 amino acids, approximately 60 amino acids to approximately 100 amino acids, approximately 60 amino acids to approximately 80 amino acids, approximately 80 amino acids to approximately 700 amino acids, approximately 80 amino acids to approximately 650 amino acids, approximately 80 amino acids to approximately 600 amino acids, approximately 80 amino acids to approximately 550 amino acids, approximately 80 amino acids to approximately 500 amino acids, approximately 80 amino acids to approximately 450 amino acids, approximately 80 amino acids to approximately 400 amino acids, approximately 80 amino acids to approximately 350 amino acids, approximately 80 amino acids to approximately 300 amino acids, approximately 80 amino acids to approximately 250 amino acids, approximately 80 amino acids to approximately 200 amino acids, approximately 80 amino acids to approximately 150 amino acids, approximately 80 amino acids to approximately 100 amino acids, approximately 100 amino acids to approximately 700 amino acids, approximately 100 amino acids to approximately 650 amino acids, approximately 100 amino acids to approximately 600 amino acids, approximately 100 amino acids to approximately 550 amino acids, approximately 100 amino acids to approximately 500 amino acids, approximately 100 amino acids to approximately 450 amino acids, approximately 100 amino acids to approximately 400 amino acids, approximately 100 amino acids to approximately 3 50 amino acids, approximately 100 amino acids to approximately 300 amino acids, approximately 100 amino acids to approximately 250 amino acids, approximately 100 amino acids to approximately 200 amino acids, approximately 100 amino acids to approximately 150 amino acids, approximately 150 amino acids to approximately 700 amino acids, approximately 150 amino acids to approximately 650 amino acids, approximately 150 amino acids to approximately 600 amino acids, approximately 150 amino acids to approximately 550 amino acids, approximately 150 amino acids to approximately 500 amino acids, approximately 150 amino acids to approximately 450 amino acids, approximately 150 amino acids to approximately 400 amino acids, approximately 150 amino acids to approximately 350 amino acids, approximately 150 amino acids to approximately 300 amino acids, approximately 150 amino acids Mino acids ~ approximately 250 amino acids, approximately 150 amino acids ~ approximately 200 amino acids, approximately 200 amino acids ~ approximately 700 amino acids, approximately 200 amino acids ~ approximately 650 amino acids, approximately 200 amino acids ~ approximately 600 amino acids, approximately 200 amino acids ~ approximately 550 amino acids, approximately 200 amino acids ~ approximately 500 amino acids, approximately 200 amino acids ~ approximately 450 amino acids, approximately 200 amino acids ~ approximately 400 amino acids, approximately 200 amino acids ~ approximately 350 amino acids, approximately 200 amino acids ~ approximately 300 amino acids, approximately 200 amino acids ~ approximately 250 amino acids, approximately 250 amino acids ~ approximately 700 amino acids, approximately 250 amino acids ~ approximately 650 amino acids,Approximately 250 amino acids to approximately 600 amino acids, approximately 250 amino acids to approximately 550 amino acids, approximately 250 amino acids to approximately 500 amino acids, approximately 250 amino acids to approximately 450 amino acids, approximately 250 amino acids to approximately 400 amino acids, approximately 250 amino acids to approximately 350 amino acids, approximately 250 amino acids to approximately 300 amino acids, approximately 300 amino acids to approximately 700 amino acids, approximately 300 amino acids to approximately 650 amino acids, approximately 300 amino acids to approximately 600 amino acids, approximately 300 amino acids to approximately 550 amino acids 0.0 amino acids, approximately 300 to 500 amino acids, approximately 300 to 450 amino acids, approximately 300 to 400 amino acids, approximately 300 to 350 amino acids, approximately 350 to 700 amino acids, approximately 350 to 650 amino acids, approximately 350 to 600 amino acids, approximately 350 to 550 amino acids, approximately 350 to 500 amino acids, approximately 350 to 450 amino acids, approximately 350 to 40 0 amino acids, approximately 400 amino acids ~ approximately 700 amino acids, approximately 400 amino acids ~ approximately 650 amino acids, approximately 400 amino acids ~ approximately 600 amino acids, approximately 400 amino acids ~ approximately 550 amino acids, approximately 400 amino acids ~ approximately 500 amino acids, approximately 400 amino acids ~ approximately 450 amino acids, approximately 450 amino acids ~ approximately 700 amino acids, approximately 450 amino acids ~ approximately 650 amino acids, approximately 450 amino acids ~ approximately 600 amino acids, approximately 450 amino acids ~ approximately 550 amino acids, approximately 450 amino acids ~ It contains approximately 500 amino acids, approximately 500 to approximately 700 amino acids, approximately 500 to approximately 650 amino acids, approximately 500 to approximately 600 amino acids, approximately 500 to approximately 550 amino acids, approximately 550 to approximately 700 amino acids, approximately 550 to approximately 650 amino acids, approximately 550 to approximately 600 amino acids, approximately 600 to approximately 700 amino acids, approximately 600 to approximately 650 amino acids, or approximately 650 to approximately 700 amino acids. In some embodiments, CP1 and / or CP2 are mature wild-type human cytokine proteins.

[0085] Each monomer construct of ACC may utilize any of the various dimerization domains. Preferred DDs include both polymeric (e.g., synthetic polymers, polypeptides, polynucleotides, etc.) and small molecule (non-polymeric moieties having a molecular weight of less than about 1 kilodalton, and possibly less than about 800 daltons). The DD pair may be any pair of moieties known in the art to bond with each other.

[0086] For example, in some embodiments, DD1 and DD2 are: a sucoid domain derived from the alpha chain (IL15Rα) of the human IL-15 receptor and soluble IL-15; barnase and barnster; PKA and AKAP; an adapter / docking tag module based on a mutant RNase I fragment; a pair of antigen-binding domains (e.g., a pair of single-domain antibodies); a soluble N-ethylmaleimide-sensitive factor-adhering protein receptor (SNARE) module based on the interaction of protein syntaxin, synaptotagmin, synaptobrevinn, and SNAP25; a single-domain antibody (sdAb) and its corresponding epitope; an antigen-binding domain (e.g. The pair members are selected from the following groups: single-chain variable fragments (scFv), single-chain antibodies (such as single-domain antibodies) and their corresponding epitopes; 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 binding pairs such as biotin and avidin or streptavidin, amines / aldehydes, lectins / carbohydrates; and pairs of polymers that can bind to each other, such as a pair of sulfur or thiol-containing polymers (e.g., a pair of Fc domains, a pair of thiolated human serum albumin polypeptides, etc.).

[0087] In some embodiments, DD1 and DD2 are non-polypeptide polymers. Non-polypeptide polymers can be covalently bonded to each other. In some embodiments, the non-polypeptide polymer may be a sulfur-containing polymer, such as sulfur-containing polyethylene glycol. In such cases, DD1 and DD2 may be covalently bonded to each other via one or more disulfide bonds.

[0088] If the DD1 and DD2 pair are members of a pair of epitopes and antigen-binding domains, the epitope may be of natural or non-natural origin. Exemplary non-natural epitopes include, for example, non-natural peptides such as poly-His peptides (e.g., His tags, etc.).

[0089] In certain embodiments, DD1 and DD2 are a pair of Fc domains. As used herein, “Fc domain” refers to a continuous amino acid sequence of a single heavy chain of immunoglobulin. The pair of Fc domains bind together to form the Fc region of the immunoglobulin.

[0090] In some embodiments, the Fc domain pair 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 contains a sequence that is at least 80% identical to Sequence ID No. 3 (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).

[0091] In some embodiments, the Fc domain pair 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 within the boundary between two Fc domains (e.g., in the CH3 domain). In one example, the modifications include amino acid substitution T366W and optionally amino acid substitution S354C in one of the antibody heavy chains, and amino acid substitution T366S, L368A, Y407V and optionally Y349C in another of the antibody heavy chains (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, as well as 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 entirety. In some embodiments, the dimerized domains each contain a sequence that is at least 80% identical to sequence number 315 or 316 (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).

[0092] In some embodiments, DD1 and / or DD2 may further comprise serum half-life extension moieties (e.g., polypeptides that bind serum proteins such as immunoglobulins (e.g., IgG)) or serum albumin (e.g., human serum albumin (HSA)). Examples of half-life extension 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 epitopes, Glu-Glu epitope, KT3 epitope, IRS epitope, Btag epitope, protein kinase C epitope and VSV epitope.

[0093] In some embodiments, DD1 and / or DD2 each contain a total of approximately 5 to 250 amino acids, approximately 5 to 200 amino acids, approximately 5 to 180 amino acids, approximately 5 to 160 amino acids, approximately 5 to 140 amino acids, approximately 5 to 120 amino acids, approximately 5 to 100 amino acids, approximately 5 to 80 amino acids, approximately 5 to 60 amino acids, approximately 5 to 40 amino acids, approximately 5 to 20 amino acids, approximately 5 to 10 amino acids, approximately 10 to 250 amino acids, and approximately 10 to 20 amino acids. 0 amino acids, approximately 10 amino acids to approximately 180 amino acids, approximately 10 amino acids to approximately 160 amino acids, approximately 10 amino acids to approximately 140 amino acids, approximately 10 amino acids to approximately 120 amino acids, approximately 10 amino acids to approximately 100 amino acids, approximately 10 amino acids to approximately 80 amino acids, approximately 10 amino acids to approximately 60 amino acids, approximately 10 amino acids to approximately 40 amino acids, approximately 10 amino acids to approximately 20 amino acids, approximately 20 amino acids to approximately 250 amino acids, approximately 20 amino acids to approximately 200 amino acids, approximately 20 amino acids to approximately 180 amino acids, approximately 20 amino acids to approximately 160 amino acids, approximately 20 amino acids to approximately 14180 amino acids, approximately 180 amino acids, approximately 180 amino acids, approximately 180 amino acids, approximately 180 amino acids, approximately 180 amino acids, approximately 180 amino acids, approximately 180 amino acids, approximately 180 amino acids, approximately 180 amino acids, approximately 180 amino acids, approximately 180 amino acids, approximately 180 amino acids, approximately 180 amino acids, approximately 180 amino acids, approximately 180 amino acids, approximately 180 amino acids, approximately 180 amino acids, approximately 180 amino Approximately 120 amino acids, approximately 20 to approximately 100 amino acids, approximately 20 to approximately 80 amino acids, approximately 20 to approximately 60 amino acids, approximately 20 to approximately 40 amino acids, approximately 40 to approximately 250 amino acids, approximately 40 to approximately 200 amino acids, approximately 40 to approximately 180 amino acids, approximately 40 to approximately 160 amino acids, approximately 40 to approximately 140 amino acids, approximately 40 to approximately 120 amino acids, approximately 40 to approximately 100 amino acids, approximately 40 to approximately 80 amino acids, approximately 40 to approximately 60 amino acids, approximately 60 to approximately 250 amino acids, approximately 60 amino acids Acid ~ approximately 200 amino acids, approximately 60 amino acids ~ approximately 180 amino acids, approximately 60 amino acids ~ approximately 160 amino acids, approximately 60 amino acids ~ approximately 140 amino acids, approximately 60 amino acids ~ approximately 120 amino acids, approximately 60 amino acids ~ approximately 100 amino acids, approximately 60 amino acids ~ approximately 80 amino acids, approximately 80 amino acids ~ approximately 250 amino acids, approximately 80 amino acids ~ approximately 200 amino acids, approximately 80 amino acids ~ approximately 180 amino acids, approximately 80 amino acids ~ approximately 160 amino acids, approximately 80 amino acids ~ approximately 140 amino acids, approximately 80 amino acids ~ approximately 120 amino acids, approximately 80 amino acids ~ approximately 100 amino acids, approximately 100 amino acids ~ approximately 250 amino acids,Contains approximately 100 to 200 amino acids, approximately 100 to 180 amino acids, approximately 100 to 160 amino acids, approximately 100 to 140 amino acids, approximately 100 to 120 amino acids, approximately 120 to 250 amino acids, approximately 120 to 200 amino acids, approximately 120 to 180 amino acids, approximately 120 to 160 amino acids, approximately 120 to 140 amino acids, approximately 140 to 250 amino acids, approximately 140 to 200 amino acids, approximately 140 to 180 amino acids, approximately 140 to 160 amino acids, approximately 160 to 250 amino acids, approximately 160 to 200 amino acids, approximately 160 to 180 amino acids, approximately 180 to 250 amino acids, approximately 180 to 200 amino acids, or approximately 200 to 250 amino acids. In some embodiments, DD1 and DD2 are Fc domains each containing a portion of a hinge region comprising 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 read from N to C (e.g., cysteine ​​226 of human IgG1 or IgG4 using EU numbering).

[0094] In some embodiments, the cleavable portion containing the protease substrate is positioned directly or indirectly (e.g., via a linker) between the CP and DD components. In some embodiments, CM1 and CM2 are ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADEMDE C1, 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 14, Cathepsin A, Cathepsin B, Cathepsin C, Cathepsin G, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V / L2, Cathepsin X / Z / P, Kymase, Cruzipain, DESC1, DPP-4, FAP, Regmine, Otsubine-2, Elastase, FVIIa, FiXA, FXa, FXIa, FXIIa, Granzyme B, Guanidino Benzoate, hepsin, HtrA1, human neutrophil elastase, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, lactoferrin, malapsin, matryptase-2, meprin, MT-SP1 / matryptase, neprilysin, NS3 / 4A, PACE4, plasmin, PSMA, PSA, BMP-1, MMP1, MMP2, MMP3, Each of these may independently contain a substrate for a protease selected from the group consisting of 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.

[0095] In some embodiments of any ACC described herein, the proteases that cleave any of the CMs described herein 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, ca 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, regmain, otsubine-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, FXI a, FXIIa, elastase, granzyme B, guanidinobenzoate, HtrA1, human neutrophil lyase, lactoferrin, malapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, tryptase, uPA, DESC1, DPP-4, FAP, hepsin, matryptase-2, MT-SP1 / matryptase, TMPRSS2, TMPRSS3, and TMPRSS4.

[0096] In some embodiments of any ACC described herein, the protease is selected from the group consisting of uPA, regmine, MT-SP1, ADAM17, BMP-1, TMPRSS3, TMPRSS4, MMP-2, MMP-9, MMP-12, MMP-13, and MMP-14.

[0097] Elevated levels of proteases with known substrates have been reported in several cancers. See, for example, 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 those more widely found in cancer cells and tissues. Accordingly, in certain embodiments, CM1 and / or CM2 each independently contain substrates for proteases more widely found in cancer-related affected tissues. 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 HER2-positive cancer. In some embodiments, the cancers include Kaposi's sarcoma, pilocytic cell leukemia, chronic myeloid leukemia (CML), follicular lymphoma, renal cell carcinoma (RCC), melanoma, neuroblastoma, basal cell carcinoma, cutaneous T-cell lymphoma, nasopharyngeal adenocarcinoma, breast cancer, ovarian cancer, bladder cancer, BCG-resistant nonmuscle-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. In some of the embodiments described above, the CM component includes a substrate for proteases that are more widely present in tumor tissue.

[0098] In some embodiments, CM1 and / or CM2 each independently include a sequence selected from the group consisting of SEQ ID NOs: 5 to 100, as well as its C-terminal and N-terminal shortened variants.

[0099] In some embodiments, CM includes a sequence selected from the group ISSGLLSGRSDNH (SEQ ID NO: 28), LSGRSDDH (SEQ ID NO: 33), ISSGLLSGRSDQH (SEQ ID NO: 54), and ISSGLLSGRSDNI (SEQ ID NO: 68).

[0100] In certain embodiments, CM1 and / or CM2 include sequences selected from the group consisting 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 their C-terminal and N-terminal shortened variants. Examples of CMs include those described in U.S. Patent Application Publications 2016 / 0289324, 2019 / 0284283, International Publications 2010 / 081173, 2015 / 048329, 2015 / 116933, 2016 / 118629, and 2020 / 118109, which are incorporated herein by reference in their entirety.

[0101] Shortened variants of the above-described amino acid sequences that are suitable for use in CM1 and / or CM2 are any variants that retain the recognition site for the corresponding protease. These include C-terminal and / or N-terminal shortened variants that include at least three consecutive amino acids of the above-described amino acid sequence, or at least four, at least five, at least six, or at least seven amino acids of the aforementioned amino acid sequence that retain the protease recognition site. In certain embodiments, the shortened variants of the above-described amino acid sequences are C and / or N-terminal shortened 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, and (1) have at least three amino acid residues and (2) retain the protease recognition site. In some of the embodiments described above, the shortened CM is a CM shortened at the N-terminus. In some embodiments, the shortened CM is a CM shortened at the C-terminus. In some embodiments, the shortened CM is a CM shortened at both the C and N-terminus.

[0102] In some embodiments of any of the activatable cytokine constructs described herein, CM1 and / or CM2 comprises a total of about 3 to about 25 amino acids. In some embodiments, CM1 and / or CM2 comprises a total of about 3 to about 25 amino acids, about 3 to about 20 amino acids, about 3 to about 15 amino acids, about 3 to about 10 amino acids, about 3 to about 5 amino acids, about 5 to about 25 amino acids, about 5 to about 20 amino acids, about 5 to about 15 amino acids, about 5 to about 10 amino acids, about 5 to about 10 amino acids, about 10 to about 25 amino acids, about 10 to about 20 amino acids, about 10 to about 15 amino acids, about 15 to about 25 amino acids, about 15 to about 20 amino acids, or about 20 to about 25 amino acids.

[0103] In some embodiments, ACC may comprise multiple CMs, each containing a substrate for a different protease. In some embodiments, CM1 and CM2 contain substrates for different proteases. In some embodiments, CM1 and CM2 contain substrates for the same protease.

[0104] The first and second monomer constructs may include one or more additional components, such as one or more linkers. In some embodiments, the first monomer may include a linker positioned between CP1 and CM1. In some embodiments, CP1 and CM1 are directly adjacent to each other in the first monomer. In some embodiments, the first monomer includes a linker positioned between CM1 and DD1. In some embodiments, the linker has a total length of 1 to about 15 amino acids. In some embodiments, CM1 and DD1 are directly adjacent to each other in the first monomer. In some embodiments, any linker positioned between CM1 and CP1 and DD1 has a combined total length of 3 to 15 amino acids, or 3 to 10 amino acids, or 3 to 7 amino acids.

[0105] In some embodiments, the second monomer includes a linker positioned between CP2 and CM2. In some embodiments, CP2 and CM2 are directly adjacent to each other in the second monomer construct. In some embodiments, the second monomer includes a linker positioned between CM2 and DD2. In some embodiments, the linker has a total length of 1 to about 15 amino acids. In some embodiments, the linker includes the sequence GGGS (SEQ ID NO: 2). In some embodiments, CM2 (e.g., any of the cleavable portions described herein) and DD2 (e.g., any of the DDs described herein) are directly adjacent to each other in the second monomer. In some embodiments, CM and any linker positioned between CP2 and DD2 have a combined total length of 3 to 15 amino acids, or 3 to 10 amino acids, or 3 to 7 amino acids.

[0106] In some embodiments, the first monomer and / or the second monomer total about 50 to 800 amino acids, about 50 to 750 amino acids, about 50 to 700 amino acids, about 50 to 650 amino acids, about 50 to 600 amino acids, about 50 to 550 amino acids, about 50 to 500 amino acids, about 50 to 450 amino acids, about 50 to 400 amino acids, about 50 to 350 amino acids, about 50 to 300 amino acids, about 50 to 250 amino acids, and about 50 amino acids. ~200 amino acids, ~50 amino acids~150 amino acids, ~50 amino acids~100 amino acids, ~100 amino acids~800 amino acids, ~100 amino acids~750 amino acids, ~100 amino acids~700 amino acids, ~100 amino acids~650 amino acids, ~100 amino acids~600 amino acids, ~100 amino acids~550 amino acids, ~100 amino acids~500 amino acids, ~100 amino acids~500 amino acids, ~100 amino acids~450 amino acids, ~100 amino acids~400 amino acids, ~100 amino acids~350 amino acids, ~100 amino acids~300 amino acids, ~100 amino acids Acid ~ approximately 250 amino acids, approximately 100 amino acids ~ approximately 200 amino acids, approximately 100 amino acids ~ approximately 150 amino acids, approximately 150 amino acids ~ approximately 800 amino acids, approximately 150 amino acids ~ approximately 750 amino acids, approximately 150 amino acids ~ approximately 700 amino acids, approximately 150 amino acids ~ approximately 650 amino acids, approximately 150 amino acids ~ approximately 600 amino acids, approximately 150 amino acids ~ approximately 550 amino acids, approximately 150 amino acids ~ approximately 500 amino acids, approximately 150 amino acids ~ approximately 450 amino acids, approximately 150 amino acids ~ approximately 400 amino acids, approximately 150 amino acids ~ approximately 350 amino acids, approximately 150 amino acids ~ approximately 300 amino acids, approximately 15 0 amino acids to approximately 250 amino acids, approximately 150 amino acids to approximately 200 amino acids, approximately 200 amino acids to approximately 800 amino acids, approximately 200 amino acids to approximately 750 amino acids, approximately 200 amino acids to approximately 700 amino acids, approximately 200 amino acids to approximately 650 amino acids, approximately 200 amino acids to approximately 600 amino acids, approximately 200 amino acids to approximately 550 amino acids, approximately 200 amino acids to approximately 500 amino acids, approximately 200 amino acids to approximately 450 amino acids, approximately 200 amino acids to approximately 400 amino acids, approximately 200 amino acids to approximately 350 amino acids, approximately 200 amino acids to approximately 300 amino acids, approximately 200 amino acids to approximately 250 amino acids,Approximately 250 amino acids to approximately 800 amino acids, approximately 250 amino acids to approximately 750 amino acids, approximately 250 amino acids to approximately 700 amino acids, approximately 250 amino acids to approximately 650 amino acids, approximately 250 amino acids to approximately 600 amino acids, approximately 250 amino acids to approximately 550 amino acids, approximately 250 amino acids to approximately 500 amino acids, approximately 250 amino acids to approximately 450 amino acids, approximately 250 amino acids to approximately 400 amino acids, approximately 250 amino acids to approximately 350 amino acids, approximately 250 amino acids to approximately 300 amino acids, approximately 300 amino acids to approximately 800 amino acids, approximately 300 amino acids to approximately 750 amino acids, approximately 300 amino acids to approximately 700 Amino acids, approximately 300 amino acids to approximately 650 amino acids, approximately 300 amino acids to approximately 600 amino acids, approximately 300 amino acids to approximately 550 amino acids, approximately 300 amino acids to approximately 500 amino acids, approximately 300 amino acids to approximately 450 amino acids, approximately 300 amino acids to approximately 400 amino acids, approximately 300 amino acids to approximately 350 amino acids, approximately 350 amino acids to approximately 800 amino acids, approximately 350 amino acids to approximately 750 amino acids, approximately 350 amino acids to approximately 700 amino acids, approximately 350 amino acids to approximately 650 amino acids, approximately 350 amino acids to approximately 600 amino acids, approximately 350 amino acids to approximately 550 amino acids, approximately 350 amino acids to approximately 600 amino acids Approximately 500 amino acids, approximately 350 to approximately 450 amino acids, approximately 350 to approximately 400 amino acids, approximately 400 to approximately 800 amino acids, approximately 400 to approximately 750 amino acids, approximately 400 to approximately 700 amino acids, approximately 400 to approximately 650 amino acids, approximately 400 to approximately 600 amino acids, approximately 400 to approximately 550 amino acids, approximately 400 to approximately 500 amino acids, approximately 400 to approximately 450 amino acids, approximately 450 to approximately 800 amino acids, approximately 450 to approximately 750 amino acids, approximately 450 to approximately 700 amino acids, approximately 450 Amino acids ~ approximately 650 amino acids, approximately 450 amino acids ~ approximately 600 amino acids, approximately 450 amino acids ~ approximately 550 amino acids, approximately 450 amino acids ~ approximately 500 amino acids, approximately 500 amino acids ~ approximately 800 amino acids, approximately 500 amino acids ~ approximately 750 amino acids, approximately 500 amino acids ~ approximately 700 amino acids, approximately 500 amino acids ~ approximately 650 amino acids, approximately 500 amino acids ~ approximately 600 amino acids, approximately 500 amino acids ~ approximately 550 amino acids, approximately 550 amino acids ~ approximately 800 amino acids, approximately 550 amino acids ~ approximately 750 amino acids, approximately 550 amino acids ~ approximately 700 amino acids, approximately 550 amino acids ~ approximately 650 amino acids,It may contain approximately 550 to 600 amino acids, approximately 600 to 800 amino acids, approximately 600 to 750 amino acids, approximately 600 to 700 amino acids, approximately 600 to 650 amino acids, approximately 650 to 800 amino acids, approximately 650 to 750 amino acids, approximately 650 to 700 amino acids, approximately 700 to 800 amino acids, approximately 700 to 750 amino acids, or approximately 750 to 800 amino acids.

[0107] In some embodiments of any ACC described herein, one or more linkers (e.g., flexible linkers) can be introduced into the activatable cytokine construct to provide flexibility to one or more junctions between domains, between parts, between parts and domains, or to any other junctions where a linker is deemed beneficial. In some embodiments where the ACC is provided as a conformationally constrained construct, flexible linkers can be inserted to facilitate the formation and maintenance of structure in the uncleaved activatable cytokine construct. Any linker described herein can provide desired flexibility to facilitate inhibition of binding to a target (e.g., a cytokine receptor) or to facilitate cleavage of CM by a protease. In some embodiments, a linker that is entirely or partially flexible is incorporated into the ACC, thereby allowing the linker to include a flexible linker and a portion that provides one or more less flexible structures in order to obtain the desired ACC. Some linkers contain cysteine ​​residues, which can form disulfide bonds and reduce the flexibility of the construct. In some embodiments, shortening the length of the linker or linking region reduces the activity of mature cytokine proteins in the ACC (see, e.g., Figures 8A, 8B and 10A, 10B). In most cases, the linker length 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 preceding component to the C-terminus of the linker adjacent to the N-terminal amino acid of the following component, in the N-to-C direction (i.e., in this case, the linker length does not include the C-terminal amino acid of the preceding component or the N-terminal amino acid of the following component). In some embodiments where a linker is used at the N-terminus of a DD containing an Fc domain, the linker length 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 preceding component to the C-terminus of the linker adjacent to the first cysteine ​​of the Fc hinge region (i.e., in this case, the linker length does not include the C-terminal amino acid of the preceding component or the first cysteine ​​of the Fc hinge region).

[0108] As is evident from this disclosure and Figure 25, the ACC of this disclosure includes a sequence of amino acids between the CP and the proximity point of interaction between the dimerized domains. This sequence of amino acids is sometimes referred to as the linking region (LR). As used herein, the terms “linking region” or “LR” refer to a sequence of amino acid residues between the C-terminus of the cytokine and the N-terminal amino acid residue adjacent to the proximity point of interaction between the dimerized domains (i.e., the linking region does not include the C-terminal amino acid of the cytokine or the N-terminal amino acid of the DD that forms the proximity point of interaction to the corresponding second monomer). For example, if the DD is a pair of Fc domains, the linking region is a sequence of amino acid residues between the C-terminus of the cytokine and the first N-terminal cysteine ​​residue contributing to the disulfide bond of the Fc (e.g., cysteine ​​226 of the IgG1 or IgG4 Fc domain, according to EU numbering). If the dimerized domain is not a peptide, the linking region is a sequence 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 sequence 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 sequence of amino acid residues between the C-terminus of the cytokine and the streptavidin molecule. In some embodiments, the linking region may contain 24, 18, 14, 12, 11, 10, 9, 8, 7, 6, 5, or 4 amino acids or less, for example, 5-14, 7-12, or 8-11 amino acids.

[0109] In some embodiments, additional amino acid sequences may be positioned at the N-terminus or C-terminus of any of the domains of ACC. Examples include, but are not limited to, a targeting moiety (e.g., a ligand for a cell receptor present in a target tissue) and a serum half-life extension moiety (e.g., a polypeptide that binds to an immunoglobulin serum protein) or serum albumin (e.g., human serum albumin (HSA)).

[0110] In some embodiments of any cytokine construct described herein, the linker comprises a total of about 1 to about 25 amino acids (e.g., about 1 to about 24 amino acids, about 1 to about 22 amino acids, about 1 to about 20 amino acids, about 1 to about 18 amino acids, about 1 to about 16 amino acids, about 1 to about 15 amino acids, about 1 to about 14 amino acids, about 1 to about 12 amino acids, about 1 to about 10 amino acids, about 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, approximately 1 to approximately 3 amino acids, approximately 1 to approximately 2 amino acids, approximately 2 to approximately 25 amino acids, approximately 2 to approximately 24 amino acids, approximately 2 to approximately 22 amino acids, approximately 2 to approximately 20 amino acids, approximately 2 to approximately 18 amino acids, approximately 2 to approximately 16 amino acids, approximately 2 to approximately 15 amino acids, approximately 2 to approximately 14 amino acids, approximately 2 to approximately 12 amino acids, approximately 2 to approximately 10 amino acids, approximately 2 to approximately 8 amino acids, approximately 2 to approximately 6 amino acids, approximately 2 to approximately 5 amino acids, approximately 2 to approximately 4 amino acids, approximately 2 to approximately 3 amino acids, approximately 4 to approximately 25 amino acids, approximately 4 to approximately 24 amino acids, approximately 4 to approximately 22 amino acids, approximately 4 to approximately 20 amino acids, approximately 4 to approximately 18 amino acids, approximately 4 to approximately 16 amino acids, approximately 4 to approximately 15 amino acids, approximately 4 to approximately 14 amino acids, approximately 4 to approximately 12 amino acids, approximately 4 to approximately 10 amino acids, approximately 4 to approximately 8 amino acids, approximately 4 to approximately 6 amino acids, approximately 4 to approximately 5 amino acids, approximately 5 to approximately 25 amino acids, approximately 5 to approximately 24 amino acids, approximately 5 to approximately 22 amino acids, approximately 5 amino acids Acid ~ approximately 20 amino acids, approximately 5 amino acids ~ approximately 18 amino acids, approximately 5 amino acids ~ approximately 16 amino acids, approximately 5 amino acids ~ approximately 15 amino acids, approximately 5 amino acids ~ approximately 14 amino acids, approximately 5 amino acids ~ approximately 12 amino acids, approximately 5 amino acids ~ approximately 10 amino acids, approximately 5 amino acids ~ approximately 8 amino acids, approximately 5 amino acids ~ approximately 6 amino acids, approximately 6 amino acids ~ approximately 25 amino acids, approximately 6 amino acids ~ approximately 24 amino acids, approximately 6 amino acids ~ approximately 22 amino acids, approximately 6 amino acids ~ approximately 20 amino acids, approximately 6 amino acids ~ approximately 18 amino acids, approximately 6 amino acids ~ approximately 16 amino acids, approximately 6 amino acids ~ approximately 15 amino acids, approximately 6 amino acids ~ approximately 14 amino acids,Approximately 6 to 12 amino acids, approximately 6 to 10 amino acids, approximately 6 to 8 amino acids, approximately 8 to 25 amino acids, approximately 8 to 24 amino acids, approximately 8 to 22 amino acids, approximately 8 to 20 amino acids, approximately 8 to 18 amino acids, approximately 8 to 16 amino acids, approximately 8 to 15 amino acids, approximately 8 to 14 amino acids, approximately 8 to 12 amino acids, approximately 8 to 10 amino acids, approximately 10 to 25 amino acids, approximately 10 to 24 amino acids, approximately 10 Mino acids ~ approximately 22 amino acids, approximately 10 amino acids ~ approximately 20 amino acids, approximately 10 amino acids ~ approximately 18 amino acids, approximately 10 amino acids ~ approximately 16 amino acids, approximately 10 amino acids ~ approximately 15 amino acids, approximately 10 amino acids ~ approximately 14 amino acids, approximately 10 amino acids ~ approximately 12 amino acids, approximately 12 amino acids ~ approximately 25 amino acids, approximately 12 amino acids ~ approximately 24 amino acids, approximately 12 amino acids ~ approximately 22 amino acids, approximately 12 amino acids ~ approximately 20 amino acids, approximately 12 amino acids ~ approximately 18 amino acids, approximately 12 amino acids ~ approximately 16 amino acids, approximately 12 amino acids ~ approximately 15 amino acids, approximately 12 amino acids ~ approximately 14 Amino acids, approximately 14 amino acids to approximately 25 amino acids, approximately 14 amino acids to approximately 24 amino acids, approximately 14 amino acids to approximately 22 amino acids, approximately 14 amino acids to approximately 20 amino acids, approximately 14 amino acids to approximately 18 amino acids, approximately 14 amino acids to approximately 16 amino acids, approximately 14 amino acids to approximately 15 amino acids, approximately 15 amino acids to approximately 25 amino acids, approximately 15 amino acids to approximately 24 amino acids, approximately 15 amino acids to approximately 22 amino acids, approximately 15 amino acids to approximately 20 amino acids, approximately 15 amino acids to approximately 18 amino acids, approximately 15 amino acids to approximately 16 amino acids, approximately 16 amino acids to approximately 25 amino acids, approximately 1 It may contain 6 to approximately 24 amino acids, approximately 16 to approximately 22 amino acids, approximately 16 to approximately 20 amino acids, approximately 16 to approximately 18 amino acids, approximately 18 to approximately 25 amino acids, approximately 18 to approximately 24 amino acids, approximately 18 to approximately 22 amino acids, approximately 18 to approximately 20 amino acids, approximately 20 to approximately 25 amino acids, approximately 20 to approximately 24 amino acids, approximately 20 to approximately 22 amino acids, approximately 22 to approximately 25 amino acids, approximately 22 to approximately 24 amino acids, or approximately 24 to approximately 25 amino acids.

[0111] In some embodiments of any ACC described herein, the linker comprises 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.

[0112] Surprisingly, the inventors discovered that ACC without any linker between CP and DD showed the most significant reduction in cytokine activity compared to wild-type mature cytokines. See Figures 8A and 10A. Furthermore, the arrangement without a linker between CP and DD still allows for effective cleavage of the CM placed between CP and DD. See Figures 12-14. Thus, in some embodiments, ACC contains no linker between CP and DD, and the CM between CP and DD contains 10, 9, 8, 7, 6, 5, 4, or 3 or fewer amino acids. In some embodiments, the total number of amino acids in the linking region is 25 amino acids or less, for example, 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 less, or any range or a specific number of amino acids selected from the range encompassed by 3 to 10 amino acids, 5 to 15 amino acids, 7 to 12 amino acids, or 3 to 25 amino acids.

[0113] In some embodiments of any ACC described herein, the linker may be rich in glycine (Gly or G) residues. In some embodiments, the linker may be rich in serine (Ser or S) residues. In some embodiments, the linker may be rich in both glycine and serine residues. In some embodiments, the linker has one or more glycine-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).

[0114] In some embodiments of any ACC described herein, the linker is GSSGGSGGSGG (SEQ ID NO: 210), GGGS (SEQ ID NO: 2), GGGSGGGS (SEQ ID NO: 211), GGGSGGGSGGGS (SEQ ID NO: 212), GGGGSGGGGSGGGGS (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), GGGGSGG This includes one or a combination of any one of the following: GGSGGGGSGS (SEQ ID NO: 218), GGSLDPKGGGGS (SEQ ID NO: 219), PKSCDKTHTCPPCPAPELLG (SEQ ID NO: 220), SKYGPPCPPCPAPEFLG (SEQ ID NO: 221), GKSSGSGSESKS (SEQ ID NO: 222), GTSTGSGKSSEGKG (SEQ ID NO: 223), GTSTGSGKSSEGSGSTKG (SEQ ID NO: 224), and GTSTGSGKPGSGEGSTKG (SEQ ID NO: 225).

[0115] Non-restrictive examples of linkers may include sequences that are 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 (sequence number 2), GSSGGSGGSGG (sequence number 210), GGGGSGGGGSGGGGGS (sequence number 213), GGGGSGS (sequence number 217), GGGGSGGGGSGGGGSGS (sequence number 218), GGGGSGGGGSGGGGSGGGGS (sequence number 214), GGSLDPKGGGGS (sequence number 215), and GTSTGSGKPGSSEGST (sequence number 226).

[0116] In some embodiments, the linker includes an array selected from the group consisting 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), GGGGSGGGGSGGGGS (SEQ ID NO: 213), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 214), GTSTGSGKPGSSEGST (SEQ ID NO: 226), and (GGGGS)n (SEQ ID NO: 216). In the formula, n is at least an integer of 1. In some embodiments, the linker includes 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 ACC described herein, the linker includes a sequence selected from the group consisting of GGGSGGGGSGGGGS (SEQ ID NO: 213), GGGSGGGGSGGGGSGGGGS (SEQ ID NO: 214), and GTSTGSGKPGSSEGST (SEQ ID NO: 226). In some embodiments of any activatable cytokine construct described herein, the linker includes a sequence selected from the group consisting of GGGSGGGGSGGGGS (SEQ ID NO: 213) or GGGGS (SEQ ID NO: 216). In some embodiments, the linker includes the sequence GGGS (SEQ ID NO: 2).

[0117] In some embodiments, ACC may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linker sequences (e.g., linker sequences that are the same as or different from any of the exemplary linker sequences described herein or known in the art). In some embodiments, the linker comprises sulfo-SIAB, SMPB, and sulfo-SMPB, and the linker reacts with a primary amine sulfhydryl.

[0118] In some embodiments of any ACC described herein, the ACC is characterized by a reduction in the activity of at least one of CP1 and / or CP2 compared to a control level of the activity of at least one of CP1 and / or CP2. In some embodiments, the control level may 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 may be the activity level of the cleavage (activation) form of the ACC. In certain embodiments, the control level may be the activation level of pegylated CP1 and / or CP2.

[0119] In some embodiments, the activity of at least one of the CP1 and / or CP2 receptors is measured using surface plasmon resonance (e.g., performed in phosphate-buffered saline at 25°C) by the binding affinity (K) of the CP1 and / or CP2 receptors. D ) In certain embodiments, at least one activity is the level of lymphoma cell proliferation. In other embodiments, at least one activity is the level of JAK / STAT / ISGF3 pathway activation in lymphoma cells. In some embodiments, at least one activity is the level of SEAP production in lymphoma cells. In further embodiments, at least one activity of CP1 and / or CP2 is the level of cytokine-stimulated gene induction using, for example, RNA sequencing (e.g., Zimmerer et al., Clin. Cancer Res. 14(18):5900-5906, 2008; Hilkens et al., J. Immunol. 171:5255-5263, 2003).

[0120] In some embodiments, the ACC is characterized by at least a 2-fold reduction in at least one CP1 and / or CP2 activity compared to a control level of at least one CP1 and / or CP2 activity. In some embodiments, the ACC is characterized by at least a 5-fold reduction in at least one CP1 and / or CP2 activity compared to a control level of at least one CP1 and / or CP2 activity. In some embodiments, the ACC is characterized by at least a 10-fold reduction in at least one CP1 and / or CP2 activity compared to a control level of at least one CP1 and / or CP2 activity. In some embodiments, the ACC is characterized by at least a 20-fold reduction in at least one CP1 and / or CP2 activity compared to a control level of at least one CP1 and / or CP2 activity. In some embodiments, the ACC is characterized by a reduction of at least 30, 40, 50, 60, 70, 80, 90, 100, 500, or 1000 times in the activity of at least one of CP1 and / or CP2 compared to a control level of the activity of at least one of CP1 and / or CP2. In some embodiments, ACC is reduced by 1 to 20 times, 200 to 500 times, 300 to 500 times, 400 to 500 times, 500 to 600 times, 600 to 700 times, 150 to 1000 times, 100 to 1500 times, 200 to 1500 times, 300 to 1500 times, 400 to 1500 times, and 50 times compared to the control level of the activity of at least one of CP1 and / or CP2. It is characterized by a reduction of 0 to 1500 times, a reduction of 1000 to 1500 times, a reduction of 100 to 1000 times, a reduction of 200 to 1000 times, a reduction of 300 to 1000 times, a reduction of 400 to 1000 times, a reduction of 500 to 1000 times, a reduction of 100 to 500 times, a reduction of 20 to 50 times, a reduction of 30 to 50 times, a reduction of 40 to 50 times, a reduction of 100 to 400 times, a reduction of 200 to 400 times, or a reduction of 300 to 400 times, a reduction of 100 to 300 times, a reduction of 200 to 300 times, or a reduction of 100 to 200 times.

[0121] In some embodiments, the control level of the activity of at least one of CP1 and / or CP2 is the activity of CP1 and / or CP2 released from ACC after cleavage of CM1 and CM2 by protease ("cleavage products"). In some embodiments, the control level of the activity of at least one of CP1 and / or CP2 is the activity of the corresponding wild-type mature cytokine (e.g., recombinant wild-type mature cytokine).

[0122] In some embodiments, incubation of ACC with protease yields an activated cytokine product in which the activity of one or more CP1 and / or CP2 in the activated cytokine product is greater than the activity of one or more CP1 and / or CP2 in untreated ACC. In some embodiments, the activity of one or more CP1 and / or CP2 in the activated cytokine product is 1-fold greater than the activity of one or more CP1 and / or CP2 in ACC. In some embodiments, the activity of one or more CP1 and / or CP2 in the activated cytokine product is 2-fold greater than the activity of one or more CP1 and / or CP2 in ACC. In some embodiments, the activity of one or more CP1 and / or CP2 in the activated cytokine product is 5-fold greater than the activity of one or more CP1 and / or CP2 in ACC. In some embodiments, the activity of one or more CP1 and / or CP2 in the activated cytokine product is 10-fold greater than the activity of one or more CP1 and / or CP2 in ACC. In some embodiments, the activity of one or more CP1 and / or CP2 of the activated cytokine product is 20 times greater than the activity of one or more CP1 and / or CP2 of the ACC. In some embodiments, the activity of one or more CP1 and / or CP2 of the activated cytokine product is at least 1 to 20 times greater, 2 to 20 times greater, 3 to 20 times greater, 4 to 20 times greater, 5 to 20 times greater, 10 to 20 times greater, 15 to 20 times greater, 1 to 15 times greater, 2 to 15 times greater, 3 to 15 times greater, 4 to 15 times greater, 5 to 15 times greater, 10 to 15 times greater, 1 to 10 times greater, 2 to 10 times greater, 3 to 10 times greater, 4 to 10 times greater, 5 to 10 times greater, 1 to 5 times greater, 2 to 5 times greater, 3 to 5 times greater, 4 to 5 times greater, 1 to 4 times greater, 2 to 4 times greater, 3 to 4 times greater, 1 to 3 times greater, 2 to 3 times greater, or 1 to 2 times greater than the activity of one or more CP1 and / or CP2 of ACC.

[0123] In some embodiments, the ACC may include sequences that are 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 sequence numbers 309 or 311. In some embodiments, the ACC may be encoded by nucleic acids that include sequences that are 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 sequence numbers 310 or 312. In some embodiments, the ACC includes such sequences, but none of them include the signal sequences of these sequences. The signal sequences are not particularly limited. Some non-limiting examples of signal sequences include, for example, residues 1-20 of sequence number 309 and corresponding residues and nucleotides in other sequences or derived from signal sequences of another species or cell line. Other examples of signal sequences include MRAWIFFLLCLAGRALA (sequence number 343) and MALTFALLVALLVLSCKSSCSVG (sequence number 344).

[0124] Various exemplary embodiments of these activatable cytokine constructs are described below and can be used without limitation in any combination by the methods provided herein. The activatable cytokine constructs and methods for producing them are described below.

[0125] In some embodiments, CM is selected for use with a specific protease. The protease may be one produced in tumor cells (for example, tumor cells may express larger amounts of proteases than healthy tissue). In some embodiments, CM is a substrate for at least one protease selected from the group consisting of cysteine ​​proteases such as ADAM17, BMP-1, cathepsin, HtrA1, regmine, matryptase (MT-SP1), matrix metalloproteinase (MMP), neutrophil elastase, TMPRSS such as TMPRSS3 or TMPRSS4, thrombin, and u-type plasminogen activator (uPA, also called urokinase).

[0126] In some embodiments, CM is a substrate for at least one matrix metalloproteinase (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, CM is a substrate for MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11, and MMP19. In some embodiments, CM is a substrate for MMP7. In some embodiments, CM is a substrate for MMP9. In some embodiments, CM is a substrate for MMP14. In some embodiments, CM is a substrate for two or more MMPs. In some embodiments, CM is a substrate for at least MMP9 and MMP14. In some embodiments, the CM includes two or more substrates for the same MMP. In some embodiments, the CM includes at least two or more MMP9 substrates. In some embodiments, the CM includes at least two or more MMP14 substrates.

[0127] In some embodiments, CM is a substrate for MMP and includes the sequence ISSGLLSS (sequence number 19); QNQALRMA (sequence number 16); AQNLLGMV (sequence number 15); STFPFGMF (sequence number 18); PVGYTSSL (sequence number 74); DWLYWPGI (sequence number 75); MIAPVAYR (sequence number 42); RPSPMWAY (sequence number 43); WATPRPMR (sequence number 44); FRLLDWQW (sequence number 45); LKAAPRWA (sequence number 76); GPSSHLVLT (sequence number 77); LPGGLSPW (sequence number 78); MGLFSEAG (sequence number 79); SPLPLRVP (sequence number 80); RMHLRSLG (sequence number 81); LAAPLGLL (sequence number 17); AVGLLAPP (sequence number 14); LLAPSHRA (sequence number 82); PAGLWLDP (sequence number 20); and / or ISSGLSS (sequence number 73).

[0128] In some embodiments, CM is a substrate for thrombin. In some embodiments, CM is a substrate for thrombin and includes the sequence GPRSFGL (SEQ ID NO: 83) or GPRSFG (SEQ ID NO: 84).

[0129] In some embodiments, CM includes an amino acid sequence selected from the group consisting of NTLSGRSENHSG (SEQ ID NO: 9); NTLSGRSGNHGS (SEQ ID NO: 10); TSTSGRSANPRG (SEQ ID NO: 11); TSGRSANP (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).

[0130] In some embodiments, CM is a substrate for neutrophil elastase. In some embodiments, CM is a substrate for serine proteases. In some embodiments, CM is a substrate for uPA. In some embodiments, CM is a substrate for legmine. In some embodiments, CM is a substrate for matryptase. In some embodiments, CM is a substrate for serine proteases. In some embodiments, CM is a substrate for cysteine ​​proteases such as cathepsin.

[0131] In some embodiments, 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 Includes the sequence GRSDNHGGVHMPLGFLGP (sequence code 267);LSGRSDNHGGSGGSISSGLLSS (sequence code 268);LSGRSGNHGGSGGSISSGLLSS (sequence code 279);ISSGLLSSGGSGGSLSGRSGNH (sequence code 269);LSGRSDNHGGSGGSQNQALRMA (sequence code 270);QNQALRMAGGSGGSLSGRSDNH (sequence code 271);LSGRSGNHGGSGGSQNQALRMA (sequence code 272);QNQALRMAGGSGGSLSGRSGNH (sequence code 273), and / or ISSGLLSGRSGNH (sequence code 274).

[0132] In some embodiments, CM1 and / or CM2 include sequences selected from the group consisting of SEQ ID NOs. 5 to 100. In some embodiments, CM includes sequences selected from the group consisting 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).

[0133] In some embodiments, the ACC includes CP1 selected from SEQ ID NOs: 1 and 101-209, CM1 selected from SEQ ID NOs: 5-100 and 263-308, and DD1 dimerized with CP2 selected from SEQ ID NOs: 1 and 101-209, CM2 selected from SEQ ID NOs: 5-100 and 263-308, and DD2. In some embodiments, the ACC may include linkers selected from SEQ ID NOs: 2 and 210-234, 245, or 250 between CP1 and CM1 and / or between CM1 and DD1, and linkers selected from SEQ ID NOs: 2 and 210-234, 245, or 250 between CP2 and CM2 and / or between CM2 and DD2. In some embodiments, ACC includes DD1 and / or DD2 having an amino acid sequence that is at least 80% identical to SEQ ID NO: 3 or SEQ ID NO: 4 (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). In some embodiments, ACC includes DD1 having an amino acid sequence that is at least 80% identical to SEQ ID NO: 315 or SEQ ID NO: 316 (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). In some embodiments, ACC includes DD2 having an amino acid sequence that is at least 80% identical to SEQ ID NO: 315 or SEQ ID NO: 316 (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).

[0134] Conjugation of drugs This disclosure also provides methods and materials for incorporating additional elements into any of the ACCs described herein, for example, a targeting moiety that facilitates delivery to cells or tissues of interest, a drug (e.g., a therapeutic agent, an antitumor agent), a toxin, or fragments thereof.

[0135] In some embodiments of any ACC described herein, the ACC can be conjugated to a cytotoxic agent, including, but not limited to, a toxin (e.g., an enzymatically active toxin or fragment thereof of bacterial, fungal, plant, or animal origin) or a radioisotope. In some embodiments of any ACC described herein, the activatable cytokine construct can be conjugated to a cytotoxic agent, including, but not limited to, a toxin (e.g., an enzymatically active toxin or fragment thereof of bacterial, fungal, plant, or animal origin) or a radioisotope.

[0136] Non-limiting examples of cytotoxic agents that can be conjugated to any of the ACCs described herein include drastatin and its derivatives (e.g., auristatin E, AFP, monomethyl auristatin D (MMAD), monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), desmethyl auristatin E (DMAE), auristatin F, desmethyl auristatin F (DMAF), drastatin 16 (DmJ), drastatin 16 (Dpv), auristatin derivatives (e.g., auristatin tyramine, auristatin quinolone)), mytansinoids (e.g., DM-1, DM-4), mytansinoid derivatives, duocalmycin, α-amanitin, turvostatin, fenstatin, hydroxyfenstatin Examples include spongistatin 5, spongistatin 7, halistatin 1, halistatin 2, halistatin 3, halocomstatin, pyrrolobenzimidazole (PBI), sibrostatin 6, doxaliform, semadin analog (CemCH2-SH), Pseudomonas toxin A (PES8) variant, Pseudomonas toxin A (ZZ-PE38) variant, ZJ-101, anthracyclines, doxorubicin, daunorubicin, bryostatin, camptothecin, 7-substituted camptothecin, 10,11-difluoromethylenedioxycamptothecin, combretastatin, debromoaprysiatoxin, KahaMide-F, discodermolide, and ecteinacidin.

[0137] Non-limiting examples of enzymatically active toxins that can be conjugated to any of the ACCs described herein include diphtheria toxin, Pseudomonas aeruginosa exotoxin A chain, lysine A chain, abrin A chain, modesin A chain, alpha-sarcin, Aleurites fordii, dianfhin protein, pokeweed (e.g., PAPI, PAPII, and PAP-8), bitter melon inhibitors, curcin, crotirs, soapwort inhibitors, geionin, mitogeliin, restrictosin, phenomycin, neomycin, and trichothecin.

[0138] Non-limiting examples of anticancer drugs that can be conjugated to any of the ACCs described herein include Adriamycin, Seruvidine, Bleomycin, Alkeran, Verban, Oncovin, Fluorouracil, Methotrexate, Thiotepa, Bisanthren, Novantrone, Thioguanine, Procarbazine, and Cytarabine.

[0139] Non-limiting examples of antiviral drugs that can be conjugated to any of the ACCs described herein include acyclovir, vir-A, and symmetrel.

[0140] An example of an antifungal agent that can be conjugated to any of the ACCs described herein is nystatin.

[0141] Non-limiting examples of conjugable detection reagents that can be conjugated to any of the ACCs described herein include fluorescein and its derivatives and fluorescein isothiocyanate (FITC).

[0142] Non-limiting examples of antimicrobial agents that can be conjugated to any of the activatable cytokine constructs described herein include aminoglycosides, streptomycin, neomycin, kanamycin, amikacin, gentamicin, and tobramycin.

[0143] Non-limiting examples of 3β,16β,17α-trihydroxycholesta-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 hemiasterin, cephalotaxin, homohalintin, pyrrolobenzodiazepine dimers (PBDs), functionalized pyrrolobenzodiazepines, calitiamycin, podophyllotoxin, taxanes, and vinca alkaloids.

[0144] Non-limiting examples of 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 mTc (technetium).

[0145] Non-limiting examples of heavy metals that can be conjugated to any of the ACCs described herein include barium, gold, and platinum.

[0146] Non-limiting examples of anti-mycoplasma drugs that can be conjugated to any of the ACCs described herein include tylosin, spectinomycin, streptomycin B, ampicillin, sulfanilamide, polymyxin, and chloramphenicol.

[0147] Those skilled in the art will understand that a wide range of possible parts can be conjugated into any of the activatable cytokine constructs described herein. Conjugation may involve any chemical reaction that joins the two molecules, insofar as ACC and the other parts retain their respective activities. Conjugation may involve many chemical mechanisms, e.g., covalent bonding, affinity bonding, intercalation, coordination bonding, and complexation. In some embodiments, the preferred bond is covalent bonding. Covalent bonding can be achieved either by direct condensation of existing side chains or by the incorporation of external crosslinking molecules. Many divalent or polyvalent linkers are useful in conjugating any of the activatable cytokine constructs of this disclosure. For example, conjugation may include organic compounds such as thioesters, carbodiimides, succinimides, glutaraldehyde, diazobenzene, and hexamethylenediamine. In some embodiments, the activatable cytokine construct may contain, or can be introduced, one or more native amino acid residues to provide a suitable site for conjugation.

[0148] In some embodiments of any ACC described herein, the drug and / or conjugate is attached to the antigen-binding domain by disulfide bonds (e.g., disulfide bonds on a cysteine ​​molecule). Since many cancers naturally release high levels of the reducing agent glutathione, the glutathione present in the cancer tissue microenvironment can reduce disulfide bonds, thereby releasing the drug and / or conjugate at the delivery site.

[0149] In some embodiments of any ACC described herein, when a conjugate binds to its target in the presence of complement within a target site (e.g., affected tissue (e.g., cancerous tissue)), the amide or bond attaching the conjugate and / or drug to the linker is cleaved, resulting in the release of the conjugate and / or drug in its activated form. Upon administration to a subject, these conjugates and / or drugs achieve delivery and release at the target site (e.g., affected tissue (e.g., cancerous tissue)). These conjugates and / or drugs are particularly effective for in vivo delivery of any of the conjugates and / or drugs described herein.

[0150] In some embodiments, the linker is not cleavable by complement system enzymes. For example, the conjugate and / or drug is released without complement activation, as complement activation would ultimately lyse the target cells. In such embodiments, the conjugate and / or drug is delivered to the target cells (e.g., hormones, enzymes, corticosteroids, neurotransmitters, or genes). Furthermore, the linker is less sensitive to cleavage by serum proteases, and the conjugate and / or drug is released slowly at the target site.

[0151] In some embodiments of any ACC described herein, the conjugate and / or drug is designed such that the conjugate and / or drug is delivered to a target site (e.g., diseased tissue (e.g., cancerous tissue)) but the conjugate and / or drug is not released.

[0152] In some embodiments of any ACC described herein, the conjugate and / or drug is attached to the antigen-binding domain 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 include functional groups that can later be used for binding to the antigen-binding domain.

[0153] In some embodiments of any ACC described herein, the ACC includes at least one conjugation site for a drug. In some embodiments, all possible conjugation sites are available for conjugation to a drug. In some embodiments, one or more conjugation sites include, but are not limited to, a sulfur atom involved in disulfide bonding, a sulfur atom involved in interchain disulfide bonding, a sulfur atom involved in interchain sulfide bonding but not in intrachain disulfide bonding, and / or a sulfur atom of cysteine ​​or other amino acid residues containing a sulfur atom. In such cases, the residues may be naturally present in the protein construct structure or may be incorporated into the protein construct by means of, but not limited to, site-directed mutagenesis, chemical transformation, or misincorporation of non-native amino acids.

[0154] This disclosure also provides methods and materials for preparing ACC for conjugation. In some embodiments of any ACC described herein, the ACC is modified to include one or more interchain disulfide bonds. For example, disulfide bonds in the ACC may be reduced after exposure to a reducing agent such as TCEP, DTT, or β-mercaptoethanol, but are not limited. In some cases, the reduction of disulfide bonds is only partial. As used herein, partial reduction of the term means a situation in which the ACC comes into contact with a reducing agent and some of all possible conjugation sites are reduced (e.g., not all disulfide bonds are reduced). In some embodiments, the activatable cytokine construct is partially reduced after contact with a reducing agent if less than 99% of all possible conjugation sites (e.g., less than 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or less than 5%) are reduced. In some embodiments, ACCs having reduction in disulfide bonds between one or more chains are conjugated to a drug that is reactive with free thiols.

[0155] This disclosure also provides methods and materials for conjugating therapeutic agents to specific sites on ACC. In some embodiments of any ACC described herein, the ACC is modified so that a therapeutic agent can conjugate to the ACC at a specific site on the ACC. For example, the ACC may be partially reduced in a manner that facilitates conjugation to the ACC. In such cases, the partial reduction of the ACC occurs in a manner that the conjugation site in the ACC is not reduced. In some embodiments, the conjugation site on the ACC is selected to facilitate conjugation of a drug at a specific site on a protein construct. Various factors may affect the “level of reduction” of the ACC when treated with a reducing agent. For example, but not limited to, the ratio of the reducing agent to the ACC, the length of incubation, the incubation temperature, and / or the pH of the reduction reaction solution may require optimization to achieve partial reduction of the ACC with the methods and materials described herein. Partial reduction of ACC (e.g., total reduction of possible conjugation sites or reduction at specific conjugation sites) can be achieved using 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).

[0156] The effective ratio of a reducing agent to ACC can be any ratio that reduces ACC at least partially in a manner that allows for conjugation to the drug (e.g., total reduction of possible conjugation sites or reduction at specific conjugation sites). In some embodiments, the ratio of reducing agent to ACC is in the range of approximately 20:1 to 1:1, approximately 10:1 to 1:1, approximately 9:1 to 1:1, approximately 8:1 to 1:1, approximately 7:1 to 1:1, approximately 6:1 to 1:1, approximately 5:1 to 1:1, approximately 4:1 to 1:1, approximately 3:1 to 1:1, approximately 2:1 to 1:1, approximately 20:1 to 1:1.5, approximately 10:1 to 1:1.5, approximately 9:1 to 1:1.5, approximately 8:1 to 1:1.5, approximately 7:1 to 1:1.5, approximately 6:1 to 1:1.5, approximately 5:1 to 1:1.5, approximately 4:1 to 1:1.5, approximately 3:1 to 1:1.5, approximately 2:1 to 1:1.5, approximately 1.5:1 to 1:1.5, or approximately 1:1 to 1:1.5. In some embodiments, the ratio is in the range of approximately 5:1 to 1:1. In some embodiments, the ratio is in the range of approximately 5:1 to 1.5:1. In some embodiments, the ratio is in the range of approximately 4:1 to 1:1. In some embodiments, the ratio is in the range of approximately 4:1 to 1.5:1. In some embodiments, the ratio is in the range of approximately 8:1 to 1:1. In some embodiments, the ratio is in the range of approximately 2.5:1 to 1:1.

[0157] The effective incubation time and temperature for treating ACC with a reducing agent may be any time and temperature that at least partially reduces the ACC in a manner that allows for the conjugation of the agent into the ACC (e.g., total reduction of possible conjugation sites or reduction at specific conjugation sites). In some embodiments, the incubation time and temperature for treating ACC is in the range of about 1 hour at 37°C to about 12 hours at 37°C (or any partial range thereof).

[0158] The effective pH for the reduction reaction to treat ACC with a reducing agent may be any pH that reduces ACC at least partially in a manner that allows for conjugation of ACC to the agent (e.g., total reduction of possible conjugation sites or reduction at specific conjugation sites).

[0159] When partially reduced ACC is brought into contact with a thiol-containing agent, the agent can conjugate to the interchain thiols in ACC. The agent can be modified to contain thiols using a thiol-containing reagent (e.g., cysteine ​​or N-acetylcysteine). For example, ACC can be partially reduced after incubation with a reducing agent (e.g., TEPC) at about 37°C for about 1 hour in a desired ratio of reducing agent to ACC. The effective ratio of reducing agent to ACC may be any ratio that partially reduces the ACC at at least two interchain disulfide bonds located in ACC in a manner that allows for the conjugation of the thiol-containing agent (e.g., total reduction of possible conjugation sites or reduction at specific conjugation sites).

[0160] In some embodiments of any ACC described herein, the ACC is reduced by a reducing agent in a manner that avoids reduction of any intrachain disulfide bonds. In some embodiments of any ACC described herein, the ACC is reduced by a reducing agent in a manner that avoids reduction of any intrachain disulfide bonds and reduces at least one interchain disulfide bond.

[0161] In some embodiments of any ACC described herein, the ACC may also include a drug conjugated to the ACC. In some embodiments, the conjugated drug is a therapeutic agent.

[0162] In some embodiments, the drug (e.g., a drug conjugated to an activatable cytokine construct) is a detectable moiety, such as a label or other marker. For example, the drug is a radiolabeled amino acid, one or more biotinyl moieties detectable by marked avidin (e.g., streptavidin containing a fluorescent marker or enzyme activity detectable by optical or calorimetry), one or more radioisotopes or radionuclides, one or more fluorescent labels, one or more enzyme labels, and / or one or more chemiluminescent agents, or comprising these. In some embodiments, the detectable moiety is attached by a spacer molecule.

[0163] In some embodiments, the drug (e.g., a cytotoxic drug 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.

[0164] In some embodiments of any ACC conjugated with a drug described herein, the drug (e.g., a cytotoxic drug conjugated with an activatable cytokine construct) is conjugated to the ACC via a linker and / or CM (also called a cleavable sequence). In some embodiments, the drug (e.g., a cytotoxic drug conjugated with an activatable cytokine construct) is conjugated to cysteine ​​or lysine in the ACC. In some embodiments, the drug (e.g., a cytotoxic drug conjugated with an activatable cytokine construct) is conjugated to another residue of the ACC, such as residues disclosed herein. In some embodiments, the linker is a thiol-containing linker. In some embodiments, the linker is a non-cleavable linker. Several non-limiting examples of cleavable moieties and linkers are provided in Table 1. [Table 1]

[0165] Those skilled in the art will understand that a wide variety of possible parts can be coupled to the ACCs of this disclosure. (See, for example, “Conjugate Vaccines”, Contributions to Microbiology and Immunology, JMCruse and RELewis, Jr(eds), Carger Press, New York, (1989), which is incorporated herein by reference in its entirety). In general, effective conjugation of a drug (e.g., a cytotoxic drug) to an ACC can be achieved by a chemical reaction that binds the drug to the ACC while simultaneously enabling both the drug and the ACC to retain their function.

[0166] In some embodiments of any ACC conjugated with a drug, the drug 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), difunctional derivatives of imide esters (e.g., dimethyladipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide 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, lysine immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). In some embodiments, radioactive nucleotides can be conjugated to ACC using a carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) chelating agent. (See, for example, International Publication No. 94 / 11026).

[0167] Suitable linkers and CMs are described in the literature. (For example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also U.S. Patent No. 5,030,719, which describes the use of halogenated acetylhydrazide derivatives coupled to ACC via an oligopeptide linker. In some embodiments, suitable linkers include (i) EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene) (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamide]hexanoate (Pierce Chem. Co.) (iv) Sulfo-LC-SPDP (Sulfosuccinimidyl 6[3-(2-Pyridyldithio)propionamide]hexanoate (Pierce Chem.Co. catalog number 2165-G)); and (v) Sulfo-NHS (N-hydroxysulfosuccinimidide: Pierce Chem.Co., catalog number 24510) conjugated to EDC. Additional linkers include, but are not limited to, SMCC, sulfo-SMCC, SPDB, or sulfo-SPDB.

[0168] The above-mentioned CMs and linkers contain components with various attributes and therefore result in conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. Linkers containing NHS esters are less soluble than sulfo-NHS esters. Furthermore, linker SMPTs contain sterically hindered disulfide bonds and can form conjugates with improved stability. Disulfide bonds are generally less stable than other bonds because they are cleaved in vitro, resulting in fewer available conjugates. Sulfo-NHS can enhance the stability of carbodiimide couplings in particular. When used with sulfo-NHS, carbodiimide couplings (such as EDC) form esters that are more resistant to hydrolysis than carbodiimide coupling reactions alone.

[0169] In some embodiments of any ACC, a drug can be conjugated to the ACC using a modified amino acid sequence contained within the amino acid sequence of the ACC. By inserting conjugable amino acids at specific positions within the amino acid sequence of the ACC, the protein construct can be designed for controlled placement and / or dosage of the conjugated drug. For example, the ACC can be modified to include a cysteine ​​amino acid residue at a position on the first monomer, second monomer, third monomer, and / or fourth monomer that provides a reactive thiol group without adversely affecting protein folding and / or construction and without altering binding properties. In some embodiments, the ACC can be modified to include one or more non-natural amino acid residues within the amino acid sequence of the ACC to provide a suitable site for conjugation. In some embodiments, the ACC can be modified to include an enzymatically activatable peptide sequence within the amino acid sequence of the ACC.

[0170] nucleic acid Provided herein are nucleic acids comprising sequences encoding a first monomer construct (or protein portion of a first monomer construct) (e.g., any of the first monomer constructs described herein) and a second monomer construct (or protein portion of a second monomer construct) (e.g., any of the second monomer constructs described herein) of any of the ACCs described herein. In some embodiments, a pair of nucleic acids together encodes the first monomer construct (or protein portion of a first monomer construct) and the second monomer construct (or protein portion of a second monomer construct). In some embodiments, the nucleic acid sequence encoding the first monomer construct (or the protein portion of the first monomer 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 monomer construct (or the protein portion of the second monomer construct).

[0171] In some embodiments, the nucleic acid encoding the protein portion of the first monomer construct encodes a polypeptide comprising CP1 and CM1 portions. In some embodiments, the nucleic acid encoding the protein portion of the second monomer construct encodes a polypeptide comprising CP2 and CM2 portions. In some embodiments, a pair of nucleic acids together encode the protein portion of the first monomer construct and the protein portion of the second monomer construct, and the protein portions are then conjugated to DD1 and DD2 portions, respectively (in a subsequent conjugation step).

[0172] In some embodiments, the nucleic acid encoding the first monomer construct encodes a polypeptide containing the DD1 moiety. In some embodiments, the nucleic acid encoding the second monomer construct encodes a polypeptide containing the DD2 moiety.

[0173] vector Provided herein are vectors and vector sets containing 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., an expression vector) for producing any of the ACCs described herein and for using the vector or vector set to express any of the ACCs described herein. For example, in the selection of a vector or vector set, cells must be taken into consideration, for the reason that the vector must be able to integrate into and / or replicate within the chromosomes of a cell. Exemplary vectors that can be used to produce ACCs are also described below.

[0174] As used herein, the term “vector” refers to a polynucleotide capable of inducing the 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” is capable of delivering nucleic acids and their fragments into a host cell and includes regulatory sequences (e.g., promoters, enhancers, poly(A) signals). Exogenous polynucleotides may be inserted into an expression vector for expression. The term “vector” also includes artificial chromosomes, plasmids, retroviruses, and baculovirus vectors.

[0175] Methods for constructing vectors containing any of the nucleic acids described herein and suitable for the transformation of cells (e.g., mammalian cells) are well known in the art. For example, Sambrook et al., Eds. “Molecular Cloning: A Laboratory Manual,” 2 ndSee Ed., Cold Spring Harbor Press, 1989 and Ausubel et al., Eds., “Current Protocols in Molecular Biology,” Current Protocols, 1993.

[0176] Non-limiting examples of vectors include plasmids, transposons, cosmids, and viral vectors (e.g., any adenovirus (e.g., pSV or pCMV vectors), adeno-associated virus (AAV) vectors, lentiviral vectors, and retroviruses), and any gateway® vectors. A vector may, for example, contain sufficient cis-acting elements for expression; other elements for expression can be supplied by host mammalian cells or in an in vitro expression system. A skilled practitioner will be able to select suitable vectors and mammalian cells for the production of any ACC described herein.

[0177] In some embodiments of any ACC described herein, the ACC may be prepared by biosynthesis using recombinant DNA technology and expression in eukaryotes or prokaryotes.

[0178] In some embodiments, the vector comprises nucleic acids encoding a first monomer and a second monomer of any of the ACCs described herein. In some embodiments, the vector is an expression vector.

[0179] In some embodiments, a pair of vectors comprises a pair of nucleic acids encoding together a first monomer and a second monomer of any of the ACCs described herein. In some embodiments, the vector pair is a pair of expression vectors.

[0180] cell Furthermore, provided herein are host cells comprising any vector or vector set described herein, which comprises any nucleic acid described herein.

[0181] 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.

[0182] Methods for introducing nucleic acids and vectors (e.g., any of the vectors or vector sets described herein) into cells are known in the art. Non-limiting examples of methods that can be used for introducing nucleic acids into cells include lipofection, transfection, calcium phosphate method, cationic polymer transfection, viral transfection (e.g., adenovirus transfection, lentivirus transfection), nanoparticle transfection, and electroporation.

[0183] In some embodiments, the introduction step includes introducing into cells a vector (e.g., any of the vectors or vector sets described herein) containing nucleic acids encoding monomers that constitute any of the ACCs described herein.

[0184] In some embodiments of any of the methods described herein, the cells may be eukaryotic cells. As used herein, the term “eukaryotic cell” refers to a cell having a distinct membrane-bound nucleus. Such cells may include, for example, mammalian (e.g., rodents, non-human primates, or humans), insect, fungal, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell, such as Saccharomyces cerevisiae (budding yeast) cell. In some embodiments, the eukaryotic cell is a higher eukaryotic cell, such as a mammalian, bird, plant, or insect cell. Non-limiting examples of mammalian cells include Chinese hamster ovary (CHO) cells and human fetal kidney cells (e.g., HEK293 cells).

[0185] In some embodiments, the cell comprises a nucleic acid encoding a first monomer and a second monomer of any of the ACCs described herein. In some embodiments, the cell comprises a nucleic acid pair encoding together the first monomer and the second monomer of any of the ACCs described herein.

[0186] Method for producing activatable cytokine constructs Provided herein is a method for producing any of the ACCs described herein, comprising (a) culturing any of the recombinant host cells described herein in a liquid medium under conditions sufficient to produce ACCs; and (b) recovering the ACCs from the host cells and / or the liquid medium.

[0187] Methods for culturing cells are well known in the art, and cells can be maintained in vitro under conditions favorable for 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 medium containing sufficient necessary growth factors and supplements to support cell survival and growth.

[0188] In some embodiments of any of the methods described herein, the method further includes 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., anionic or cationic), and hydrophobic interaction chromatography.

[0189] In some embodiments, cells can produce protein moieties of a first monomer construct containing CP1 and CM1 and protein moieties of a second monomer construct containing CP2 and CM2, which are then conjugated to DD1 and DD2 moieties, respectively.

[0190] The compositions and methods described herein may involve the use of non-reducing or partially reducing conditions that cause disulfide bonds to form between dimerized domains in order to form and maintain dimerization of ACC.

[0191] In some embodiments of any of the methods described herein, the method further includes formulating an isolated ACC into a pharmaceutical composition. Various formulations are known in the art and are described herein. Any isolated ACC described herein can be formulated for any route of administration (e.g., intravenous, intratumoral, subcutaneous, intradermal, oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, or intramuscular).

[0192] Furthermore, this specification provides ACC manufactured by any of the methods described herein. Furthermore, this specification provides compositions (e.g., pharmaceutical compositions) comprising any ACC manufactured by any of the methods described herein. Furthermore, this specification provides kits comprising at least one dosage of any of the compositions (e.g., pharmaceutical compositions) described herein.

[0193] Treatment methods Provided herein is a method for treating a disease of interest (e.g., cancer (e.g., any of the cancers described herein)) comprising administering to the subject a therapeutically effective dose of any of the ACCs described herein.

[0194] As used herein, the term “subject” refers to any mammal. In some embodiments, the subject is a cat (e.g., cat), a dog (e.g., dog), a horse (e.g., horse), a rabbit, a pig, a rodent (e.g., mouse, rat, hamster, or guinea pig), a non-human primate (e.g., monkey (e.g., baboon, golden macaque)), or ape (e.g., chimpanzee, gorilla, orangutan, or gibbon)), or a human. In some embodiments, the subject is a human.

[0195] In some embodiments, the subject has been previously identified or diagnosed with a disease (e.g., cancer (e.g., any of the cancers described herein)).

[0196] As used herein, the term “treat” includes reducing the severity, frequency, or symptoms or signs of one or more (e.g., one, two, three, four, or five) diseases (e.g., cancer (e.g., any of the cancers described herein)) of a subject (e.g., any of the subjects described herein). In some embodiments where the disease is cancer, treating results in a reduction of cancer growth, inhibition of cancer progression, inhibition of cancer metastasis, or a reduction in the risk of cancer recurrence in the cancer subject.

[0197] In some embodiments of any of the methods described herein, the disease is cancer. Provided herein is a method of treating a subject in need (e.g., any exemplary subject described herein or known in the art) by administering a therapeutically effective amount of any ACC or any composition described herein (e.g., a pharmaceutical composition) to the subject.

[0198] In some embodiments of these methods, the subject is identified or diagnosed with cancer. Non-limiting examples of cancer include solid tumors, hematological malignancies, sarcomas, osteosarcomas, glioblastomas, neuroblastomas, melanomas, rhabdomyosarcomas, Ewing's sarcoma, osteosarcomas, B-cell neoplasms, multiple myeloma, lymphomas (e.g., B-cell lymphoma, B-cell non-Hodgkin lymphoma, Hodgkin lymphoma, cutaneous T-cell lymphoma), and leukemias (e.g., pilocytic cell leukemia, chronic lymphocytic leukemia (CLL), acute myeloid leukemia). AML (AML), Chronic Myeloid Leukemia (CML), Acute Lymphoblastic Leukemia (ALL), Myelodysplastic Syndrome (MDS), Kaposi's Sarcoma, Retinoblastoma, Gastric Cancer, Urothelial Carcinoma, 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 Carcinoma of the Head and Neck, Endometrial Cancer, Bladder Cancer, Cervical Cancer, Liver Cancer Examples include hepatocellular carcinoma. In some embodiments, the cancer is lymphoma. In some non-limiting embodiments, the lymphoma is Burkitt lymphoma. In some embodiments, the subjects are identified or diagnosed with familial cancer syndromes such as Lie-Fraumeni syndrome, familial breast and ovarian cancer (BRCA1 or BRCA2 mutation). The disclosed methods are also useful for treating non-solid tumors. Exemplary solid tumors include malignant tumors of various organ systems such as the lung, breast, lymphoid, gastrointestinal (e.g., colon), and urogenital (e.g., kidney, urothelial, or testicular tumors), pharynx, prostate, and ovaries (e.g., sarcomas, adenocarcinomas, and carcinomas). Exemplary adenocarcinomas include colorectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, and small intestine cancer.

[0199] Exemplary cancers described by the National Cancer Institute include: acute lymphoblastic leukemia (adult), acute lymphoblastic leukemia (child), acute myeloid leukemia (adult), adrenocortical carcinoma (adrenocortical carcinoma) (child), AIDS-associated lymphoma (AIDS-associated malignant tumor), anal cancer, astrocytoma (pediatric cerebellar), astrocytoma (pediatric brain), cholangiocarcinoma (extrahepatic), bladder cancer (pediatric), bone cancer, osteosarcoma / malignant fibrous histiocytoma, brainstem glioma (child), brain tumor (adult), brain tumor (brainstem glioma) (child), brain tumor (cerebellar astrocytoma) (child), brain tumor (cerebral astrocytoma / malignant glioma) (child), brain tumor (ependymoma) (child), brain tumor (medulloblastoma), Children, brain tumors, supratentorial primitive neuroectodermal tumors, children, brain tumors, visual pathway and hypothalamic gliomas, children, brain tumors, children (other), breast cancer, breast cancer and pregnancy, breast cancer, children, breast cancer, men, bronchial adenoma / carcinoid, children, carcinoid tumors, children, carcinoid tumors, gastrointestinal, carcinoma, adrenal cortex, carcinoma, islet cells, carcinoma of unknown primary origin, central nervous system lymphoma, primary, cerebellar astrocytoma, children, cerebral astrocytoma / malignant glioma, children, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, clear cell sarcoma of the tendon sheath, colon cancer, colorectal cancer, children, cutaneous T Cellular lymphoma, endometrial cancer, ependymoma, pediatric, epithelial carcinoma, ovarian, esophageal cancer, esophageal cancer, pediatric, Ewing family tumors, extracranial germ cell tumors, pediatric, extragonadal germ cell tumors, extrahepatic cholangiocarcinoma, ocular cancer, intraocular melanoma, ocular cancer, retinoblastoma, gallbladder cancer, gastric (stomach) cancer, gastric (stomach) cancer, pediatric, gastrointestinal carcinoid tumors, germ cell tumors, extracranial, pediatric, germ cell tumors, extragonadal, germ cell tumors, ovarian, gestational trophoblastoma, glioma, pediatric brainstem, glioma, pediatric visual pathway and hypothalamus, hairy cell leukemia, head and neck cancer, hepatocellular (liver) carcinoma, adult (primary), hepatocellular (liver) carcinoma, pediatric (primary), ho Dikin lymphoma, adult; Hodgkin lymphoma, pediatric; Hodgkin lymphoma during pregnancy; hypopharyngeal cancer; hypothalamic and visual pathway glioma, pediatric; intraocular melanoma; islet cell carcinoma (pancreatic islets); Kaposi's sarcoma; renal cancer; laryngeal cancer; laryngeal cancer, pediatric; leukemia, acute lymphoblastic; adult; leukemia, acute lymphoblastic; pediatric; leukemia, acute myeloid; adult; leukemia, acute myeloid; pediatric; leukemia, chronic lymphocytic; leukemia, chronic myeloid; leukemia, hairy cell; lip and oral cancer; liver cancer, adult (primary); liver cancer, pediatric (primary); lung cancer, non-small cell; lung cancer, small cell; lymphoblastic leukemia, adult acuteLymphoblastic leukemia, acute in children, lymphocytic leukemia, chronic, lymphoma, AIDS-related, lymphoma, central nervous system (primary), lymphoma, cutaneous T-cells, lymphoma, Hodgkin, adult, lymphoma, Hodgkin, pediatric, lymphoma, Hodgkin during pregnancy, lymphoma, non-Hodgkin, adult, lymphoma, non-Hodgkin, pediatric, lymphoma, non-Hodgkin during pregnancy, lymphoma, primary central nervous system, macroglobulinemia, Waldenström, male breast cancer, malignant mesothelioma, adult, malignant mesothelioma, pediatric, malignant thymoma, medulloblastoma, pediatric, melanoma, melanoma, intraocular, Merkel cell carcinoma, mesothelioma, malignant, primary Unknown metastatic squamous cell carcinoma of the neck, multiple endocrine neoplasia syndrome, pediatric, multiple myeloma / plasmacytic neoplasm, mycosis fungoides, myelodysplastic syndrome, myeloid leukemia, chronic, myeloid leukemia, acute pediatric, multiple myeloma, myeloproliferative disorder, chronic, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, nasopharyngeal cancer, pediatric, neuroblastoma, non-Hodgkin lymphoma, adult, non-Hodgkin lymphoma, pediatric, non-Hodgkin lymphoma during pregnancy, non-small cell lung cancer, oral cancer, pediatric, oral and lip cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, pediatric, ovarian epithelial carcinoma, ovarian germ cell tumor, low-grade ovarian tumor, pancreatic cancer, pancreatic Pancreatic cancer, pediatric, pancreatic cancer, islet cell carcinoma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pheochromocytoma, pineal and supratentorial primitive neuroectodermal tumors, pediatric, pituitary tumors, plasma cell tumors / multiple myeloma, pleuroblastoma, pregnancy and breast cancer, pregnancy and Hodgkin lymphoma, pregnancy and non-Hodgkin lymphoma, primary central nervous system lymphoma, primary liver cancer, adult, primary liver cancer, pediatric, prostate cancer, rectal cancer, renal cell (kidney) carcinoma, renal cell carcinoma, pediatric, renal pelvis and ureter, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, pediatric, salivary gland cancer, salivary gland cancer, pediatric, sarcoma, Ewing family of tumors, sarcoma, carcinoma Positive, sarcoma (osteosarcoma) / malignant fibrous histiocytoma of bone, sarcoma, rhabdomyosarcoma, pediatric, sarcoma, soft tissue, adult, sarcoma, soft tissue, pediatric, Sézary syndrome, skin cancer, skin cancer, pediatric, skin cancer (melanoma), skin cancer, Merkel cells, small cell lung cancer, small intestine cancer, soft tissue sarcoma, adult, soft tissue sarcoma, pediatric, squamous cell carcinoma of unknown primary origin in the neck, metastatic, gastric (stomach) cancer, gastric (stomach) cancer, pediatric, supratentorial primitive neuroectodermal tumor, pediatric, T-cell lymphoma, skin, testicular cancer, thymoma, pediatric, thymoma, malignant, thyroid cancer, thyroid cancer, pediatric, transitional cell carcinoma of the renal pelvis and ureter, choriocarcinoma, pregnancy-related,These include cancers of unknown primary site in children, rare cancers in children, ureteral and renal pelvis, transitional cell carcinoma, urethral cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, pediatric vulvar cancer, Waldenström macroglobulinemia, and Wilms' tumor.

[0200] Further exemplary cancers include diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL).

[0201] The aforementioned cancer metastases can also be treated and prevented by the methods described herein.

[0202] In some embodiments, these methods may result in a reduction in the number, severity, or frequency of one or more symptoms of the target cancer (for example, compared to the number, severity, or frequency of one or more symptoms of the target cancer before treatment).

[0203] In some embodiments of any of the methods described herein, the method further includes administering an additional therapeutic agent (e.g., one or more therapeutic agents listed in Table 2) to the target. [Table 2] TIFF2026086743000004.tif242160TIFF2026086743000005.tif242162

[0204] Composition / kit Also provided herein are compositions (e.g., pharmaceutical compositions) comprising any of the ACCs described herein and one or more (e.g., 1, 2, 3, 4, or 5) pharmaceutically acceptable carriers (e.g., any of the pharmaceutically acceptable carriers described herein), diluents, or excipients.

[0205] In some embodiments, a composition containing any of the ACCs described herein (e.g., a pharmaceutical composition) may be placed in a sterile vial or a pre-filled syringe.

[0206] In some embodiments, a composition comprising any of the ACCs described herein (e.g., a pharmaceutical composition) may be formulated for different routes of administration (e.g., intravenous, subcutaneous, intramuscular, intraperitoneal, or intratumor administration).

[0207] In some embodiments, any of the pharmaceutical compositions described herein may include one or more buffers (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 pharmaceutically acceptable carrier (e.g., bacteriostatic water, PBS, or saline).

[0208] As used herein, the term “pharmaceutically acceptable carrier” refers to any solvent, dispersion, coating, antimicrobial agent, isotonic agent, and absorption retarder that is suitable for pharmaceutically acceptable administration. Suitable carriers include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and about 5% human serum albumin.

[0209] In some embodiments of any of the pharmaceutical compositions described herein, any of the ACCs described herein are prepared together with carriers that prevent rapid removal from the body, for example, as sustained and controlled-release formulations including implants and microencapsulation delivery systems. Biodegradable and biocompatible polymers, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Methods for preparing such pharmaceutical compositions and formulations will be obvious to those skilled in the art.

[0210] Furthermore, this specification provides a kit comprising any ACC described herein, any composition comprising any ACC described herein, or any pharmaceutical composition comprising any ACC described herein. Also provided are kits comprising, in addition to the ACC described herein, one or more second therapeutic agents selected from Table 2. The second therapeutic agent may be provided in a dosage form separate from the ACC, or the second therapeutic agent may be prescribed together with the ACC.

[0211] Any kit described herein may include instructions for using any of the compositions described herein (e.g., pharmaceutical compositions) and / or any of the ACCs. In some embodiments, the kit may include instructions for carrying out any of the methods described herein. In some embodiments, the kit may include at least one dose of any of the compositions described herein (e.g., pharmaceutical compositions). In some embodiments, the kit may provide a syringe for administering any of the pharmaceutical compositions described herein.

[0212] Examples The present invention is further illustrated by the following embodiments, which do not limit the scope of the invention as described in the claims. Example 1: Production of Activatable Cytokine Constructs The activatable cytokine construct IFNα2b-1204DNIdl-hIgG4 was prepared by recombinant DNA. The first and second monomer constructs of this ACC are identical, and each is a polypeptide having the amino acid sequence (SEQ ID NO: 309) shown in Figure 3. Each of the first and second monomer constructs contains, from the N-terminus to the C-terminus, a signal sequence derived from the mouse IgG kappa signal sequence (residues 1-20 of SEQ ID NO: 309), a mature cytokine protein corresponding to human interferon α2b (SEQ ID NO: 1), a cleavable moiety having the amino acid sequence of SEQ ID NO: 99, a linker with 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 host cells with a polynucleotide having the sequence of SEQ ID NO: 310, and the resulting recombinant host cells were subsequently cultured. Dimerization of the expressed polypeptide yielded the activatable cytokine construct, IFNα2b 1204DNIdl-hIgG4.

[0213] The activatable cytokine construct IFNα2b 1490DNI-hIgG4 was also prepared by recombinant DNA. The first and second monomer constructs of this ACC are also identical, and each is a polypeptide having the amino acid sequence (SEQ ID NO: 311) shown in Figure 4. Each of the first and second monomer constructs of this ACC contains, from the N-terminus to the C-terminus, a signal sequence derived from the mouse IgG kappa signal sequence (residues 1-20 of SEQ ID NO: 309), a mature cytokine protein corresponding to human interferon α2b (SEQ ID NO: 1), a cleavable moiety 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 host cells with a polynucleotide having the sequence of SEQ ID NO: 312, and the resulting recombinant host cells were subsequently cultured. Dimerization of the resulting expressed polypeptide produced the activatable cytokine construct, IFNα2b 1204dl-hIgG4.

[0214] An additional activatable cytokine construct containing five additional amino acid residues in the linker was prepared.

[0215] Electrophoresis was performed on the activatable cytokine construct and the protease-treated activatable cytokine construct. Figure 6 shows the gel, which, from left to right, is: (1) ACC IFNα2b-1204DNIdl-hIgG4 ("1204"); (2) MT-SP1 treated IFNα2b-1204DNIdl-hIgG4 ("1204 MT-SP1"); (3) uPA treated IFNα2b-1204DNIdl-hIgG4 ("1204 uPA"); (4) IFNα2b-1204DNIdl-hIgG4 (1204+1) with 5 amino acid residues added to the linker; (5) MT-SP1 treated IFNα2b-1204DNIdl-hIgG4 ("1204+1 MT-SP1"); (6) uPA treated IFNα2b-1204DNIdl-hIgG4 ("1204+1 uPA"); (7) IFNα2b The results for (8) MT-SP1 treated IFNα2b 1490DNI-hIgG4 ("1490 MT-SP1") and (9) uPA treated IFNα2b 1490DNI-hIgG4 ("1490 uPA") are shown. The results suggest that the protease was effective in cleaving the cleavable portion of the activatable cytokine construct.

[0216] Example 2. IFNα2b activity of the activatable cytokine construct The activity of ACC described in Example 1 was tested using a cell-based reporter assay against human type I interferon. IFN-responsive HEK293 cells were generated by stably transfecting with human STAT2 and IRF9 genes to obtain a fully active type I IFN signaling pathway. The cells also possess 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 blastosidine, 100 μg / mL zeosin, and 100 μg / mL normosine. Addition of type I IFN to these cells activated the JAK / STAT / ISGF3 pathway, subsequently inducing SEAP production, which can be readily assessed in the supernatant using colorimetric detection of alkaline phosphatase activity with Quanti-Blue solution. Using this reporter assay, the activity of IFNα2b-containing ACC was compared to the activity of Cilatron® (pegylated interferon α2b). The data in Figure 7 shows that the IFNα2b activity of ACC was significantly reduced compared to that of Cilatron® (pegylated interferon α2b).

[0217] Furthermore, the data in Figures 8A and 8B demonstrate that the activity of (uncleaved) ACC could be regulated by altering the length of the linker or linking region. The data in Figures 8A and 8B show the results of IFNα2b-hIgG4 Fc fusion constructs with or without a variable linker length between IFNα2b and hIgG4 Fc, tested in the HEK293 reporter assay. The fusion proteins tested in this experiment consist of a mature IFNα2b cytokine sequence, an optional linker and / or cleavable moiety, and the Fc domain of human IgG4 of SEQ ID NO: 4 (including a full hinge region such that the N-terminus of the Fc sequence begins with the amino acid sequence ESKYGPPCPPC···). The first construct (linking region = 7) has no linker or cleavable moiety; its sequence consists of SEQ ID NO: 1 fused to SEQ ID NO: 4, from N to C-terminus. The second construct (linking region = 12) has a 5-amino acid linker SGGGG (SEQ ID NO: 335); its sequence consists of SEQ ID NO: 1 fused to SEQ ID NO: 4, which is then fused to SEQ ID NO: 4, and then fused to SEQ ID NO: 1. The third construct (linking region = 18) contains a 7-amino acid CM (SGRSDNI) and a 4-amino acid linker GGGS; its sequence consists of SEQ ID NO: 1 fused to SEQ ID NO: 100, which is then fused to SEQ ID NO: 4, which is then fused to SEQ ID NO: 4, and then fused to SEQ ID NO: 100, and then fused to SEQ ID NO: 4, and then fused to SEQ ID NO: 1. The fourth construct (linking region = 23) contains a 5-amino acid linker, a 7-amino acid CM, and a 4-amino acid linker; its sequence consists of SEQ ID NO: 1 fused to SEQ ID NO: 335, which is then fused to SEQ ID NO: 335, which is then fused to SEQ ID NO: 100, which is then fused to SEQ ID NO: 4, which is then fused to SEQ ID NO: 4, and then fused to SEQ ID NO: 2. The fifth construct (linking region = 24) contains a 13-amino acid CM (ISSGLLSGRSDNI) and a 4-amino acid linker; its sequence consists of SEQ ID NO: 1 fused to SEQ ID NO: 68 fused to SEQ ID NO: 2 fused to SEQ ID NO: 4 fused to SEQ ID NO: 4, and SEQ ID NO: 1 fused to SEQ ID NO: 4

[0218] Example 3: In vitro antiproliferative effect of ACC on cancer cells The antiproliferative effects of IFNα2b and IFNα2b-containing ACC were tested using Daudi cells, a cell line of human B-cell lymphoblastic origin. Daudi cells were placed in RPMI-1640 medium supplemented with 10% FBS in 2x10⁶ units. 5 The solution was prepared at a concentration of cells / mL, and 50 μL of the solution was pipetteed into the wells of a white, flat-bottomed, 96-well plate (10K / well). The ACC to be tested or the control was diluted in RPMI-1640 medium supplemented with 10% FBS. A double 5-fold serial dilution was prepared, and 50 μL of this was added to each well. After incubation at 37°C for 3 days, intracellular ATP levels were measured using a viability kit as an estimate of the number of remaining viable cells. 100 μL of cell-titer go was added directly to the plate and then placed in an orbital shaker for 10 minutes. After this incubation, the luminescence signal was measured directly using an Envision plate reader. Dose-response curves were generated, and EC50 values ​​were obtained by sigmoid fitting of nonlinear regression using GraphPad Prism software. Specific activity was determined by comparing the EC50 values ​​with recombinant IFNα2b or pharmaceutical-grade cilatron® (pegylated interferon α2b).

[0219] The antiproliferative activity of IFNα2b-containing ACC in Daudi lymphoma cells showed that the IFNα2b activity of uncleaved ACC was reduced compared to that of Cilatron® (pegylated interferon α2b) (Figure 9).

[0220] The data in Figures 10A and 10B also showed that (uncleaved)ACC activity could be regulated by altering the linker length. IFNα2b-hIgG4 Fc fusion protein constructs with varying linker lengths between species IFNα2b and hIgG4 Fc, or with no linker at all, were tested in vitro using Daudi cells. The data indicate that the length of the flexible linker and the linking region (LR) between the cytokine and the Fc domain affected (uncleaved)ACC activity. Constructs with zero linker, or short linker, and correspondingly short LR, showed reduced cytokine activity, while constructs with longer linker and therefore longer LR exhibited higher levels of cytokine activity. The results are shown in Figures 10A and 10B. The fusion protein constructs are the same as those described in Example 2 above with respect to Figures 8A and 8B.

[0221] Example 4: Activity of protease-treated ACC Protease-treated IFNα2b-containing ACC was tested for antiproliferative response using Daudi lymphoma cell-based assays and cell-based reporter assays to determine whether its activity could be restored. To cleave the dimerization domain, IFNα2b-containing ACCs were treated overnight at 37°C with recombinant human proteases such as urokinase-type plasminogen activator (uPA) or matryptase (MT-SP1). A cocktail of protease inhibitors was added to neutralize the proteases before testing for activity as described in Examples 2 and 3. The results from these assays demonstrate that treatment of IFNα2b-containing ACCs with proteases can restore activity to levels comparable to recombinant cytokines. The EC50 values ​​for ACC IFNα2b-1204DNIdl-hIgG4, ACC IFNα2b-1204DNIdl-hIgG4+uPA, and Stem Cell IFNα2b (human recombinant IFNα2b, available from StemCell Technologies, Catalog #78077.1) were calculated from the Daudi apoptosis assay results and are shown in Table 3 below. [Table 3] The EC50 values ​​for ACC IFNα2b-1204DNIdl-hIgG4, ACC IFNα2b-1204DNIdl-hIgG4+uPA, and Stem Cell IFNα2b were calculated from the IFNα / β assay results and are shown in Table 4 below. [Table 4]

[0222] These results indicate that in the absence of activated protease, the activity of IFNα2b-1204DNIdl-hIgG4 is significantly reduced compared to the IFNα2b control.

[0223] Example 5: Universal ProIFN An ACC according to this disclosure, possessing a universal interferon sequence (ProC859) (IFNαAD 0AA 1204DNIdL 0AA IgG4) active against both human and mouse cells, was prepared by recombinant method. The first and second monomeric constructs of this ACC are identical and are polypeptides, each having an amino acid sequence (SEQ ID NO: 323 and a signal sequence at its N-terminus). Each of the first and second monomeric constructs contains, from N-terminus to C-terminus, a signal sequence, a mature cytokine protein corresponding to the universal interferon molecule which is a hybrid of IFNα1 and IFNα2a (SEQ ID NO: 324), a cleavable moiety having the amino acid sequence of SEQ ID NO: 100, and a dimerization domain corresponding to human IgG Fc (SEQ ID NO: 3). The activity of the universal ProIFN was tested in vitro using IFN-responsive HEK293 cells and B16 mouse melanoma cells.

[0224] The activity of ProC859 was reduced by at least 150X compared to mouse IFNα4. Protease activation by uPa restored activity to a level comparable to that of mouse IFNα4, as shown in Figure 19. The EC50 values ​​of ACC ProC859, ACC ProC859+uPA, and mouse IFNα4 were calculated from the assay results and are shown in Figure 19. [Table 5]

[0225] Example 6: In vitro characterization of lead compound ACC ProC440: The activatable cytokine construct ProC 440 (N IFNα2b 0 1204DNIdL 0AA Fc) was also prepared by recombinant method. The first and second monomer constructs of this ACC are identical, each being a polypeptide having the amino acid sequence of SEQ ID NO: 313 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, a mature cytokine protein corresponding to human interferon α2b (SEQ ID NO: 1), a cleavable moiety having the amino acid sequence of SEQ ID NO: 100, and a dimerization domain corresponding to human IgG Fc (SEQ ID NO: 3).

[0226] The activity of ProC440 was tested in vitro using IFN-responsive HEK293 cells and Daudi cells as described above. In both assays, the activity of ProC440 was reduced by at least 1,000X compared to Stem Cell IFNα2b (Figure 13). Protease activation by uPA restored activity to a level comparable to that of recombinant cytokine (IFNα2b shown in Figure 13). The EC50 of ACC ProC440, ACC ProC440+uPA, and Stem Cell IFNα2b was calculated from the IFNα / β assay results and is shown in Table 5 below. [Table 6]

[0227] The EC50 values ​​for ACC ProC440, ACC ProC440+uPA, and Stem Cell IFNα2b were calculated from the results of the Daudi apoptosis assay and are shown in Table 6 below. [Table 7]

[0228] Mass spectrometry confirmed uPA-mediated cleavage at the predicted site during CM (Figures 14A and 14B). In addition to sensitivity to uPA activation, ProC440 is also cleaved by MMP4 (Figures 14A and 14B). Mass spectrometry identified the MMP14 cleavage site at the tip of the C-terminus of IFNα, near the cleavable region (Figure 14B). Protease activation by MMP14 restored activity to levels comparable to recombinant cytokines. Together, this indicates that ACC ProC440 can fully recover its activity after cleavage of the cleavable region, which is uniquely modified by at least uPA and MMP14.

[0229] ACC ProC657 (N IFNα2b 0AA 1204DNIdL 0AA IgG4 KiHSS) was also prepared by recombinant method. The first monomer construct of this ACC is a polypeptide having the amino acid sequence of SEQ ID NO: 314 and a signal sequence at its N-terminus. From the N-terminus to the C-terminus, this first monomer construct of ACC includes, from the N-terminus to the C-terminus, a signal sequence, a mature cytokine protein corresponding to human interferon α2b (SEQ ID NO: 1), a cleavable moiety having the amino acid sequence of SEQ ID NO: 68, and a dimerization domain corresponding to human IgG Fc with a knob mutation (SEQ ID NO: 315). The second monomer construct of this ACC is a polypeptide having the amino acid sequence of SEQ ID NO: 322 and a signal sequence at its N-terminus. From the N-terminus to the C-terminus, this second monomer construct includes, from the N-terminus to the C-terminus, a signal sequence, a stub moiety (SEQ ID NO: 317), and a dimerization domain corresponding to human IgG Fc with a hole mutation (SEQ ID NO: 316).

[0230] The activity of ProC657 was tested in vitro using IFN-responsive HEK293 cells as described above. The activity of ProC657 was decreased compared to Stem Cell IFNα2b or uPA-activated ProC440, but increased compared to ProC440 (Figure 15). Therefore, this disclosure provides various structures of ACC that allow for the controllability of the level of decrease in ACC activity.

[0231] Example 7: In vivo antiproliferative activity of ACC: The in vivo antiproliferative effect of IFNα2b ACC ProC440 was tested using the Daudi xenograft tumor model. Beige mice were given 10x10 in serum-free medium (1:1 Matrigel). 6 Daudi cells were subcutaneously transplanted. The average tumor volume was approximately 60-120 mm². 3 Once mice reached a certain threshold, they were randomized and administered ProC440 once weekly for 5 weeks. Body weight and tumor measurements were recorded twice weekly during the study period. The data in Figure 16 show that IFNα2b-containing ACC ProC440 induced complete tumor regression at a low dose of 0.1 mg / kg and reduced the rate of tumor growth at a dose of 0.02 mg / kg (top panel), and the antiproliferative effect of cilatron® is shown for comparison (bottom panel).

[0232] Example 8: In vivo tolerance activity of ACC Human IFNα2b has been previously shown to cross-react with the hamster IFNα receptor and be active in hamsters (Altrock et al, Journal of Interferon Research, 1986). To evaluate the tolerability of IFNα2b-containing ACC ProC440, Syrian Gold Hamsters were administered a starting dose of 0.4 mg / kg. Animals received one dose of the test product and were kept tested for up to 7 days post-administration unless unacceptable toxicity (DLT, meaning dose-limiting toxicity) was observed. The starting dose (0.4 mg / kg ("mpk")) represents the equivalent amount of INFα-con (recombinant interferon α, non-naturally occurring type I interferon, manufactured by Amgen under the name Infergen®) that is expected to induce weight loss, reduced food intake, and myelosuppression in hamsters (125g). In cynomolgus macaques (cyno), 0.1 mg / kg / day of INFα-con is associated with weight loss, decreased food intake, and bone marrow suppression (1.25-2.5 x 10 for a 125g hamster). 7 (Equivalent to U). If the initial dose was acceptable, the animals were advanced to an "intermediate dose" of 2 mg / kg and received three doses of the test substance unless unacceptable. If acceptable, the animals were advanced to a "high dose" of 10 mg / kg and received three doses of the test substance unless unacceptable. If acceptable, the animals were advanced to an "even higher dose" of 15 mg / kg. At each stage, if the test dose was unacceptable, the animals were withdrawn to the next lower dose. If the initial dose was unacceptable, the animals were withdrawn to an "even lower dose" of 0.08 mg / kg. The animals were administered ACC(ProC286) having an N-to-C-terminal structure of the DD-CM-CP dimer. As a negative control, the animals were administered human IgG4. As expected, the negative control showed no toxicity in the animals.

[0233] ProC286 (ChIgG4 5AA 1204DNIdL IFNα2b) was also prepared by recombinant method. The first and second monomer constructs are identical, each being 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 contains, 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 moiety 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 α2b (SEQ ID NO: 1).

[0234] ProC291 (NhIgG4 5AA 1204DNIdL IFNα2b) was also prepared by recombinant method. The first and second monomer constructs are identical. Each of the first and second monomer constructs contains, from N-terminus to C-terminus, a human IgG4 Fc region (SEQ ID NO: 4) containing a mature cytokine protein corresponding to human interferon α2b (SEQ ID NO: 1), a linker (SEQ ID NO: 321), a CM (SEQ ID NO: 100), a linker (GGGS), and a complete hinge sequence.

[0235] The activity of ProC286 and ProC291 was compared to that of Cilatron® (PEG-IFNα2b) in the Daudi apoptosis assay (Figures 17A and 17B). In this assay, ProC286 and Cilatron® showed similar levels of activity, as shown in Figure 17A. This indicates that ProC286 has similar activity to pegylated IFNα2b and can be used as a substitute for the Cilatron® control to assess IFNα2b tolerance in hamster studies. ProC291 showed reduced activity compared to ProC286 and Cilatron®, indicating that the structural arrangement of the IFN N-terminus to the Fc is important for the reduction in activity. That is, when the DD has a pair of Fc domains, positioning the cytokine at the N-terminus relative to the DD (as in ProC291) can result in a greater decrease in cytokine activity than when the cytokine is positioned at the C-terminus relative to the DD (as in ProC286).

[0236] Animals were administered an initial dose of 0.4 mg / kg on day 1. The study was continued for one week unless non-tolerable toxicity (DLT) was reached. Clinical observation, body weight, and temperature measurements were performed for each animal before administration, and at 6, 24, 72 hours, and 7 days after administration. Blood samples for hematological and chemical analysis were collected for each animal at 72 hours and 7 days after administration. Hematological and chemical analyses were performed immediately after sampling. For hematological analysis, blood smears, variance in blood cell counts, hematocrit, hemoglobin, mean corpuscular hemoglobin, mean cytological volume, platelet count, erythrocyte count, erythrocyte distribution width, reticulocyte count, and leukocyte count were evaluated. The clinical chemistry panel included measurements of alanine aminotransferase, albumin, albumin / globulin ratio, alkaline phosphatase, aspartate aminotransferase, calcium, chloride, cholesterol, creatine kinase, creatine, γ-glutamyltransferase, globulin, glucose, inorganic phosphorus, potassium, sodium, total bilirubin, total protein, triglycerides, urea, nitrogen, and C-reactive protein. Evidence of toxicity in tolerability testing is summarized in Figures 22-24.

[0237] Overall, animals administered with the unmasked ProC286 construct showed a mean weight loss of 5% when administered at 2 mpk and 15% when administered at 10 mpk and 15 mpk (Figure 22). One animal administered with ProC286 at 15 mpk showed a 20% weight loss 7 days after administration (at the end of the study). This is considered an unacceptable dose. In contrast, animals administered with ProC440 at 2 mpk and 10 mpk did not show weight loss.

[0238] Animals administered ProC440 at 15 mpk showed an average weight loss of 5% (Figure 22). This unexpectedly demonstrates that the ACC of this disclosure, having a dimerized structure beginning at the N-terminus, CP-CM-DD, suppresses IFNα2b-mediated weight loss. While not intended to be theoretically bound, it is thought that placing the interferon at the N-terminus of DD and using a relatively short LR suppresses cytokine activity in the case of ProC440, thereby reducing interferon toxicity compared to pegylated IFNα2b (Cilatron®) or ProC286.

[0239] From a clinical chemistry perspective, animals administered with ProC286 showed a significant increase in alkaline phosphatase (ALP) at all doses (0.4 mpk, 2 mpk, 10 mpk, and 15 mpk) 7 days after administration (end of the study) (Figure 23). No significant increase in ALP was measured when animals were administered 10 mpk or 15 mpk of ProC440 (Figure 23). Elevated ALP is a marker of hepatotoxicity. IFNα2b has been shown to induce hepatotoxicity. This suggests that ACC of this disclosure, which has a dimerized structure starting at the N-terminus, CP-CM-DD, unexpectedly suppresses IFNα2b-mediated hepatotoxicity.

[0240] From a hematological perspective, animals administered ProC286 at 2mpk, 10mpk, and 15mpk showed significant decreases in reticulocyte, neutrophil, and white blood cell (WBC) counts at 3 and 7 days post-administration (end of the study) (Figure 24). These decreases are reminiscent of IFNα2b-mediated myelotoxicity. Animals administered ProC440 at 3 days post-administration also showed decreases in reticulocyte, neutrophil, and white blood cell (WBC) counts (Figure 24). Overall, the level of hematopoietic cell decrease observed in animals administered ProC440 was not as pronounced as the level of decrease observed in animals administered ProC286. Seven days after administration (end of the study), in animals administered ProC440, overall levels of reticulocyte count, neutrophil count, and white blood cell (WBC) count returned to normal levels or to levels similar to those observed in animals administered the negative control IgG4 (Figure 24). In animals administered ProC286, reticulocyte count, neutrophil count, and white blood cell (WBC) count remained low. This indicates that the ACC of this disclosure, which has a dimerized structure starting at the N-terminus, CP-CM-DD, unexpectedly suppresses IFNα2b-mediated myelotoxicity.

[0241] Example 4. In vitro characterization of additional cytokine constructs Additional activatable cytokine constructs 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, from the N-terminus to the C-terminus, included a signal sequence derived from the mouse IgG kappa signal sequence (residues 1-20 of SEQ ID NO: 309), a mature cytokine protein corresponding to human interferon α2b (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). Additionally, these ACCs included, or did not include, a linker having the amino acid sequence SGGGG between the CP and the CM. These ACCs included, or did not include, a linker having the amino acid sequence GGGS between the CM and the DD. These ACCs also included, or did not include, a portion of the hinge of the DD from the N-terminus to cysteine 226. These additional activatable cytokine constructs are described in Table 6 (see SEQ ID NOs: 336-342 and SEQ ID NO: 313).

Table 8

[0242] The activity of ProC440, an ACC having an Fc region truncated to cysteine 226 without a flexible linker, 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 of ProC440 (e.g., the anti-proliferative effect) was reduced compared to all other ACCs containing various additional sequences between the cytokine and the first amino acid (i.e., cysteine 226) that binds the DD to the corresponding second monomer. The EC50 values of the ACCs were calculated from the IFNα / β assay results and are shown in Table 7 below.

Table 9

[0243] The EC50 values of the ACCs were calculated from the Daudi apoptosis assay results and are shown in Table 8 below.

Table 10

[0244] While not intending to be bound by theory, based on the results shown herein, the inventors predict that placing the cytokine at the N-terminus of DD and using a relatively short LR will suppress cytokine activity for various cytokines in addition to the interferon α cytokine exemplified in the specific embodiments described above. As described above, the invention described herein encompasses activatable cytokine constructs comprising the various cytokine proteins discussed herein. By way of non-limiting example, the CP used in the ACC of the present invention can be any of those enumerated in SEQ ID NOs: 101-209, and their variants. In particular, monomeric cytokines are suitable for use in the ACC described herein. Based on the results provided herein, the ACC of the present invention exhibits reduced cytokine activity compared to the corresponding wild-type cytokine, and upon cleavage of the ACC by the relevant protease, the cleavage products are thought to recover cytokine activity similar to the corresponding wild-type cytokine.

[0245]

Table 11

[0246] Other Embodiments While the present invention has been described in detail, it should be understood that the foregoing description is intended to illustrate, and not to limit, the scope of the invention as defined by the attached claims. Other aspects, advantages, and modifications fall within the scope of the following claims.

Claims

[Claim 1] An activatable cytokine construct (ACC) comprising a first monomer construct and a second monomer construct, (a) The first monomer construct comprises a first mature cytokine protein (CP1), a first cleavable portion (CM1), and a first dimerization domain (DD1), (b) The second monomer 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 comprising CP1, CM1, and DD1 in the direction from N to the C terminus, wherein the first monomer construct is characterized in that CP1 and DD1 are linked by a linking region having 18 amino acids or less, and the linking region having 18 amino acids or less includes CM1. (d) Furthermore, (i) The second monomer construct is the same as the first monomer construct, (ii) The DD1 and DD2 are a pair of human IgG Fc domains, (e) The DD1 and DD2 are covalently bonded to each other via at least one disulfide bond, thereby forming dimers of the first monomer construct and the second monomer construct, and (f) The ACC is characterized in that it has at least one reduced level of activity of CP1 and CP2 compared to at least one corresponding control level of activity of CP1 and CP2. Activatable cytokine constructs (ACCs).

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